Compound mouse mutants of bZIP transcription factors MafG and MafK reveal a regulatory network of non-crystallin genes linked to cataract

Although majority of the genes linked to pediatric cataract exhibit lens fiber cell-enriched expression, our understanding of gene regulation in these cells is limited to function of just eight transcription factors and largely in the context of crystallins. Here, we identify small Maf transcription factors MafG and MafK as regulators of several non-crystallin human cataract genes in fiber cells and establish their significance to cataract. We applied a bioinformatics tool for cataract gene discovery iSyTE to identify MafG and its co-regulators in the lens, and generated various null-allelic combinations of MafG:MafK mouse mutants for phenotypic and molecular analysis. By age 4-months, MafG-/-:MafK+/- mutants exhibit lens defects that progressively develop into cataract. High-resolution phenotypic characterization of MafG-/-:MafK+/- lens reveals severe defects in fiber cells, while microarrays-based expression profiling identifies 97 differentially regulated genes (DRGs). Integrative analysis of MafG-/-:MafK+/- lens-DRGs with 1) binding-motifs and genomic targets of small Mafs and their regulatory partners, 2) iSyTE lens-expression data, and 3) interactions between DRGs in the String database, unravels a detailed small Maf regulatory network in the lens, several nodes of which are linked to human cataract. This analysis prioritizes 36 highly promising candidates from the original 97 DRGs. Significantly, 8/36 (22%) DRGs are associated with cataracts in human (GSTO1, MGST1, SC4MOL, UCHL1) or mouse (Aldh3a1, Crygf, Hspb1, Pcbd1), suggesting a multifactorial etiology that includes elevation of oxidative stress. These data identify MafG and MafK as new cataract-associated candidates and define their function in regulating largely non-crystallin genes linked to mouse and human cataract. Microarray comparision of lenses from mixed background (129Sv/J, C57BL/6J, and ICR) control (MafG+/-:MafK+/-; no-cataract) and compound (MafG-/-:MafK+/-; cataract) mouse mutants尽管多数与儿童白内障（pediatric cataract）相关的基因均呈现晶状体纤维细胞（lens fiber cell）富集表达的特征，但目前我们对这类细胞内基因调控的认知仅局限于8种转录因子的功能，且大多围绕晶状体蛋白（crystallins）展开。本研究首次将小Maf转录因子（small Maf transcription factors）MafG与MafK鉴定为纤维细胞中若干非晶状体蛋白（non-crystallin）相关人类白内障基因的调控因子，并明确了二者与白内障的关联意义。我们借助用于白内障基因挖掘的生物信息学工具iSyTE，鉴定了晶状体中的MafG及其共调控因子，并构建了MafG:MafK的多种无效等位基因组合小鼠突变体，用于表型与分子层面的分析。至4月龄时，MafG-/-:MafK+/- 突变体即出现晶状体异常，并随时间进展最终形成白内障。对MafG-/-:MafK+/- 晶状体的高分辨率表型表征显示，其纤维细胞存在严重异常；而基于基因芯片（microarrays）的表达谱分析则鉴定出97个差异调控基因（differentially regulated genes, DRGs）。针对MafG-/-:MafK+/- 晶状体差异调控基因开展三项整合分析：1）小Maf及其调控搭档的结合基序与基因组靶标；2）iSyTE晶状体表达谱数据；3）STRING数据库中差异调控基因间的互作关系，最终揭示了晶状体中一套完整的小Maf调控网络，其中多个节点均与人类白内障相关。本次分析从最初的97个差异调控基因中优先筛选出36个极具研究价值的候选基因。值得注意的是，36个候选基因中有8个（占比22%）分别在人类（GSTO1、MGST1、SC4MOL、UCHL1）或小鼠（Aldh3a1、Crygf、Hspb1、Pcbd1）中与白内障相关，这提示白内障的发病机制包含氧化应激升高在内的多因素参与。本研究数据证实MafG与MafK为全新的白内障相关候选因子，并明确了二者主要调控与小鼠及人类白内障相关的非晶状体蛋白基因的功能。对混合遗传背景（129Sv/J、C57BL/6J及ICR）下的对照组（MafG+/-:MafK+/-；无白内障表型）与复合突变组（MafG-/-:MafK+/-；白内障表型）小鼠晶状体开展的基因芯片对比分析