autoregulation of pax6 transcriptional activation by two distinct dna-binding subdomains of the...

Autoregulation of Pax6 transcriptional activation by twodistinct DNA-binding subdomains of the paired domain

Yuki Yamaguchi1, Jun-ichi Sawada1, Masao Yamada2, Hiroshi Handa1,* and NoriyukiAzuma3

1Department of Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho,Yokohama 226, Japan2National Children’s Medical Research Centre; and 3 Department of Ophthalmology, National Children’s Hospital, 3-35-31 Taishido,Setagaya 154, Japan

Abstract

Background: Pax6 is a transcription factor that plays acentral role in eye development. Pax6 contains aDNA-binding domain called paired domain, whichconsists of a highly conserved N-terminal subdo-main and a variable C-terminal subdomain. Recentfindings have suggested that both subdomainspossess distinct DNA-binding activities.

Results: To understand the mechanism of DNA-binding and transcriptional activation by Pax6 viathese subdomains, we employed Pax6 paireddomain mutants previously isolated from patientswith ocular disorders. Analysis of these mutants bygel shift assay revealed that the N-terminal and C-terminal subdomains can independently bind to

their respective cognate sites, P6CON and 5aCON.Results from a luciferase assay, however, showedthat the two functional subdomains negativelyregulate their transactivation potentials each other.Wild-type Pax6 and its hyperactive variants showdifferent patterns of DNA contact.

Conclusion: These results support a new model forthe regulation of Pax6 transactivation: When oneDNA-binding subdomain binds to its cognate site,the other subdomain also interacts with the flankingsequences nonspecifically, and this interaction con-strains its structure to give a reduced level oftransactivation.

IntroductionThe Pax family is a set of transcription factors isolatedfrom humans and mouse, which share a conservedpaired domain and homeodomain, and are thought toplay important roles in development (Gruss & Walter1992; Noll 1993; Strachan & Read 1994). Pax6, amember of the Pax family, plays a central role in eyedevelopment (Hanson & van Heyningen 1995). PAX6was initially identified as a candidate gene for aniridia, arare human congenital defect of the eye, and as a generesponsible for mouse Small eye mutation (Hill et al.1991; Ton et al. 1991). Subsequently, the DrosophilaPAX6 homologue eyeless was isolated, and the mutationwas also shown to cause a defect of compound eyes(Quiring et al. 1994), which are structurally very

different from the vertebrate eye. More strikingly,targeted expression of the eyeless gene in Drosophilainduced the formation of ectopic eyes on the wing andleg (Halder et al. 1995). Thus, PAX6 is now proposed tobe a master control gene for eye development (Gehring1996).

Pax6 contains a DNA-binding domain called the‘paired’domain at its N-terminus. The paired domain isfound in various transcription factors, including theDrosophila segmentation gene product paired (Bopp et al.1986). This 128 amino acid domain consists of ahighly conserved N-terminal subdomain (NTS) and avariable C-terminal subdomain (CTS). The former hasgenerally been considered to be important for specificDNA-binding (Chalepakis et al. 1991; Treisman et al.1991). Recent X-ray structural analysis of paired hasrevealed that these subdomains contain helix-turn-helixmotifs resembling the Hin recombinase, and are separatedby a b-turn, and that the NTS and the b-turn make

q Blackwell Science Limited Genes to Cells (1997) 2, 255–261 255

Communicated by: Masaki Iwabuchi*Correspondence: E-mail: [email protected]

contacts with DNA (Xu et al. 1995). In contrast to theother paired domain-containing proteins, however, bothNTS and CTS of the Pax6 paired domain are suggestedto have distinct DNA-binding activities (Epstein et al.1994b). Human and mouse PAX6 genes are alternativelyspliced, to produce a Pax6-5a variant which has a 14amino acid insertion into NTS encoded by exon 5a. Thehigh affinity binding sites for Pax6 and Pax6-5a weredetermined by a PCR-based method, and are calledP6CON and 5aCON, respectively (Epstein et al. 1994a,b).According to this model, NTS is involved in the P6CONbinding, and the 5a insertion serves as a molecular switchto unmask the cryptic DNA-binding activity of CTS to5aCON (Epstein et al. 1994b).

In this paper, we analysed the mechanism of DNA-binding and transcriptional activation by Pax6, by usingthe paired domain mutants previously isolated frompatients with ocular disorders (Hanson et al. 1994;Azuma et al. 1996). Extending previous observations(Epstein et al. 1994b), we clearly demonstrate that thetwo subdomains can bind independently to theircognate sites. However, the two functional subdomainsare shown to negatively regulate their transactivationpotentials to each other. From these and other results,we propose a new model for the regulation of Pax6trans-activation. Furthermore, based on phenotype-genotype correlations of the Pax6 mutants, a possiblelink between the bipartite function of Pax6 and eyedevelopment is discussed.

Results

DNA-binding activity of the C-terminalsubdomain of Pax6 paired domain

In a search for the PAX6 mutation in patients withocular disorders other than aniridia, we previouslyidentified a missense mutation (R128C) of PAX6 inisolated foveal hypoplasia—a defect in an excavated areaof the retina at the posterior end of the eye thatfacilitates good visual acuity (Azuma et al. 1996). Theaffected family members have poorly defined fovealregions (Fig. 1, arrow), but have normal anteriorsegments including the cornea and the iris (Azuma et al.1996). The mutation changes the Arg-128 residuelocated in the CTS of the paired domain to Cys (Fig.2a).

The CTS has recently been suggested to form aDNA-binding domain distinct from that of the NTS(Epstein et al. 1994b), but this functional importance isnot understood. To examine the CTS function, weprepared wild-type and R128C mutant paired domain

(PrD) proteins fused to GST, and analysed their bindingto P6CON and 5aCON by gel shift assay. The C-terminal mutation had only a slight effect, if any, onP6CON binding (Fig. 2b, lanes 2–7), but abolished5aCON binding almost completely (Fig. 2c). Asreported previously (Epstein et al. 1994b), the 5ainsertion abolished P6CON binding (Fig. 2b, lanes8–13), but did not affect 5aCON binding (Fig. 2c). Weobtained virtually identical results using the full-lengthPax6 proteins (data not shown). We estimated theequilibrium dissociation constant (Kd) of these protein–DNA interactions by quantitative gel shift assays, andobtained Kd values of 5.0 nM for PrD vs. P6CON, 16nM for PrD-R128C vs. P6CON, 2.1 nM for PrD vs.5aCON, and 5.2 nM for PrD-5a vs. 5aCON. The otherinteractions were too weak to calculate the Kd values. Acomparison of these values supports the above results.Thus, the CTS was shown to play a primary role in the5aCON binding, which is abolished by the singleamino acid substitution.

How does this mutation affect 5aCON binding? TheArg-128 residue may stabilize the protein–DNAinteraction by directly contacting DNA, which isconsistent with computer modelling (Xu et al. 1995),while the mutated Cys residue may destabilize theirinteraction, for instance, by disulphide bond formation.To distinguish between these possibilities, we mutatedthe Arg-128 to residues other than Cys. As shown inFig. 2d, the conservative mutant R128K showed areduced but persistent 5aCON binding (lanes 7–9).However, the Ala and Ser substituents could not bind5aCON as well as the R128C mutant (lanes 4–6 and10–15). Thus, it is concluded that CTS can bind

Y Yamaguch et al.

256 Genes to Cells (1997) 2, 255–261 q Blackwell Science Limited

Figure 1 The fundus of an 8-year-old boy with fovealhypoplasia. The foveal reflex is absent and capillary vessels runinto a slight residue of luteal pigment. The presumed fovealregion is indicated by an arrow.

5aCON by itself, independently on NTS in the contextof both Pax6 and Pax6-5a. This conclusion is slightlydifferent from the previous model, in that the 5ainsertion serves as a molecular switch to unmask thecryptic DNA-binding activity of CTS (Epstein et al.1994b).

Pax6 transactivation is negatively regulated bythe two DNA-binding subdomains

We next compared the transactivation potential of theR128C mutant with that of the wild-type. The wild-type or mutant Pax6 expression vector was co-transfected with either P6CON- or 5aCON-luciferasereporter vector into mouse embryonal carcinoma P19cells. In parallel with the results of the gel shift assay,the R128C mutation abolished its transactivationvia 5aCON (Fig.3b). Unexpectedly, however, themutation hyperactivated the transcription directedfrom P6CON (Fig. 3a, lanes 8–13). In contrast, the5a insertion abolished its transactivation ability via

P6CON (Fig. 3a, lanes 14–25), but hyperactivatedthe transcription directed from 5aCON (Fig. 3b,lanes 8–10). We cannot explain these hyperactivationsby the differences in their DNA-binding activities, sincethe affinity of PrD-R128C vs. P6CON is comparableto, or slightly weaker than that of PrD vs. P6CON,as noted above. The affinity of PrD-5a vs. 5aCONis also comparable to that of PrD vs. 5aCON. Ittherefore seems likely that the presence of onefunctional DNA-binding subdomain represses trans-activation mediated by the other DNA-bindingsubdomain.

To explain this phenomenon, we have developedthe model shown in Fig. 4a. When one subdomainbinds to its cognate site, the other subdomain maynonspecifically interact with the flanking DNAsequences as well, and this interaction may constrainits structure to give a reduced level of transactivation.To test this model, we performed DNase I andchemical footprinting experiments to analyse thepossible differences in the mode of DNA-binding

Functional analysis of Pax6 paired domain


Figure 2 Effect of a point mutation in the C-terminal subdomain of Pax6 paired domain on its DNA-binding activity. (a) Structure ofPax6 paired domain. The Pax6 paired domain consists of an N-terminal subdomain (NTS) and a C-terminal subdomain (CTS). Alsoshown are the positions of point mutations analysed in this paper, and a 14 amino acid insertion generated by alternative splicing of exon5a. (b) R128C mutation affects P6CON binding only slightly. Bindings of GST-paired domain (PrD) and its derivatives (10, 50 and200 ng) to P6CON were examined in a gel shift assay. (Lane 1) no protein; (lanes 2–4) GST-PrD wild-type; (lanes 5–7) GST-PrDR128C mutant; (lanes 8–10) GST-PrD5a wild-type; (lanes 11–13) GST-PrD5a R128C mutant. (c) R128C mutation markedly reduces5aCON binding. Bindings of GST-PrD and its derivatives to 5aCON were examined as in (b). Since the 5aCON sequence contains twoimperfect direct repeats, two differently migrating complexes are observed, which result from the monomeric and dimeric binding ofPrD (Epstein et al. 1994b). (d) To examine the role of Arg-128, this residue was mutated to Ala (lanes4–6), Lys (lanes7–9), and Ser (lanes10–12) in the context of PrD. The conservative Arg-to-Lys mutant shows a stronger 5aCON binding than the other mutant.

(Fig. 4b). As shown in lanes 3–7, PrD protected awider region than PrD-5a in 5aCON-binding, indicat-ing that the NTS of PrD interacts with the sequenceflanking the region bound by CTS. Similarly, PrDprotected a wider region than PrD-R128C in P6CON-binding (lanes 9–19), indicating the interactionbetween CTS and the sequence flanking the regionbound by NTS. Since their Kd values are comparable,these interactions are probably weak enough not tocontribute to overall affinity. These results stronglysupport the above model.

Functional analysis of Pax6 R26G mutant

To substantiate this model, we employed anothermissense mutation of the PAX6 paired domain,R26G, which was isolated from patients with Peters’

Y Yamaguch et al.


Figure 3 Transactivation potentials of wild-type and R128Cmutant Pax6 in vivo. (a) The R128C mutant hyperactivatestranscription from P6CON. pCMV-Pax6 and its derivatives (1,3, 10, 30, 100 and 300 ng) were co-transfected with P6CON-luc(5 mg) into mouse embryonal carcinoma P19 cells. 5a insertionabolishes the transactivation from P6CON. (Lane 1) emptyvector; (lanes 2–7) Pax6 wild-type; (lanes 8–13) Pax6 R128Cmutant; (lanes 14–19) Pax6-5a wild-type; (lanes 20–25) Pax6-5aR128C mutant. (b) The R128C mutation abolishes transactiva-tion from 5aCON, whereas 5a insertion hyperactivates thetranscription. pCMV-Pax6 and its derivatives (100, 300 and1000 ng) were cotransfected with 5aCON-luc (5 mg) as in (a).(Lane 1) empty vector; (lanes 2–4) Pax6 wild-type; (lanes 5–7)Pax6 R128C mutant; (lanes 8–10) Pax6-5a wild-type; (lanes 11–13) Pax6-5a R128C mutant.

Figure 4 Examination of a model for the autoregulationmechanism of Pax6 transactivation by the two DNA-bindingsubdomains. (a) A model for the autoregulation of Pax6transactivation. The NTS and CTS of Pax6 are marked as Nand C. A dot and a bar indicates the R128C mutation and the 5ainsertion, respectively. See text for details. (b) To test this model,bindings of GST-PrD and its derivatives (50 and 200 ng) to eitherP6CON (lanes 8–20) or 5aCON (lanes 1–7) were comparedusing DNase I (lanes 1–14) and chemical footprinting (lanes 15–20). PrD protects a wider region than PrD-5a or PrD-R128C in5aCON- or P6CON-binding, respectively. (Lanes 1, 14 and 20)G ladder of the probe; (lanes 2 and 8) no DNase I; (lanes 3, 9 and15) no protein; (lanes 4, 5, 10,11, 16 and 17) GST-PrD wild-type; (lanes 6, 7) GST-PrD5a wild-type; (lanes 12, 13, 18 and 19)GST-PrD R128C mutant.

anomaly, a congenital defect of the anterior segment ofthe eye (Hanson et al. 1994; see also Fig. 2a). Thismutation changes Arg-26—a highly conserved residuein NTS—to Gly. The residue is thought to beimportant for P6CON binding from previous X-raystructural analysis of the paired (Xu et al. 1995). Asexpected, the R26G mutation reduced P6CON-binding (Fig. 5a, lanes 15–17), but had only a slighteffect, if any, on 5aCON binding (lanes 5–7). Next weexamined its transactivation potential in the luciferaseassay (Fig. 5b). The R26G mutant showed a reducedtransactivation via P6CON (lanes 8–13), but ithyperactivated the transcription directed from

5aCON (lanes 18–20). These results are in goodagreement with the above model.

Discussion

Autoregulation of Pax6 transcriptionalactivation

In this paper, we have demonstrated that the two DNA-binding subdomains of Pax6 paired domain negativelyregulate its transactivation potentials to each other. Thisautoregulatory mechanism is disrupted by the insertion ofexon 5a or the missense mutations analysed here. In fact,the R26G mutant seems to possess an intermediate levelof transactivation potential between Pax6 and Pax6-5a.The R26G mutant can weakly activate transcription fromP6CON (Fig. 5b, lanes 8–13), whereas its hyperactivationof transcription from 5aCON is moderate (about 180-fold) compared to that of Pax6-5a (about 370-fold) (Figs3b and 5b). This may be attributable to the fact that themutant can bind P6CON, although weakly (Fig. 5a, lanes5–7). In this regard, 5a insertion is thought to function asa natural mutation which completely disrupts theautoregulatory mechanism.

Eye development is very susceptible to a reduction inPAX6 gene dosage (Glaser et al. 1994; Hanson & vanHeyningen 1995). It has recently been shown thatincreased levels of PAX6 expression also interfere withnormal eye development (Schedl et al. 1996). Thus, theautoregulation of Pax6 may be quite important for themaintenance of the appropriate level of Pax6 transcrip-tional activity.

Phenotype-genotype correlations of Pax6mutants

Taken together, the R128C mutation that hyperacti-vates transcription directed from P6CON and abolishesthe transactivation via 5aCON causes a defect of theposterior part of the eye (Azuma et al. 1996), while theR26G mutation that reduces the transactivation viaP6CON and hyperactivates the transcription directedfrom 5aCON is associated with a defect in the anteriorpart (Hanson et al. 1994). Another point mutation thatoccurs in the exon 5a splice acceptor of PAX6 andincreases the ratio of Pax6-5a variant has been reportedwhich also causes a defect of the anterior part (Epstein etal. 1994b). This mutation is thought to result indecreased P6CON transcriptional activity and inincreased 5aCON transcriptional activity. In contrast,in aniridia patients who are haploinsufficient for PAX6,both the anterior and posterior parts are affectedgenerally (Glaser et al. 1992; Hanson et al. 1993;



Figure 5 Analysis of a point mutation in the NTS of the paireddomain. (a) DNA-binding activity of the R26G mutant. Thebinding of the wild-type and R26G mutant GST-PrD (10, 30and 100 ng) to either P6CON (lanes 11–20) or 5aCON (lanes1–10) are compared. The N-terminal mutation reduces P6CONbinding, whereas it affects 5aCON binding only slightly. (Lanes 1and 11) no protein; (lanes 2–4 and 12–14) GST-PrD wild-type;(lanes 5–7 and 15–17) GST-PrD R26G mutant; (lanes 8–10 and18–20) GST-PrD5a wild-type. (b) Transactivation potential ofR26G mutant. Pax6 wild-type and R26G mutant (lanes 2–13, 1,3, 10, 30, 100 and 300 ng; lanes 15–20, 100, 300 and 1000 ng)were co-transfected with either P6CON-luc (5 mg, lanes 1–13)or 5aCON-luc (5 mg, lanes 14–20). The mutation reducestransactivation via P6CON, whereas it hyperactivates transcrip-tion directed from 5aCON.

Martha et al. 1994; Hanson & van Heyningen 1995).These phenotype–genotype correlations suggest theinteresting idea, that genes governed by P6CON and5aCON play distinct roles in the eye development, andthat a small change in the ratio of P6CON- to 5aCON-transcriptional activity leads to the developmentaldisorder. Possibly, the genes directed by P6CON and5aCON may be involved in the formation of anteriorand posterior parts of the eye, respectively.

It should be noted that the 5a alternative splicevariant is only found in vertebrate PAX6 (Walter &Gruss 1991; Glaser et al. 1992; Puschel et al. 1992;Dozier et al. 1993), and that the corresponding exon isapparently missing in the Drosophila PAX6 homologueeyeless or sea urchin PAX6 (Quiring et al. 1994; Czerny& Busslinger 1995). Thus, the bipartite function of Pax6might have evolved in order to regulate the develop-ment of the highly complex vertebrate eye.

P6CON-like sequences have been found in thepromoter of crystalline genes, which encode structuralproteins of the lens (Cvekl et al. 1995a,b; Richardson etal. 1995; Cvekl & Piatigorsky 1996). However, theirfunctional relevance has not been fully determined. Onthe other hand, no 5aCON-like sequence has beenreported to date. Thus, in order to understand thepathways by which Pax6 induces eye development, itwill be necessary to determine the genes activated byPax6 via P6CON-like and 5aCON-like sequences.

Experimental procedures

Gel shift assay

The Pax6 cDNA fragment encoding the paired domain (aminoacids 1–130) was amplified by PCR, and inserted into GSTexpression vector pGEX-4T1 (Pharmacia). The R26G andR128C mutations were created by changing the correspondingcodons CGG to GGG, and CGC to TGC, using the appropriatemutagenic PCR primers (Hanson et al. 1994; Azuma et al. 1996).The nucleotide sequences were verified by dideoxy sequencing.P6CON (50-GGAATTCAGGAAAAATTTTCACGCTTGAGTTCACAGCTCGAGT-30) and 5aCON (50-AAATCTGAACATGCTCAGTGAATGTTCATTGACTCTCGAGGTC-30)sequences—previously determined by a PCR based method(Epstein et al. 1994a,b)—were used as probes. The indicatedamount of GST-PrD proteins were incubated with eitheroligonucleotide probe in the buffer containing 50 mM Tris(pH7.9), 20% glycerol, 50 mM KCl, 0.1 mM EDTA, 5 mg of BSAand 1 mg of poly dIdC for 30 min at 30 8C, and subjected to 4%native PAGE (in 0.5xTBE).

DNase I and chemical footprinting

GST-PrD proteins were incubated with a 130 bp-long probe

labelled at one end by Klenow for 30 min at 30 8C as in the gelshift assay. The probes were then digested with 0.24 U of DNase I(Worthington) for 1 min at 0 8C, or with Cu-phenanthrolinereagent (Kuwabara & Sigman 1987) for 10 min at 30 8C. Thereactions were stopped by phenol-chloroform extraction,ethanol precipitated, and subjected to 8% urea-PAGE.

Transfection and luciferase assay

Mouse embryonal carcinoma P19 cells were maintained by a-MEM (Gibco) supplemented with 10% FBS. P6CON-luc and5aCON-luc were constructed by inserting six copies of P6CONor two copies of 5aCON just upstream of the adenovirus E4minimal promoter (Watanabe et al. 1988), which directs theexpression of luciferase gene. Pax6 expression vectors wereconstructed by inserting the respective cDNAs into the plasmidpCAGGS, which contains cytomegalovirus immediate-earlyenhancer and chicken b-actin promoter (Niwa et al. 1991).Indicated amounts of pCMV-Pax6 and its derivatives were co-transfected with 5 mg of either P6CON-luc or 5aCON-luc into5 ×104 cells by a standard calcium phosphate method. Thetransfectants were harvested 42 h post-transfection, and theluciferase activities measured. These values were adjusted byprotein amount. The results shown are the means and standarddeviations from three experiments.

AcknowledgementsWe thank K. Yokoyama (Riken, Tsukuba) for the P19 cells, andK. Nagata (Tokyo Institute of Technology) for a critical readingof the manuscript. This work was supported by a Grant-in-Aidfor Scientific Research on Priority Areas from the Ministry ofEducation, Science, Sports and Culture of Japan to H.H., and bya grant from Research Committee on Chorioretinal Degenera-tions, the Ministry of Health and Welfare of Japan to N.A.

ReferencesAzuma, N., Nishina, S., Yanagisawa, H., Okuyama, T. &

Yamada, M. (1996) PAX6 missense mutation in isolated fovealhypoplasia. Nature Genet. 13, 141–142.

Bopp, D., Burri, M., Baumgertner, S., Frigerio, G. & Noll, M.(1986) Conservation of a large protein domain in thesegmentation gene paired and in functionally related genes ofDrosophila. Cell 47, 1033–1040.

Chalepakis, G., Fritsch, R., Fickenscher, H., Deutsch, U.,Goulding, M. & Gruss, P. (1991) The molecular basis of theundulated/Pax-1 mutation. Cell 66, 873–884.

Cvekl, A. & Piatigorsky, J. (1996) Lens development andcrystallin gene expression: many roles for Pax–6. BioEssays18, 621–630.

Cvekl, A., Kashanchi, F., Sax, C.M., Brady, J.N. & Piatigorsky, J.(1995a) Transcriptional regulation of the mouse aA-crystallingene: activation dependent on a cyclic AMP-responsiveelement (DE1/CRE) and a Pax-6-binding site. Mol. Cell.Biol. 15, 653–660.

Y Yamaguch et al.


Cvekl, A., Sax, C.M., Li, X., McDermott, J.B. & Piatigorsky, J.(1995b) Pax-6 and lens-specific transcription of the chickend1-crystallin gene. Proc. Natl. Acad. Sci. USA 92, 4681–4685.

Czerny, T. & Busslinger, M. (1995) DNA-binding andtransactivation properties of Pax-6: Three amino acids in thepaired domain are responsible for the different sequencerecognition of Pax-6 and BSAP (Pax-5). Mol. Cell. Biol. 15,2858–2871.

Dozier, C., Carriere, C., Grevin, D., et al. (1993) Structure andDNA-binding properties of Pax-QNR, a paired box- andhomeobox-containing gene. Cell Growth Diff. 4, 281–289.

Epstein, J., Cai, J., Glaser, L., Jepeal, L. & Maas, R. (1994a)Identification of a Pax paired domain recognition sequenceand evidence for DNA-dependent conformational changes. J.Biol. Chem. 269, 8355–8361.

Epstein, J.A., Glaser, T., Cai, J., Jepeal, L., Walton, D.S. & Maas,R.L. (1994b) Two independent and interactive DNA-bindingsubdomains of the Pax6 paired domain are regulated byalternative splicing. Genes Dev. 8, 2022–2034.

Gehring, W.J. (1996) The master control gene for morphogenesisand evolution of the eye. Genes Cells 1, 11–15.

Glaser, T., Jepeal, L., Edwards, J.G., Young, S.R., Favor, J. &Maas, R.L. (1994) PAX6 gene dosage effect in a family withcongenital cataracts, aniridia, anophthalmia and centralnervous system defects. Nature Genet. 7, 463–471.

Glaser, T., Walton, D.S. & Maas, R.L. (1992) Genomic structure,evolutionary conservation and aniridia mutations in thehuman PAX6 gene. Nature Genet. 2, 232–239.

Gruss, P. & Walter, C. (1992) Pax in development. Cell 69, 719–722.

Halder, G., Callaerts, P. & Gehring, W.J. (1995) Induction ofectopic eyes by targeted expression of the eyeless gene inDrosophila. Science 267, 1788–1792.

Hanson, I. & van Heyningen, V. (1995) Pax6: more than meetsthe eye. Trends Genet. 11, 268–272.

Hanson, I.M., Fletcher, J.M., Jordan, T., et al. (1994) Mutationsat the PAX6 locus are found in heterogeneous anteriorsegment malformations including Peters’ anomaly. NatureGenet. 6, 168–173.

Hanson, I.M., Seawright, A., Hardman, K., et al. (1993) PAX6mutations in aniridia. Hum. Mol. Genet. 2, 915–920.

Hill, R.E., Favor, J., Hogan, B.L., et al. (1991) Mouse small eyeresults from mutations in a paired-like homeobox-containinggene. Nature 354, 522–525.

Kuwabara, M.D. & Sigman, D.S. (1987) Footprinting DNA-protein complexes in situ following gel retardation assays using,

1,10-phenanthroline-copper ion: Escherichia coli RNA poly-merase-lac promoter complexes. Biochemistry 26, 7234–7238.

Martha, A., Ferrell, R.E., Mintz-Hittner, H., Lyons, L.A. &Saunders, G.F. (1994) Paired box mutations in familial andsporadic aniridia predicts truncated aniridia proteins. Am. J.Hum. Genet. 54, 801–811.

Niwa, H., Yamamura, K. & Miyazaki, J. (1991) Efficientselection for high expression transfectants with a noveleukaryotic vector. Gene 108, 193–199.

Noll, M. (1993) Evolution and role of Pax genes. Curr. Opin.Genet. Dev. 3, 595–605.

Puschel, A.W., Gruss, P. & Westerfield, M. (1992) Sequence andexpression pattern of pax-6 are highly conserved betweenzebrafish and mice. Development 114, 643–651.

Quiring, R., Walldorf, U., Kloter, U. & Gehring, W.J. (1994)Homology of the eyeless gene of Drosophila to the small eye genein mice and aniridia in humans. Science 265, 785–789.

Richardson, J., Cvekl, A. & Wistow, G. (1995) Pax-6 is essentialfor lens-specific expression of z-crystallin. Proc. Natl. Acad. Sci.USA 92, 4676–4680.

Schedl, A., Ross, A., Lee, M., et al. (1996) Influence of PAX6gene dosage on development: Overexpression causes severeeye abnormalities. Cell 86, 71–82.

Strachan, T. & Read, A.P. (1994) PAX genes. Curr. Opin. Genet.Gev. 4, 427–438.

Ton, C.C.T., Hirvonen, H., Miwa, H., et al. (1991) Positionalcloning and characterization of a paired box- and homeobox-containing gene from aniridia region. Cell 67, 1059–1074.

Treisman, J., Harris, E. & Desplan, C. (1991) The paired boxencodes a second DNA-binding domain in the pairedhomeodomain protein. Cell 67, 1059–1074.

Walter, C. & Gruss, P. (1991) Pax-6, a murine paired box gene, isexpressed in the developing CNS. Development 113, 1435–1449.

Watanabe, H., Imai, T., Sharp, P.A. & Handa, H. (1988)Identification of two transcription factors that bind to specificelements in the adenovirus early region 4. Mol. Cell. Biol. 8,1290–1300.

Xu, W., Rould, M.A., Jun, S., Desplan, C. & Pabo, C.O. (1995)Crystal structure of a paired domain-DNA complex at 2.5 Aresolution reveals structural basis for Pax developmentalmutations. Cell 80, 639–650.

Received: 6 February 1997Accepted: 18 March 1997



autoregulation of pax6 transcriptional activation by two distinct dna-binding subdomains of the...

Documents