Functional characterization of small Maf transcription factors MafG and MafK in mammalian lens homeostasis

Lachke, Salil A.In my thesis, I have identified and characterized a new function of the small Maf transcription factors MafG and MafK in regulating gene expression in mouse lens fiber cells, disruption of which causes lens defects including cataract. I used the bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery, http://bioinformatics.udel.edu/Research/iSyTE) to first identify MafG as a candidate gene with highly enriched expression in the lens. I analyzed iSyTE for expression of MafG, MafK , and MafF and performed real time quantitative RT-PCR to test expression of these genes in postnatal lens tissue. While MafG exhibits highly enriched expression in embryonic and postnatal lens, MafK is expressed in these stages, albeit at low levels and MafF expression is undetected in the lens, indicating that MafG and MafK may function in lens development or homeostasis. In situ expression analysis of wild type embryonic mouse lens confirmed the highly enriched expression of MafG transcripts in lens fiber cells. Thus, to investigate the function of MafG and MafK in the lens, I bred previously generated germline knock-out mouse mutants to derive various combinations of MafG and MafK compound mutant mice. MafG-/- :MafK +/- compound mouse mutants exhibit fully penetrant lens defects, including smaller lens (evident postnatal day 60 (P60) and later), a subset of which develop severe cataract (evident P120 and later). To gain insight into the molecular changes underlying these lens defects in MafG-/-:MafK+/- mice, I performed microarray-based transcript profiling analysis on MafG-/- :MafK+/- mutant and control lens at 2 month age, prior to development of cataract phenotype, and identified altered expression of several genes functional in distinct cell response pathways such as DNA damage response, cell cycle regulation, and apoptosis. At 1.5-fold levels, 949 genes were found to be up- or down-regulated in MafG -/- :MafK+/- mutant lens. Among other pathways, stress response genes such as Hsp27, Hmox1 and Ddit3 were mis-regulated in MafG-/- :MafK +/- mutant lens. In silico analyses of genomic regions surrounding a subset of these target genes identified conserved ARE (Antioxidant Response Element) cis-regulatory binding sites recognized by MafG/K and Cap n' Collar (CNC)/Bach heterodimers. Furthermore, I performed an integrated analysis based on iSyTE data on lens-enrichment or lens expression, as well as previously published ChIP (Chromatin Immuno-Precipitation) data on Nrf2, a small Maf binding partner, to identify genes with anti-oxidant response elements (ARE) to prioritize candidates from the extensive list of differentially regulated genes in MafG-/- :MafK+/- mutant lens. This analysis led to the identification of 24 genes that are promising direct targets of MafG/MafK and their co-regulatory protein partners in the lens. In sum, my thesis research has led to the identification and functional characterization of the transcription factors MafG and MafK in regulation of gene expression that control diverse pathways functional in stress response, apoptosis, and cell cycle regulation in lens fiber cells. Moreover, my research demonstrates that deficiency of these genes in a compound mouse mutant model causes severe lens fiber cell defects, including cataract. These findings imply MafG and MafK as important new candidate genes for further examination in human cataract patients. (Abstract shortened by UMI.)University of Delaware, Department of Biological SciencesM.S