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Ales Cvekl, Qing Xie, Rebecca McGreal, Raven Harris, Saima Limi, Chun Gao, Wei Liu, Lixing W Reneker, Linda Musil; The molecular basis of crystallin gene expresssion in lens: Role of FGF signaling and c-MAF. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
Embryonic tissue development requires intricate temporal and spatial control of gene expression that is executed through specific gene regulatory networks (GRNs). GRNs are comprised of individual modules that contain inputs form signal transduction pathways, action of specific DNA-binding transcription factors via binding to the promoters and enhancers, regulatory functions of non-coding RNAs (miRNAs and lncRNAs), and information about global and local chromatin structure and dynamics including spatial organization of co-regulated genes inside of the cell nuclei.
Expression of αA-crystallin in individual nuclei of the developing mouse lens was analyzed by RNA-FISH. To dissect gene control of the mouse c-Maf promoter, we generated a series of 5’- and 3’-deletion constructs. The reporters were transfected into the primary cultures of embryonic chick lens epithelial cells (DCDMLs) grown in the absence/presence of FGF2. The wild type c-Maf promoter and its internal deletion of 202 bp (-272/-70) were coupled with lens-specific enhancer and fused to the EGFP reporter. Transgenic mice were generated and expressions of EGFP were evaluated in mouse embryos. Expression of c-Jun and c-Maf, and Etv5/ERM was evaluated by immunofluorescence and in situ hybridizations, respectively. Localization of these proteins was determined by chromatin immunoprecipitation (ChIP) using the c-Maf and Cryaa loci and mouse lens chromatin. Cotransfection experiments with c-Jun and Etv5 were conducted using c-Maf promoter-based constructs and αA-crystallin promoter/enhancer system.
We identified and characterized a novel FGF2-responsive region in the c-Maf promoter (-272/-70, FRE). Both c-Maf and Cryaa regulatory regions contain arrays of AP-1 and Ets-binding sites. ChIP assays established binding of c-Jun (an AP-1 factor) and Etv5/ERM (an Ets factor) to these regions in lens chromatin. Analysis of temporal and spatial expression of c-Jun, phospho-c-Jun, and Etv5/ERM in wild type and pERK1/2 deficient lenses supports their roles as nuclear effectors of FGF signaling in mouse embryonic lens.
Collectively, these studies show that FGF signaling up-regulates expression of αA-crystallin both directly and indirectly via up-regulation of c-Maf. These molecular mechanisms are applicable for other crystallins and genes highly expressed in terminally differentiating lens fibers.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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