Gene Regulation by Pax6 in Mammalian Lens Development

Abstract

Gene regulatory networks (GRNs) play critical roles during embryonic development and evolution. Lens development represents an advantageous system to identify specific GRNs as each stage of lens formation is characterized by unique morphology of lens cells and tight genetic control executed by specific DNA-binding transcription factors. Pax6, a paired domain and homeodomain-containing transcription factor, is essential for both lens placode formation and subsequent stages of lens morphogenesis while c-Maf, a basic leucine zipper-containing factor, controls terminal differentiation of lens fiber cells. During lens cell differentiation, Pax6 appears to regulate expression of a small group of regulatory proteins. My hypothesis is that Pax6 employs a range of combinatorial DNA-binding mechanisms in directing transcriptional control of key genes (e.g. c-Maf, Foxe3, Prox1, Sox1, and Hsf4) and novel non-regulatory genes required for lens development..;We performed protein-DNA binding selection assays and identified nine novel DNA-binding motifs for Pax6. Electrophoretic mobility shift assays (EMSAs) confirmed the binding of these DNA motifs with Pax6. Luciferase reporter assays showed that Pax6 differentially activates or represses the reporter gene expression through those DNA-binding sequences.;Our studies of transcriptional regulation of c-Maf identified a 1.6kb lens/retinal pigment epithelium (RPE)-preferred enhancer, CR1. A combination of CR1 with a 1.3kb c-Maf promoter supported EGFP expression in lens and RPE. Expression of EGFP was delayed and reduced in Pax6+/- transgenic lenses. Chromatin immunoprecipitation assays revealed binding of Pax6 and c-Maf to multiple regions of the c-Maf locus in lens. A pair of Pax6-binding sites in the CR1 was identified by newly-identified Pax6 DNA-binding motifs. Mutagenesis of these Pax6-binding sites inactivated transgenic expression of EGFP in lens but not in RPE.;Comparative data analysis including RNA expression profiling in Pax6 +/- lenses and Pax6 ChIP-on-chip data lead to the identification of six novel Pax6-regulated genes, Gaa, Isl1, Kif1b, Mtmr2, Pcsk1n, and Snca. By EMSAs and transfection/reporter assays, direct regulation of Kif1b and Snca by Pax6 was established.;These data show multiple novel regulatory roles for Pax6 during lens development, link together the Pax6/c-Maf/crystallin regulatory network, and suggest a novel type of GRN sub-circuit that controls a major part of embryonic lens development.