Human Pax6 Paired-Domain

Alice Tillman '21 and Alanta Budrys '21 

Contents:

I. Introduction

The human paired box 6 protein or Pax6 is a Prd-domain containing transcription factor that serves as a master regulator of development. Originally discovered for its role in Drosophila segmentation, Pax6 expression has since been implicated in eye, pancreas, and central nervous system development. During the first few weeks of embryonic development, Pax6 expression is thought to drive the maturation of embryonic stem cells into the neuroectoderm by repressing genes associated with pluripotency, including Oct4, Nanog, and Myc. It is also believed to simultaneously activate ectodermal transcription factors and genes, including NeuroD1, Neurog2, Ift74, and Pou3f2, and repress the expression of mesodermal and endodermal genes. As development proceeds, Pax6 helps coordinate cell fate, patterning, and circuit formation in the CNS.

The importance of Pax6 is illustrated by loss-of-function phenotypes in Pax6 mutants. For example, heterozygous Pax6 mutants can cause eye disorders such as aniridia and Small eye (Sey), while homozygous mutations are associated with complete eye failure. Pax6 mutations are also known to decrease insulin, glucagon, and somatostatin production in the pancreas and are therefore associated with a higher risk for diabetes. . 

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Figure 1: Pax6 as a master regulator of development. It is responsible for the activation of genes and other transcription factors involved in neurogenesis, as well as the Notch signaling pathway. Pax6 drives differentiation of ectodermal cells in the developing embryo by repressing mesodermal and endodermal genes (Thakurela et. al., 2016).

Pax6 belongs to a family of nine mammalian paired box proteins, Pax1-9, which are characterized by the evolutionary-conserved paired-box DNA-binding domain. It is thought that the nine Pax proteins arose from two whole-genome duplication events, followed by a series of gene deletions to refine the function of each protein. Pax6, along with Pax3, 7, and 4 also contain a DNA-binding homeodomain at the C-terminus. Unlike many other Pax proteins, Pax6 does not contain an octapeptide region, which normally functions as a binding site for downstream corepressors. All Pax proteins, however, contain a transactivation domain at the C-terminus, which serves as a binding site for other proteins. Pax6 can also be alternatively spliced to produce variants with different homeodomain and transactivation domain binding sites. The functional diversity of Pax6 is essential to its activity, allowing it to drive the development of a wide-range of processes.

This page is focused on the role of the DNA-binding paired-domain in Pax6 function.

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Figure 2: General structure of a Pax protein. Pax6 includes (1) a DNA-binding paired domain, (2) a DNA-binding homeodomain, and (3) a transactivation domain. Our JMOL is focused on the paired-domain of Pax6. (Blake and Ziman, 2014)

II. General Structure

The paired domain of is composed of two subdomains, the N-subdomain and the C-subdomain, that are connected by a fifteen residue polypeptide chain linker.

The N-subdomain is made up of three alpha helices and two beta sheets that form a hairpin, while the C-subdomain forms three alpha helices and lack any beta sheets. This describes the of the two subdomains.

DNA contact is established by amino acids in the two sub-domains and the linker. DNA contact is established by amino acids in the two sub-domains and the linker. The paired-domain or Pax6 recognizes and binds a conserved 20 base-pair of DNA (Fig. 3). This sequence is primarily composed of B-DNA with an average helical twist of 34.7 degrees. The helical twist between bases 11 and 12, however, is 15 degrees, but opens up to 48 degrees between bases 12 and 13.

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Figure 3: A comparison of DNA-binding sequences between human Pax6, human Pax5, and Drosophila Prd proteins (Xu et al., 1999).

IV. The N-Subdomain

In the N-subdomain, the three alpha helices interact with the major groove of the DNA and the beta sheets interact with the minor groove. The first amino acid of the , Asn 47, interacts with thymine 2 and thymine 4. Asn 47 first interacts with the methyl group of thymine 4 through . Asn 47 also forms a water-mediated with the phosphate of thymine 2 on the 5’ to 3’ strand. This helps the transcription factor recognize and bind the at position 4 of the paired-domain binding site. Ser 46, Cys 49, and Arg 56 all form hydrogen bonds with phosphates on the 3’ to 5’ strand.

The beta strands also make important contacts with the minor groove of the DNA. Leu 18 forms a with the DNA backbone at guanine 8. In a similar manner, Asp 14 forms a hydrogen bond with the N2 of guanine 9. Gly 15 forms a hydrogen bond with the N2 of guanine 10.

V. The Linker Region

The extended polypeptide chain makes contacts with minor groove DNA over an 8bp region . Ile-68 is an important residue, seen in paired family domains, and it makes with Thymine 11 and 12, and the sugar of Guanine 10, by fitting directly into the minor groove. Another key residue is Pro 73: the side chain packs with the sugar of Guanine 15, this alters the directionality of the linker, and allows for subsequent hydrogen bonds to occur. This includes a hydrogen bond between two amino acid residues: the carbonyl oxygen of Lys 72 to an NE of Arg 74 This illustrates a few of the interactions that contribute to the stability for the binding of the two subdomains.

III. The C-Subdomain

Helix 5 and helix 6 of C-subdomain form an . The DNA is recognized via of helix 6, which fits directly into the major groove. Hydrogen bonds between phosphate groups of the occur with Ser 119, Ser 116 and Arg 125. Similarly, Tyr 97, Ala 96 and Arg 125 form hydrogen bonds with the . Additionally, water-mediated are made between Ser-118 and the N7 of guanine 17, as well as the Od of Asn-121 and the N7 of guanine 20, Asn-121 also makes water-mediated contacts with two . The C-subdomain also exhibits forces between DNA bases and Arg 122, Arg 125 and Phe 95. These interactions illustrate the bonding between DNA bases and the C-domain.


VI. References

Blake JA and Ziman MR. (2014) Pax genes: regulators of lineage specification and progenitor cell maintenance Development 141 737-751

Duan D, Fu Y, Paxinos G, & Watson C. (2013) Spatiotemporal expression patterns of Pax6 in the brain of embryonic, newborn, and adult mice. Brain Struct. Funct. 218, 353–372

Epstein JA, Glaser T, Cai J, Jepeal L, Walton DS, Maas RL. (1994). Two independent and interactive DNA-binding subdo- mains of the Pax6 paired domain are regulated by alternative splicing. Genes Dev. 8:2022–34

Gosmain Y, Katz L, Masson MH, Cheyssac C, Poisson C, and Philippe J. (2012) Pax6 Is Crucial for ?-Cell Function, Insulin Biosynthesis, and Glucose-Induced Insulin Secretion. Molecular Endocrinology, 26: 696-709

Hart AW, Mella S, Mendrychowski J, van Heyningen V, Kleinjan DA. (2013) The developmental regulator Pax6 is essential for maintenance of islet cell function in the adult mouse pancreas. PLoS One. 8: 1-10

Ooki A, Dinalankara W, Marchionni L, et al. (2018) Epigenetically regulated PAX6 drives cancer cells toward a stem-like state via GLI-SOX2 signaling axis in lung adenocarcinoma. Oncogene 37, 5967–5981

Paixγo-Cτrtes VR ,Salzano FM, Bortolini MC. (2013). Evolutionary history of chordate PAX genes: dynamics of change in a complex gene family. PLOS ONE, 8.

Short S and Holland LZ. (2008) The Evolution of Alternative Splicing in the Pax Family: The View from the Basal Chordate Amphioxus. Mol Evol 66: 605.

Thakurela S, Tiwari N, Schick S, Garding A, Ivanek R, Berninger B, and Tiwari V (2016) Mapping gene regulatory circuitry of Pax6 during neurogenesis. Cell discovery 2:15045.

Xu HE, Rould MA, Xu W, Epstein JA, Maas RL, et al. (1999) Crystal structure of the human Pax6 paired domain-DNA complex reveals specific roles for the linker region and carboxy-terminal subdomain in DNA binding. Genes Dev 13: 1263–1275.

Yasuda T, Kajimoto Y, Fujitani Y, Watada H, Yamamoto S, Watarai T, Umayahara Y, Matsuhisa M, Gorogawa S, Kuwayama Y, Tano Y, Yamasaki Y, Hori M (2002) PAX6 mutation as a genetic factor common to aniridia and glucose intolerance. Diabetes 51:224–230

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