anna saramÄki regulation of the p21cip1/waf1 (cdkn1a) … · myc v-myc myelocytomatosis viral...

ANNA SARAMÄKI

Regulation of the p21CIP1/WAF1 (CDKN1A)Gene at the Chromatin Level by

1α,25-dihydroxyvitamin D3

JOKAKUOPIO 2008

KUOPION YLIOPISTON JULKAISUJA C. LUONNONTIETEET JA YMPÄRISTÖTIETEET 246KUOPIO UNIVERSITY PUBLICATIONS C. NATURAL AND ENVIRONMENTAL SCIENCES 246

Doctoral dissertation

To be presented by permission of the Faculty of Natural and Environmental Sciences

of the University of Kuopio for public examination in Auditorium L21,

Snellmania building, University of Kuopio,

on Friday 5th December 2008, at 12 noon

Department of BiosciencesUniversity of Kuopio

Distributor : Kuopio University Library P.O. Box 1627 FI-70211 KUOPIO FINLAND Tel. +358 40 355 3430 Fax +358 17 163 410 http://www.uku.fi/kirjasto/julkaisutoiminta/julkmyyn.html

Series Editors: Professor Pertti Pasanen, Ph.D. Department of Environmental Science

Professor Jari Kaipio, Ph.D. Department of Physics

Author’s address: Department of Biosciences University of Kuopio P.O. Box 1627 FI-70211 KUOPIO FINLAND Tel. +358 40 355 3084 E-mail : [email protected]

Supervisors: Professor Carsten Carlberg, Ph.D. Department of Biosciences University of Kuopio

Dos. Sami Väisänen, Ph.D. Department of Biosciences University of Kuopio

Reviewers: Luciano Adorini, M.D. Intercept Pharmaceuticals Corciano (Perugia), Italy Professor Alberto Muñoz, Ph.D. Instituto de Investigaciones Biomédicas “Alberto Sols” Madrid, Spain

Opponent: Professor Roger Bouil lon, M.D. Afd. Exp. Geneeskunde – Endocrinologie Katholieke Universiteit Leuven Leuven Belgium

ISBN 978-951-27-1184-0ISBN 978-951-27-1099-7 (PDF)ISSN 1235-0486

KopijyväKuopio 2008Finland

Saramäki, Anna. Regulation of the p21CIP1/WAF1 (CDKN1A) gene at the chromatin level by 1α,25-dihydroxyvitamin D3. Kuopio University Publications C. Natural and Environmental Sciences 246. 2008. 100 p. ISBN 978-951-27-1184-0 ISBN 978-951-27-1099-7 (PDF) ISSN 1235-0486

Abstract Ligand-activated nuclear receptors (NRs) are transcription factors that link the regulation of gene expression directly to the body’s hormonal and nutritional status. The vitamin D receptor (VDR) is a NR activated by the hormone 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In the organism, 1α,25(OH)2D3 synthesis mostly results from the initial stimulus from the UV portion of sunlight. This solar-powered transcription factor primarily regulates mineral homeostasis but is also an important modulator of the immune-system and cellular proliferation, thereby counteracting tumorigenesis.

Regulation of the cell cycle by the cyclin-dependent kinase-inhibitor p21CIP1/WAF1 (encoded by the human gene CDKN1A, hereafter called p21) has been suggested as one of the anti-proliferative mechanisms of 1α,25(OH)2D3. Our results further establish p21 gene as a primary target of 1α,25(OH)2D3 by characterizing three novel 1α,25(OH)2D3-responsive regions residing up to 7 kbp upstream of the p21 transcription start site (TSS). These regions are ligand-dependently enriched with VDR, as assayed by chromatin-immunoprecipitation. Interestingly, the tumor suppressor p53 also binds two of these regions. Comparison of co-activator association of VDR and another NR, the peroxisome proliferator-activated receptor β/δ (PPARβ/δ) on the regulatory regions of the p21 gene and on those of the pyruvate dehydrogenase kinase (PDK) gene family, respectively, supports the stricter ligand-dependency of the VDR. The modulation of 1α,25(OH)2D3 response by co-repressors and associated histone deacetylases (HDACs) showed that combined treatment of 1α,25(OH)2D3 and the HDAC inhibitor trichostatin A (TSA) achieved maximal induction of the p21 gene and the other cyclin-dependent kinase inhibitor (CDKI) genes studied. Assays using small-inhibitory RNA (siRNA) knockdown revealed that HDAC3 and HDAC7 restricted the response to 1α,25(OH)2D3 whereas nuclear co-repressor 1 (NCoR1) and un-liganded VDR attenuated the response to TSA.

We also provide evidence that the p21 gene derived steady state mRNA expression responds to 1α,25(OH)2D3 in a cyclical manner. This results from sequential and cyclical association of VDR, RNA polymerase II (Pol II) and co-factors on the regulatory regions of the p21 gene with simultaneous chromatin looping that connects the 1α,25(OH)2D3-responsive regions to the TSS. Inhibition of either HDAC4, the histone-specific lysine demethylase LSD1 or the MED1 subunit of the Mediator complex disturbs both the ligand-induced changes in histone patterns and the chromatin looping, which lead to the attenuation and distortment of the cyclical accumulation of mRNA transcripts. These results suggest strong interplay between histone modifications, chromatin looping and transcriptional response in gene regulation by NRs.

Concisely, this thesis further establishes the human p21 gene as a primary 1α,25(OH)2D3 target and provides insight into the mechanism of NR-dependent activation of transcription with special emphasis on chromatin modifying enzymes and chromatin looping.

Universal Decimal Classification: 575.113, 577.214, 577.218

National Library of Medicine Classification: QU 56, QU 141, QU 173, QU 470, QU 475

Medical Subject Headings: Genes; Gene Expression Regulation; Transcription Factors; Chromatin; Receptors, Cytoplasmic and Nuclear; Response Elements; Calcitriol; Receptors, Calcitriol; Histones; Histone Deacetylases; Gene Silencing; Cyclin-Dependent Kinase Inhibitor p21; Cell Cycle; Peroxisome Proliferator-Activated Receptors

Acknowledgements I would like to express my gratitude to all the people who supported and helped me to complete

this thesis.

I acknowledge the University of Kuopio and the Department of Biosciences for the opportunity

to study and to do research. Funding from the European Union Structural Fund, the County

Administrative Board of Eastern Finland, the Academy of Finland and BioXell SpA supported

this research.

I would like to express my gratitude to my principal supervisor, Prof. Carsten Carlberg for his

excellent guidance and advice during these years, which motivated me to mature into a scientist.

I thank my second supervisor and co-author Dos. Sami Väisänen for guidance to the ultimate

method, ChIP, and help with especially methodological and technical problems during all the

projects.

I would like to thank my reviewers, M.D. Luciano Adorini and Prof. Alberto Muñoz, for

improving my thesis with critical, yet helpful comments and insights.

I acknowledge Dr. Tom Dunlop for critical reading of this thesis and my manuscripts, and

especially for fruitful and winding discussions where I, eventually, learned to understand the

Scottish accent.

I am grateful to all of my co-authors. To Dr. Claire Banwell, from whom I adopted my target

gene p21 and hence the whole project, and her supervisor Prof. Moray Campbell. To Marjo

Malinen, with whom I share a big part of my mortal soul in addition to our joint publication,

which was probably the loudest manuscript ever made. To Tatjana Degehardt for discussions on

science and scientific career as well as strong peer-support. To Antti Ropponen, Markus Rieck,

Anne Huotari, Prof. Karl-Heinz Herzig and Prof. Rolf Müller for their contribution in the

publications.

I thank all the current and former group members Anssi, Antti, Christian, Claire, Ferdi, Harri L.,

Harri M., Heidi, Juha, Jussi, Katri, Lasse H., Lasse S., Laura, Maija, Mari, Marjo, Merja, Mikko,

Osku, Petra, Ruth, Sami H., Sami V., Sabine, Sarah, Sari, Sophia, Suvi, Taru, Tatjana and Tom

for all the informative, fun and motivating (and sometimes quite absurd) discussions that created

a productive atmosphere for research. Especially I want to express my gratitude to all my

students, Laura, Sari, Sarah, Ruth and Heidi, who helped me in the lab sacrificing blood, sweat

and tears, and to Maija for her indispensable technical support.

Finally I want to thank people who kept me mostly sane during my Ph. D. studies. I am most

grateful for my friends Jenni, Sanna, Anu, Katja and Jutta for the support and unwinding they

provided me with. Leena, thank you for being the best, most brainless and supporting sister one

can have. Äiti ja isä: kiitos ehdottomasta tuesta ja rakkaudesta, vaikka huomenna lyötäiskin pää

kivveen. And finally I want to express my endless gratitude towards Erno, who has encouraged

and loved me every step of the way, even and especially on times I have not deserved it.

Abbreviations

1α,25(OH)2D3 1α,25-dihydroxyvitamin D3

3C chromosome conformation capture 5-FU 5-fluorouracil AF-2 Activating Function 2 ATM ataxia-telangiectasia, mutated ATR ATM and Rad3-related AR androgen receptor BAZ1A bromodomain adjacent to zinc finger domain, 1A; also called hAcf1 or WSTF BPTF bromodomain plant homeodomain finger transcription factor cAMP Cyclic adenosine monophosphate CBP CREB-binding protein CDK cyclin-dependent kinase CDKI cyclin-dependent kinase inhibitor C/EBPα CCAAT enhancer binding protein-alpha

ChIP chromatin immuno-precipitation CoA co-activator CoR co-repressor CORO2A coronin, actin binding protein, 2A, also known as IR-10 CREB cAMP response element binding CYP24A1 cytochrome P450, family 24, subfamily A, polypeptide 1 gene CYP27A1 cytochrome P450, family 27, subfamily A, polypeptide 1 gene CYP27B1 cytochrome P450, family 27, subfamily B, polypeptide 1 gene DBD DNA-binding domain DMSO dimethyl sulfoxide DR direct repeat ER everted repeat ERα estrogen receptor α

ERRα estrogen-related receptor α

FAM 6-carboxyfluorescein

FBS fetal bovine serum GADD45A growth arrest and DNA-damage-inducible α gene

GPS2 G protein pathway suppressor 2 H3K4me histone H3, methylated at the lysine 4 position H3K9ac histone H3, acetylated at the lysine 9 position H3K14ac histone H3, acetylated at the lysine 14 position H4ac acetylated histone H4 HAT histone acetyltransferase HDAC histone deacetylase HIF-1 hypoxia-induced factor-1 hKSR-2 human kinase suppressor of Ras 2 HMT histone methyltransferase HNF4α Hepatocyte nuclear factor 4α

IGF insulin-like growth factor IGFBP insulin-like growth factor binding protein IL interleukin ING2 the inhibitor of growth family, member 2 IR inverted repeat JNK1 c-Jun N-terminal kinase 1 KAT2A lysine acetyltransferase 2A KLF Krüppel-like factor LBD ligand-binding domain LCoR ligand dependent nuclear receptor corepressor LSD1 lysine-specific histone demethylase 1, encoded by the gene AOF2 MAP3K5 mitogen-activated protein kinase kinase kinase 5 MAPK mitogen-activated protein kinase MDM2 mouse double minute 2 homolog MED1 Mediator subunit 1, also called TRAP220 or DRIP205 MRN MRE11–RAD50–NBS1 myc myc proto-oncogene protein MYC v-myc myelocytomatosis viral oncogene homolog (avian) gene NCOA1 Nuclear receptor coactivator 1, also called SRC-1

NCOA2 Nuclear receptor coactivator 2, also called SRC-2, TIF2 or GRIP1 NCOA3 Nuclear receptor coactivator 3, also called SRC-3, ACTR or RAC3 NCoR1 nuclear co-repressor 1 NER nucleotide excision repair NRIP1 nuclear receptor interacting protein 1, also called RIP140 p18 cyclin-dependent kinase inhibitor 2C (CDKN2C) gene p19 cyclin- dependent kinase inhibitor 2D (CDKN2D) gene p21 cyclin- dependent kinase inhibitor 1A (CDKN1A) gene p27 cyclin- dependent kinase inhibitor 1B (CDKN1B) gene p300 E1A binding protein p300 (EP300) p53 tumor protein p53 (TP53) PBS phosphate buffered saline PCNA proliferating nuclear antigen PDK pyruvate dehydrogenase kinase PGC-1α peroxisome proliferator-activated receptor gamma, co-activator 1α

PHD plant homeodomain PIC preinitiation complex Pol II RNA polymerase II p-Pol II phosphorylated RNA polymerase II PPAR peroxisome proliferator-activated receptor PPRE peroxisome proliferator-activated receptor response element P-TEFb Positive Transcription Elongation Factor b PTH parathyroid hormone Rb retinoblastoma 1 RE response element RELB v-rel reticuloendotheliosis viral oncogene homolog B gene ROR RAR-related orphan receptor ROS reactive oxygen species RPLP0 ribosomal protein, large, P0 gene RAR retinoic acid receptor RT room temperature RXR retinoid X receptor

siRNA small inhibitory RNA SKP45 S-phase kinase-associated protein 2 (p45) SMRT Silencing Mediator of Retinoid and Thyroid Receptors, also called NCoR2 SNW1 SNW domain containing 1, also called NCOA-62 or SKIP TBL1 transducin-β-like 1 TBL1R1 TBL1 receptor 1 TCF3 transcription factor 3 TCF/LEF T-cell factor/lymphoid enhancer factor TR thyroid hormone receptor TRPV6 transient receptor potential vanilloid type 6 gene TRRAP transformation/transcription domain-associated protein TSA trichostatin A TSS transcription start site UV ultraviolet VDR vitamin D receptor VDRE vitamin D response element VEGF vascular endothelial growth factor gene WINAC WSTF including nucleosome assembly complex YY1 YY1 transcription factor

List of original publications

This thesis is based on the following publications referred to in the text by

their roman numerals (I-IV):

I. Saramäki, A., Banwell, C.M., Campbell, M.J. and Carlberg, C. (2006) Regulation of the

human p21(waf1/cip1) gene promoter via multiple binding sites for p53 and the vitamin D3

receptor. Nucleic Acids Res. 34:543-54

II. Degenhardt, T., Saramäki, A., Malinen, M., Rieck, M., Väisänen, S., Huotari, A., Herzig,

K.H., Müller, R. and Carlberg, C. (2007) Three members of the human pyruvate

dehydrogenase kinase gene family are direct targets of the peroxisome proliferator-

activated receptor β/δ. J Mol Biol. 372:341-55.

III. Malinen, M.*, Saramäki, A.*, Ropponen, A., Degenhardt, T., Väisänen, S. and Carlberg

C. (2008) Distinct HDACs regulate the transcriptional response of human cyclin-

dependent kinase inhibitor genes to trichostatin A and 1α,25-dihydroxyvitamin D3.

Nucleic Acids Res. 36:121-32.

* Shared authorship

IV. Saramäki, A. and Carlberg, C. Cyclical chromatin looping and transcription factor

association on the regulatory regions of the p21 (CDKN1A) gene in response to 1α,25-

dihydroxyvitamin D3, Submitted

Table of contents

Abstract .........................................................................................................................1 Acknowledgements........................................................................................................3 Abbreviations ................................................................................................................5 List of original publications ..........................................................................................9 1. Introduction..........................................................................................................13 2. Review of the literature ........................................................................................17

2.1 Regulation of gene expression by nuclear receptors..........................................17 2.1.1 Classification of NRs................................................................................17 2.1.2 VDR and 1α,25(OH)2D3...........................................................................18

2.1.2.1 1α,25(OH)2D3 metabolism ...............................................................18 2.1.2.2 Structure and function of the VDR ....................................................19 2.1.2.3 Target genes and physiological role of 1α,25(OH)2D3 .......................20

2.1.3 PPARβ/δ ..................................................................................................25 2.1.4 NR co-factors ...........................................................................................26

2.1.4.1 Co-activators.....................................................................................27 2.1.4.2 Co-repressors ....................................................................................30

2.1.5 Role of chromatin in NR-mediated transcription.......................................32 2.1.5.1 Histone modifications .......................................................................32 2.1.5.2 Chromatin looping ............................................................................33

2.1.6 Models for NR-induced gene expression ..................................................34 2.2 Cyclin-dependent kinase inhibitor p21 .............................................................35

2.2.1 Functions of p21.......................................................................................35 2.2.1.1 Regulation of the cell cycle by cyclin-dependent kinase inhibitors ....35

2.2.1.1.1 p21, p16 and senescence............................................................36 2.2.1.2 Anti- and pro-apoptotic p21 functions ...............................................37 2.2.1.3 p21 in DNA repair and replication.....................................................37 2.2.1.4 Transcriptional control by p21...........................................................38 2.2.1.5 Knockout models of Cip/Kip family members...................................39 2.2.1.6 Bivalency of p21 in tumorigenesis ....................................................39

2.2.2 Regulation of p21 expression....................................................................40 2.2.3 Regulation of cell cycle by 1α,25(OH)2D3 ................................................43

3. Aims of the study ..................................................................................................47 4. Materials and Methods.........................................................................................49

4.1 Materials..........................................................................................................49 4.1.1 Cell culture...............................................................................................49 4.1.2 Ligands ....................................................................................................50 4.1.3 DNA constructs ........................................................................................50

4.2 Methods ...........................................................................................................51 4.2.1 In vitro methods .......................................................................................51

4.2.1.1 siRNA interference ...........................................................................51 4.2.2 Ex vivo analyses .......................................................................................52

4.2.2.1 RNA extraction.................................................................................52 4.2.2.2 cDNA synthesis ................................................................................52

4.2.2.3 Real-time PCR ..................................................................................53 4.2.2.4 Chromatin immuno-precipitation assay .............................................53 4.2.2.5 Chromatin conformation capture assay..............................................55 4.2.2.6 PCR of chromatin templates..............................................................56

5. Results ...................................................................................................................59 5.1 The human p21 gene is regulated by p53 and VDR via multiple response elements...........................................................................................................59 5.2 Association of nuclear receptor complexes on regulatory regions of the human PDK gene family and the human p21 gene............................................60 5.3 Role of HDACs in regulation of human CDKI genes by 1α,25(OH)2D3

and TSA in mammary cell lines .......................................................................62 5.4 The cyclical response of the human p21 gene to 1α,25(OH)2D3 at the level of transcription factor binding, chromatin looping and transcription .................65

6. Discussion..............................................................................................................71 6.1 Human p21 is a primary 1α,25(OH)2D3 target gene..........................................71 6.2 Regulation of the human p21 gene by p53 and its role in 1α,25(OH)2D3 response ...........................................................................................................72 6.3 Divergent CoA profiles associated with VDR and PPARβ/δ ............................72 6.4 Role of HDACs in regulation of CDKI genes and convergence with 1α,25(OH)2D3 response....................................................................................73 6.5 1α,25(OH)2D3 induces cyclical chromatin looping and transcription factor association on the regulatory regions of p21 .....................................................75

7. Summary and conclusions....................................................................................81 8. Future aspects.......................................................................................................83 9. References .............................................................................................................85

Kuopio Univ. Publ. C. Nat. and Environ. Sci. 246: 1-100 (2008) 13

1. Introduction

Genes carry the inheritable information in the form of DNA (or in the case of certain viruses, in

the form of RNA), which is first transcribed to a messenger molecule, mRNA. RNA in turn is

translated into an effector molecule, a protein. This Central Dogma, with its distinctive polarity,

presented by Francis Crick in 1958 suggests that the genome of an organism forms its biological

blueprint (Crick, 1958). The process of reading the blueprint, however, is far from simple. First

of all, one gene can code for multiple distinct RNA molecules via alternative exon splicing, a

phenomenon that occurred in 30 % of expressed sequences in a human embryonic kidney cell

line (Sultan et al., 2008). Secondly, a substantial portion of RNA molecules does not code for

proteins but participates in regulation of transcription, splicing, degradation or translation of

mRNA (Amaral et al., 2008). Additionally, less than 2% of the human genome sequence consists

of protein-coding genes, leaving the large majority of the genome for multiple regulatory

functions. Finally, the human genome codes for roughly the same number of genes than that of

the nematode Caenorhabditis elegans, therefore stating that the number of protein coding genes

does not correlate with an organism’s corporeal complexity (Clamp et al., 2007 These facts

emphasize the importance of the non-protein coding part of the genome, stressing the role of

regulation of gene expression.

The process of gene expression is tightly controlled at multiple levels with increasing complexity

in higher organisms. In eukaryotes, the packaging of DNA into chromatin forms the first barrier

against aberrant transcription. Chromatin structure is formed of nucleosomes, where 146 bp of

DNA is wrapped almost twice around the protein octamer that consists of core histones H2A,

H2B, H3 and H4. An additional 20 to 90 bp linker region of DNA that straddles from one

nucleosome core to another is associated with histone H1. Disrupting the nucleosomes over the

core promoter of a yeast gene is sufficient to activate transcription in the absence of activating

signals (Zhang and Reese, 2007). In addition to the barrier function, the purpose of the packaging

Anna Saramäki: Regulation of the p21 (CDKN1A) gene at the chromatin level by 1α,25-(OH)2D3

14 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 246: 1-100 (2008)

is to achieve maximal compactivity while maintaining DNA adequately accessible to regulatory

proteins and transcription (Wu et al., 2007).

The mRNA synthesis is carried out by Pol II in eukaryotes, which also transcribes a large number

of non-protein coding RNAs. The first step in the activation of transcription is the recognition of

specific DNA sequences by transcription factors that trigger the recruitment of co-activators,

basal transcription machinery, and Pol II to form a preinitiation complex (PIC). This process is

dependent on the presence of the Mediator complex, through which sequence-specific

transcription factors are able to contact the basal transcription factors and Pol II. The assembly of

the PIC begins with binding of TFIID complex to core promoter recognition sequence, such as

the TATA-box. Histone modifications on the core promoter strongly affect TFIID binding

(Vermeulen et al., 2007). Subsequently TFIIA, TFIIB and the non-phosphorylated Pol II-TFIIF

complex join the TFIID. Finally, TFIIE recruits TFIIH, which phosphorylates the serine at

position 5 in the carboxy-terminal domain of the largest subunit of Pol II (serine 5 phophorylated

Pol II). The TFIIE-TFIIH complex melts the DNA double strand on the promoter to enable the

synthesis of RNA on the DNA template (Lin and Gralla, 2005). Interestingly, the Mediator

complex promotes both the recruitment and the catalytic activity of TFIIH, leading to increased

serine 5 phophorylated Pol II. This form of Pol II is not able to bind the Mediator complex and

eventually phosphorylation leads to dissociation of the Mediator from the complex (Esnault et al.,

2008). The serine 5 phosphorylated Pol II also recruits the enzymes that cap the nascent transcript

at its 5' end. Pol II then pauses until the Positive Transcription Elongation Factor b (P-TEFb)

elongation complex phosphorylates serine 2 of the largest Pol II subunit, which is required for

both productive elongation as well as the recruitment of mRNA splicing and polyadenylation

complexes (Peterlin and Price, 2006). This multi-faceted process offers a vast amount of potential

regulatory targets, from chromatin context and its interplay with the sequence-specific and basal

transcription factors right through to the splicing, modification and transport of mRNA.

Control of the translational efficacy and post-translational modifications that modulate the

activity and stability of proteins, together with their discrete cellular compartmentalization,

constitute the last steps in the regulation of gene expression.

Introduction


In summary, the immense complexity of this process enables the multiplicity of adaptable

expression patterns that ultimately leads to distinct cell and tissue type states.


2. Review of the literature

2.1 Regulation of gene expression by nuclear receptors

The NR family of transcription factors controls all aspects of animal physiology: homeostasis,

differentiation, development, sexual maturation and the response to stress. The activity of most

NRs is regulated by small hydrophobic ligands, such as sex steroids, nutrition-derived fatty acids,

xenobiotic lipids or fat-soluble vitamins A and D, whereas other NRs appear not to have ligands.

NRs generally contain two highly conserved zinc finger motifs that recognize specific sequences

on the genome, called response elements (REs). Regulation of gene expression by NRs provides

multi-cellular animals with the ability to modify the metabolism, development and growth of

tissues based on developmental or environmental stimuli in a synchronized manner.

2.1.1 Classification of NRs

NRs form a superfamily of transcription factors with 48 members in the human genome

(Robinson-Rechavi et al., 2001). They can be classified based on ligand sensitivity (Chawla et

al., 2001), evolution of NR genes (Bertrand et al., 2004) or their physiological role as interpreted

from tissue-specific expression patterns (Bookout et al., 2006). The ligand sensitivity approach

suggests three classes: endocrine receptors with high-affinity hormonal lipids, such as the

estrogen receptors α and β (ERα, β), the androgen receptor (AR) and VDR; adopted orphan

receptors that binds to dietary lipids and xenobiotics with low affinity. This group includes the

PPARs α, β/δ and γ. As a final class, by this ligand driven definition, the orphan receptors, for

which a physiological ligand has not yet been identified, is represented by such examples as

estrogen-related receptor α (ERRα).

When the sequences of NRs are compared, the grouping significantly differs from the ligand-

centered view, e.g., VDR belongs to the same group with PPARs (group NR1), and highly

ligand-sensitive ERs and orphan ERRs are both in group NR3. On the basis of transcriptomes



derived from multiple mouse tissues, NRs can be divided into clades with distinct physiological

roles (Bookout et al., 2006). In this classification, PPARs are linked to lipid metabolism and

energy homeostasis, whereas VDR is grouped with bile acid and xenobiotic metabolism based on

its high expression in gastroentric tissues.

2.1.2 VDR and 1α ,25(OH)2D3

2.1.2.1 1α ,25(OH)2D3 metabolism

Vitamin D3 (cholecalciferol) is formed in response to sunlight in skin, where ultraviolet (UV) B

radiation converts 7-dehydrocholesterol to pre-vitamin D3, which is isomerized to vitamin D3 in a

heat-dependent process in epidermal basal layers (Holick, 2003b). Vitamin D can also be

acquired from the diet, either as D2 from plants or D3 derivatives obtained from animals. These

sources can either be part of the traditional diet or be in the form of supplements. However at

high latitudes (such as Finland) the basal requirement is not met despite vitamin D,

fortification of dairy products (Lehtonen-Veromaa et al., 2008) and this leads to sub-optimal

levels of the hormone in the general human population. Vitamin D3 is, itself, biologically inactive

and two hydroxylation steps are needed for the synthesis of the physiologically active form,

1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). Vitamin D3 is first hydroxylated by the 25-

hydroxylase (encoded by the gene CYP27A1) in liver, and the resulting 25-hydroxyvitamin D3

(25(OH)D3), the major circulating form of vitamin D3, is subsequently hydroxylated to the active

form by the 1α-hydroxylase enzyme (encoded by the gene CYP27B1) in the kidney. However,

the latter enzyme is also expressed in colon, brain, skin and breast tissues, and this may provide

tissue-specific control of 1α,25(OH)2D3 levels (Deeb et al., 2007). The physiologically active

form is inactivated by the 24-hydroxylase (encoded by the gene CYP24A1), which can also

hydroxylate 25(OH)D3 and is the rate-limiting enzyme in 1α,25(OH)2D3 catabolism. This

metabolism is regulated by 1α,25(OH)2D3, which strongly induces the expression of the

CYP24A1 gene and decreases the expression of CYP27B1 (Anderson et al., 2003). Moreover,

linked to the physiological role of 1α,25(OH)2D3, high Ca2+ and Pi concentration inhibit the

synthesis of the nuclear hormone, whereas parathyroid hormone (PTH) induces it via increasing

CYP27B1 gene expression (Bland et al., 1999).

Review of the literature


2.1.2.2 Structure and function of the VDR

The VDR protein is composed of domains that allow it to translocate to the nucleus, bind its

ligand, heterodimerize with the retinoid X receptor (RXR), bind to DNA, and finally, to interact

with co-factors (Carlberg, 2003). In the absence of ligand, VDR is partitioned between the

cytoplasm and nucleus, whereas ligand induces interaction of VDR with importin β via its

nuclear localization signal regions. This increases nuclear translocation of VDR, as well as the

translocation of the VDR-RXR complexes (Yasmin et al., 2005). The ligand-binding domain

(LBD) in the C-terminal of the protein binds 1α,25(OH)2D3 at sub-nanomolar concentrations and

also contains the transactivation domain that includes helix 12, which is also called AF-2

(Carlberg, 2003). This transactivation domain is essential for the ability of NR to activate target

gene transcription, as the change of positioning of helix 12 upon ligand binding creates a binding

surface that favors the interaction with co-activators (CoAs) instead of co-repressors (CoRs)

(Nagy and Schwabe, 2004).

The dimerization domains in the LBD and the DNA-binding domain (DBD) allow association of

the VDR with RXR. This interaction is induced by 1α,25(OH)2D3 (Cheskis and Freedman, 1996).

The VDR-RXR heterodimer binds to specific DNA target sequences in DNA, termed vitamin D

response elements (VDREs), that consist of two half-sites with the sequence RGKTCA (R = A or

G, K = G or T) separated by three to four spacing nucleotides (Wang et al., 2005). The DBD on

the N-terminal of VDR binds to the 3’ half-site of VDREs, while the corresponding DBD of RXR

binds the 5’ half-site (Kurokawa et al., 1993). VDR-RXR heterodimers prefer direct repeats (DR)

with 3 spacing nucleotides (DR3), although they also bind to DR4-type VDRE, and to everted

repeats (ER) with 6, 7, 8 or 9 spacing nucleotides (ER6, ER7, ER8 and ER9) (Schräder et al.,

1995, Wang et al., 2005, Tavera-Mendoza et al., 2006). VDR homodimers have also been

characterized both in vitro and in vivo (Carlberg 1993), but their physiological relevance remains

to be under debate as the heterodimer complexes are considerably more stable (Jurutka et al.,

2002; Kahlen and Carlberg, 1994). The heterodimer is able to bind DNA also in non-liganded

form, but ligand enhances this interaction (Ross et al., 1993).

Certain responses to 1α,25(OH)2D3 are very rapid (seconds to minutes) and take place in the

presence of transcription inhibitors. Examples of this type of behavior are the opening of voltage-



gated Ca2+ and Cl– channels, the activation of phospholipase A and C as well as the induction of

several signaling cascades, including protein kinase C and mitogen-activated protein kinase

(MAPK) pathways (Norman, 2006). These non-genomic actions are suggested to be exerted by

VDR associated with caveolae present in the plasma membrane and they may interact with the

genomic actions, for instance, by the modulation of the transcriptional response to liganded VDR

by its phosphorylation.

2.1.2.3 Target genes and physiological role of 1α ,25(OH)2D3

The effect of 1α,25(OH)2D3 on the transcriptome (the total mRNA expressed in a cell or tissue at

a given point in time) has been assayed by multiple microarray experiments, with, for example,

over 900 genes responding after 12 h of ligand treatment in the presence of the protein synthesis

inhibitor cycloheximide in the human head and neck squamous cell carcinoma line SCC25

(Wang et al., 2005). As the transcriptional response is highly dependent on cells or tissue used,

duration of the treatment, concentration of the ligand and co-treatments, there are an infinite

number of putative responding transcriptomes, resulting in various physiological outcomes. This

section describes selected physiological effects of 1α,25(OH)2D3 along with examples of target

genes that are suggested to be responsible for the effects.

Ca2+ and Pi homeostasis and bone mineralization

The most striking effect of severe vitamin D deficiency is rickets. Rickets can also by inflicted by

mutations in the gene of the 1α,25(OH)2D3 synthesizing enzyme 1αOH-ase, CYP27B1, or in the

VDR gene itself. 1α,25(OH)2D3 is essential for adequate Ca2+ and Pi absorption from the intestine

and hence for bone formation (Renkema et al., 2008). Liganded VDR has been shown to induce

expression of the gene encoding for the major Ca2+ channel in intestinal epithelial cell, transient

receptor potential vanilloid type 6 (TRPV6), by direct binding on a functional VDRE at -1.2 kbp

from the TSS (Meyer et al., 2006). A phosphate transporter is also induced, but the response on

chromatin level is less characterized (Xu et al., 2002). 1α,25(OH)2D3 also down-regulates the

expression of the PTH gene that opposes 1α,25(OH)2D3 in regulation of serum Ca2+ and Pi levels,

but up-regulates FGF23, which, like PTH, lowers serum Pi levels (Liu et al., 1996a, Saito et al.,

2005). The induction of the RANKL gene by liganded VDR via multiple distant VDREs (up to 70



kbp from the TSS) leads to stimulation of osteoclast precursors to fuse and form new osteoclasts,

resulting in enhanced resorption of the bone (Kim et al., 2007).

Feedback loop regulation of 1α,25(OH)2D3 metabolism

Liganded VDR down-regulates the last step of ligand synthesis by inhibiting CYP27B1

expression in the kidney. This is suggested to occur via a negative RE or a mixture of positive

end negative elements (Murayama et al., 2004; Turunen et al., 2007). In the absence of

1α,25(OH)2D3, the negative element binds the transcriptional activator transcription factor 3

(TCF3, also called VDIR) associated with CoAs, whereas the liganded VDR-RXR complex binds

to TCF3 and attracts CoRs (Murayama et al., 2004). Association of VDR-RXR heterodimers to

TCF3 binding sites may also occur through ligand-dependent chromatin looping from more distal

regions that directly bind the VDR (Turunen et al., 2007). 1α,25(OH)2D3 highly induces

expression of the CYP24A1 gene through multiple binding sites, thus increasing the inactivating

hydroxylation step and its own catabolism (Väisanen et al., 2005). Liganded VDR also induces

its own expression (Brown et al., 1995).

Modulation of the immune system

Already in the early 20th century sunlight exposure was used as a treatment for tuberculosis and in

recent years some of the molecular mechanisms involved in the immuno-modulatory actions of

1α,25(OH)2D3 have been revealed. 1α,25(OH)2D3 enhances the innate immune response, such as

in the case of tuberculosis, and modulates the adaptive immunity towards self-tolerance and

inhibition of autoimmune diseases (Adorini and Penna, 2008). The anti-autoimmune effect of

1α,25(OH)2D3 is evidenced by epidemiological studies where the vitamin D system reduces the

risk of type I diabetes, multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis and

systemic lupus erythematosus (Adorini and Penna, 2008). At the cellular level, 1α,25(OH)2D3 has

been shown to induce myeloid differentiation and phagocytosis by macrophages as well as inhibit

the development and responses of proinflammatory TH1 cells, proinflammatory responses of

pathogenic TH17 cells, proliferation, plasma-cell differentiation and immunoglobulin production

of B-cells and to modulate antigen presenting dendritic cells to act tolerogenically, to induce the

differentiation and expression of regulatory T cells (Baeke et al., 2008; Adorini and Penna, 2008;

Mora et al., 2008). Regulatory T cells suppress the effector functions of other immune cells and



are crucial in maintenance of peripheral self-tolerance (Mora et al., 2008). There are also adverse

immuno-suppressive effects of 1α,25(OH)2D3 signaling, as suggested by increased resistance of

VDR knockout mice to the intracellular protozoan Leishmania major when compared to wild-

type littermates (Ehrchen et al., 2007).

At the molecular level, the curing power of sunlight is explained by a 1α,25(OH)2D3-dependent

process in the innate immunity system: Toll-like receptors in macrophages recognize

Mycobacterium tuberculosis-derived ligands and induce expression of VDR and CYP27B1 genes,

leading to increased liganded VDR that in turn augments the expression of the anti-microbial

peptide cathelicidin (Gombart et al., 2005; Liu et al., 2006). Induction of cathelicidin by

1α,25(OH)2D3 occurs also in keratinocytes in response to injury, where TGF-β induces CYP27B1

expression, leading to increased 1α,25(OH)2D3 concentration that then triggers expression of the

genes coding for pattern recognition receptors Toll-like receptor 2 and CD14 in addition to that

coding for cathelicidin (Schauber et al., 2007).

1α,25(OH)2D3 inhibits maturation and cytokine production of dendritic cells by repressing the

expression of the v-rel reticuloendotheliosis viral oncogene homolog B (RELB) gene, that codes

for a subunit of NF-κB, via binding of VDR-RXR heterodimer on two VDREs upstream of TSS,

the mouse equivalent of which ligand-dependently recruits HDAC3 to the heterodimer (Dong et

al., 2003; Dong et al., 2005). In T cells 1α,25(OH)2D3 inhibits the expression of TH1 –type

cytokines interferon-γ and interleukin-2 (IL-2) as well as the expression of IL12B that codes for

the p40 subunit of IL-23, which induces IL-17 production linked to inflammation and

autoimmune diseases (Adorini and Penna, 2008). A new candidate for the ability of

1α,25(OH)2D3 to induce myeloid cell differentiation is an inhibitor of the proliferative ERK

pathway, the human kinase suppressor of Ras 2 (hKSR-2), which contains VDREs with VDR-

RXR heterodimer association and is up-regulated by ligand treatment (Wang et al., 2007).

Regulation of cell proliferation and tumorigenesis

The role of 1α,25(OH)2D3 as an anti-proliferative and anti-cancer agent is supported by research

at multiple levels from epidemiological studies to cell culture models depicting molecular

mechanisms. Epidemiological studies show a positive correlation between low serum 25(OH)D3



levels and increased risk for colorectal, breast and prostate cancers (Deeb et al., 2007).

Additionally, VDR knockout mice show hyper-proliferation in the colon, accelerated growth and

induced branching in the mammary gland and are more prone to develop carcinogen-induced skin

tumors and in situ hyperplasia of the mammary gland (Kallay et al., 2001; Zinser et al., 2002a;

Zinser et al., 2002b; Zinser et al., 2005). At the cellular level, 1α,25(OH)2D3 induces

differentiation, apoptosis and cell cycle arrest at G0/G1, and inhibits metastatic and angiogenic

pathways. The effects of 1α,25(OH)2D3 to cell growth are mild when compared to the

chemotherapeutic agents currently in use and 1α,25(OH)2D3 itself causes hypercalcemia when

used in high, anti-cancer quantities. Therefore, neither 1α,25(OH)2D3 nor its non-calcemic

analogs are currently used as a standard treatment for cancer (Bouillon et al., 2006). As a discreet

and safe modifier of proliferation, 1α,25(OH)2D3 (originating either from sunlight exposure or

from food supplements) may be used to prevent cancer. Alternatively, its non-calcemic analogs

may be used in combination with established chemotherapeutic drugs, a strategy used in many

ongoing clinical trials, e.g. by Novacea and Hybrigenics (Bouillon et al., 2006; Deeb et al.,

2007).

The pro-apoptotic effect of 1α,25(OH)2D3 is highly variant among cell and tissue types, as

1α,25(OH)2D3 also showed anti-apoptotic potential in some studies (Marcinkowska et al., 2001).

Among apoptosis-related targets, the anti-apoptotic protein BCL-2 is downregulated and the pro-

apoptotic proteins BAX and BAK are upregulated by the ligand (Diaz et al., 2000; Wagner et al.,

2003). Whether the genes encoding these targets respond primarily to 1α,25(OH)2D3 remains to

be elucidated, since no VDREs have yet been identified in their regulatory regions. Also a

mechanism involving destabilization of telomerase reverse transcriptase (TERT) gene mRNA by

1α,25(OH)2D3 has been suggested (Jiang et al., 2004). In addition, a role for caspases,

intracellular Ca2+ and μ-calpain as well as cathepsins in 1α,25(OH)2D3-mediated apoptosis has

been proposed, but putative direct genomic targets still remain to be characterized (Byrne and

Welsh, 2007).

1α,25(OH)2D3 is also able to hinder the first steps of metastasis by modulation of cellular

adhesion and epithelial to mesenchymal transition. A hallmark of transition from adenoma to

carcinoma is the down-regulation of the adhesion molecule E-cadherin, accompanied with the

loss of the adhesive and polarized phenotype and the release of cells from parent epithelial tissue



(Jamora and Fuchs, 2002). E-cadherin binds β-catenin at adherent junctions, thereby inhibiting its

ability to bind the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription

factors that activate genes involved in proliferation, invasiveness and angiogenesis. Liganded

VDR-RXR complexes inhibit this transition both by competing with TCF/LEF transcription

factors for β-catenin binding and by inducing expression of E-cadherin gene (also known as

CDH1). However, in tumors these actions are counteracted by the transcriptional repressor

SNAIL that down-regulates the expression of the VDR gene (Pálmer et al., 2001; Pálmer et al.,

2004).

Anti-angiogenic effects of 1α,25(OH)2D3 have been shown in cell culture and in nude mouse

tumor transplant models (Mantell et al., 2000). One suggested explanation for this phenomenon is

the down-regulation of the hypoxia-induced factor-1 (HIF-1) via reduced protein translation, as

neither mRNA expression (only measured at one time point) nor the degradation of HIF-1 α

protein was affected by ligand treatment (Ben-Shoshan et al., 2007). 1α,25(OH)2D3 treatment

was also shown to reduce the expression of HIF-1 target genes, such as vascular endothelial

growth factor (VEGF), in a HIF-1-dependent manner.

The most ubiquitous and well-characterized anti-cancer effect of 1α,25(OH)2D3 is the cell-cycle

arrest at G0/G1. The factors behind this effect are discussed in the last section of this literature

review after the general review of cell cycle regulation.

Anti-cancer activities of 1α,25(OH)2D3 are counteracted by various mechanisms in tumors. In

addition to the mentioned down-regulation of VDR by increased SNAIL expression in tumors,

disturbances in expression of genes coding the metabolic enzymes CYP24A1 and CYP27B1 are

common (Bouillon et al., 2006). Moreover, the ubiquitously overexpressed oncogenic H-ras

(encoded by HRAS) decreases the stability of VDR mRNA (Rozenchan et al., 2004). Besides

VDR expression and ligand availability, the transcriptional activation potential of liganded VDR-

RXR complex is compromised in cancer by post-translational modifications of the receptors and

aberrant expression of co-factors. For example, phosphorylation of RXR at serine 260 by the

MAPK pathway inhibits co-activator recruitment by liganded VDR-RXR complex, and hence the

transcriptional activation potential of 1α,25(OH)2D3 (Macoritto et al., 2006). The responsiveness

to 1α,25(OH)2D3 is also affected by ratio of VDR expression to that of the NR-associated co-



repressors NCoR1 and/or the Silencing Mediator of Retinoid and Thyroid Receptors (SMRT or

NCoR2) (Abedin et al., 2006). Ectopic overexpression of NCoR1 abolished the anti-proliferative

response of a non-malignant mammary cell line (MCF-12A) to 1α,25(OH)2D3 (Abedin et al.,

2006). Additionally, chemical inhibition of chromatin modifiers that associate with co-repressors,

such as HDACs and DNA methyltransferases, increased or even restored the anti-proliferative

effect of 1α,25(OH)2D3 or its analogue, 1α,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor

vitamin D3 in malignant cell mammary cell lines on the level of proliferation, cell cycle phase

distribution and target gene expression (Banwell et al., 2006). These findings indicate that co-

repressors and associated chromatin modifiers play a significant role in determination of

1α,25(OH)2D3 response, and that modification of their activity provides means to augment the

anti-proliferative effects of 1α,25(OH)2D3.

2.1.3 PPARβ/δ

PPARβ/δ forms the PPAR subfamily of NRs with its siblings PPARα and PPARγ. Their

endogenous ligands are native and oxidized poly-unsaturated fatty acids and arachidonic acid

derivatives, such as prostaglandins and prostacyclins, which selectively bind the PPAR subtypes

and stimulate their transcriptional activity (Desvergne et al., 2004).

Like VDR, PPARs also heterodimerize with RXRs, but they bind to the 5’ half site and prefer

DR1-type REs (Bardot et al., 1995; IJpenberg et al., 1997). The structure of PPARs resembles

that of other NRs, such as VDR, but they have a larger ligand-binding cavity as well as

differences in the AF-2 domain function, that result in high basal activity of PPARs with the

ability to associate with CoAs even in the absence of ligand (Molnar et al., 2005; Zoete et al.,

2007). The concept of high basal activity of PPARs is still under debate though, because a natural

ligand was found from the LBD in a re-analysis of the “non-liganded” PPARβ/δ crystal structure,

raising the question whether naturally occurring ligands could play a significant role in the

constitutive activity of PPARs (Fyffe et al., 2006).

PPARβ/δ shows the widest expression pattern among the sub-family and is highly expressed in

various tissues, indicating a broad role in physiology (Bookout et al., 2006). Because PPARs

bind ubiquitous nutrition-derived ligands, they regulate various metabolic processes as sensors



for the nutritional status. PPARβ/δ controls many aspects of glucose homeostasis and fatty acid

synthesis/storage, mobilization and catabolism. It increases lipogenesis and reduces glucose

output in liver, but also increases fatty acid oxidation in adipocytes and oxidative metabolism in

muscle. In addition, PPARβ/δ increases energy expenditure both in adipocytes and in muscle, and

controls the fiber type switch in muscle, promoting endurance (Reilly and Lee, 2008). The latter

effect was seen in ‘marathon mice’ that express a constitutively active PPARβ/δ–VP16 hybrid

transcription factor in skeletal muscles, which results in an increased exercise endurance with

muscle type switching as well as in increased resistance to obesity (Wang et al., 2004).

Convergence of PPAR and VDR pathways has been reported e.g. in adipogenesis, where VDR

inhibits PPARγ-mediated gene expression. Putative mechanisms for this attenuation are the

decreased expression of CCAAT enhancer binding protein-alpha (C/EBPα) and PPARγ, reduced

synthesis of natural PPAR ligands, and/or competition over the common heterodimerization

partner RXR by VDR (Wood, 2008). Contrary to the PPARγ gene, expression of the PPARβ/δ

gene is induced by 1α,25(OH)2D3 via a functional VDRE 366 bp upstream of the PPARβ/δ TSS

(Dunlop et al., 2005).

2.1.4 NR co-factors

As the genomic DNA of eukaryotes is packaged into chromatin, NR co-factors are needed to

alleviate this packaging. Moreover, they interpret and leave messages to the chromatin structure,

as well as bridge the NRs to the basal transcriptional machinery. Co-regulators also modify

components of the transactivation complex as well as NRs themselves, control splicing of

transcripts and regulate the assembly, recruitment and release of co-regulatory complexes

(Lonard and O'Malley, 2005; Perissi and Rosenfeld, 2005). CoAs that directly bind NRs share a

common structural motif that interacts with the AF-2 motif of NRs, the nuclear-receptor box with

a LXXLL composition, where L equals leucine and X equals any amino acid (Heery et al., 1997).

The interactions of CoAs and CoRs with NRs can be controlled at several levels, including

expression and post-translational modifications of co-factors and NRs, ligand binding by the NRs

and histone or DNA modifications in the targeted chromatin, providing integration of multiple

signaling pathways to the NR transcriptional response (Rosenfeld et al., 2006). Examples of co-

regulators are categorized in the following sections to CoAs and CoRs, although the strict



division is artificial and possibly misleading, as multiple factors are essential in both

transcriptional repression and activation. For example, the well-established CoA p300 acts as a

repressor for the v-myc myelocytomatosis viral oncogene homolog (avian) gene (MYC gene),

which encodes for myc proto-oncogene protein (myc), expression in co-operation with HDAC3

(Sankar et al., 2008).

2.1.4.1 Co-activators

The multi-subunit Mediator complex acts as a bridge between NRs and the Pol II machinery and

thus enables the activation of transcription by NRs and other transcription factors. Although the

importance of the Mediator complex is well established, its exact mode of function remains

undefined. The complex is believed to act predominantly during the assembly of the PIC as it is

recruited to target chromatin prior to Pol II. Putative mechanisms include induction of the PIC

assembly, recruitment of Pol II or members of the basal transcription machinery and docking of

other CoAs, such as histone acetyltransferases (HATs) (Casamassimi and Napoli, 2007). The

MED1 subunit of the Mediator complex is essential for transcriptional activation by many NRs,

such as VDR, and it interacts with the AF-2 activation domain of NRs through its LXXLL motif

and thus recruits the entire Mediator complex to the VDR (Casamassimi and Napoli, 2007;

Rachez et al., 1998). Phosphorylation of MED1 is stimulated by NR ligands and promotes its

association with the Mediator complex. Phosphorylation of MED1 also enhances NR-dependent

transcription in vitro, providing additional mechanisms for integration of NR and cell membrane

receptor pathways (Belakavadi et al., 2008).

HATs, such as the family of NR CoAs (NCOA1, NCOA2 and NCOA3, also known as SRC-1, -2

and -3), E1A binding protein p300 (EP300 or p300) and CREB-binding protein (CBP or

CREBBP), catalyze acetyl group transfer from acetyl-coenzyme A to histones forming coenzyme

A and acetyl-histones. HATs can also acetylate non-histone targets, such as transcription factors,

and modify their activity and/or stability. NCOA proteins possess only weak intrinsic HAT

activity and their main function is the recruitment of histone modifying CoAs to ligand-activated

and DNA-bound NRs, since, e.g. CBP only weakly binds NRs. The modifiers include CoAs with

HAT activity, such as p300, CBP and p/CAF, as well as those with histone methyltransferase

(HMT) activity, such as CARM1 and PRMT1 (Chen et al., 1997; Chen et al., 1999; Sheppard et



al., 2001). The actions of HATs and HMTs lead to more permissive chromatin and, more

importantly, provide docking sites for acetyl-lysine binding bromodomain proteins and methyl-

lysine binding chromodomain proteins with other properties such as chromatin remodeling

activity.

The PPARγ co-activator 1 α (PGC-1α) functions as CoA to many NRs, including PPARs and

VDR (Liang and Ward, 2006; Savkur et al., 2005). It does not possess HAT activity itself, but is

able to recruit HATs, such as CBP and p300, as well as the hSWI/SNF-remodeling complex via

its BAF60a subunit (Li et al., 2008; Liang and Ward, 2006).

Both lysine-specific HMTs, such as G9a, SET7/9 and MLL, and arginine-specific HMTs, such as

PRMT1 and CARM1, have been shown to interact with NRs (Subramanian et al., 2008). The

function of these co-factors on NR-regulated transcription remains quite poorly defined. In a

study on ER, SET7/9, the lysine methyltransferase that methylates histone H3 at lysine 4, was

shown to methylate ER, leading to more stable receptor, increased recruitment to target genes as

well as increased transactivation potential (Subramanian et al., 2008). CARM1 methylates

histone H3 at arginine 2, 17 and 26 and co-operates with HATs and chromatin remodeling

complexes to enhance transcriptional activation by NRs. However, it still remains unclear, to

which extent the co-operation depends on methylation of histone residues, since CARM1 also

methylates NCOAs. Methylation of NCOAs leads to both increased activation and, paradoxically,

to disassembly of the CoA complex (Feng et al., 2006).

Lysine demethylation by the lysine-specific histone demethylase 1 (LSD1, encoded by AOF2

gene) has been linked to both repression and activation of genes. LSD1 catalyzes the

demethylation of both the activity-linked H3K4me1/2 and the repression-linked H3K9me,

although the latter activity has not been detected in vitro (Forneris et al., 2008). LSD1 was

originally identified from a repressor complex and thus identified as a CoR (Forneris et al.,

2008). However, in a genome-wide analysis, LSD1 was shown to occupy 20 % of all promoters,

84 % of which were associated with Pol II and histone modifications typical for transcriptionally

active regions, such as H3K4me2, indicating a role as a CoA (Garcia-Bassets et al., 2007). LSD1

is also essential for AR-induced gene expression and H3K9me1/2 demethylation, whereas the

JMJC domain-containing protein is needed for the removal of the trimethylation modification of



H3K9 (Wissmann et al., 2007). LSD1 seems also to play an important part in NR ligand-induced

chromatin looping, which is discussed in more detail in a following section.

Chromatin remodeling complexes contain an ATP-dependent DNA helicase / ATPase that

induces or represses transcription by using the energy of ATP to dissociate core histones, slide

nucleosomes or relocate entire histone octamers within the chromatin structure. These remodeling

complexes are divided into four major sub-families based on their central catalytic subunit: the

SWI/SNF, ISWI, CHD/Mi-2 and INO80 complexes with BRG1/hBRM, hSNFL2/hSNF2h,

CHD1/CHD3/CHD4 and SCRAP/p400 as catalytic units, respectively (Lall, 2007). The

components of the sub-families can also form joint multi-complexes, such as the WSTF including

nucleosome assembly complex (WINAC), which includes members of the hSWI/SNF and ISWI

complexes and promotes both the assembly and disruption of nucleosomal structure in an ATP-

dependent manner via BRG1 and hBrm (Trotter and Archer, 2008). The WINAC complex is able

to interact ligand-dependently with VDR via the WSTF component and activates and represses

gene transcription by creating either an open or a closed chromatin structure (Kitagawa et al.,

2003). VDR has also been shown to associate with the PBAF and hSWI/SNF complexes in vitro,

and in repressive role with the hSNF2 complex (Ewing et al., 2007; Lemon et al., 2001). The

rationale for chromatin remodeling in transcription was illustrated by in vitro transcription

reactions performed by Lemon and colleagues (Lemon et al., 2001). In their studies, the

component of the basal transcription machinery, TFIID, and the Mediator complex were essential

and sufficient for transcriptional activation by VDR-RXR heterodimers on naked DNA, where

the ligand and ATP-dependent chromatin remodeling complex PBAF completely lacked effect. In

transcription from chromatin, however, the remodeling complex PBAF was essential in addition

to TFIID and the Mediator complex with immense induction by ligand. This assay suggests that

the chromatin structure restrains transcription by the basal transcriptional machinery and the

Mediator complex, but that this obstacle can be overcome by chromatin remodeling that is

activated by liganded NRs.

VDR can also modify transcription in events subsequent to initiation of transcription. SNW

domain containing 1 (SNW1, also called NCOA-62 or SKIP) is a CoA that can bind to the LBD

of VDR and retinoic acid receptors (RARs) to enhance ligand-mediated gene expression

(MacDonald et al., 2004). SNW1 interacts with the spliceosome and expression of a dominant



negative SNW1 resulted in a ligand-dependent transient accumulation of un-spliced transcripts

generated from a 1α,25(OH)2D3-responsive mini-gene cassette (Zhang et al., 2003). Therefore,

SNW1 may couple regulation of transcription by VDR to splicing and thus determining the

overall response of ligand-regulated genes.

2.1.4.2 Co-repressors

CoRs can be classified according to their function to basal CoRs, such as NCoR1 or SMRT, that

function as platforms for the recruitment of several sub-complexes that often contain HDAC

activity, ATP-dependent chromatin remodeling complexes and other CoRs that recruit general

CoRs upon ligand induction, such as the ligand dependent nuclear receptor corepressor (LCoR)

and NR interacting protein 1 (NRIP1 or RIP140) (Perissi and Rosenfeld, 2005).

In the absence of ligand NRs, such as thyroid hormone receptors (TRs), RARs and VDR, reside

in the nucleus and interact with basal CoR complexes containing the NCoR1 or SMRT, which

mediate repression by deacetylation of histones. The complex for either of them contains

HDAC3, transducin-β-like 1 (TBL1 encoded by the TBL1X gene), TBL1 receptor 1 (TBL1R1

encoded by the TBL1XR1 gene), G protein pathway suppressor 2 (GPS2) and coronin, actin

binding protein, 2A (CORO2A or IR-10) (Yoon et al., 2003). In the complex NCoR1 and SMRT

serve as docking sites between NRs and the CoR complex and are also required for the HDAC

activity of HDAC3 (Guenther et al., 2001). Although other HDACs have been connected to the

complex, HDAC3 is essential at least for the repression by un-liganded TR (Yoon et al., 2003).

TBL1/TBL1R1 are essential for the recruitment of the ubiquitin conjugating/19S proteasome

complex, where TBL1R1 selectively mediates the exchange of the NCoR1/SMRT complex to

CoAs upon ligand binding, and they are also essential in targeting the CoR complex to hypo-

acetylated chromatin (Perissi et al., 2004; Yoon et al., 2005). Of the remaining complex subunits,

CORO2A has homology to actin-binding proteins, and thus may serve in repositioning the target

chromatin inside the nucleus. The GPS2 subunit may serve as a link between NR and cellular

signaling pathways, as GPS2 inhibits the transcription activation potential of the c-Jun N-terminal

kinase 1 (JNK1) pathway (Zhang et al., 2002). The role of NCoR1/SMRT complexes is not only

restricted to deacetylation, since recently NCoR1 was shown to recruit a H2A ubiquitin ligase



that inhibits the elongation by Pol II beyond the first nucleosome of the transcribed region (Zhou

et al., 2008).

HDACs are an important group of effectors in NR-mediated repression of gene transcription,

forming a counterforce to HATs. There are currently 18 known mammalian HDACs, grouped

into four classes based on sequence similarity: class I HDACs include HDAC1, 2, 3 and 8, class

II HDACs include HDAC4, 5, 6, 7, 9 and 10, class III refers to seven unrelated sirtuin

deacetylases, whereas HDAC11 alone forms the class IV (Gallinari et al., 2007). In addition to

the permanent subunit HDAC3, NCoR1/SMRT complex interacts with class II HDACs.

However, the HDAC activity of HDAC4 and HDAC5 depends on presence of HDAC3 in a

repressor complex (Fischle et al., 2002). The HDAC activity of purified class II HDACs is low in

vitro, suggesting that their main role is deacetylation of non-histone targets rather than of

histones. Interestingly, HDAC3 and HDAC4 were shown to bind NRs also directly and

independently of the CoR complex in vitro, but the in vivo relevance of this finding remains

unclear (Franco et al., 2003). Despite the broad role of HDACs in repression, the use of HDAC

inhibitors leads to induction of bulk levels of histone acetylation, but to very limited

transcriptional response with equal numbers of genes activated and repressed by HDAC

inhibition (Smith, 2008). HDACs may modulate transcription via deacetylation of transcription

factors and CoAs in addition to histones. As acetylation of a protein may either increase or

decrease its activity, the net transcriptional outcome of deacetylation of non-histone targets may

be activation as well as repression.

In addition to NCoR1/SMRT, Hairless and Alien have been reported to be VDR-associated CoRs

(Tagami et al., 1998; Polly et al., 2000a; Hsieh et al., 2003). Also NRIP1 has been shown to

associate with VDR (Masuyama et al., 1997). Interestingly, NRIP1 and LCoR can bind to NRs in

a ligand-dependent manner and compete with CoAs by displacing them. However, the latter

phenomenon has not been characterized with VDR bound CoRs (Perissi and Rosenfeld, 2005).

The nucleosome remodeling complex with strongest association to repression is NuRD, which

possesses HDAC1 and 2 in addition to the ATP-dependent chromatin remodelers CHD3 or

CHD4. The complex is recruited to chromatin by sequence-specific transcription factors, such as

un-liganded RAR and TR and/or the MBD2 protein, which binds to methylated DNA (Crook et



al., 2006). The importance of this complex in creation and maintenance of repressed chromatin is

illustrated by knockdown of its components, which impaired histone deacetylation, chromatin

compaction, DNA and histone methylation as well as stable silencing, favoring cellular

differentiation of leukemic cells (Morey et al., 2008). The repression of target genes by un-

liganded VDR has been shown to be dependent on the bromodomain adjacent to the zinc finger

domain, 1A (BAZ1A or hAcf1), which is a part of the hSNF2h chromatin remodeling complex

and improves the ability of this complex to mobilize nucleosomes (Ewing et al., 2007). BAZ1A

directly associates with NCoR1 and stabilizes both the CoR complex association and repressive

chromatin architecture at selected 1α,25(OH)2D3 target gene promoters. These studies suggest

that ATP-dependent nucleosome remodeling is an essential part of repression by NRs, possibly

via establishing and maintenance of repressive chromatin and inhibition of nucleosome eviction

from target promoters.

2.1.5 Role of chromatin in NR-mediated transcription

2.1.5.1 Histone modifications

Both DNA and histones on the chromatin structure can be modified and increasing evidence

supports the idea that these modifications serve as specific signals for chromatin binding proteins,

i.e. that the modifications form a ‘histone code’ read by associated factors (Strahl and Allis, 2000;

Turner et al., 1992). The modifications include DNA methylation, variant isotypes of the core

histones themselves and various covalent modifications of N-terminal tails of core histones. The

modifications of the tail residues include acetylation (ac), mono-, di- or trimethylation

(me/me2/me3), ubiquitylation (ub) or sumoylation (su) of lysines (K), mono- or dimethylation

(me/me2) or citrullination (cit) of arginines (R) and phosphorylation (phos) of serines (S) and

threonines (T) (Ruthenburg 2007, Lall 2007).

As yet, we are far from fluent reading of the histone code, but we can try to approach it by

defining, in which processes the modifications are involved, which proteins bind them and which

proteins can alter them. The reading of the code is further complicated by the different

combinations of modifications, which lead to distinct outcomes (Fischer et al., 2008; Garske et

al., 2008). As an example of a single modification, H3K4me2, on a genome-wide scale, is



enriched to approximately the 1 kbp area around active TSSs and within active enhancer regions

(Barski et al., 2007; Heintzman et al., 2007). H3K4me2 is bound by several chromodomain or

plant homeodomain (PHD) containing proteins, such as the ATP-dependent chromatin-

remodeling factor chromodomain helicase DNA binding protein 1 (CHD1) that can alter the

accessibility of DNA for transcription (Pray-Grant et al., 2005), the inhibitor of growth family,

member 2 (ING2), which is part of a transcriptionally repressive complex (Peña et al., 2006) and

bromodomain PHD finger transcription factor (BPTF), which is part of the ATP-dependent

chromatin-remodeling complex (Li et al., 2006). Methylation of H3K4 also prevents the binding

of the CoR complex NuRD to the histone H3 tail (Zegerman et al., 2002). These binding partners

indicate important regulatory functions for this modification, such as induction of chromatin

remodeling needed for transcription initiation and, paradoxically, potential repression of gene

expression by histone deacetylation.

Another example of modification linked to transcriptional activity is H3K9ac, which is also

enriched close to TSSs and enhancers and is induced upon activation of transcription (Garcia-

Bassets et al., 2007; Heintzman et al., 2007). It is bound by bromodomain containing proteins

that recognize this specific acetylated residue, such as the HAT CBP that acetylates H3K14 and

TAF1, a subunit of the general transcription factor TFIID (Agalioti et al., 2002; Zeng et al.,

2008). Interestingly, acetylations at H3K9 and H3K14 are both essential for the recruitment of

TAFIID in an in vitro model with mutant histones, pointing out a key role for H3K9ac as an

initial signal for the assembly of transcriptional machinery (Agalioti et al., 2002).

2.1.5.2 Chromatin looping

Since the establishment of the chromosome conformation capture (3C) assay by Dekker and

colleagues (Dekker et al., 2002), chromatin looping has been widely studied as a mode of

interaction between genomic elements distal to each other. The bending of chromatin provides a

physical contact between NRs residing on REs and the basal Pol II machinery bound to the TSS

of target genes, enabling transcriptional activation from distal REs. As the method is still quite

new, the mechanisms that mediate chromatin looping remain poorly understood. Recently, Perillo

and colleagues suggested a molecular basis for the formation of chromatin loops involving

histone demethylation (Perillo et al., 2008). Based on their studies with the ERα, they propose a



model, where chromatin-bound liganded receptor induces demethylation of H3K9me2 by the

lysine demethylase LSD1. A side product of this demethylation is the strictly local production of

hydrogen peroxide, which, in turn, oxidizes DNA to 8-oxo-guanine. This is then recognized and

removed by base excision repair enzymes. The resulting single-stranded DNA breaks are

subsequently recognized by topoisomerase IIb that acts to relax DNA strand topology, enabling

the bending of chromatin to form loops between enhancers and the transcription initiation

complex. In addition to relaxation of chromatin, the reactive oxygen species (ROS) produced by

LSD1 inhibit proteasome activity and hence ERα degradation. ROS was also shown to be

essential for accumulation of phosphorylated Pol II to target gene promoters. Transcription cycles

could then be set to pace by local ROS concentrations that enable both the flexibility of the DNA

strand and timely degradation of the NR as ROS concentration reduces without new

demethylation events. It still remains a mystery, though, whether increased flexibility of DNA

leads to increased random movement and hence to increased chance for the factors on enhancers

and on TSS to encounter and bind to each other, or whether looping requires an active network

that brings the elements together, such as a hypothetical dynamic nuclear matrix directed by

ATP-dependent chromatin remodeling complexes (Varga-Weisz and Becker, 2006).

2.1.6 Models for NR-induced gene expression

In vitro, binding of a ligand to a NR induces a structural change in the receptor that favors

interactions with CoAs instead of CoRs bound by an unliganded receptor. The application of

these in vitro results to depict the situation in vivo lead to the on/off model, where an agonistic

ligand acts as a switch between CoR and CoA recruitment. This model has restrictions in vivo.

For instance, it does not provide an explanation for the termination of transcription. With kinetic

data from chromatin immuno-precipitation (ChIP), the on-off model has been replaced by a

cyclical one, where the NR, CoAs and CoRs are recurrently recruited to and released from the

target promoter in an ordered manner to repeatedly first induce and then limit transcription of

responsive genes (Metivier et al., 2003; Shang et al., 2000). The release from the promoter is

suggested to occur via proteasomal degradation and/or chaperone-mediated disassembly of the

regulators (Reid et al., 2003; Freeman and Yamamoto, 2002; Bikle et al., 2007). In addition to

the dynamics of transcription regulators, the role of chromatin modifications and conformation as

well as the dynamics of both has become an issue of interest. Already the first cyclical models



included recurrent changes in histone acetylation, and since then a ligand-dependent cyclical

response in DNA methylation via DNA methyltransferases has been introduced (Metivier et al.,

2008).

2.2 Cyclin-dependent kinase inhibitor p21

2.2.1 Functions of p21

2.2.1.1 Regulation of cell cycle by cyclin-dependent kinase inhibitors

In multi-cellular organisms, the division of cells is tightly controlled by protein-kinases of the cell

cycle machinery that are regulated by extraneous signals such as growth factors, enabling co-

ordinated division of individual cells to meet the needs of the organism as a whole. A signal to

proliferate, such as growth factor stimulation, allows a quiescent cell to re-enter the cell cycle at

G0/G1, after which, in the absence of disturbances, damage or stress, the rest of the cell cycle is a

highly co-ordinated but nonetheless automatic process (Pajalunga et al., 2008). Growth factors

induce the expression of D-type cyclins, which form complexes with cyclin-dependent kinases

(CDKs) 4 or 6 to phosphorylate the pocket proteins retinoblastoma 1 (Rb, encoded by RB1), p107

and p130 at multiple amino acid residues. This releases E2F family transcription factors, which

activate the expression of numerous effectors of DNA synthesis. E2F factors also drive

expression of cyclin E, which activates CDK2 to further phosphorylate the pocket proteins and, in

a kinase-independent manner, loads MCM helicase complexes onto DNA replication origins

(Geng et al., 2007). The cyclin-CDK complexes are inhibited by proteins of the family of cyclin-

dependent kinase inhibitors (CDKIs), composed of the INK4 sub-family including p15 (INK4B,

encoded by the human gene CDKN2B), p16 (INK4A, encoded by the CDKN2A gene), p14 (ARF,

also encoded by the CDKN2A gene), p18 (INK4C, encoded by the CDKN2C gene) and p19

(INK4D, encoded by the CDKN2D gene), and the Cip/Kip family with p21 (WAF1/Cip1 encoded

by the CDKN1A gene), p27 (Kip1, encoded by the CDKN1B gene) and p57 (Kip2 encoded by the

gene CDKN1C). The members of the INK4 family specifically bind and inhibit the CDKs 4 and

6, thus blocking the entry to the S phase, whereas CIP/KIP inhibitors bind to both cyclin and



CDK subunits and can modulate the activities of cyclin D-, E-, A-, and B-CDK complexes

(Pajalunga et al., 2008).

In addition to their inhibitory function for CDK activity, the role of p21 and p27 is complicated

by their function as a bridge between cyclin D and CDK4 to promote their association and

nuclear translocation (Coqueret, 2003). In the nucleus cyclin-D-CDK complexes titrate p21 and

p27 from CDK2, which leads to cell cycle progression. When the amount of CIP/KIP proteins

increases to a saturated level, they bind to cyclin-E-CDK2, in order to block the catalytic activity

of the kinase and cause cell-cycle arrest. CDK2 is dispensable for cell cycle arrest at the G1-S

transition and in CDK2-/- cells p21 induces cell cycle arrest via inhibition of CDK1

(Satyanarayana et al., 2008). A further complication to the system is provided by an assembly

factor of the pre-replicative complex Cdc6, which is able to activate CDK2 despite the presence

of active p21 (Kan et al., 2008).

2.2.1.1.1 p21, p16 and senescence

Senescence is the physiological process of terminal growth arrest, accompanied by specific

changes in cell shape, adhesion and gene expression. It can be divided into replicative

senescence, mediated primarily by telomere shortening, and accelerated senescence that occurs in

response to DNA damage or introduction of oncogenic RAS and permanently stops the

proliferation of cells with potentially carcinogenic damage (Roninson, 2002). DNA damage or

telomere shortening beyond a critical length activates p53, leading to a transient peak in p21

expression. After the peak, p21 expression settles to a slightly induced level, whereas p16

becomes constitutively upregulated, presumably via stress-related MAPK pathways and

maintains terminal growth arrest by quenching CDK4/6 activity (Herbig and Sedivy, 2006;

Roninson, 2002). Overexpression of either p21 or p16 induces senescence both in normal and

malignant cells in an Rb-dependent manner, confirming that the senescence promoting function

of these CDKIs is a consequence of the cell cycle block (Chen et al., 2006). Induction of

senescence does not require activity of both CDKIs, as overexpression of p21 with knockdown of

p16 and vice versa also leads to increased senescence. There is also a downside of the senescence

promoting function of p21, as p21 gene knockout prolongs the lifespan of telomerase knockout

mice (with dysfunctional telomeres) and improves stem cell function when compared to wild type



p21 gene expressing littermates, but not in telomerase expressing mice (Choudhury et al., 2007).

Although p21 gene deletion did not induce chromosomal instability or cancer formation in mice

lacking telomerase, p21 gene deletion induced both the number of transformed foci in oncogene-

transfected mouse embryonic fibroblasts and macroscopic tumor incidence in mice with

heterozygous telomerase expression.

2.2.1.2 Anti- and pro-apoptotic p21 functions

Both overexpression and deletion of p21 have been shown to sensitize cells towards apoptosis.

For example, overexpression of the p21 gene enhances apoptosis in response to cisplatin in

glioma and ovarian carcinoma cell lines, potentiates bile acid-induced MAPK-dependent

apoptosis in hepatocytes, induces pro-apoptotic BAX in presence of C6-ceramide and sensitizes

granulocytes to differentiation-induced apoptosis (Gartel, 2005). Conversely, p21 has been shown

to protect cells from apoptosis, since the protein blocks the processing of pro-caspase-3 into its

active form, which, in turn, can cleave and thus inactivate p21 (Levkau et al., 1998; Suzuki et al.,

1998). The CDK inhibition activity of the p21 protein itself may inhibit apoptosis, because CDK

activity may be essential for an efficient activation of the caspase cascade (Besson et al., 2008).

Moreover, cytosolic p21 protein interferes with apoptosis-linked mitogen-activated pathways,

e.g., by inhibition of a death receptor target and the pro-apoptotic MAPK kinase kinase 5

(MAP3K5 or ASK1) via direct binding and of stress-activated protein kinases (Asada et al.,

1999; Shim et al., 1996). In some cases the anti-apoptotic function of the p21 protein is a

consequence of its growth-inhibitory function. For example, the increased apoptosis with DNA-

damaging agents or anti-microtubular drugs in cells lacking a p21 gene may be merely a

consequence of lack of DNA repair prior to mitosis, leading to mitotic catastrophe and finally

apoptosis (Roninson, 2002).

2.2.1.3 p21 in DNA repair and replication

The proliferating nuclear antigen (PCNA) is a co-factor of DNA polymerases that encircles DNA

and tethers polymerases firmly to it, increasing the processivity of DNA polymerases from tens to

thousands of nucleotides per minute (Moldovan et al., 2007). Apart from playing an essential role

in DNA replication it is also involved in DNA synthesis during UV-induced nucleotide excision

repair (NER) (Montecucco et al., 1998). Direct binding of the p21 protein to PCNA inhibits DNA



replication in vitro and in vivo by blocking the activity of PCNA to stimulate DNA polymerases,

although the role of the p21 protein in DNA repair remains controversial (Moldovan et al., 2007).

Human fibroblasts lacking a p21 gene are more sensitive to UV-induced DNA damage and show

defects in NER, with prolonged PCNA association on the DNA repair sites (Stivala et al., 2001).

On the other hand, the p21 protein bound to PCNA inhibits NER in vitro. In vivo accumulation of

endogenous p21 protein also inhibits NER. The inhibition of NER in cells lacking growth arrest

and DNA-damage-inducible α (GADD45A) gene or a gene encoding subunit of the damaged-

DNA-binding protein DDB, DDB2, is restored by the inhibition of p21 gene transcription

(Stoyanova et al., 2008). Whether with positive or negative role on NER, p21 protein is

immediately recruited to DNA damage sites, together with repair factors, such as DNA

polymerase δ, XPG and CAF-1 (Perucca et al., 2006). A recent model suggested a positive role

for p21 in NER and included interactions with PCNA and p300 (Cazzalini et al., 2008). The HAT

activity of p300 may provide chromatin accessibility to the NER machinery on the non-

transcribed regions, however, paradoxically, PCNA inhibits the HAT activity of p300. DNA

damage induces PCNA recruitment to regions harvesting p300, with subsequent recruitment of

p21 that leads to disruption of association of PCNA and p300, thus promoting accessibility of

PCNA-dependent NER machinery in surrounding chromatin regions.

2.2.1.4 Transcriptional control by p21

In addition to indirect modulation of E2F transcription factors via regulating pocket protein

phosphorylation, CIP/KIP proteins can also modulate the activity of transcription factors via

direct binding. For example, the p21 protein is recruited to the promoters of the human MYC and

CDC25A genes, the latter of which encodes for a phosphatase that activates Cdc2. It is essential

for progression from G1 to the S phase and, together with the STAT3 and E2F1 transcription

factors, leads to reduced expression of target genes (Vigneron et al., 2006). The p21 protein also

binds to the product of the MYC gene, myc, thereby blocking myc-MAX complex formation and

subsequently suppressing the transcriptional activation by this complex (Kitaura et al., 2000). On

the other hand, p21 protein can modulate p300/CBP HAT complex-mediated transcriptional

activation via binding to p300, which disrupts the activity of the transcriptional-repression

domain of p300 and de-represses transcription of target genes (Garcia-Wilson and Perkins, 2005).

Recently, this CDKI was found to directly interact with un-liganded ERα in a complex that



includes also CDK2 and cyclin E (Maynadier et al., 2008). Loss of the p21 gene abolished the

growth inhibition by un-liganded ERα, although the mechanism of the action of this complex is

unclear as the ability of ERα to bind DNA is not needed for the growth inhibitory response.

2.2.1.5 Knockout models of Cip/Kip family members

Despite the similar binding preferences to cyclins and CDKs shared by the Cip/Kip family of

CDKIs, they respond to distinct anti-proliferative signals and are involved in different

physiological pathways. DNA damage leads to activation of p53 that directly binds to the p21

gene promoter and up-regulates its transcription, whereas p27 gene expression is induced in

quiescent cells lacking mitogenic signals and p27 protein is rapidly degraded as cells reenter the

cell cycle (Besson et al., 2008). On the other hand, p57 is mainly expressed during

embryogenesis and regulated by embryogenic transcription factors, such as Notch/Hes1, MyoD,

BMP-2 and -6. Knockout of the Cip/Kip family member genes in model organisms further

illuminate their distinct physiological roles. p27 gene null mice have an increased body size and

multiple organ hyperplasia, as the p27 protein is essential in limiting growth. Mouse embryos null

for the p57 gene also have organ hyperplasia and delayed differentiation but die at birth,

confirming p57 as an essential regulator of embryonic growth (Besson et al., 2008). Mice lacking

p21 develop normally, although p21-/- murine embryonic fibroblasts are deficient in their ability

to arrest in G1 in response to DNA damage and show aberrant growth achieving a higher

saturation density than wild-type cells (Deng et al., 1995).

2.2.1.6 Bivalency of p21 in tumorigenesis

The role of p21 in tumorigenesis appears controversial, suggesting both tumor suppressive and

oncogenic functions for its product. Although p21 null mice are susceptible to spontaneous

tumors of hematopoietic, endothelial and epithelial origin at an average age of 16 months, loss of

p21 delays tumor development in ataxia-telangiectasia, mutated negative (ATM-/-), p53-/- or

irradiated wild-type mice, presumably due to the anti-apoptotic activity of p21 (Martin-Caballero

et al., 2001). Moreover, PDGF-induced gliomagenesis is dramatically reduced in mice lacking

the p21 gene, because the p21 protein induces the nuclear localization of cyclin D1. Inactivating

mutations of the p21, as well as the p27 gene, are exceedingly rare. While loss of the p21 gene is

a negative prognostic marker in some cancer types, it appears that overexpression or cytoplasmic



localization of p21 is a marker of poor prognosis and aggressive tumors in carcinomas of the

pancreas, breast, prostate, ovary and cervix, as well as in glioblastomas. These results suggest

that, while nuclear p21 protein acts as a tumor suppressor by the induction of G1 arrest,

cytoplasmic p21 is oncogenic. Indeed, phosphorylation of p21 by the proliferative kinases

PKB/Akt and PKC promotes cytoplasmic localization. The phosphorylation status of the p21

protein also modifies the outcome of the interaction with cyclin-CDK complexes. For example,

phosphorylation of p21 by CDK2 converts it from an inhibitory subunit to a positive regulatory

factor that assists cyclin B1-CDK1 activation at the G2/M phase, thus accelerating the progression

of the cell cycle (Dash and El-Deiry, 2005).

2.2.2 Regulation of p21 expression

The major transcription factor regulating p21 expression is the tumor suppressor protein p53

(encoded by the TP53 gene) that mediates the information of DNA damage to a cell cycle block

via direct transcriptional activation of the p21 gene to prevent replication of damaged DNA. The

damaged DNA is recognized either by the MRE11–RAD50–NBS1 (MRN) complex that recruits

and activates ATM and Rad3-related (ATR) and ATM kinases, or by the kinases themselves. The

kinases in turn activate the p53 protein by direct phosphorylation on the serine at position 15 and

by activating the CHK2 kinase, which further phosphorylates p53 at another serine at position 20

(Cann and Hicks, 2007). These phosphorylations interrupt association of p53 with its negative

regulator mouse double minute 2 homolog (MDM2), an ubiquitin ligase that both sterically

blocks the transcriptional activity of p53 and mediates the degradation of the p53 protein. As a

result from the phosphorylations, the half-life of the p53 protein is increased from minutes to

hours (Riley et al., 2008). ATM can also initiate a phosphorylation cascade that leads rapidly to

CDK2 protein inactivation. The acetylation and methylation of p53 by HATs (CBP, p300, PCAF)

and methyltransferases (SET9), respectively, can further stabilize the protein by inhibiting

interactions with MDM2 and increase its affinity for DNA.

The p53 tetramer binds to specific REs with two or more RRRCWWGYYY (R = A or G, W = A

or T, Y = C or T) half-sites in various orientations, separated by 0 to 21 spacer nucleotides (Riley

et al., 2008). The co-factors involved and the mechanisms of transcription activated by p53

resemble those of NRs, with HATs (p300, CBP), arginine methyltransferases (CARM1 and



PRMT1), the lysine demethylase LSD1, HDACs, chromatin remodelers (hSWI/SNF via BAF60)

and the MED1 subunit of the Mediator complex acting to promote transcription (An et al., 2004;

Huang et al., 2007; Donner et al., 2007; Oh et al., 2008). There are also differences between p53-

and NR-mediated transcription, such as protein stabilization of p53 versus ligand activation of

NRs, the ability of p53 to recruit the p300/CBP HAT complex directly through the same domain

in CBP that is bound by NCOA1 in the NR response, and finally, a shortage of reported contacts

between p53 and primary NR co-factor complexes, such as NCoR1/SMRT or NCOAs

(Livengood et al., 2002).

Several members of the Sp/Krüppel-like factor (KLF) family of transcription factors, such as

Sp1, Sp3, KLF2, KLF4, KLF5, KLF6 and KLF7, induce p21 gene transcription via binding to

common GC-rich sequences termed as GC-boxes at proximal promoter of p21 (Rowland and

Peeper, 2006). The six GC-boxes of the p21 gene promoter are crucial for both basal and induced

p21 gene transcription, as mutation of a selected GC-box abolished the activity of a 2.3 kbp p21

gene promoter construct, and knockdown of Sp1 greatly decreases p21 protein levels in a p53-/-

genetic background (Cen et al., 2008; Koutsodontis et al., 2002). Sp proteins interact with a wide

variety of transcriptional regulators, such as NRs, E2F transcription factors, NCoR1/SMRT

CoRs, HATs and other chromatin modifiers. Sp proteins are considered as constitutively active,

but their activity is also regulated by post-translational modifications from cell signaling

pathways, such as phosphorylation by the epidermal growth factor-activated MAP kinase, ERK2,

or DNA damage-activated kinase ATM (Iwahori et al., 2008; Merchant et al., 1999). It has been

suggested that one mechanism for HDAC inhibitors is modulation of Sp1 activity via increased

acetylation as opposed to unspecific de-repression of chromatin (Huang et al., 2005; Lee et al.,

2005).

As the p21 protein is an important regulator of cell proliferation, its expression is under direct

regulation of several cellular pathways that either promote or in inhibit proliferation. For

example, the Notch and TGF-β/Smad3/4 pathways counteract each other on multiple CDKI gene

promoters, where Notch and Smad3 are enriched to a shared chromatin template and inhibit each

other’s binding (Carlson et al., 2008). Notch inhibits CDKI gene expression and promotes muscle

stem cell regeneration in young muscle, whereas mainly TGF-β is expressed in old muscle, with

Smad3 activating CDKI gene expression that leads to block in muscle stem cell regeneration.



Thus, increased expression of CDKI protein is linked to senescence on the whole organism level

through inhibition of stem cell regeneration, which compromises normal tissue maintenance.

Several NRs, such as Hepatocyte nuclear factor 4α (HNF4α), ERRβ, AR, RARs, RXRs, REV-

ERBα /β, RAR-related orphan receptors α and γ (RORα and -γ), and VDR have been shown to

associate with regulatory regions of the mammalian p21 gene and modulate its expression (Lu et

al., 1999; Tanaka et al., 2007; Yu et al., 2008; Hwang-Verslues and Sladek, 2008; Grechez-

Cassiau et al., 2008). For many NRs, including HNF4α, PPARγ and progesterone receptor, the

association with the p21 gene promoter and/or modulation of p21 expression has been suggested

to occur via Sp proteins (Owen et al., 1998; Han et al., 2004; Hwang-Verslues and Sladek, 2008).

This may partially be an artifact, as Sp REs reside close to the TSS, which may be occupied by

the NRs in ChIP assays via chromatin looping, and lack of REs may result from restrictions in the

promoter area searched and/or the in silico method used.

The transcription factor myc inhibits the expression of the p21 gene via interaction with either

Sp1 or Miz-1, both of which up-regulate p21 gene transcription in absence of myc (Gartel and

Radhakrishnan, 2005). As for Sp1-mediated repression, myc reduces the transcriptional activity

of Sp1 without influencing its binding to the p21 gene promoter, whereas Miz-1-mediated

repression depends on DNA methylation by the DNA methyltransferase Dnmt3, that also forms a

complex with myc and Miz-1 (Brenner et al., 2005; Vaque et al., 2005). Additionally, the

ubiquitously expressed YY1 transcription factor (YY1) downregulates p21 gene expression via

association with Sp1, although in vitro YY1 can bind directly to the distal p53 RE of the p21

gene promoter (Santiago et al., 2007; Yakovleva et al., 2004). In addition, YY1 induces p53

degradation by stimulating MDM2-mediated p53 ubiquitination via a direct physical interaction

(Sui et al., 2004). YY1 is able to repress transcription via interaction with the

NCoR1/Sin3A/HDAC3 complex, as well as via HDACs 4 and 5 (Le May et al., 2008; Sucharov

et al., 2008; Wang et al., 2008). YY1 also attenuates 1α,25(OH)2D3-responsive transcription

either by binding to YY1 RE within VDREs or by sequestering the CoAs TFIIB and CBP from

VDR (Guo et al., 1997; Raval-Pandya et al., 2001).



2.2.3 Regulation of cell cycle by 1α ,25(OH)2D3

The main anti-proliferative effect of 1α,25(OH)2D3 on cells is cell cycle block at the G1 phase.

This can be explained by changed expression of multiple cell cycle regulator genes. Among the

first targets described, expressions of the p21 and p27 genes were found to be upregulated by

ligand treatment (Jiang et al., 1994; Wang et al., 1996). For the p21 gene a VDRE on the

proximal promoter was characterized, thus establishing p21 as a direct 1α,25(OH)2D3 target gene

(Liu et al., 1996b). Later on it has been questioned, whether p21 truly is a primary target gene or

a secondary target via up-regulation of TGF-β or of the insulin-like growth factor binding protein

3 (IGFBP3), and whether the described VDRE is truly functional (Danielsson et al., 1997;

Verlinden et al., 1998; Boyle et al., 2001). The credibility of the p21 gene as the anti-proliferative

target of 1α,25(OH)2D3 has also been eroded by studies showing lack of induction of p21

expression upon to 1α,25(OH)2D3 exposure, and the demonstration that 1α,25(OH)2D3 has anti-

proliferative effects also in p21 knockdown cells (Akutsu et al., 2001; Wade et al., 2002;

Ryhänen et al., 2003). However, siRNA against the p21 transcript abolished the growth inhibition

of LNCaP malignant prostate cells by 1α,25(OH)2D3, thereby supporting the role of p21 as an

important anti-proliferative target of 1α,25(OH)2D3 (Rao et al., 2004).

In general, the regulation of the quantity of the p27 occurs via protein stabilization and this is

how 1α,25(OH)2D3 appears to increase this protein’s abundance as well. Firstly, 1α,25(OH)2D3

represses cyclin E gene expression. As a result, cyclin E cannot activate CDK2 to phosphorylate

p27 on threonine 187, a modification needed for the binding of the ubiquitin ligase complex that

includes S-phase kinase-associated protein 2 (p45) (SKP45). Secondly, 1α,25(OH)2D3 represses

the transcription of SKP45 gene itself, thus inhibiting p27 degradation by the proteasome (Liu et

al., 2002; Lin et al., 2003; Li et al., 2004). Also a transient mRNA response of p27 gene has been

reported, but the significance of the transcriptional response as opposed to protein stabilization

remains unclear (Muto et al., 1999). The importance of p27 as an anti-proliferative target of

1α,25(OH)2D3 is as controversial as that of p21: ligand arrested only wild-type mouse embryo

fibroblasts cells in the G1 phase, whereas p27 null cells were not arrested, although when

immortalized, p27 null mouse embryo fibroblasts remain responsive to growth inhibition by

1α,25(OH)2D3 (Li et al., 2004; Wade et al., 2002).



Other CDKIs, such as p15, p16, p18 and p19, show transcriptional response to 1α,25(OH)2D3, but

for p16 the response is secondary as it can be blocked by inhibition of protein synthesis (Liu et

al., 1996b; Tavera-Mendoza et al., 2006). In addition to the already mentioned repression of

cyclin E, transcripts of cyclin D1 and CDK2 are downregulated by 1α,25(OH)2D3 (Alagbala et

al., 2006; Verlinden et al., 1998). It remains to be elucidated, whether these effects are primary

and occur via functional VDREs on the regulatory regions of these genes. On the other hand, the

cyclin C transcript levels are induced by 1α,25(OH)2D3 and the cyclin C promoter has functional

VDREs (Polly et al., 2000b; Sinkkonen et al., 2005). On protein level, 72 h treatment with

1α,25(OH)2D3 analogs has been shown to downregulate cyclin C expression, whereas shorter

treatment (4 to 12 h) with 1α,25(OH)2D3 induces cyclin C protein levels (Verlinden et al., 2000;

Polly et al., 2000b). Cyclin C is linked to cell cycle regulation via binding to Cdk3 protein and to

transcriptional regulation via binding to Cdk8 protein (Hoeppner et al., 2005). The complex

formed by cyclin C and Cdk3 phosphorylates pRb at serine 807/811, and ectopic cyclin C

expression promotes G0 exit (Ren and Rollins, 2004). The cyclin C –Cdk8 pair complex with

MED12 and MED 13 form an accessory subcomplex of the Mediator, where Cdk8 inhibits PIC

assembly via phosphorylation of the carboxy-terminal of the largest subunit of Pol II in vitro (Sun

et al., 1998, Meyer et al., 2008). Cyclin C-Cdk8 complex also phosphorylates and inactivates

cdk7-cyclin H subunits of the TFIIH complex (Akoulitchev et al., 2000). In complex with lysine

acetyltransferase 2A (KAT2A, also called GCN5L), transformation/transcription domain-

associated protein (TRRAP) and the mediator subcomplex Cdk8 phosphorylates serine-10 of

histone H3, which in turn stimulates H3K14 acetylation by KAT2A, and Cdk8 knockdown

substantially reduces these histone modifications globally (Meyer et al., 2008). Interestingly,

Cyclin C-Cdk8 complex also functions as a co-activator for p53: it induces the transcriptional

activation potential of p53 as shown by reduced p21 mRNA upon CDK8 knockdown and

increased the p21 promoter reporter gene construct activity upon ectopic Cyclin C and Cdk8

expression (Donner et al., 2007).

In addition to p21, also other p53 target genes, such as the GADD45A, members of the IGFBP

gene family and MYC, also respond to 1α,25(OH)2D3 treatment (Simpson et al., 1987; Baudet et

al., 1996; Colston et al., 1998; Akutsu et al., 2001; Prudencio et al., 2001; Pálmer et al., 2003;

Matilainen et al., 2005; Ho et al., 2005). GADD45A plays an essential role in DNA repair and



GADD45 proteins also bind and inhibit Cdc2, displacing cyclin B1 and thus inhibit the formation

of M phase-promoting factor that is essential for G2/M transition (Zhan et al., 1999). GADD45A

has been shown to be a direct transcriptional target of 1α,25(OH)2D3 with a functional VDRE

found in the fourth exon of the gene (Jiang et al., 2003). Jiang and colleagues also showed a

specific role for GADD45 in regulation of the cell cycle, as GADD45 was essential for the G2/M

block by 1α,25(OH)2D3, whereas its knockdown did not influence the ligand-induced G1/S block.

IGFBPs modulate the activity of the circulating growth factors named insulin-like growth factors

(IGF) I and II by sequestering them and may also regulate cell growth and apoptosis in an IGF-

independent manner (Meinbach and Lokeshwar, 2006). The IGFBP-3 gene was first discovered

to be upregulated by 1α,25(OH)2D3 and contains a functional VDRE (Colston et al., 1998; Peng

et al., 2004). Subsequently, the transcript levels of IGFBP-1, -2, -5 and -6 genes respond to

1α,25(OH)2D3, of which functional VDREs have been characterized for IGFBP-1 and -5 (Pálmer

et al., 2003; Matilainen et al., 2005). In LNCaP cells, IGFBP-3 was shown to be essential for the

induction of p21 protein expression by of 1α,25(OH)2D3 (Boyle et al., 2001). A possible

mechanism for the p21 response to IGFBP-3 is the activation of TGF-β/Smad pathway by

IGFBP-3, as it induces phosphorylation of Smad2 and Smad3, which in turn are able to activate

p21 gene expression (Baxter, 2001; Moustakas et al., 2002). MYC was one of the earliest found

proliferation-associated targets of 1α,25(OH)2D3: the transcription rate of MYC decreases upon

1α,25(OH)2D3 treatment (Simpson et al., 1987), however no VDREs have been characterized, as

yet. Since 1α,25(OH)2D3 affects many regulators of MYC expression, such as the E-cadherin- β-

catenin–TCF4 pathway and the homeobox B4 transcription factor, indirect effects may contribute

to downregulation of MYC by 1α,25(OH)2D3 (Pálmer et al., 2001; Pan and Simpson, 2001).

Overall, 1α,25(OH)2D3 restricts cell cycle progression in several phases via multiple and partially

redundant targets on parallel pathways, which when combined, provide robustness for the anti-

proliferative effect.


3. Aims of the Study

1. To investigate whether p21 is a primary 1α,25(OH)2D3 target gene and whether VDR

directly binds to the p21 promoter.

2. To study the ligand-dependent co-factor associations of VDR and PPARβ/δ ex vivo.

3. To assess the role of HDACs in the response of CDKI genes to 1α,25(OH)2D3.

4. To build a dynamic model of the response of p21 to 1α,25(OH)2D3 with special emphasis

on chromatin modifications and looping.


4. Materials and Methods

This section provides the materials and methods for the parts of the research contained with the

presented research articles that I have conducted myself. This also applies for the section with

results, except for the in silico characterization of REs, performed by Dr. Christian Frank, and the

execution of gel shift and reporter gene assays, performed by Prof. Carsten Carlberg.

4.1 Materials

4.1.1 Cell culture

MCF-7 is a human luminal mammary cancer cell line derived from pleural effusion of a 69-year

old woman with invasive ductal carcinoma (Neve et al., 2006). MCF-7 cells are responsive to

estrogen and express wild-type p53, although at a lower level than normal breast epithelium

(Neve et al., 2006; Raman et al., 2000). MDA-MB453 mammary luminal metastatic

adenocarcinoma cells were derived from a pericardial effusion of a 48-year woman with breast

cancer that had metastasized to the lymph nodes, brain and both pleural and pericardial cavities

(Neve et al., 2006). MDA-MB453 cells do not express ERs and have mutated p53 protein with

aberrations in the DBD (Lacroix et al., 2006; Neve et al., 2006). MCF-12A is a spontaneously

immortalized mammary epithelial non-tumorigenic epithelial cell line established from residual

tissue samples resulting from a reduction mammoplasty from a 60-year old woman with

fibrocystic breast disease containing focal areas of intraductal hyperplasia (Neve et al., 2006).

This cell line expresses wild-type p53, but lacks ERs and unlike MCF-7 and MDA-MB453 cells,

shows basal lineage-like gene expression patterns instead of luminal (Neve et al., 2006). The

HEK293 cell line was generated by transformation of normal human embryonic kidney cells with

adenoviral DNA (Graham et al., 1977).

MCF-7 cells were used in experiments for (I) and (III), MDA-MB453 cells for (III) and (IV),

MCF-12A cells for (III) and HEK293 cells for (II). MCF-7 and MDA-MB453 cells were grown



in α-MEM containing 5 or 10 % fetal bovine serum (FBS), respectively, HEK293 cells in

DMEM with 10 % FBS and MCF-12A cells were grown in a mixture of DMEM and Ham’s F12

medium (1:1) with 20 ng/ml of epidermal growth factor, 100 ng/ml of cholera toxin, 10 mg/ml

insulin, 500 ng/ml hydrocortisone and 5 % horse serum. All cell lines were grown in a humidified

incubator (atmosphere 95 % air / 5 % CO2) and media was supplemented with 2 mM L-

glutamine, 0.1 mg/ml of streptomycin and 100 U/ml of penicillin. For experiments, phenol red-

free DMEM with 5 % charcoal-treated FBS and antibiotics was used, except for MCF-12A cells,

for which above mentioned culture medium was used but supplemented with 5 % charcoal-

treated FBS instead of horse serum. For real-time PCR, ChIP and chromosome conformation

capture (3C) assays cells were grown overnight prior to treatments. In siRNA experiments,

DMEM supplemented only with only glutamine was used.

4.1.2 Ligands

In cellular experiments, the physiologically active ligand of VDR, 1α,25(OH)2D3, was used at a

final concentration of 10 nM, diluted in ethanol (I, III) or in ethanol and DMEM (IV) (with either

0.1 % or 0.001 % of solvent in cell culture medium, respectively). The hormone was kindly

provided by Dr. Lise Binderup (LEO Pharma, Ballerup, Denmark) (I and III) or by Dr. Milan

Uskokovic (BioXell Inc., Nutley, NJ, USA) (IV). The thymidylate synthase inhibitor 5-

fluorouracil (5-FU), that induces p53 protein levels, was used at 5 mM concentration diluted in

dimethyl sulfoxide (DMSO) with 0.1 % of solvent in cell culture medium, and was purchased

from Sigma-Aldrich (St. Louis, MO, USA) (I). The specific PPARβ/δ agonist GW501516 was

purchased from Alexis Biochemicals (San Diego, CA, USA) and used at a final concentration of

100 nM diluted in DMSO with 0.1 % of solvent in cell culture medium (II). The HDAC inhibitor

trichostatin A (TSA) (Sigma Aldrich, St. Louis, MO, USA) was used in a concentration of 15

nM, diluted in ethanol with 0.1 % of solvent in cell culture medium (III).

4.1.3 DNA constructs

The locations and sequences of the human p21 gene promoter fragments are provided in the

original articles (I) and (III). The fragments were generated by PCR from human genomic DNA

and sub-cloned into a plasmid containing the thymidine kinase minimal promoter and the

Materials and Methods


luciferase reporter gene (pGL3) with XbaI restriction enzyme (I, III) or into a plasmid (pGL4.10,

Promega, Madison, WI, USA) that contains only the luciferase reporter gene with expression

vector using XbaI and NheI restriction enzymes (IV). Point mutations to selected constructs were

generated using the QuickChange site-directed mutagenesis kit (Stratagene) according to the

manufacturer’s instructions. The locations of the mutations are indicated in the original

publication (I). All constructs were verified by sequencing. For 3C experiments in (III) and (IV),

positive controls were made from plasmids containing p21 promoter fragments as mentioned (III

and IV). 1 µg of plasmid covering the responsive target region and 1 µg of plasmid covering the

TSS were digested with 50 U of MvaI, Hpy8I or SchI (Fermentas, Vilnius, Lithuania) overnight at

37 °C and ligated with T4 ligase (Fermentas) for 2 h at room temperature (RT).

4.2 Methods

4.2.1 In vitro methods

4.2.1.1 siRNA interference

Before siRNA transfection for (III), cells were grown to 30-40 % confluency in 6-well plates.

Cells were the transfected with 200 pmol of each double-stranded siRNA oligonucleotides

(Eurogentec, Liege, Belgium) with 5 µl of Lipofectamine 2000 (used for MCF-12A cells) or

Lipofectamine RNAiMAX (MCF-7 and MDA-MB453) (Invitrogen, Carlsbad, CA, USA). The

transfection reagent was diluted to DMEM supplemented only with glutamine and incubated for

5 min at RT, during which time the oligonucleotides for each target were mixed and diluted to

medium. Following this, the reagent and oligonucleotide mixture was mixed, followed by 20 min

incubation at RT, after which the lipofectamin-oligonucleotide mixture was pipeted on medium-

aspirated cells. After 4 to 6 h from transfection, medium with charcoal-treated 5 % FCS was

added to final volume of 2 ml. RNA extraction was performed 48 h after the start of the

transfection.

For (IV) MDA-MB453 cells were reverse transfected with Lipofectamine RNAiMAX

(Invitrogen) with the aforementioned double-stranded siRNA oligonucleotides. In reverse

transfection, the oligonucleotides were first diluted to medium, into which the transfection

reagent was subsequently added and the mixture was incubated for 15 min at RT, followed by



addition of cells in medium. For RNA extractions 650,000 cells, for 3C assays 750,000 cells and

for ChIP assays 4,500,000 cells were used. Cell treatments were started 24 h after plating, after

which cells were used for RNA extraction, ChIP assays or 3C assays.

4.2.2 Ex vivo analyses

4.2.2.1 RNA extraction

Cells were seeded on 6-well plates and grown overnight to reach 50 to 60 % confluency (I, III)

or, for (IV) 650,000 cells for each well were seeded approximately 24 h prior to collection. Total

RNA was extracted using the Mini RNA Isolation II kit (Zymo Research, HiSS Diagnostics,

Freiburg, Germany). Prior to collection, cells were treated as indicated in the publications and

washed twice (I, III) or once (IV) with ice-cold phosphate buffered saline (PBS, 140 mM NaCl,

2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4.2H2O), after which cells were lysed and RNA

was extracted using 600 µl of ZR buffer. The RNA lysate was collected and adsorbed to matrix

of the ZYMO Spin Columns and washed twice with RNA wash buffer (350 µl each time) with 1

min of 14,000 x g centrifugation and discarding of the flow-through after each step. RNA was

eluted to 50 µl of sterile H2O and the purity and RNA concentration was measured by a

NanoDrop ND-1000 (NanoDrop, USA).

4.2.2.2 cDNA synthesis

To reverse-transcribe 1 µg of total RNA to cDNA in (I) and (III), 100 pmol of oligodT18 primer,

20 nmol of dNTPs, 200 pmol of DTT, reverse transcriptase buffer (50 mM Tris–HCl, pH 8.3, 50

mM KCl, 4 mM MgCl2, 10 mM DTT), 40 U of reverse transcriptase and 40 U of RNAse

Inhibitor (buffer and enzymes from Fermentas) were incubated for 1 h at 37 °C in a 40 µl final

volume. Following synthesis the reverse transcriptase was inactivated for 5 min at 95 °C and

cDNA was diluted 1:10 in sterile H2O before further use.

For (IV) cDNA synthesis was performed using Transcriptor first strand cDNA synthesis kit

(Roche Diagnostics, Mannheim, Germany). Here, 1 µg of total RNA was pre-incubated with 50

pmol of anchored oligodT18 (targeting the 5’ end of the 3’ poly(A) tail) for 10 min at 65 °C in

volume of 13 µl and the subsequent synthesis was performed for 30 min at 55 °C with dNTP mix



(20 nmol of each), Transcriptor Reverse Transcriptase Reaction Buffer, 10 U of reverse

transcriptase and 20 U of RNAse Inhibitor in 20 µl total volume. Following synthesis, the reverse

transcriptase was inactivated for 5 min at 85 °C and the cDNA was diluted 1:20 with sterile H2O.

4.2.2.3 Real-time PCR

Real-time quantitative PCR (for I and III) was performed in an IQ-cycler (BioRad, Hercules, CA,

USA) using the dye SybrGreen I (Molecular Probes, Leiden, The Netherlands) that binds to

double stranded DNA. Per reaction, 1 U Hot Start Taq polymerase (Fermentas) (I) or FastStart

polymerase (Roche) (III), 0.18 mM of MgCl2, 1 x HotStart or FastStart buffer, respectively,

dNTPs (0.2 mM of each), 1:2000 diluted SybrGreen stock and 2 % DMSO was used in a total

volume of 20 µl. In the PCR, DNA was pre-denaturated at for 5 min 95 °C, amplified with 40

cycles of 30 s of denaturation at 95 °C, 30 s of primer annealing at primer specific temperatures

and 30 s of elongation at 72 °C, followed by further elongation of 10 min at 72 °C.

In (IV), the real-time quantitative PCR for cDNA was performed using a LightCycler® 480

System (Roche) and FastStart SYBR Green Master mix (Roche). Each reaction was performed

using 4 pmol of each specific primer, 4 µl of cDNA template and 1x supermix in a volume of 10

µl and the PCR cycling conditions were: pre-incubation for 10 min at 95 °C, 38 cycles of 20 s at

95 °C, 15 s at 60 °C and 15 s at 72°C.

Fold inductions were calculated using the formula 2(ΔΔCt), where ΔΔCt is the ΔCt(stimulus)-

ΔCt(solvent), ΔCt is Ct(target gene)-Ct(RPLP0), Ct is the cycle at which the threshold is crossed

and RPLP0 is the housekeeping gene ribosomal protein, large, P0. Quality of the PCR product

was monitored using post-PCR melt curve analysis.

4.2.2.4 Chromatin immuno-precipitation assay

Nuclear proteins were cross-linked to genomic DNA by adding formaldehyde directly to the

medium to a final concentration of 1 % with 10 (I) or 5 min (II-IV) incubation at RT. Cross-

linking was stopped by adding glycine to a final concentration of 0.125 M and incubating for 5

min at RT on a rocking platform. The medium was removed and the cells were washed twice

with ice-cold PBS. The cells were collected by scraping into ice-cold PBS supplemented with a



protease inhibitor cocktail (Roche) (I) or into plain ice-cold PBS (II-IV). Cells were pelleted by

centrifugation (700 g for 5 min at 4 °C), pellets were resuspended in lysis buffer (1 % SDS, 10

mM EDTA, protease inhibitors and 50 mM Tris-HCl, pH 8.1) and the lysates were sonicated to

result in the majority of DNA fragments being 300 to 1000 bp in length.

Cellular debris was removed by centrifugation (16,000 g for 15 min at 4 °C), 1:40 volume of the

supernatant was taken for inputs that serve as a control for the target DNA amount for each

sample. For outputs, the remaining supernatant was diluted 1:10 in ChIP dilution buffer (0.01 %

SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 16.7 mM NaCl, protease inhibitors and 16.7 mM

Tris–HCl, pH 8.1). The input sample was diluted in ChIP Dilution buffer and further processed

like outputs from step with reverse-crosslinking onwards. Non-specific background was removed

by incubating the chromatin resuspension with a salmon sperm DNA/protein A agarose slurry

(Upstate Biotechnology, Lake Placid, NY, USA) for 30 min at 4 °C with agitation (I, III).

Addition of 2.4 µg of salmon sperm DNA directly to the samples or pre-incubation with salmon

sperm beads was left out and instead protein A agarose bead slurry (Upstate) was blocked

overnight with 10 mg/ml of bovine serum albumin and 0.1 mg/ml of salmon sperm DNA at 4 °C

(IV). The samples were centrifuged (I-III) and the recovered chromatin solutions were incubated

with 1 µg of purified antibodies or with 1.8 µl of anti-sera, both indicated in individual

publications, overnight at 4 °C with rotation.

The immuno-complexes were collected with 60 µl of protein A agarose slurry for 2 h (I) or 1 h

(II-IV) at 4 °C with rotation. The beads were pelleted by centrifugation for 1 min at RT at 100 g

and washed sequentially (each time for 5 min) by rotation with 1 ml of the following buffers: low

salt wash buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 150 mM NaCl and 20 mM Tris–

HCl, pH 8.1) (used twice in IV), high salt wash buffer (0.1 % SDS, 1 % Triton X-100, 2 mM

EDTA, 500 mM NaCl and 20 mM Tris–HCl, pH 8.1) and LiCl wash buffer (0.25 mM LiCl, 1 %

Nonidet P-40, 1 % sodium deoxycholate, 1 mM EDTA and 10 mM Tris–HCl, pH 8.1). Finally,

the beads were washed twice with 1 ml TE buffer (1 mM EDTA and 10 mM Tris–HCl, pH 8.0)

and then eluted twice with 250 µl of elution buffer (1 % SDS and 100 mM NaHCO3) by

incubation for 15 min at RT with rotation. For (IV) chromatin bound to agarose beads was eluted

with a different elution buffer (25 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.5 % SDS) for 30 min

at 65 °C followed by second elution for 2 min at RT.



After centrifugation, the supernatants were collected and combined, and the cross-linking was

reversed by adding NaCl to final concentration of 200 mM (I) and incubating overnight at 65 °C.

The remaining proteins were digested by proteinase K either following reverse crosslinking (I) by

incubation for 1 h at 45 °C with proteinase K from Invitrogen (Carlsbad, CA, USA; final

concentration 40 µg/ml) or by proteinase K from Fermentas (Vilnius, Lithuania; final

concentration 38 µg/ml) during the reverse crosslinking at 65 °C (II-IV). The DNA was

recovered by phenol/chloroform/isoamyl alcohol (25/24/1) extraction and precipitated with 0.1

volumes of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol using glycogen as a carrier.

Input and output samples were dissolved in 200 µl and 60 µl of H2O, respectively. The difference

in dilution volumes, in addition to the difference in the amount of starting material used were

taken into account when calculating PCR results from ChIP template.

For re-ChIP (I-III) the immuno-complexes were eluted by adding 100 µl re-ChIP elution buffer

(10 mM DTT) for 30 min at RT with rotation, the supernatant was diluted 1:40 in ChIP dilution

buffer and the antibody against the second protein of interest was added, the new immuno-

complexes were allowed to form by incubating overnight at 4 °C on a rocking platform, the

immuno-complexes were collected by incubating with 120 ml protein A agarose slurry for 2 h at

4 °C on a rocking platform and finally washed as indicated above. Elution and DNA purification

were then carried as described above for regular ChIP.

4.2.2.5 Chromatin conformation capture assay

In 3C assays, chromatin is crosslinked in living cells as in ChIP assays, but instead of shearing

and antibody-based selection, chromatin is restricted and then ligated in circumstances favoring

intramolecular interactions. Hence the probability that two distant chromatin regions separated by

multiple restriction sites will form a ligation product increases, if they are looped together. To

exclude the contribution of random chromatin associations, we used non-responsive regions

between the TSS and the responsive regions as negative controls as the probability of random

interactions increases with decreasing distance (Simonis et al., 2007). For (III) 1:10 of chromatin

in a 60 to 70 % confluent cell culture flask (175 cm2) or for (IV), chromatin from two wells of a

6-well plate (750,000 cells plated per well), was collected, crosslinked and lysed as for ChIP

assays, except for the sonication time, which was reduced to two (III) or one (IV) 15 s pulse to



complete cell lysis without shearing the DNA. After removal of cellular debris by centrifugation,

100 (III) or 25 μl of chromatin was diluted in 345 μl (III) or 75 μl (IV) of ChIP dilution buffer

and digested overnight at 37 °C with the restriction enzymes MvaI (50 U for (III), 25 U for (IV)),

Hpy8I (25 U) or SchI (25 U, only used in (III)) and Red buffer (with MvaI) or Tango buffer (with

Hpy8I or SchI) (enzymes and buffers from Fermentas, Vilnius, Lithuania). Digested chromatin

was diluted 1:1.5 (III) or 1:6 (IV) to T4 ligation buffer supplemented with 0.5 mM ATP and

ligated with 50 (III) or 15 U (IV) of T4 DNA ligase (Fermentas, Vilnius, Lithuania) for 2 h at 16

°C (III) or for 4 h at RT (IV). Samples were reverse crosslinked and DNA was recovered as

described for ChIP assays.

4.2.2.6 PCR of chromatin templates

Semi-quantitative PCR for (I), (II) and (III) was performed in an IQ- and the dye SybrGreen I

(stock diluted 1:2000) with 1 U Hot Start Taq polymerase (Fermentas) (I) or FastStart polymerase

(Roche) (II, III), 0.09 mM MgCl2, 1 x HotStart or FastStart buffer, respectively, dNTPs (0.2 mM

of each) and 2 % DMSO was used in a volume of 25 µl. PCR program used was the same as

described for real-time PCR in section 4.2.3.3. The PCR products were separated by

electrophoresis through 2 % agarose and shown as representive gels (I, II) or with strong protein

association (I, III), relative enrichment levels were calculated using the formula 2-ΔCt, where ΔCt

is Ct(output) - Ct(input) normalized by the amount of chromatin used for preparation of input

versus output. Gel images were scanned on a FLA3000 reader (Fuji) using ScienceLab99

software (Fuji).

In (IV) genomic primers indicated in the original publication were designed for each of the three

VDRE carrying regions and the TSS of the p21 gene and for their quantification 6-

carboxyfluorescein (FAM)-modified probes (Eurogentec, Liege, Belgium) were used. Similarly,

primers were designed for the detection of 3C ligation products and their TaqMan quantification

(Eurogentec, Liege, Belgium). To achieve quantifiable product specificity, the probes used for 3C

ligation products were targeted against the ligation site, as described previously for quantification

of chromatin looping (Jing 2008). Oligonucleotides were obtained from Eurogentec. Real-time

quantitative PCR was performed with the Maxima Probe qPCR master mix (Fermentas), 4 µl of

purified DNA template, 4 pmol primers and 1 pmol labeled probe on a LightCycler® 480 System



(Roche). The PCR cycling conditions for 2-step PCR used were: pre-incubation for 10 min at 95

°C, 50 cycles of 20 s at 95 °C and 60 s at 60 °C. The PCR products were also resolved on 2 %

agarose gels to control for correct product size. Relative association of chromatin bound proteins

or histone modifications were calculated using the formula 2-ΔCt, where ΔCt is Ct(output)-

Ct(input), output is the immuno-precipitated DNA and input is the purified genomic DNA from

starting material of the ChIP assay. For the 3C assay relative chromatin looping was calculated

using the formula 2-ΔCt, where ΔCt is Ct(3C ligation product)-Ct(positive control plasmid) and

then divided by the 2-ΔCt value of non-treated p21-2 for Hpy8I restricted template or p21-3 for

MvaI restricted template. Positive control plasmids are described in original publications and in

section 4.1.3.


5. Results

5.1 The human p21 gene is regulated by p53 and VDR via multiple response

elements

The p21 has been characterized as the main target gene of p53 in order to block the cell cycle

upon exposure to cellular stresses, such as DNA damage, hypoxia or oncogene activation. The

regulation of p21 expression by p53 occurs via two REs at positions 1400 and 2300 bp upstream

from the p21 TSS, and the p53 response can be activated, e.g., by inducing translation of p53

protein with the application of the cytotoxic anti-cancer drug 5-FU (Espinosa 2001, Ju 2007).

1α,25(OH)2D3 has been suggested to up-regulate p21 gene transcription via a VDRE at position

750 bp upstream of the TSS, but the functionality of this RE has been questioned, as well as the

role of p21 as a primary 1α,25(OH)2D3 target gene (Liu et al., 1996b; Danielsson et al., 1997;

Verlinden et al., 1998). Most established REs on the p21 promoter have been characterized based

on mutation/deletion analysis of promoter constructs with a length of 2.5 kbp or less, thus

ignoring putative distal REs. Additionally, the transcriptional response is often measured only in

one or two time points neglecting the possible dynamic nature of the response.

To assess the transcriptional response of the p21 gene to 1α,25(OH)2D3 or to induce p53 in MCF-

7 breast cancer cells, the cells were treated with 1α,25(OH)2D3 or 5-FU, respectively, or solvent

and used as a template for real-time PCR and ChIP assays. The induction of p53 by 5-FU caused

a steady increase of p21 mRNA up to a level of 5.2-fold after 360 min of stimulation, whereas

1α,25(OH)2D3 induced p21 mRNA expression with a maximum of only 1.6-fold induction after

120 min of stimulation (I, Figs. 1A and B).

In order to get an extensive view of binding of VDR and p53 on the p21 promoter, we screened

7.1 kbp of chromosomal DNA upstream of the TSS by overlapping ChIP regions. In addition to

the two established p53 binding sites, we discovered a novel but relatively weak site residing 4 to



4.5 kbp upstream of the TSS, as well as (indirect) binding onto the TSS, with between 3.7- to 70-

fold inductions by 5-FU (I, Figs. 2B and C). Surprisingly, VDR was found to associate with two

regions with p53 binding sites, residing at -2 to -2.5 kbp and the newly identified p53 RE at 4 to

4.5 kbp upstream the TSS (I, Fig. 3). In addition, VDR was binding to a region 6.7 to 7.1 kbp

upstream of the TSS, but not to the region with the previously described VDRE at position -750

bp. In in silico screening for VDR and p53 binding sites on the p21 promoter regions that were

positive in ChIP assays, eight and five putative REs were found (I, Fig. 4), of which five and

three were found positive in in vitro gel shift assay, respectively. The functionality of putative

REs was confirmed by reporter gene assays, where both the previously established p53 binding

sites and the novel p53 site as well as the five newly identified VDREs residing on three ChIP

regions were confirmed to be functional in activation of transcription.

In conclusion, these data confirm p21 as primary 1α,25(OH)2D3 target gene with three functional

responsive regions that bind VDR both in vitro and ex vivo, residing at 2, 4.5 and 7 kbp upstream

of the TSS. The two proximal 1α,25(OH)2D3 target regions also harvest functional p53 REs,

putatively representing regulatory hotspots on the human p21 gene promoter.

5.2 Association of nuclear receptor complexes on regulatory regions of the

human PDK gene family and the human p21 gene

As NRs, the PPARs and VDR have similarities in their strategy to induce target gene expression:

they share a common heterodimerization partner RXRα, bridge to the basal transcription

machinery and to Pol II via the MED1 subunit of the Mediator complex (Rachez et al., 1999; Zhu

et al., 1997) and associate with a multitude of shared co-factors (Mukherjee et al., 2002; Deeb et

al., 2007; Feige and Auwerx, 2007). On the other hand, PPARs possess intrinsic, ligand-

independent ability to associate with CoAs thus resembling orphan NRs, whereas VDR is able to

recruit CoAs only in the presence of its ligand (Molnar et al., 2005).

In HEK293 cells treated with the selective PPARβ/δ agonist GW501516, the association of

PPARβ/δ with co-factors and phosphorylated Pol II (p-Pol II) was studied on the TSSs and on six

responsive regions containing functional PPAR REs (PPREs) of the genes pyruvate

Results


dehydrogenase kinase (PDK) 1 to 4 by re-ChIP assays (II, Fig. 5). Of the regions studied, six

showed p-Pol II enrichment suggesting their direct transcriptional contribution. This association

was also ligand-dependent, except for the region 2 of PDK4. The association with RXRα was

more divergent in response to ligand and even repressed by the ligand on region 2/TSS and on

region 3 of the PDK2 and PDK3 genes, respectively. Association of the CoA PGC-1α was

induced except for the TSSs of PDK 2 and 4 genes, whereas MED1 (TRAP220 in Fig. 5 of II)

association was reduced by the ligand on the PDK2 gene TSS/region 2 and on the PDK3 gene

region 1. The ligand-free association with MED1 may reflect the aforementioned ability of

PPARs to associate with CoAs in the apo-form.

Composition of NR complexes by re-ChIP was also studied in MDA-MB453 (III) and MCF-7 (I)

breast cancer cells targeting VDR-associated factors on the TSSs of all expressed CDKI genes

(III, Fig. 2D) and the 1α,25(OH)2D3-responsive regions of p21 (I, Fig. 7; III, Fig. 3E). Although

both p18 and p21 TSSs harvested VDR-Pol II complexes, they were not induced with 60 min of

ligand treatment (III, Fig. 2D), unlike seen for the responsive regions (III, Fig. 3E). In contrast to

the PPAR response of the PDK genes, MED1 association with VDR was not present on the

1α,25(OH)2D3-responsive regions of the p21 gene without ligand, supporting the low CoA

binding ability of non-liganded VDR (Molnar 2005). Also, VDR associated with CBP and

especially with NCOA1 (referred as SRC-1 in I) in MCF-7 cells in treatment-time dependent

manner starting from 15 to 30 min of treatment (I, Fig. 7). CBP showed pronounced basal

association on regions that harvest also p53, presumably due to the ability of CBP to associate

with p53 in addition to NRs (Lill 1997, Avantaggiati 1997).

In conclusion, both PPARβ/δ and VDR form complexes with Pol II and MED1 on responsive

regions of their target genes, where VDR is dependent on the presence of its ligand and PPARβ/δ

shows higher basal ability to induce bridging via MED1.



5.3 Role of HDACs in regulation of human CDKI genes by 1α ,25(OH)2D3 and

TSA in mammary cell lines

In addition to mutations in DNA sequence, chromatin alterations leading to aberrant gene

expression patterns are crucial in both onset and progression of cancer (Baylin and Ohm, 2006).

The reversal of aberrant histone modifications and hence, the following aberrant gene expression

patterns in cancer cells motivates the research on HDAC inhibitors, which have been shown to

possess potent anti-cancer activities in pre-clinical studies (Bolden et al., 2006). Among the anti-

proliferative targets of HDAC inhibitors are several members of the CDKI family, namely p15

(CDKN2B), p16 (CDKN2A), p18 (CDKN2C), p19 (CDKN2D) and p21 (CDKN1A) (Richon et al.,

1996; Hitomi et al., 2003; Yokota et al., 2004a; Yokota et al., 2004b; Wu et al., 2008). ). As

several of the CDKI genes have been shown to respond to 1α,25(OH)2D3 as well (Liu et al.,

1996b), we wanted to explore the modulation of this response by the HDAC inhibitor TSA and

the roles of individual HDACs in this response.

The mRNA expression and TSS histone acetylation status of the four CDKI genes p18, p19, p21

and p27 were monitored by real-time PCR (III, Figs. 2B and S2A) and ChIP assays (III, Figs. 2B

and 3C) in three mammary cell lines with different stages of malignancy, MCF-12A, MCF-7 and

MDA-MB453 cells, which were treated with either 10 nM 1α,25(OH)2D3, 15 nM TSA or their

combination. The three remaining CDKI gene family members, p15, p16/p14 (both transcribed

from CDKN2A) and p57 (CDKN2C) were not expressed to detectable level and hence were

excluded from further analysis. The highest inductions were seen with TSA on the expression of

p18 and p19 mRNA in MCF-7 cells (11- and 6-fold at 4 h) (III, Figs. 2B and S2B), accompanied

by inductions in the acetylation levels of the TSSs regions as seen in ChIP assays with 1 h of 15

nM TSA treatment (2-fold for H3K14ac, called AcH3K14 in III, and 4-fold for H4ac, called

AcH4 in III, in MCF-7 cells; III, Fig. 2A). However, the TSS induction of the acetylation at 1 h

cannot explain the high mRNA inductions, since the acetylation was similarly induced on the

p18-TSS in MDA-MB453 cells and on the p21-TSS in MCF-7 cells, in neither of which the

change was transmitted to high transcript levels.

In all three cell lines the established 1α,25(OH)2D3 target gene p21 (Liu et al., 1996b) was

significantly induced by 1α,25(OH)2D3 (1.6- to 3.6-fold). Additionally, the other studied genes

Results


showed inductions, but less consistently. However, in most cases these mRNA fold inductions

were not reflected by changes of the acetylation levels of the respective CDKI gene TSS regions

(III, Fig. 2A), unlike on the 1α,25(OH)2D3-responsive regions of p21, namely p21-1 (p21-7 in

(I)), p21-2 (p21-12C in (I)) and p21-3 (p21-19 in (I)) (see Fig. 3A of (III) for location). On the

ligand-responsive regions, the effects of 1α,25(OH)2D3 on the acetylation status were more

pronounced than seen on the TSS (III, Fig. 3C), while TSA still induced both of the studied

modifications. In general, the combined treatment with TSA and 1α,25(OH)2D3 resulted in all

three cell lines in a higher mRNA expression level than either of the substances alone (an

exception was the p27 gene in MCF-7 cells, see Fig. 2B and S2B of III), but not in additional

histone acetylation on the TSSs (III, Fig. 2A). In a number of cases, these co-operative effects

were also statistically significant, pointing out the potential of combined use in the attenuation of

proliferation (III, Fig. S2B). On one responsive region, p21-2 at -4.5 kbp from the TSS, the

combination of the ligand and TSA even synergistically induced H3K14ac on in MDA-MB453

cells (III, Fig. 3C).

To elucidate the role of individual HDACs on acetylation changes on the regulatory regions of

CDKI genes, we analyzed, by ChIP assays, the occupancy of the HDAC1 to 7 and NCoR1 on the

CDKI gene TSSs and the 1α,25(OH)2D3-responsive regions of the p21 gene (III, Figs. 2C and

3D). HDAC3 was the most pre-dominant deacetylase on the TSSs except for p27-TSS (III, Fig,

2C), which was also inert to induction of acetylation by TSA (III, Fig. 2A). HDAC3 was only

associated on ligand-responsive regions, which also bind p53 (I, Fig. 2B) and only in p53 positive

cell lines. NCoR1 was also widely present at the TSSs and to lesser extent on ligand-responsive

regions of the p21 gene. HDAC5 was enriched especially on the TSSs of the Cip/Kip subfamily

genes p21 and p27 and to ligand-responsive regions of p21. HDACs 6 and 7 showed less

enrichment, while the binding of HDACs 1 and 2 was highly infrequent and the latter were not

binding to responsive regions at all. In conclusion, the HDAC association pattern on the

1α,25(OH)2D3-responsive regions of the p21 gene differed clearly from that of the CDKI TSS

regions, with less overall HDAC occupancy, lower HDAC3 enrichment and more dominant

HDAC4 and HDAC5 (III, Fig. 3D).

The effect of 1 h treatment with either TSA, 1α,25(OH)2D3 or their combination on the VDR-

HDAC or VDR-NCoR1 complexes were elucidated by re-ChIP assays (III, Figs. 2D and 3E), in



which HDAC3 to 7 were chosen based on their occupancy on the studied regions and MDA-

MB453 cell line to see p53-independent patterns. HDAC4, 6 and 7 showed most consistent co-

occupancy with VDR on all p21 regions, with reduction upon ligand, whereas HDAC3 showed

lower and more fragmented basal enrichment and was even induced by the ligand. HDAC5 was

strongly co-immuno-precipitating with VDR only to the p21-TSS and responded only to TSA,

showing no change with 1α,25(OH)2D3. The p18-TSS also showed ligand-dependent VDR-

HDAC complex enrichment accompanied by a mild mRNA fold induction (III, Figs. 2B and D).

The putative direct contact between the 1α,25(OH)2D3-responsive regions and the TSS of p21

was studied by 3C assays, in which 1α,25(OH)2D3, but not TSA, induced looping of the two more

distal regions at positions -4.5 kbp and -6.9 kbp (III, Fig. 3B). Surprisingly, DNA looping could

not be detected from the more proximal region at position -2.3 kbp to the TSS, although ligand

was able to induce both histone acetylation and VDR–Pol II complex formation on this region

(III, Figs. 3B, C and E).

In order to define the contribution of selected HDACs to both basal expression and response to

1α,25(OH)2D3 and/or TSA, the three mammary cell lines were transfected with siRNAs against

VDR, NCoR1 or HDAC3, 4, 6 or 7 (III, Figs. 3A, 3B, S4A and B). The effects of the siRNA-

treatments to the ligand-responsiveness of the highly 1α,25(OH)2D3 inducible CYP24A1 gene in

MCF-7 cells (III, Fig. S3C) was also assayed to see how general the contributions of HDACs in

ligand response to the ligand are. HDAC5 was left out based on the low occupancy on VDR

complexes on the ligand-responsive regions. Effects of siRNA on response to treatments in MCF-

12A cells were hardly significant and are not addressed here. Inhibition of expression of HDAC6

or 4 broadly induced the basal expression of CDKI genes, while HDAC6 siRNA did not inflict

the effects of the treatments. Knockdown of HDAC4 attenuated the high induction of p18 to TSA,

but further induced the response of the CYP24A1 gene to 1α,25(OH)2D3 in MCF-7 cells. Release

from HDAC3 repression strongly and specifically induced the basal expression of the INK4 sub-

family genes p19 and especially p18, and also attenuated the induction of these genes by TSA. In

addition to the inhibition of HDAC4, siRNA against HDAC3 and 7 also increased the observed

fold induction of the CYP24A1 gene as well as the p21 gene by 1α,25(OH)2D3 in MCF-7 cells.

NCoR1, and surprisingly, un-liganded VDR, repress CDKI genes, as their removal induced a

significant rise in CDKI basal expression. Knockdown of VDR or NCoR1 also intensified the

Results


response of p18 and partly p19 to TSA and attenuated the response of the p21 gene to

1α,25(OH)2D3, whereas of the proteins studied, only VDR seemed to be needed for the ligand

response of the CYP24A1 gene. Overall, HDACs attenuate CDKI gene expression and response

to 1α,25(OH)2D3, and combining an HDAC inhibitor with 1α,25(OH)2D3 treatment provides

means of augmenting the effect of the ligand via increased induction of CDKI genes.

5.4 The cyclical response of human p21 gene to 1α ,25(OH)2D3 at the level of

transcription factor binding, chromatin looping and transcription

In a classical model of NR activation, the ligand acts as an on-off switch of transcription as it

leads to displacement of CoRs and recruitment of co-factors to the receptor, which in turn leads

to active transcription of the target gene (Deeb et al., 2007). Recently, models have been

presented for the activation of transcription by NRs, where ligand-dependent transcription is seen

as a cyclical process with alternating activating and repressive actions on chromatin, as proposed

for ERα on the trefoil factor-1 gene (Metivier et al., 2003).

To assay the dynamics of the p21 induction, real-time quantitative PCR analysis of p21 mRNA

expression in response to 1α,25(OH)2D3 was performed in a detailed time course of 300 min with

15 min intervals on un-synchronized MDA-MB453 cells (IV, Fig. 1). In the early phase, the

accumulation of p21 mRNA peaked first at 60 min and at 105 min resulting in significant but low

(20 to 30 %) increase in transcript levels. At 150 min of 1α,25(OH)2D3 treatment time, p21

mRNA peaked to 1.7-fold when compared to vehicle, followed by a decrease in transcript level

close to that of untreated cells within 45 min. Subsequently, a fourth peak appeared at 210 min

and a fifth peak at 255 to 270 min. After each of these peaks, the accumulation of p21 mRNA

ceased resulting in a decrease of p21 mRNA levels. The peaks of p21 steady state mRNA levels

appeared in cycles of 45 to 60 min with the longest lag-time after the major peak at 150 min.

These data indicate that 1α,25(OH)2D3 induces the transcription of the p21 gene only for

repeated, short periods at selected time points, and that between these time points transcription is

actively restricted.



In order to elucidate the events on chromatin that precede and putatively lead to cyclical mRNA

accumulation, we performed quantitative ChIP assays using FAM-labeled real-time PCR probes.

We assessed the enrichment of VDR, serine 5 phosphorylated Pol II, MED1, HDAC3, HDAC4,

CBP, NCoR1, YY1, the lysine demethylase LSD1 as well as activity-linked histone

modifications H3K4me2 and H3K9ac on the p21 TSS and the three 1α,25(OH)2D3-responsive

regions p21-1, -2 and 3, targeting the initial phase of transcription from 0 to 150 min with 15 to

30 min intervals (IV, Figs. 2A-C). The chromatin response of the p21 gene to 1α,25(OH)2D3

begun with an initial rise in MED1 association on both 1α,25(OH)2D3-responsive regions and

TSS, LSD1 induction on TSS and release of HDAC3 from the responsive regions. This was

followed by induction of H3K9ac and H3K4me2 on both distal enhancers and on the TSS,

leading to increased p-Pol II association on both the TSS and REs and VDR association on the

REs. The peak in CBP, H3K4me2 and H3K9ac on the TSS at 30 min was followed by a drastic

decrease in association of histone modifications, and putatively of nucleosomes, at 45 to 60 min

preceding the initial peak in mRNA accumulation.

At 90 min binding of VDR to responsive regions was enhanced with re-association of MED1 and

p-Pol II and a peak in H3K9ac, preceding the second mRNA accumulation peak at 105 min.

Subsequently there was an increase in CBP, H3K4me2 and H3K9ac on the TSS, after which, at

150 min, VDR, p-Pol and MED1 were enriched on 1α,25(OH)2D3-responsive regions, with a

time point which displayed increased p21 mRNA accumulation. Basal repression of the REs by

HDAC3, NCoR1 and YY1 on the p21-1 and p21-3 regions was decreased by ligand treatment

within 15 to 30 min, but was re-initiated at 120 min and on responsive regions, even induced

when compared to non-treated cells to decrease activating histone modifications and hence

diminish transcription after it has peaked. In short, these results show distinct cyclicity in

especially VDR, p-Pol II and MED1 associations as well as suggest a clearance of histone

modifications from the TSS upon transcription initiation.

To unravel the spatial prerequisites as to how the 1α,25(OH)2D3-responsive regions, up to 7 kbp

away from the TSS dynamically harvest p-Pol II and hence contribute to enhanced p21 gene

transcription, we performed 3C assays. As for ChIP, we used FAM-labeled real-time PCR probes

to gain maximum specificity of the ligation products. Association of two most proximal

1α,25(OH)2D3-responsive regions to the TSS was studied using the restriction enzyme Hpy8I,

Results


whereas for most distal region p21-3, MvaI was used as Hpy8I has a target sequence between the

two VDREs on this region (IV, Fig. 3A). For 3C assays MDA-MB453 cells were treated

identically as for ChIP assays. On the basal state both the p21-1 and p21-3 regions looped to the

TSS, suggesting a role for these regions in repression by un-liganded VDR, as these regions also

harvested NCoR1 and HDAC3 without the ligand (IV, Figs. 3B and 2B). The increase seen in

MED1 association at 15 to 30 min (IV, Fig. 2B) did not manifest significantly on the association

of ligand-responsive regions to the TSS, although the first peak on chromatin association

appeared from p21-2 to the TSS after 30 min of treatment. On 1α,25(OH)2D3-responsive regions

chromatin looping to the TSS was the highest after 90 min of treatment, with 2- to 15-fold

induction when compared to non-treated cells. This induction of looping from 1α,25(OH)2D3-

responsive regions to the TSS was concomitant to p-Pol II enrichment on these regions, thus

explaining its presence on regions up to 7 kbp away from the TSS. In conclusion, basal looping

from 1α,25(OH)2D3-responsive regions to the TSS in non-treated cells is associated with

enrichment of CoRs to the responsive regions. Introduction of ligand to the cells induces

chromatin loops from all three 1α,25(OH)2D3-responsive regions of the p21 promoter to the TSS

in a dynamic manner. Via looping they contribute to initiation of transcription cycles, which is

supported by concomitant enrichment of p-Pol II on these regions.

ChIP results implied an important role for MED1, CoRs and histone modifying enzymes in

transcriptional response of the p21 gene to 1α,25(OH)2D3. In order to assess this, the expression

of MED1, CBP, HDAC3, HDAC4, LSD1, NCoR1 or YY1 was diminished by siRNA. As

expected, removal of MED1 completely abolished induction of p21 gene expression and

transcription cycles, whereas removal of LSD1 decreased p21 basal expression and the peak at

240 min was lost. Even though the knockdown of HDAC4 and HDAC3 both induced the p21

basal expression, further induction and cycling of p21 expression was abolished by loss of

HDAC4, whereas without HDAC3 the first induction at 150 min was abolished but the

transcriptional cycling remained at later time points. Inhibition of either NCoR1 or YY1 caused

an analogous change to the p21 transcript response showing lower inductions and deviated

timing. Inhibition of CBP expression had no effect on transcription even though 1α,25(OH)2D3

induced significant changes in CBP enrichment on all regions studied. This implies that CBP,



although present on the regulatory regions of p21, is not essential to response of p21 to

1α,25(OH)2D3 and may be replaced by other histone acetylating CoAs.

The drastic effects of removal of a Mediator complex subunit MED1, HDACs or the demethylase

LSD1 on the 1α,25(OH)2D3 influenced cycling of p21 steady state transcript levels, suggest a

crucial role of histone modifications in this phenomenon. Hence, we assayed the effect of

removal of MED1, HDAC4 or LSD1 on acetylation of H3K9 or dimethylation of H3K4 by

combined siRNA and ChIP assays (IV, Fig. 5). Surprisingly, inhibition of HDAC4 did not

systematically induce basal H3K9 acetylation levels and even reduced this modification on two

regions. The effects of siLSD1 were also mild on both modifications, even though LSD1 has the

potential to remove H3K4me2 and indirectly induce H3K9ac by removing of the methyl group in

the same residue. In spite of the low effects seen on the basal level, inhibition of either HDAC4

or LSD1 inhibited the clearance of both H3K4me2 and H3K9ac from TSS region, suggesting an

important role for these modifiers in removal of nucleosomes from TSS in preparation for

transcription. On the ligand-responsive regions MED1 and HDAC4 were essential for the

induction of H3K4 dimethylation on the proximal region p21-1, whereas on the most distal

1α,25(OH)2D3-responsive region of the p21 promoter, p21-3, MED1, HDAC4 and LSD1 were

similarly indispensable for induction of H3K9 acetylation. These results imply a strong interplay

between H3K9ac and H3K4me2 as the removal of the demethylase inhibits the induction in

acetylation and the removal of the deacetylase blocks the enhancement of dimethylation.

As MED1, HDAC4 or LSD1 are crucial to ligand-dependent dynamical changes in histone

modifications and resulting mRNA accumulation, a factor-specific siRNA transfection followed

by a 3C assays were performed to study their role in chromatin looping (IV, Fig. 6). On the

proximal region p21-1, 1α,25(OH)2D3 only mildly induced looping and the effects of above

mentioned siRNAs on ligand-induced looping from p21-1 to the TSS were not significant, even

though inhibition of LSD1 increased levels of association of this region to the TSS containing

one. On the contrary, on the two distal elements removal of either MED1 or LSD1 blocked the 90

min peak in association to the TSS, but did not effect the basal looping significantly. For the most

distal region at -7 kbp, also HDAC4 was essential for looping. These results confirm that, as

expected, bridging from VDR to the basal transcriptional machinery by MED1 is essential for the

dynamic looping of the distal ligand-responsive regions of the p21 gene to its TSS. Significantly,

Results


we found that also ordered and specific deacetylation by HDAC4 and demethylation by LSD1 are

essential for optimal chromatin looping.


6. Discussion

6.1 Human p21 is a primary 1α ,25(OH)2D3 target gene

The hypothesis of p21 as a direct 1α,25(OH)2D3 transcriptional target has been repeatedly

questioned but our studies present firm evidence to support it. First of all, we characterized three

novel functional binding sites associated with VDR-Pol II complexes ex vivo in both of the

studied mammary cancer cell lines used in this study (I, III). Secondly, we show coherent

transcriptional response of p21 to 1α,25(OH)2D3 in three mammary cell lines with different

stages of malignancy, and thirdly, we show ligand-dependent chromatin looping that provides a

physical contact between 1α,25(OH)2D3-responsive regions and the p21 gene TSS. Induction of

TGF-β/Smad, IGFBP-3 and MAPK pathways have been proposed as targets for 1α,25(OH)2D3

upstream of the human p21 gene, as their inhibition has lead to abolishment of p21 induction

(Verlinden et al., 1998; Boyle et al., 2001; Wu et al., 2007). Inductions of TGF-β and IGFBP-3

are linked to each other and may provide extension in the p21 response time, as their inhibition

blocked the p21 mRNA or protein response in 24 or 72 h, respectively. MAPK pathways

represent a non-genomic target of 1α,25(OH)2D3 and seemed to abolish also p21 basal

expression, so their role in 1α,25(OH)2D3 response remains obscure (Wu et al., 2007).

Nevertheless, our results do not preclude the role of TGF-β or MAPK pathways in upregulation

of p21 by 1α,25(OH)2D3, although we do have evidence of VDR presence on the regulatory

regions of the p21 gene. The intracellular signaling pathways could contribute to or even be

essential in the transcriptional response via phosphorylation of VDR or its co-factors. Therefore,

our results are not in conflict with the indirect effects of 1α,25(OH)2D3 to p21 gene expression,

but instead, the indirect effects can further boost the transcriptional response to the ligand, thus

making the response more robust and less error-prone.



6.2 Regulation of the human p21 gene by p53 and its role in 1α ,25(OH)2D3

response

The adjacent location of the p53 and VDR binding sites on two regions suggests interplay

between these two factors. The VDR gene is also a transcriptional target of p53 and its related

proteins p63 and p73 (Welsh, 2007). However, no synergistic or even additional effect of

1α,25(OH)2D3 and 5-FU on p21 expression was observed, and p21 is highly responsive to

1α,25(OH)2D3 in the p53 negative MDA-MB453 cells (unpublished data, III and IV). One could

assume that the chromatin remodeling by one of the factors would ease transcription initiation by

the other, but the lack of even additional effects upon simultaneous activation suggests that the

agitation of transcriptional machinery on the p21 gene TSS by liganded VDR-RXR heterodimers

is hindered during high p53 induction. This could occur via co-factor squelching or steric

interference. Nevertheless, the regions that contain multiple REs constitute hotspots on the p21

promoter and show high regulatory potential on the whole-genome in silico prediction for

functional elements based on conservation, predicted deoxyribonuclease hypersensitivity and

transcription factor training sets (Taylor et al., 2006 and the linked data in the UCSC genome

browser).

In the skin the differential response to p53 and 1α,25(OH)2D3 translates to UV-response of

different intensities: activation of p53 pathway alerts cells to significant levels of DNA damage

and highly induces p21 to eliminate proliferation, while moderately increased 1α,25(OH)2D3 may

only inform cells about (and possibly protects them from) UV exposure and the possibility of

damage (Holick, 2003a) via moderately increasing p21 expression, which leads to slower

progression of the cell cycle. During the alarm, the subtle message from 1α,25(OH)2D3 becomes

unnecessary, but otherwise 1α,25(OH)2D3 constitutes a modest but stable signal for cells to

restrict proliferation.

6.3 Divergent CoA profiles associated with VDR and PPARβ /δ

The basal transcriptional activity of PPARs resulting from the semi-orphan structure of the

receptors and/or presence of endogenous ligands poses different settings for agonist induced

Discussion


dynamics of co-factor recruitment when compared to VDR (Fyffe et al., 2006; Molnar et al.,

2005). The on/off model of co-factor exchange proposed for other NR1 group NRs has also been

suggested for PPARs (Michalik et al., 2006). In vivo, PPARα readily associates with CoAs, such

as p300, also in the absence of ligand, whereas ligand induces this interaction only by 40 %

(Tudor et al., 2007). We saw an interaction between PPARβ/δ and MED1 on some PPREs of the

PDK genes also in the absence of exogenously added ligand, although PPARγ has been shown to

interact with the Mediator complex in a strictly ligand-dependent manner in vitro (Ge et al.,

2002). The PDK PPREs also exhibit p-Pol II association in the absence of MED1 association.

Recently, the MED1 subunit of the Mediator complex was shown to be dispensable for PPARγ

target gene transactivation and Mediator recruitment to target promoters in undifferentiated

mouse embryonic fibroblasts (Ge et al., 2008). VDR lacks the constitutive activity displayed by

PPARs (Molnar 2005), and MED1 protein association with VDR appears strictly ligand

dependent. Surprisingly, the VDR shows association with other CoA, such as CBP, also in the

absence ligand, either due to interactions with other transcription factors on these regions or

uncharacterized basal association. The latter is supported by our quantitative data from MDA-

MB453 cells in (IV), where CBP association on ligand-responsive regions is significantly

reduced upon 15 min of 1α,25(OH)2D3 treatment, whereas MED1 protein association is

increased. In general, our data support differential association of co-factors, and especially

MED1, of un-liganded VDR and PPARβ/δ, where the latter possesses ability to interact with

CoAs also in the absence of ligand.

6.4 Role of HDACs in regulation of CDKI genes and convergence with

1α ,25(OH)2D3 response

The p21 gene has been suggested as one of the main targets of HDAC inhibitors in executing

their anti-proliferative activity, but in mammary cell lines the INK4 family CDKI genes p18 and

p19 displayed a pronounced response to TSA, whereas the p21 gene was only mildly induced,

terms of fold change (III). The transcriptional response of the CDKI genes to TSA correlated

neither with the basal association of specific HDACs nor with changes in histone acetylation on

the TSS and proximal promoter. HDAC patterns and acetylation changes showed some



congruence, since HDAC3 was only enriched on TSSs that showed induced acetylation upon

TSA treatment, but not on the p27 gene TSS. Nevertheless, the knockdown of HDAC3 mimics

the effects of TSA resulting in high induction of p18 expression. Interestingly, the knockdown of

HDAC3 attenuates the response to TSA, suggesting HDAC3 as one of the main targets of TSA.

The effects of HDAC3 were not dependent on NCoR1, as inhibition of NCoR1 gene expression

failed to produce similar effects on the steady state expression of the p18 gene and even further

induced the response to TSA. These results rule out increased histone acetylation resulting from

inhibition of NCoR1 dependent HDAC3 as the cause for high transcriptional induction of p18 by

TSA or siRNA inhibition of HDAC3. The mechanism of HDAC inhibitor induced gene

expression is yet far from well-characterized, as they produce a general increase in histone

acetylation that is not transmitted to a broad transcriptional activation (Smith 2008). The

transcription factor Sp1 is considered as a none-histone target for HDACs, and hence a putative

provider of target gene selectivity in response to HDAC inhibitors (Huang 2005, Lee 2005). But

as all studied CDKI genes contain GC-boxes and are regulated by Sp proteins irrespective of their

responsiveness to HDAC inhibitors, a change in the transcriptional activity potential of Sp

proteins would not alone provide an explanation for differential responses to TSA.

Besides that of the p21 gene, transcript levels of other CDKI encoding genes were also up-

regulated by 1α,25(OH)2D3 in the mammary cell lines studied here, although in a less coherent

manner (III). As previously reported with the p21 gene, a combination of an HDAC inhibitor and

1α,25(OH)2D3 resulted in higher induction of CDKI gene expression than either substance given

alone (Gaschott et al., 2001; Abedin et al., 2006). The additional or in some cases synergistic

effect appears already at 2 h, and thus is unlikely a result of de novo protein synthesis.

Modulating activity of VDR, NCoR1 and HDACs by siRNA interference results in a more

restricted response: inhibition of either VDR or NCoR1 increases the 3 h TSA response of INK4

genes in two cell lines and inhibition of either the HDAC3 or HDAC4 genes increases the

transcriptional response of both p21 and CYP24A1 genes to 3 h of 1α,25(OH)2D3 treatment only

in MCF-7 cells (III, Fig 4 and S4). These results suggest that un-liganded VDR and NCoR1

attenuate the transcriptional response to TSA, while HDACs attenuate the response to

1α,25(OH)2D3 and combined use of TSA and 1α,25(OH)2D3 can overcome these barriers. Very

recently the anti-proliferative potential of 1α,25(OH)2D3 and TSA has been combined to one

Discussion


molecule that simultaneously acts as a VDR agonist and an HDAC inhibitor (Tavera-Mendoza et

al., 2008).

6.5 1α ,25(OH)2D3 induces cyclical chromatin looping and transcription

factor association on the regulatory regions of p21

The cyclical transcription models provided by multiple studies pose a significant challenge for

the examination of gene regulation by NRs, making it extremely difficult to generalize results

from one time point to describe the whole response. Hence, we used ChIP, 3C and real-time PCR

assays with a dense time course to understand how association of the transcription factor (VDR),

co-factors and chromatin modifications on multiple REs and the TSS as well as the chromatin

looping from REs to TSS leads to transcriptional response to the ligand. Based on our results we

created a model presented in Figure 1 and discussed below. In this model, the response on

chromatin was initiated by MED1 and LSD1, which showed associated strongly with the TSS

and proximal 1α,25(OH)2D3-responsive region, although the binding of VDR only begun to rise

at this point. Based on the general principle of NR function, that the ligand can only affect

transcription through the receptor, the recruitment of LSD1 and MED1 must be dependent on

VDR. Either a very low level of liganded VDR is sufficient to recruit these factors, or VDR peaks

already before the 15 min time point for the first time to recruit the factors. Nevertheless, the

peak in LSD1 binding on TSS at this early time point, when H3K4me2 is still rising, implies that

its main substrate on the p21 promoter is H3K9me1/2, and that LSD1 is acting as a CoA rather

than CoR, as its inhibition leads to lower basal expression of the p21 gene. This would also

explain the subsequent H3K9ac peak that is enabled by removal of H3K9me1/2 by LSD1.

Controversially, both basal and ligand-induced H3K9ac are significantly higher after LSD1

knockdown, a phenomenon possibly linked to the repressive H3K4 demethylase activity of the

enzyme.

CBP is able to recognize H3K9ac through its bromodomain and acetylates H3K14, and

accordingly, CBP peaks at 30 min on the TSS, after the increase in H3K9ac (Zeng et al., 2008).

H3K9ac is also recognized by the TAF1 subunit of the basal transcriptional machinery, which

enables initiation of transcription (Agalioti et al., 2002). The lack of effect upon inhibition of



CBP on transcriptional response of p21 to 1α,25(OH)2D3, though, suggests that it can be replaced

by another HAT. The putative H3K14ac created by CBP is recognized by the bromodomain of

BRG1 that is the catalytic subunit of PBAF chromatin remodeling complex. The abolishment of

H3K4me2 and H3K9ac from the TSS following the peak in CBP and in the histone

modifications, preceding initiation of transcription, suggests ligand-dependent histone eviction

from the TSS. Histone eviction by 1α,25(OH)2D3-responsive transcription has also been reported

previously for the RANKL gene, where eviction also occurred on an VDRE containing region

(Ewing 2007). On regulation of the p21 gene, histone modification clearance only occurs at the

TSS containing region, whereas H3K4me2 and especially H3K9ac are induced by the ligand,

suggesting that effective receptor binding and looping do not require nucleosome eviction from

VDREs.

Besides modulation of binding surfaces of chromo- and bromodomain proteins, LSD1 has been

indicated to be essential in a molecular mechanism of chromatin looping as mentioned in the

literature part of this thesis (see section 2.1.5.2). In short, Perillo and colleagues proposed a

model, where H3K9 demethylation by LSD1 causes localized oxidative damage on DNA, which

is then repaired by base-excision repair complex that contains a topoisomerase that catalyzes the

transport of one DNA double-helix through another, relaxing the tortional stress on the DNA

strand and thereby enable chromatin looping by making the DNA strand more bendable (Perillo

et al., 2008). In correlation with earlier data on steroid NRs, siRNA knockdown of LSD1 inhibits

chromatin looping from distal 1α,25(OH)2D3-responsive regions to the TSS of the p21 gene.

LSD1 knockdown also attenuates the transcriptional response to 1α,25(OH)2D3 resulting in

similar level of the p21 transcript than does MED1 knockdown, and disturbs the changes in

histone modifications. The causal relationship between all the effects of LSD1 gene knockdown is

complex: the influence of 1α,25(OH)2D3 on the chromatin modifications on the TSS requires

looping from distal VDREs, but on the other hand, a certain pattern of chromatin modifications or

as proposed by Perillo and colleagues, certain side products from chromatin modifying enzymes

may be required for looping. In contrast to results by Perillo and colleagues, though, we observed

drastic ligand-dependent changes in LSD1 association especially on the TSS, whereas they

reported that LSD1 is constantly bound to both TSS and the ERα enhancer and does not respond

to ligand. Besides fitting to the cyclical model of NR-mediated gene regulation better than

Discussion


constant presence of LSD1, the dynamic association of LSD1 on both REs and TSS provides

stricter regulation of looping.

Fig. 1: Model of activation of p21 transcription by 1α ,25(OH)2D3. Simplified model of transcription factor and co-factor association, histone modifications, chromatin looping and transcript production in transcriptional response of p21 to 1α,25(OH)2D3. Transparent ovals indicate low association. Direct associations between individual proteins on this figure are incidental.

Histone octamer Histone octamer with increased H3K4me2 Histone octamer with increased H3K9ac Histone octamer with increased H3K4me2 and H3K9ac Histone octamer with high H3K4me2 and H3K9ac

VDR-RXR

Ser5-p-Pol II

p-MED1

NCoR

YY1

LSD1

HDAC3

HDAC4

CBP PIC, assumed

Mediator complex, assumed

Elongating Pol II complex, assumed

pre-mRNA

p21 TSS

0 min 15 min 30 min 45 min 60 min 90 min 120 min 150 min



Surprisingly, and unlike that of CBP, the siRNA knockdown of HDAC3 and especially HDAC4

severely obscured the transcriptional response. Knockdown of HDAC4 induces basal expression

of p21 to 2-fold, although the effects on basal H3K9ac levels are relatively mild. This suggests

that HDAC4 deacetylates either other lysine residues or non-histone targets participating in

transcriptional response. The binding pattern of HDAC4 is also more aberrant than that of

HDAC3, as it rises relatively late and incoherently on distinct regions. Surprisingly, HDAC4 is

essential for both induction of H3K9ac on 1α,25(OH)2D3-responsive regions of the p21 gene

studied here, the abolishment of histone modifications from the TSS containing region as well as

for optimal chromatin looping from the distal region to the TSS. As binding and substrate

specificity as well as protein associations of HDAC4 are still largely unknown, the reason for its

crucial importance in the 1α,25(OH)2D3 response of the p21 gene remains unresolved.

The enrichment patterns of HDAC3, NCoR1 and YY1 on the TSS resemble those of H3K9ac and

H3K4me2, with a drastic decrease at 45 to 60 min. On the contrary, on the 1α,25(OH)2D3-

responsive regions of p21 the association of the repressors decreases during the first 15 to 30 min

and increases again at later phases, while the histone modifications show an opposite pattern.

First of all, the similarity in binding patterns of NCoR1 and YY1, as well as the similarity in

effect of their removal on 1α,25(OH)2D3-responsive steady state accumulation of p21 transcripts

suggest that they work in concert to moderate p21 transcription. Secondly, these results imply

that the CoRs repress mostly the responsive regions during the transcription cycles, whereas the

low amount of histone modifications on the TSS containing regions does not provide substrates

for the CoR associated HDACs. Interestingly, two of the responsive regions show low but

significant basal looping to TSS and association of certain CoRs, which suggests that they

actively connect to the TSS region in order to restrict transcription when 1α,25(OH)2D3 is not

present.

Our data shows periodicity in association of VDR, Pol II, and certain co-factors, as well as in

looping and the accumulation of the steady state p21 gene transcripts, where events are repeated

every 45 to 60 min without any other synchronization besides the ligand treatment. Metivier and

colleagues presented similar duration but far more symmetrical transcription cycles on estrogen-

induced and α-amanitin synchronized cells, suggesting that it represents general timing of NR

mediated transcription cycles through a universal mechanism (Metivier et al., 2003). Irrespective

Discussion


whether the cyclical removal of transcriptional regulators occurs from protein degradation or

chaperone-mediated disassembly, it provides means to stringently regulate the transcriptional

response to endure only during the presence of the ligand and in the absence of contradictory

messages from cellular signaling pathways.

The in vitro based model of VDR-RXR-mediated transcription reviewed in the literature part of

this thesis, describes the PBAF remodeling complex as essential for ligand-responsive

transcription from chromatin template, whereas transcription from naked DNA is not responsive

to ligand and requires only VDR-RXR, Pol II-TAFIID complex and the Mediator complex. This

implies that the main function of the ligand is to promote chromatin remodeling, enabling VDR

to activate Pol II on an open chromatin structure. In vivo, this is further complicated by

requirement of chromatin looping from distal REs to the TSS to provide physical contact between

VDR-RXR and the basal transcription machinery, respectively, via the Mediator complex. Based

on our results, ligand-responsive dynamic association of VDR, co-factors and histone

modifications, chromatin looping and transcript accumulation are highly interlinked, and

disturbance of either the Mediator complex or distinct histone modifiers will alter all levels of

regulation. Removal of either HDAC4, LSD1 or MED1 impairs the ligand-dependent clearance

of histone modifications from the TSS as well as chromatin looping from distal REs, both of

which are essential for the induction of p21 expression by 1α,25(OH)2D3.


7. Summary and conclusions

The four scientific papers that constitute this thesis further establish p21 as a primary

1α,25(OH)2D3 target gene and provide insight into the mechanism of NR-dependent activation of

transcription.

The novel 1α,25(OH)2D3-responsive regions contribute to transcription of p21

By screening 7 kbp of the p21 promoter by ChIP assays, we characterized three regions with 5-

FU-induced p53 enrichment, one of which had not been previously characterized, and three novel

regions with 1α,25(OH)2D3-induced VDR enrichment, two of which harvested also p53. The

direct role of the characterized 1α,25(OH)2D3-responsive regions on regulation of p21 expression

is illustrated by the their association with VDR-Pol II complexes as well as by their ligand-

dependent looping to the p21 TSS. We show coherent transcriptional response of the p21 gene to

1α,25(OH)2D3 in three mammary cell lines with different stages of malignancy.

CoA association of VDR

Comparison of CoA association of VDR and PPARβ/δ on regulatory regions of p21 and genes of

the PDK family, respectively, supports the stricter ligand-dependency of the former. The

modulation of 1α,25(OH)2D3 response by CoRs and associated HDACs showed that combined

application of 1α,25(OH)2D3 and the HDAC inhibitor TSA achieved maximal induction of p21

and the other CDKI genes steady state mRNA levels, relative to the control treatment. Subsequent

assays using gene selective siRNA knockdown revealed that this was due to the attenuation of

1α,25(OH)2D3 response by HDAC3 and HDAC7 and attenuation of TSA response by the NCoR1

and un-liganded VDR.

Recurrent association of regulatory factors, chromatin looping and p21 transcript accumulation

in response to 1α,25(OH)2D3

By ChIP and 3C assays, we demonstrate a sequential association of VDR, p-Pol II and co-factors

on regulatory regions of the p21 gene accompanied by dynamic chromatin looping. The response



of the p21 gene to 1α,25(OH)2D3 is initiated by induction of MED1 on both the TSS and the

proximal ligand-responsive regions. Subsequently H3K9ac, H3K4me2 and the CBP become

highly enriched on the TSS with simultaneous or slightly delayed association of VDR and Pol II

on the 1α,25(OH)2D3-responsive regions. This is followed by an initial peak in mRNA

accumulation. Prior to the second peak in p21 gene steady state mRNA levels, VDR, MED1, p-

Pol II and LSD1 are strongly enriched within the 1α,25(OH)2D3-responsive regions accompanied

by a high level of chromatin looping to the TSS. Concurrently with transcriptional activation,

binding of HDAC3, NCoR1 and YY1 to ligand-responsive regions decreases, but at later time

points, these factors are region-specifically re-introduced. Inhibition of HDAC4, LSD1 or MED1

disturbs both the ligand-induced changes in histone patterns and the chromatin looping, leading to

attenuation and distortion of cyclical transcript accumulation. In conclusion, our results suggest

strong interplay between histone modifications, chromatin looping and transcriptional response in

gene regulation by NRs.


8. Future aspects

The data presented in this thesis opens up some interesting questions. First of all, the study on the

role of HDACs on CDKI regulation suggested that HDAC3 is the major target for TSA, but did

not provide any explanation of the selectivity of TSA on CDKI induction. This could be studied

by combined TSA treatment and knockdown of transcription factors that are known to regulate

CDKI genes, such as the Sp proteins, to see which factors are essential for the effect.

The individual histone modifications seem to play a crucial part in gene regulation, but the

interactions between the modifications and the modifiers are not well characterized. To analyze

the roles of distinct HDACs in specific histone modifications, their expression could be

individually knocked down by siRNA followed by ChIP assay or Western blot with antibodies

against distinctly modified histones. Also, the histone modification pattern preferred for binding

by distinct HDACs could be studied by ex vivo co-immunoprecipitation or two-hybrid assays.

The importance of the histone modifying enzymes on the regulation of the human p21 gene by

1α,25(OH)2D3 and the reduction of the activity-linked modifications from the TSS containing

region implies that they might serve as markers for bromo-, chromo- or PHD-containing

chromatin modifiers. First of all, the putative nucleosome eviction could be studied by

micrococcal nuclease sensitivity assays and/or ChIP using antibodies against pan-histone H3 and

H4. Secondly, individual knockdown of the catalytic ATPase subunits of mammalian chromatin

remodeling complexes by siRNA and the effect on ligand response provides a screening method

to find the essential complexes. The timing of their association could be studied using ChIP, and

the their role in establishment of chromatin modifications and looping by combined siRNA and

ChIP or 3C.

As the amount of information on gene regulation on chromatin level is exponentially increasing,

the complete and all-explaining model of metazoan transcription seems un-attainable with close

to infinite level of complexity. NRs were once thought to represent straightforward regulators of

gene expression that could be turned on by introduction of lipophilic ligand and thus were



thought to provide a simple model system for the regulation of transcription. Recent data presents

this regulation as anything but simple, where correctly timed and targeted NR mediated responses

are provided by dynamic interactions within the chromatin context via co-factors and

incorporation of messages from cellular signaling pathways to meet the needs of the cell and

ultimately the whole organism.


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Appendix: Original publications

Kuopio University Publications C. Natural and Environmental Sciences C 218. Madetoja, Elina. Novel process line approach for model-based optimization in papermaking. 2007. 125 p. Acad. Diss. C 219. Hyttinen, Marko. Formation of organic compounds and subsequent emissions from ventilation filters. 2007. 80 p. Acad. Diss. C 220. Plumed-Ferrer, Carmen. Lactobacillus plantarum: from application to protein expression. 2007. 60 p. Acad. Diss. C 221. Saavalainen, Katri. Evaluation of the mechanisms of gene regulation on the chromatin level at the example of human hyaluronan synthase 2 and cyclin C genes. 2007. 102 p. Acad. Diss. C 222. Koponen, Hannu T. Production of nitrous oxide (N2O) and nitric oxide (NO) in boreal agricultural soils at low temperature. 2007. 102 p. Acad. Diss. C 223. Korkea-aho, Tiina. Epidermal papillomatosis in roach (Rutilus rutilus) as an indicator of environmental stressors. 2007. 53 p. Acad. Diss. C 224. Räisänen, Jouni. Fourier transform infrared (FTIR) spectroscopy for monitoring of solvent emission rates from industrial processes. 2007. 75 p. Acad. Diss. C 225. Nissinen, Anne. Towards ecological control of carrot psyllid (Trioza apicalis). 2008. 128 p. Acad. Diss. C 226. Huttunen, Janne. Approximation and modellingerrors in nonstationary inverse problems. 2008. 56 p. Acad. Diss. C 227. Freiwald, Vera. Does elevated ozone predispose northern deciduous tree species to abiotic and biotic stresses? 2008. 109 p. Acad. Diss. C 228. Semenov, Dmitry. Distance sensing with dynamic speckles. 2008. 63 p. Acad. Diss. C 229. Höytö, Anne. Cellular responses to mobile phone radiation: proliferation, cell death and related effects. 2008. 102 p. Acad. Diss. C 230. Hukkanen, Anne. Chemically induced resistance in strawberry (Fragaria × ananassa) and arctic bramble (Rubus arcticus): biochemical responses and efficacy against powdery mildew and downy mildew diseases. 2008. 98 p. Acad. Diss. C 231. Hanhineva, Kati. Metabolic engineering of phenolic biosynthesis pathway and metabolite profiling of strawberry (Fragaria × ananassa). 2008. 80 p. Acad. Diss. C 232. Nissi, Mikko. Magnetic resonance parameters in quantitative evaluation of articular cartilage: studies on T₁ and T₂ relaxation time. 2008. 83 p. Acad. Diss.

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