The longterm goal of this program is to elucidate those genetic and epigenetic regulatory components of the 1Acrystallin (Cryaa) locus that control expression of 1Acrystallin in the ocular lens. A loss of 1Acrystallin expression or expression of its mutants in lens compromises lens transparency and results in lens opacification. Expression of 1Acrystallin occurs both in the lens epithelium (lens precursor cells) and in the lens fiber cells (terminally differentiated lens cells). Due to its upregulation in differentiating lens primary fibers, it is an excellent marker for fiber cell differentiation and studies of upstream signaling pathways regulating this process. The goal of this work is to identify these regulators of the 1Acrystallin locus and to understand the molecular mechanisms that govern their functions. Among the many regulators of 1Acrystallin gene expression, we have now identified a 16 kb domain of acetylated histone H3 K9ac that harbor the Cryaa locus in lens chromatin. We have also found that the 5'/3'borders of the Cryaa locus are generated by two developmentally controlled enhancers, DCR1 and DCR3. We have shown that DCR1 functions as an FGF regulated enhancer. A DCR1/1.9 kb 1Acrystallin promoter coupled with a EGFP reporter gene virtually recapitulated the expression pattern of 1Acrystallin in lens epithelium and lens fibers. Chromatin immunoprecipitation (ChIP) assays showed that high levels of 1Acrystallin expression correlate with increased binding of c Maf to the promoter, recruitment of histone acetyltransferase CBP to the promoter, and stable presence of Pax6. In order to carry out this longterm goal, the following specific aims are proposed: (1) To identify and characterize FGFresponsive ciselements in DCR1 using gene reporter assays in primary lens explants and in vivo by ChIP assays. The function of DCR1 will be also assessed in transgenic mice through its deletion from a Cryaaharboring bacterial artificial chromosome (BAC) clone with an EGFP integrated reporter, (2) To identify those FGF dependent and FGF independent regulatory elements responsible for cMaf expression in the lens, and (3) To analyze the function of CBP and its p300 homologue gene during lens development by conditional inactivation of these genes in mouse followed by molecular analysis of core histone acetylations and ATP dependent chromatin remodeling enzymes Brg1 and Snf2h associated with the Cryaa locus.
This application is relevant to human health as lens cataract is a major cause of worldwide blindness. The 1Acrystallin is the most abundant structural component of the human lens;its abnormal function and/or expression causes lens opacification. Mutations in genes encoding lens regulatory proteins such as PAX6, c MAF and CBP studied here are known to cause human congenital cataracts.
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|Wang, Wei-Lin; Li, Qingtian; Xu, Jianming et al. (2010) Lens fiber cell differentiation and denucleation are disrupted through expression of the N-terminal nuclear receptor box of NCOA6 and result in p53-dependent and p53-independent apoptosis. Mol Biol Cell 21:2453-68|
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|Ninkovic, Jovica; Pinto, Luisa; Petricca, Stefania et al. (2010) The transcription factor Pax6 regulates survival of dopaminergic olfactory bulb neurons via crystallin ?A. Neuron 68:682-94|
|Cvekl, Ales; Wang, Wei-Lin (2009) Retinoic acid signaling in mammalian eye development. Exp Eye Res 89:280-91|
|Wolf, Louise; Yang, Ying; Wawrousek, Eric et al. (2008) Transcriptional regulation of mouse alpha A-crystallin gene in a 148kb Cryaa BAC and its derivates. BMC Dev Biol 8:88|
|Yang, Ying; Wolf, Louise V; Cvekl, Ales (2007) Distinct embryonic expression and localization of CBP and p300 histone acetyltransferases at the mouse alphaA-crystallin locus in lens. J Mol Biol 369:917-26|
|Cvekl, Ales; Duncan, Melinda K (2007) Genetic and epigenetic mechanisms of gene regulation during lens development. Prog Retin Eye Res 26:555-97|
|Yang, Ying; Cvekl, Ales (2007) Large Maf Transcription Factors: Cousins of AP-1 Proteins and Important Regulators of Cellular Differentiation. Einstein J Biol Med 23:2-11|
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