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Focus on Molecules: Pax-6, the Eye Master

Panagiotis A. Tsonis a,*, Ernesto J. Fuentes b,c

a Department of Biology, University of Dayton, Dayton, OH 45469-2320, USAb Department of Biochemistry and Biophysics, University of North Carolina, NC 27599-7260, USAc Lineberger Comprehensive Cancer Center, University of North Carolina, NC 27599-7260, USA

Available online 23 March 2006

Keywords: Pax-6; structure; eye development

1. Structure

Pax-6 belongs to the family of paired box genes that contain

both the hallmark paired box domain (PD) and a homeo box domain

(HD), followed by a prolineeserineethreonine rich domain (PST)

(Fig. 1A). The Pax-6 protein contains 422 aa and at least one tran-

script variant, Pax-6-5a that contains a 14 aa insert in the PD (at

amino acid position 47). The PD binds DNA in a bipartite fashion us-

ing the N-terminal and C-terminal subdomains. The 5a insert abro-

gates DNA binding by the N-terminal subdomain suggesting that

the C-terminal subdomain dictates target specificity in this variant.

The structure of the paired box domain in complex with a 26-bp op-timal DNA duplexhas been determined (Xuet al.,1999) (Fig.1B).This

structure provides a detailed model of the interactions between Pax-6

PD and DNA, and in particular how the N-, C-terminal subdomains

and linker region combine to achieve DNA binding specificity. Specif-

ically, both the N- and C-terminal subdomains fold into a helix-turn-

helix motif, reminiscent of the homeo box domain fold (Xu et al.,

1999). The primarysites of DNA interaction occur by the so-calledrec-

ognition helicesa3anda6 (Fig.1B).Indeed,residue47 ina3 (and res-

idues 42 and 44 to a lesser degree) dictates DNA specificity within the

Pax family. Interestingly, the linker between the N- and C-terminal

domains is also involved in DNA recognition and specificity. Finally,

the structure also provides a framework for understanding the effect of

mutations known to be involved in disease (Fig. 1C, see below).

2. Function

Pax-6 is a transcriptional factor involved in the development of

the central nervous system and eye development. The corresponding

gene in Drosophila is the eyeless, a mutation in the eyeless gene re-

sults in animals with no eyes. However, mutations in Pax-6 cause no

eye or small aye phenotype in mammals as well. Pax-6 has been

considered as the master gene for eye development. Indeed, initial

studies showed that ectopic expression of Pax-6 in Drosophila could

produce ectopic eyes. Pax-6, however, seems to be involved in

a feedback loop with another homeo box-containing gene, Six-3,

in order to control development of the eye. Pax-6 also interacts

with the homeo box-containing genes Pbx1 and HoxB1 and this in-

teraction enhances its transcriptional activity. Except for being a mas-

ter gene for eye development, Pax-6 plays significant roles during

the induction of the lens and retina differentiation. Inactivation of

Pax-6 in the surface ectoderm after E9.5 resulted in arrest of lens

development. Pax-6 affects differentiation of lens fibers cells by

controlling crystallin gene expression. Inactivation of Pax-6 affects

the retinogenic potential of retinal progenitor cells (RPCs). RPCs be-come restricted to one cell fate that of amacrine interneurons. Also,

regulation of Pax-6 expression controls the identity and differentia-

tion potential of retinal pigment epithelial cells (for reviews see

Gehring, 2002; Treisman, 2004).

3. Disease involvement

As wasmentioned above, mutationsin Pax-6 lead to eyeless or small

eye phenotypes in mice. In humans, Pax-6 mutations are associated

with aniridia (Fig. 1C). Other mutations in Pax-6 have been associated

with foveal hypoplasia, presenile cataract, aniridia-related keratopathy,

cranial and CNS malformations. Most of the mutations appear to cause

loss of function (van Heyningen and Williamson, 2002).

4. Future studies

While the function of Pax-6 has been studied quite extensively and its

developmental role is known, its cooperation with other genes needs fur-

ther investigation. It is known than Pax-6 is part of a loop that involves

Six-3 as well. Identification of more downstream targetsfor this pathway

willhelp delineate the mechanismsof the different actionsof Pax-6. Also

research on genes whose Pax-6is thetargetmight shed lighton how mas-

ter genes are activated in undifferentiated cells. Recent work indicates

thatthe PD and HD canbind DNA in a cooperative fashion. Furthermore,

the PST domain seems to be important in transactivation and several* Corresponding author. Tel.: 1 937 229 2579; fax: 1 937 229 2021.

E-mail address: panagiotis.tsonis@notes.udayton.edu (P.A. Tsonis).

0014-4835/$ - see front matter 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.exer.2005.11.019

Experimental Eye Research 83 (2006) 233e234www.elsevier.com/locate/yexer

mailto:panagiotis.tsonis@notes.udayton.edumailto:panagiotis.tsonis@notes.udayton.eduhttp://www.elsevier.com/locate/yexerhttp://www.elsevier.com/locate/yexermailto:panagiotis.tsonis@notes.udayton.edu

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mutations are known to occur in this region that result in disease. The

structural rationale for both of these observations remains elusive.

References

Gehring,W.J., 2002. Thegeneticcontrolof eye developmentand its implications

for the evolution of the various eye-types. Int. J. Dev. Biol. 46, 65e73.

van Heyningen, V., Williamson, K.A., 2002. PAX6 in sensory development.

Hum. Mol. Genet. 11, 1161e1167.

Treisman, J.E., 2004. How to make an eye. Development 131, 3823e3827.

Xu, H.E., Rould, M.A., Xu, W., Epstein, J.A., Maas, R.L., Pabo, C.O., 1999.

Crystal structure of the human Pax6 paired domaineDNA complex reveals

specific roles for the linker region and carboxy-terminal subdomain in

DNA binding. Genes Dev. 13, 1263e1275.

1

23

R26G

I87R

R128C

4

N-term

subdomain

C-term

subdomain

N

C

V126DV78A

P76L

R38W

S43P

A33P

V53L

T63P

N14SG15W

56

Q47(5a)

(A)

(B)

(C) Paired box domain

N-terminal subdomain C-terminal subdomain

1 2 65431 2

PAX6 MQNSHSGVNQLGGVFVNGRPLPDSTRQKIVELAHSGARPCDISRILQVSNGCVSKILGRYYETGSIRPRAIGGSKPRVATPEVVSKIAQYKRECPSIFAWEIRDRLLSEGVCTNDNIPSVSSINRVLRNLASEKQQ

mutation ----------------SW-------G--V---P----W---SPQ--R-----L---------PV-----------L-AE-------R------------------------------RR------D-C--------

R S

DNA contact ...P....PP....P...P.P....P...........P.P........P..P.PP...P........PPSS.SS.PPPP..................PPP..................P..P.PP..P........

groove ............mm..................................MMMM..................mmmm..m...........................................M...M..M........

1 807060 1205040302010 11010090 130

PAX6 LQRNRTSFTQEQIEALEKEFERTHYPDVFARERLAAKIDLPEARIQVWFSNRRAKWRREEKLR

DNA contact .SP.SP..................P.....P..........P.P..S..SS.....P......

groove .m..m......................................M..M..MM.....M......

210 270260250240230220

1 32

Homeoboxdomain

* ****** # # # # #

Paired LinkerHomeo Box

Domain (HD)Pro-Ser-Thr (PST)

14 270209131 422

5a Box Domain(PD)

5a

Fig. 1. The mutations in Pax6 responsible for disease map primarily to the paired box domain. (A) A schematic representation of the human Pax-6 protein

(Swiss-Prot/TrEMBL accession number: P26367). The primary alternate transcript, Pax6-5a, is indicated. (B) The structural model of the Pax-6 PD in complex

with a 26-bp DNA duplex (PDB code: 6PAX). A ribbon representation of Pax-6 is shown where the helices are colored red, beta sheets in cyan, and coil in yellow.Amino acid mutations that result in disease phenotype are highlighted. Residues colored black are residues that interact with DNA, while those colored green most

likely disrupt structure or stability of the PD fold. (C) The amino acid sequence of the paired box and homeo box domains. The secondary structure is based on the

known structure of the Pax6 paired box-DNA (PDB code 6PAX) or Paired homeo boxeDNA complex (PDB code 1FJL). Protein interactions with DNA are

indicated below the amino acid sequence: P, phosphate and S, sugar. The interaction with the minor (m) or major (M) groove is also indicated. Mutations shown to

display a disease phenotype are indicated. An asterisk (*) indicates a site where the protein interacts with DNA while the pound symbol (#) indicates a mutation

not directly involved in DNA recognition.

234 P.A. Tsonis, E.J. Fuentes / Experimental Eye Research 83 (2006) 233e234