Transcriptional Regulation of Eukaryotic Genes

真核基因的转录调控

Reference ： Genes IX (Benjamin Lewin)

郭红卫 2008.11.20 PKU

1. RNA polymerase II

2. promoter and enhancers

3. transcription factors

Eukaryotic gene expression is usually controlled at the level of initiation of transcription.

· All eukaryotic RNA polymerases have ~12 subunits and are aggregates of >500 kD.

· Some subunits are common to all three RNA polymerases.

· The largest subunit in RNA polymerase II has a CTD (carboxy-terminal domain) consisting of multiple repeats of a heptamer. The CTD can be highly phosphorylated on serine or threonine residues; this is involved in the initiationreaction and RNA processing.

RNA polymerase II synthesizes mRNA in the nucleoplasm （核胞质）

Key Terms

A basal (general) factor is a transcription factor required by RNA polymerase II to form the initiation complex at all promoters. Factors are identified as TFIIX, where X is a number.

The basal transcription apparatus is the complex of transcription factors that assembles at the promoter before RNA polymerase is bound.

An enhancer is a cis-acting sequence that increases the utilization of (some) eukaryotic promoters, and can function in either orientation and in any location (upstream or downstream) relative to the promoter.

Activators and their many targets

1. TBP

2. TFIID

3. TFIIB

4. TFIIA

5. TFIIH

6. TFIIE

7. Pol II

8. Mediator

9. SAGA/Chromatin modifiers

10. Many more …

• Holoenzyme --- a supramolecular complex comprising Pol II, most GTFs, and Mediator/Srb complex

• In yeast, a 2MDa holoenzyme + TBP suffices for transcription

Ordered Assembly vs Pol II Holoenzyme

TFIIDTFIID

one-step

multiple-step

Binding of TFIID (TBP + 11 TAFs, 800KD) to the TATA box is the first step in initiation.

Other transcription factors bind to the complex in a defined order, extending thelength of the protected region on DNA.

When RNA polymerase II binds to the complex, it is ready to initiate transcription.

Sequential Assembly

TBP: TATA binding proteinTAFs: TBP associated factors

TFIIB binds to DNA and contacts RNA polymerase near the RNA exit site and at the active center, and orients it on DNA.

+25bp

Q: prok -10bp vs euk -25bp?

Phosphorylation of the CTD by the kinase activity of TFIIH may be needed to release RNA polymerase to start transcription.

· TFIIE and TFIIH are required to melt DNA to allow polymerase movement.

· Phosphorylation of the CTD (by TFIIH and other kinases) is required for elongation to begin—fire the Pol II.

· The CTD may coordinate processing of RNA with transcription.

CTD ： RNA Pol II C-terminal domain

CTD Pi

RNA Pol II

Basal transcriptional

apparatus(TBP, TFIIs)

The CTD may also be involved in processing RNA after it has been synthesized.

The capping enzyme binds to the phosphorylated CTD: this may be important in enabling it to modify the 5’ end as soon as it is synthesized.

A set of proteins called SCAFs bind to the CTD, and they may in turn bind to splicing factors. This may be a means of coordinating transcription and splicing.

Some components of the cleavage/ polyA apparatus also bind to the CTD. so RNA polymerase is all ready for the 3’ end processing reactions as soon as it sets out!

CTD may be a general focus for coupling other processes (mRNA maturation) with transcription

Factors involved in gene expression include RNA polymerase and the basal apparatus, activators that bind directly to, co-activators that bind to both activators and the basal apparatus, and regulators that act on chromatin structure (chromatin remodeling complex).

Many Transcriptional Activators

i.e. CAATGC-box

Modular organization of Transcription Factors

DNA binding Domain: Zn fingers, Homeodomains, bHLH, bZip, MADS, …

Oligomerization Domain: homo/hetero oligomers ---usually dimers, though trimers are not uncommon : HSF, NF-Y…

Regulatory Domain: Activation -acidic, Q-rich, P-rich, RNA-binding, …

Repression -basic, HDAC binding peptides, …

Other Domains (not in all factors): including nuclear localization, protein-interaction domains, ligand binding, signal-responsive sites …

Zinc Finger

bHLH

bZIP

Homeodomain

SP1 stimulates transcription in presence of TAFII110

• GC boxes bound by DNA binding protein SP1• SP1 recruits TFIID by binding TAFII110• Partially reconstituted complex (TBP and 3 TAFs) in addition to

other GTFs, Pol II leads to high levels of transcription

SV40 early promoter

Mediator complex is targeted by an activator

• Mediator is a stable complex containing several proteins (20-50)• Mediator binds to the RNA pol II and transcription factors (activators

or repressors) and ‘mediates’ the regulatory signals to pol II

( 中介复合体）

Tethering the Mediator complex

•

In yeast: SRB complex (suppressors of RNP B).

It contains factors that are necessary for transcription from many or most promoters.

It provides interaction surfaces for many transcriptional activators or repressors, thus mediates both activation and repression of transcription.

tat protein of HIV can stimulate transcription initiation without binding DNA at all

The activating domain of the tat protein can stimulate transcription if it is tethered in the vicinity of promoter by binding to the RNA product (tar sequence) of a previous round of transcription.

tar

tat

DNA-binding domain is to bring the activation domain into the vicinity of the startpoint. And activation is independent of the means of tethering.

we can think of DNA-binding (or RNA-binding in the case of tat) domain as providing a "tethering" function, whose main purpose is to ensure that the activation domain is in the vicinity of the initiation complex.

The notion of tethering is a more general idea that initiation requires a high concentration of transcription factors in the vicinity of the promoter.

This may be achieved when activators bind to enhancers, upstream promoter elements, or in an extreme case by tethering to a newly-made RNA product.

Interchangeable Modules(Activation domain is interchangeable)

Interaction AssaysDesign of Two-hybrid / Three-hybrid /etc…

separable functional domains

Two-hybrid assay(protein-protein)

Tri-hybrid assay(protein-RNA)

Summary The principle that governs the function of all activators is that a DNA-binding domain determines specificity for the target promoter or enhancer.

The DNA-binding domain is responsible for localizing a transcription-activating domain in the proximity of the basal apparatus.

An activator that works directly has a DNA-binding domain and an activating domain. An activator that does not have an activating domain may work by binding a coactivator that has an activating domain.

Several factors in the basal apparatus are targets with which activators or coactivators interact.

RNA polymerase may be associated with various alternative sets of transcription factors in the form of a holoenzyme complex.

What is the mechanism of activation?

Two models:

1.Tethering holoenzyme (recruitment)2.Activating holoenzyme (allosteric)

(interaction activation)??

In favor of recruitment model

activators recruit transcription machinery

• Gal4-binding sites are bound by Gal4

• Pre-binding by Gal4 is necessary for recruitment of the machinery• Role in re-initiation as well

‘Synergy’ High levels of transcription induced by multiple factors

• Transcription factors can enhance transcription in a non-linear manner• Synergisitic activation occurs due to multiple contacts with the machinery• Multiple copies of the same activator also induce synergistic activation

Interferon ß enhancer

• Enhancers often have binding sites for several transcription factors• Transcription factors can bind cooperatively at adjacent sites• Architectural factors (with no regulatory domains, i.e. HMG1) can assist assembly• Remarkably increase binding affinity for both DNA and machinery

HMG1

肩并肩、手挽手，根基稳、魅力足

香肩并立、玉指紧扣，脚如磐石、面若桃花

Regulatory mechanism from a distance

Compaction

Sliding

Looping

Why do enhancers act independent of distance and orientation?

An enhancer may function by bringing proteins into the vicinity of the promoter. An enhancer does not act on a promoter at the opposite end of a long linear DNA, but becomes effective when the DNA is joined into a circle by a protein bridge. An enhancer and promoter on separate circular DNAs do not interact, but can interact when the two molecules are catenated.

Two experiments support the looping model--The essential role of the enhancer is to increase the concentration of activator in the vicinity of the promoter---

(Signal transduction and gene regulation)

How is a gene regulated by signals?

The regulatory region of a human metallothionein gene contains regulator elements in both its promoter and enhancer. The promoter has elements for metal induction; an enhancer has an element for response to glucocorticoid. Promoter elements are shown above the map, and proteins that bind them are indicated below.

Each gene contains multiple response elements

The activity of a regulatory transcription factor may be controlled by synthesisof protein, covalent modification of protein, ligand binding, or binding of inhibitors that sequester the protein or affect its ability to bind to DNA.

Steroid receptors are transcription factors

Steroid receptors are examples of ligand-responsive activators that are activated by binding a steroid.

There are separate DNA-binding and ligand-binding domains.The DNA binding domain is a type of zinc finger that has Cys but not His residues. They bind to DNA as dimers.

Glucocorticoid and estrogen receptors each have two zinc fingers, the first of which determines the DNA target sequence.

Binding of ligand to the C-terminal domain increases the affinity of the DNA-binding domain for its specific target site in DNA.

Receptors for many steroid and thyroid hormones have a similar organization, with an individual N-terminal region, conserved DNA-binding region, and a C-terminal hormone-binding region

The first finger of a steroid receptor controls which DNA sequence is bound (positions shown in red); the second finger controls spacing between the sequences (positions shown in blue).

Discrimination between GRE and ERE target sequences is determined by two amino acids at the base of the first zinc finger in the receptor.

GRE ERE

Glucocorticoids regulate gene transcription bycausing their receptor to transport into the nucleus and bind to an enhancer whose action is needed for promoter function.

(or dexamethasone)

Activation of Glucocorticoid Receptor (GR)

Nuclear shuttling

Glucocorticoid receptor (GR) fusion protein induction

CortisolDexamethasone

GRGR

GR

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XX

X

Dex binds to GR, exposes nucleus localization signal (NLS), allows fusion protein to transport into the nucleus to activate transcription

----A good system to activate the function of nuclear-localized proteins (including transcription factors)

Activation Tagging approach in plants

Plant transformation

1. phenotypic characterization of mutants 2. locate T-DNA insertion site in Arabidopsis genome (how?)

3. identify the right gene conferring mutant phenotype (how?)

4. functional study of the gene

Genetic screen

XVE ， was assembled by fusion of the DNA-binding domain of LexA (X), the acidic activation domain of VP16(V), and the regulatory region of the human estrogen receptor (E)

A chemical-inducible activation tagging vector pER16 in plants

T-DNA fragment (can integrate into plant genome)

其工作原理为：在 G10-90 启动子控制下， XVE 融合转录因子组成型表达；当加入雌激素，雌激素和其受体结合，导致 XVE 融合蛋白构象发生变化，并由细胞质转移进入核内；在细胞核内 ,LexA 的 DNA 结合域特异性识别 LexA 操纵子区，以至使 VP16 的转录激活域激活 -46

35S 启动子，高水平表达其下游可能的目标基因。

TR and RAR bind the SMRT corepressor in the absence of ligand. The promoter is not expressed. When SMRT is displaced by binding of ligand, the receptor binds a coactivator complex. This leads to activation of transcription by the basal apparatus.

Corepressor/coactivator switch

Summary:1) Cis-elements mark the sites of transcription

2) Some bound by GTFs and others by Regulators (TFs)

3) Regulatory TFs are trigged by cellular signals

4) Regulatory TFs can be positive or negative

5) TFs are modular:

- DBD binds Enhancers and defines genes that are targeted

- Regulatory domain controls the expression of the gene(s)

6) Simple architecture of regulatory proteins has led to the creation of powerful tools (2-hybrid and ATFs)

诺贝尔化学奖得主 Roger Kornberg 北大演讲 被授予“北大名誉教授”

罗杰•科恩伯格 , 斯坦福大学教授，因对“真核转录的分子基础所作的研究”而荣获 2006 年诺贝尔化学奖。

科恩伯格发现了构成染色体的基本单位——核小体

If nucleosomes form at a promoter, transcriptionfactors (and RNA polymerase) cannot bind. If transcription factors (and RNA polymerase) bind to the promoter to establisha stable complex for initiation, histones are excluded.

The dynamic model for transcription of chromatin relies upon factors that can use energy provided by hydrolysis of ATP to displace nucleosomes from specific DNA sequences.

Chromatin remodeling

Chromatin remodeling is undertaken by large complexes that use ATP hydrolysis to provide the energy for remodeling.

The heart of the remodeling complex is its ATPase subunit. Remodeling complexes are usually classified according to the typeof ATPase subunit

two major types of chromatin remodeling complex: SWI/SNF and ISW (imitation SWI)

Remodeling complexes can cause nucleosomes to slide along DNA, can displace nucleosomes from DNA, or can reorganize the spacing between nucleosomes.

Remodeling complexes are recruited to promoters by sequence-specific activators.

A genomic survey suggested that most sites that bind transcription factors are free of nucleosome.

A remodeling complex binds to chromatin via anactivator (or repressor)

Hormone receptor and NF1 cannot bindsimultaneously to the MMTV promoter in the form of linear DNA, but can bind when the DNA is presented on a nucleosomal surface.

The MMTV promoter requires a change in rotational positioning of a nucleosome to allow an activator to bind to DNA on the nucleosome.

组蛋白的化学修饰

组蛋白化学修饰发生在组蛋白 N 端尾部，尤其是组蛋白H3 和 H4 的修饰起始了 染色质结构的变化。组蛋白尾部由20 个氨基酸组成，并且从 DNA 转弯处的核小体间延伸出来。

组蛋白化学修饰的类型：

组蛋白（去）乙酰化 De/Acetylation

组蛋白甲基化 Methylation

组蛋白磷酸化 Phosphorylation

组蛋白泛素化 Ubiquitination

组蛋白糖基化 ADP-Rybosilation

组蛋白与 Swi/Snf 复合体的结合 Swi/Snf complex, which, in vitro, uses the energy of ATP hydrolysis to disrupt histone-DNA interactions

多数化学修饰的化学基团可以减少组蛋白的正电性，从而使其与DNA 结合变疏松，使染色质结构发生变化。

Acetylation of H3 and H4 is associated with active chromatin, while methylation is associated with inactive chromatin.

Histone modification is a key event

Sites of post-translational modifications on the histone tails

Zhang Y., Reinberg D. Genes Dev. 2001;15:2343-2360

Most modified sites in histones have a single, specific type ofmodification, but some sites can have more than one type of modification. Individual functions can be associated with some of the modifications.

Histone Code

K K

Acetylation = Transcriptional CompetenceMethylation = Transcriptionally Silenced

· Histone acetylation occurs transiently at replication.· Histone acetylation is associated with activation of gene expression.

Histone acetylation occurs in two circumstances

Histone acetyltransferase (HAT) enzymes modify histones by addition of acetyl groups; some transcriptional coactivators have HAT activity.

A deacetylase is an enzyme that removes acetyl groups from proteins.

Histone deacetyltransferase (HDAC) enzymes remove acetyl groups from histones; they may be associated with repressors of transcription.

组蛋白乙酰化酶复合体的特点：

HAT 是一个大复合体的一部分 ( 如 SAGA 复合体中的 GCN5) ，且多种 HAT可同时存在于一个辅助激活复合体中，并具有不同的特异性 ( 如 P300, ACTR与 PCAF 的共同作用 )

PCAF

ACTR

P300

TF

EF

组蛋白乙酰化过程是动态的，被乙酰化的组蛋白位点可以通过组蛋白去乙酰化酶（ histone deacetylase, HDAC ）将乙酰基去掉。

组蛋白去乙酰化酶 (HDAC) 可分为 4 类。

(1) 第一类：为酵母转录调控子 RPD3 蛋白的同类物，这是第一个被鉴定出来的 HDAC 。它包括 HDAC1~3 、 6 、 7 、 9 、 18 ， 19，可与转录因子结合。 (2) 第二类：为酵母 HDAl 类蛋白质，包括 HDAC4 、 5 、 10 。它们的催化区域与酵母的 HDAl 蛋白同源，特异性地与细胞分化有关。

(3) 第三类：最近发现酵母中的沉默信息调节因子 2(silent information regulator 2 ， SIR 2) 也是一种组蛋白去乙酰化酶，人细胞中的 SIR 2 同源物也被鉴定出来。因此， SIR 2 及其相关蛋白构成了高等真核生物的第三类组蛋白去乙酰化酶。

(4) 第四类： HD2 类，仅在植物当中发现。

A repressor complex contains three components: a DNA binding subunit, a corepressor, and a histone deacetylase.

HDAC 是许多转录共抑制复合物中的活性成分。 HDACl和 HDAC2 均与介导转录抑制的 mSin3A 有关。通过与许多序列特异性的转录因子相互作用，可使 HDAC-mSin3 复合物结合到特异性的启动子，抑制相关基因的表达。这些转录因子包括未结合配体的核激素受体、 Mad ／ Max 异二聚体、 MeCP2 、 p53 等。

组蛋白甲基化

组蛋白甲基化是由组蛋白甲基化转移酶 (histonemethyl transferase ， HMT) 完成的。

甲基化位点：甲基化可发生在组蛋白的赖氨酸和精氨酸残基上，而且赖氨酸残基能够发生单、双、三甲基化，而精氨酸残基能够单、双甲基化，这些不同程度的甲基化极大地增加了组蛋白修饰和调节基因表达的复杂性。

甲基化的作用位点在赖氨酸 (Lys) 、精氨酸 (Arg) 的侧链N 原子上。组蛋白 H3 的第 4 、 9 、 27 和 36 位， H4 的第 20 位 Lys ， H3 的第 2 、 l7 、 26 位及 H4 的第 3 位Arg 都是甲基化的常见位点。

精氨酸甲基化：是一种相对动态的标记，精氨酸甲基化与基因激活相关，而 H3 和 H4 精氨酸的甲基化丢失与基因沉默相关。

赖氨酸甲基化：是基因表达调控中一种较为稳定的标记。例如， H3第 4 位的赖氨酸残基甲基化与基因激活相关，而第 9 位和第 27 位赖氨酸甲基化与基因沉默相关。此外， H4K20 的甲基化与基因沉默相关， H3K36 和 H3K79 的甲基化与基因激活有关。但应当注意的是，甲基化个数与基因沉默和激活的程度相关。

组蛋白甲基化与 DNA 甲基化密切相关：

Acetylation of histones activates chromatin, and methylation of DNA and histones inactivates chromatin.

Methylation of DNA and of histones is associated with heterochromatin.

The two types of methylation event may be connected.

Summary of histone acetylation, histone methylation and DNA methylation

Promoter activation involves binding of a sequence-specific activator, recruitment and action of a remodeling complex, and recruitment and action of an acetylating complex.

Promoter activation involves an ordered

series of events

组蛋白的其他修饰方式

相对而言，组蛋白的甲基化修饰方式是最稳定的，所以最适合作为稳定的表观遗传信息。而乙酰化修饰具有较高的动态，另外还有其他不稳定的修饰方式，如磷酸化、腺苷酸化、泛素化、 ADP 核糖基化等等。这些修饰更为灵活的影响染色质的结构与功能，通过多种修饰方式的组合发挥其调控功能。所以有人称这些能被专识别的修饰信息为组蛋白密码。这些组蛋白密码组合变化非常多，因此组蛋白共价修饰可能是更为精细的基因表达方式。 另外，研究发现 H2B 的泛素化可以影响 H3K4 和 H3K79的甲基化，这也提示了各种修饰间也存在着相互的关联。

Mechanisms of Transcriptional Activation

1) The Components of the Gene Expression Machinery

2) Ordered versus One-Step Recruitment

3) Recruitment versus Allosteric Regulation

4) Nature of the Activation Domain

5) Transcriptional Activators and Chromatin Remodeling

6) Future of Transcriptional Regulation

Future of Transcriptional Regulation

A) From regulation of one gene to the study of all at once

B) Importance of Genomics

C) The role of informatics

D) Tools to dissect the immediate from the secondary

E) Expression profiling as a fundamental biological and a diagnostic tool

F) Understanding the genomic access code