Tissue-specific Regulation of the Mouse αA-crystallin Gene in Lens via Recruitment of Pax6 and c-Maf to its Promoter

Abstract

Pax6 is a lineage-restricted DNA-binding transcription factor regulating the formation of mammalian organs including brain, eye and pancreas. Pax6 plays key roles during the initial formation of lens lineage, proliferation of lens progenitor and precursor cells and their terminal differentiation. In addition to Pax6, lens fiber cell differentiation is regulated by c-Maf, Prox1 and Sox1. Crystallins are essential lens structural proteins required for light refraction and transparency. Mouse alphaA-crystallin represents about 17% of all crystallins at the protein level and ranks as one of the most abundant tissue-specific proteins. Lens-specific expression of this gene is regulated at the level of transcription. A promoter fragment of -88 to +46 is capable of driving lens-specific expression in transgenic mouse. Here we provide data suggesting that this lens-specific promoter fragment is comprised of multiple Pax6 and Maf-binding sites. Site-directed mutagenesis of regions within these sites resulted in partially or completely reduced promoter activities in lens cells. Co-transfections using Pax6 and c-Maf alone revealed moderate and strong activations of this promoter, respectively. In contrast to synergistic activation of alphaB-crystallin by Pax6 and c-Maf, Pax6 has a neutral effect on c-Maf-mediated alphaA-crystallin promoter activation. Chromatin immunoprecipitations established in vivo interactions of Pax6 and c-Maf with the alphaA-crystallin promoter in lens cells. Collectively, the present data support a molecular model in which tissue-specific expression of alphaA-crystallin is regulated by recruitment of Pax6 and c-Maf, two proteins regulating multiple processes of lens differentiation, to its promoter. In addition, the data suggest a molecular model of temporal and spatial regulation of alphaB, alphaA and gamma-crystallin genes in mouse embryonic lens by using variants of the Pax6/Maf regulatory module.