The Pax and large Maf families of genes in mammalian eye development

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1 The Pax and large Maf families of genes in mammalian eye development Vertebrate eye development is dependent on the coordinated action of thousands of genes. A specific group of over one hundred of regulatory genes is both responsible for ocular cell lineage specification and differentiation. Some of these genes also regulate development of central nervous system and other organs, e.g. kidney and pancreas. The regulatory genes can be classified into two large groups: 1. A group of genes encoding transcription factors with specific DNA binding transcription factors and transcriptional co activators and corepressors; and 2. A group of genes encoding proteins that participate in the process of signal transduction between individual cells, communities of cells and tissues. Many genes of the first group encode genes with tissue specific and tissue preferred expression patterns. In contrast, genes encoding components of signal transduction pathways are found ubiquitously expressed or expressed during the development of other tissues and organs. Herein, we will focus on two families of genes, the Pax and large Maf genes, as they encode important regulatory genes required for the formation of lens and retinal progenitor cells and for their terminal differentiation. The mammalian family of Pax genes is comprised of nine members (see Fig. 1) (for recent reviews, see Chi and Epstein, 2003). The Pax proteins (paired box proteins) share a common 128 amino acid domain, the paired domain (PD). The PD acts as a bipartite specific DNAbinding domain. Each PD is comprised of two helix turn helix subdomains, PAI and RED, tethered together by a linker region. The crystal structure of the PD in complex with DNA (Xu et al. 1999) and DNase I footprinting studies (Epstein et al. 1994) showed interactions between the protein and DNA over a distance of 20 base pairs. Four Pax proteins, Pax3, Pax4, Pax6 and Pax7 contain an additional DNA binding domain, the paired type homeodomain (HDPrd) (see Fig. 1). The isolated Pax HD binds cooperatively to DNA as a HD dimer (Wilson et al. 1995). It also interacts with the PD resulting in novel foldings of these domains. Spectroscopic studies of isolated PD revealed that a protein in solution has different secondary structure compared to the PD bound to DNA (Epstein et al.1994). Thus, binding of Pax3, Pax4, Pax6 and Pax7, to DNA is more complex than as described by the models based on interactions of isolated PD and HD with DNA. Additional studies are required to fully address the molecular mechanism Pax protein binding to DNA. The Pax genes play important roles in the development of multicellular organisms by regulating cell fate specification and terminal differentiation. Mutations in Pax genes cause developmental abnormalities in mouse and congenital defects in humans (see Semenza 1999). Mutations in a single Pax allele cause relatively modest defects. In contrast, homozygous mutations result in severe defects suggesting gene haploinsufficiency (see van Heyningen and Williamson, 2002). Dosage effect of a specific regulatory gene are explained by the model

2 in which a certain group of its specific target genes do not respond properly to the protein levels that are below a threshold value (Cvekl and Tamm, 2004). Haploinsufficiency of the mouse Pax2 gene results in kidney and retinal defects. Similar human PAX2 mutations cause renal coloboma syndrome. A series of natural mouse mutations, small eye (Sey), originates from mutations in Pax6 locus. Mutations in human PAX6 locus cause panocular disorder, aniridia, characterized by the reduced size of the iris, congenital cataract, corneal opacities and foveal hypoplasia (see van Heyningen and Williamson, 2002). Homozygous mutations in Pax6/PAX6 result in anophthalmia caused by failure in lens formation and subsequent arrest in eye development. Thus, studies of Pax genes provide important insight into lens formation. Specific roles of the Pax genes in the development of various organs including the brain, eye, inner ear, kidney, pancreas and thyroid are determined by the genetic networks operating both downstream and upstream from the particular Pax gene. Genetic studies of muscle, pituitary and retinal development identified a gene cassette comprised of members of Pax/Six/Eya/Dach families. Pax3 in combination with Six1, Eya2 and Dach2 was shown to regulate vertebrate muscle development (see Relaix and Buckingham, 1999). Pax6/Six6/Eya1 3/Dach1 2 were shown to regulate proliferation and differentiation of pituitary and retinal precursor cells (Li et al. 2002). The Pax/Six/Eya/Dach gene cassettes function through the formation of multiprotein complexes encoded by these genes assembled at promoters and enhancers of genes that control cell proliferation and differentiation. Examples of these genes include the inhibitors of cyclin dependent kinases, p21 and p57. In addition, expression of Pax genes is both directly regulated by at least two key signal transduction pathways (BMP/TGFβ, hedgehog, Notch, FGF/receptor tyrosine kinases and Wnt) and Pax gene control functions of these or other signal transduction pathways (e.g. retinoic acid and integrin/fibronectin signaling). Finally, Pax proteins can physically interact with other specific DNA binding proteins such as members of the large Maf family (see below), Etsfactors and Sox factors occupying an array of adjacent/overlapping binding sites. In our laboratory, the focus is on the function of Pax6 during murine eye development (see Cvekl and Piatigorsky, 1996; Cvekl et al. 2004). Pax6 is required for the establishment of lens precursor cells from the head ectoderm (Ashery Padan et al. 2000). Pax6 is also required for the multipotency of retinal progenitor cells (Marquardt et al. 2001). It is likely that Pax6 plays similar roles for the formation of corneal cell lineages and other ocular cells. Pax6 also plays late roles in the differentiation of lens and retina by regulating expression of other regulatory (Six3, Mab21 like1, Prox1 etc.) and structural genes such as lens crystallins, N cadherin and specific integrins (Lang, 2004). Pax6 is an evolutionary conserved gene found in all higher eukaryote genomes including worms, sea urchin and flies. The mammalian Pax6 locus encodes two major forms: Pax6 and

3 Pax6(5a) (see Fig. 2). The Pax6(5a) variant is encoded by an alternatively spliced exon 5a (Epstein et al. 1994). Biochemical studies of Pax6(5a) revealed that this insertion disrupts the ability of the PAI subdomain to bind DNA (Epstein et al. 1994; Kozmik et al. 1997). In the majority of tissues, Pax6 is the dominant form. In human fovea, iris and cornea, in contrast, both variants are expressed at comparable levels. Chicken and frog genomes also encode Pax6 loci with exon 5a. Drosophila melanogaster genome contains two genes, eyeless (ey) and twin of eyeless (toy) (Fig. 2) that are considered orthologues of Pax6 (Czerny et al. 1999). Another pair of genes, eye gone (eyg) and twin of eye gone (tog), code for proteins that lack functional PAI subdomain (Yao and Sun, 2005). Thus, their binding to DNA resembles mammalian Pax6(5a). In contrast, C. elegans genome contains one Pax6 like gene without exon 5a and is encoded by the Mab18 locus (Zhang and Emmons, 1995). Sea urchin supax6 also lacks the exon 5a (Czerny and Busslinger, 1995). The human PAX6 locus is located on chromosome 11 (see Fig. 3A). Functional studies of the mouse Pax6 locus estimated its length at 450 kb, although the area with coding exons is less than 40 kb. Important regulatory elements are found at the 3 region. Disruption of these enhancers in human PAX6 locus leads to aniridia and cataract. The Pax6 locus contains a number of additional tissue specific and tissue restricted enhancers, some of them shown in Fig. 3C. Studies of transcriptional regulation of the Pax6 locus may identify novel complex interactions between these enhancers and discover epigenetic regulatory mechanisms. In addition to Pax2 and Pax6, vertebrate eye development also requires genes from the large Maf family of transcription factors. Large Mafs form a special class of basic leucine zipper genes (bzip) (Yang and Cvekl, 2005). MafA, MafB, c Maf and NRL (see Fig. 4A) are specific DNA binding transcription factors, related to the AP 1 proteins (e.g. c Jun and c Fos). Although large Mafs are similar (see Fig. 4B), their is a major difference in their pattern of expression during development (see Yang and Cvekl, 2005). MafA, MafB and c Maf are expressed in the eye, pancreas and other organs. In contrast, NRL expression is restricted to the eye: high levels of expression are found in rod photoreceptors. Significantly reduced amounts of NRL were found in lens. Mutations in human c MAF and NRL also impair the lens and retina, respectively. Studies in mice have found that c Maf is required for terminal differentiation of lens primary fiber cells by directly controlling the expression of many, if not all, crystallin genes. Inactivation of NRL in mouse resulted in compromised photoreceptor development associated with reduced expression of the rhodopsin gene (Yoshida et al. 2004). Lens development in this model appeared normal. The roles of MafA and MafB in mouse eye development remain to be elucidated in vivo. There are 15 crystallin loci in the mouse. In humans, three of the six γ crystallins do not encode functional proteins (see Fig. 4). Transcriptional studies of αa, αb, βb1 and γfcrystallins identified binding sites for both Pax6 and large Maf proteins (Cvekl et al. 2004).

4 Transcriptional studies in cultured cells suggested that Pax6 and large Mafs engage in three types of relationships. Synergistic interactions between Pax6 and c Maf/MafA were shown on the αb crystallin promoter. Antagonistic interactions between these proteins were established on the βb1 and γf crystallin promoters. Finally, the αa crystallin locus is occupied in vivo by both Pax6 and c Maf. However, the transcriptional activation of this gene appears to depend on the strong activity of c Maf. This result agrees with the initial finding that αacrystallin gene expression is reduced to 1% of its normal level in c Maf null mouse lens (Ring et al. 2000). References Ashery Padan et al. (2000) Genes Dev. 14: Chi and Epstein (2002) Trends Genet. 18: Cvekl and Piatigorsky (1996) Bioessays 18: Cvekl and Tamm (2004) Bioessays 26: Cvekl et al. (2004) Int. J. Dev. Biol. 48: Czerny and Busslinger (1995) Mol. Cell. Biol. 15: Czerny et al. (1999) Mol. Cell 3: Epstein et al. (1994) J. Biol. Chem. 269: Epstein et al. (1994) Genes Dev. 8: Kozmik et al. (1997) EMBO J. 16: Lang (2004) Int. J. Dev. Biol. 48: Li et al. (2002) Science 297: Marquardt et al. (2001) Cell 105: Relaix and Buckingham (1999) Genes Dev. 13: Ring et al. (2000) Development 127: Semenza (1998) Transcription factors and human disease, Oxford University Press, New York. van Heyningen and Williamson (2002) Human Mol. Genet. 11: Wilson et al. (1995) Cell 82: Yang and Cvekl (2005) Einstein J. Biol. Med.: in press. Yao and Sun (2005) EMBO J. 24: Yoshida et al. (2004) Human Mol. Genet. 13: Xu et al. (1999) Genes Dev. 13: Zhang and Emmons (1995) Nature 377:55 59.

5 Figure1. Diagrammatic representation of nine murine Pax proteins. Paired domain (PD), yellow; octamer domain, blue,; homeodomain (HD), red; and transcriptional activation domain (if experimentally characterized), green. Variants of Pax3, Pax6, Pax7 and Pax8 originating from alternate

6 splicing in the PD are shown in black. Note, that alternative splicing generates additional forms, like in C-terminal domains of Pax5 and Pax8.

7 Figure 2. Mammalian and Drosophila Pax6 genes. Mammalian Pax6 locus encodes two Pax6 variants: Pax6 and Pax6(5a). Drosophila genome encodes four Pax6-like genes: eyeless (ey), twin of eyeless (toy), eye gone (eyg) and twin of eye gone (tog).

8 Figure 3. Diagram of human PAX6 locus. (A) PAX6 is part of WAGR locus. (B) Lens (L) and retinal (R) enhancers in the 3 -region. DNaseI hypersensitive sites, HS, are consistent with regulatory elements. (C) Tissue-specific enhancers in the 5 -regions and two introns. Three promoters, P0, P1 and Pα, were mapped in specific tissues.

9 Figure 4A. Schematic diagram of mammalian large Maf proteins. The individual structural domains are in colored boxes. The DNA-binding domain is comprised of the basic (blue) and ancillary (green) domains. The lengths AND chromosomal localizations are shown for mouse.

10 Figure 4B. Comparison of amino acid sequences of murine MafA, MafB, c-maf and NRL. The conserved and equivalent amino acid residues are shown in red and blue fonts, respectively. 2006