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Qing Xie, Louise Wolf, Ying Yang, Ales Cvekl; Transcriptional Regulation Of c-Maf By Pax6 During Embryonic Lens Development In Mouse. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2769.
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Pax6 is an essential gene that regulates formation of lens lineage and its subsequent differentiation. In contrast, c-Maf regulates terminal differentiation of lens fiber cells as a potent activator of crystallin gene expression. Previous studies suggested that Pax6 directly regulates c-Maf expression in cultured cell lines. In this study we aimed to elucidate molecular mechanisms in the in vivo transcriptional control of c-Maf in embryonic lens.
A series of EGFP reporter transgenic mice were generated and analyzed. A 1.3 kb promoter fragment, highly active in transient transfections, was tested in the absence or presence of distal non-coding evolutionary conserved regions (CRs). Expression of EGFP in lens at different embryonic stages was analyzed by fluorescence microscope, immunostaining and qRT-PCR. ChIP-on-chip and EMSA experiments were performed to identify and confirm Pax6-binding sites.
A 1.3 kb c-Maf promoter was not sufficient for expression of the EGFP reporter in transgenic mouse lens. In contrast, a 1.6 kb 5’-upstream region CR1, in combination with the c-Maf promoter, augmented expression of the EGFP reporter in lens from E10.5. This onset of c-Maf expression corresponded to the expression of endogenous c-Maf protein. CR1/promoter-driven EGFP expression was both delayed and reduced in Pax6+/- background. By ChIP-on-Chip and EMSA experiments, two head-to-head Pax6 binding sites were identified and confirmed within CR1. Mutagenesis/deletion of these Pax6 binding sites in CR1/promoter/EGFP transgenic mice decreased and/or abrogated the EGFP expression in transgenic lenses.
These studies showed that CR1 region serves as both essential and sufficient distal enhancer of c-Maf expression in lens. Genetic experiments demonstrate that c-Maf expression was sensitive to Pax6 haploinsufficiency. A bipartite Pax6 binding module within CR1 serves as a critical cis-acting region for its lens-specific enhancer activity. Collectively, these data establish a novel regulatory role for Pax6 during lens fiber cell differentiation and add new mechanistic data into the function of Pax6/c-Maf/crystalline regulatory network.
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