Pax6 as a key regulator of lens development

描述(由申请人提供)： 该计划的长期目标是阐明由DNA结合转录因子Pax6直接控制的哺乳动物晶状体发育和动态平衡的分子机制。先前的研究表明，Pax6对于建立晶状体谱系和调控晶状体蛋白基因的表达是必不可少的。然而，Pax6‘S功能的完整光谱和范围及其影响晶状体发育的机制仍有待确定。利用染色质免疫沉淀(ChIP)技术，我们现在已经确定了包括Mab2111和DNase II2在内的大量新基因可能是Pax6的直接靶点。Mab21l1在晶状体胎盘中高度表达，其启动子区域含有另一个重要的晶状体系特异性调控基因Six3的多个结合位点。DNA酶II2在晶状体纤维细胞去核前表达最高。有证据表明，该基因的透镜偏好表达受Pax6、AP-21和Foxe3的调控。相反，HSF4在DNase II2分化晶状体纤维细胞的过程中起着直接的作用。我们还发现，Pax6不仅促进晶状体发育，还同时抑制其他发育程序，如晶状体谱系中神经源性基因的表达。这些发现表明，Pax6控制着表观遗传机制，这些机制控制着胚胎发育中单个细胞谱系的形成。为了实现这一长期目标，提出了以下具体目标：(1)通过转基因小鼠的Pax6、Six3等因子和细胞培养实验，阐明晶状体祖细胞生存所必需的基因Mab21l1的转录调控。(2)通过蛋白-DNA结合研究和基于细胞培养的报告实验，阐明Pax6、AP-21、Foxe3和HSF4在转基因小鼠中对晶状体纤维细胞去核所需的DNase II2的转录调控。(3)鉴定通过5‘和3’末端增强子调控的新的直接Pax6靶点，并利用大规模平行DNA测序(CHIP-SEQ)分析染色质免疫沉淀，在正常组织和Pax6突变组织中进行RNA表达谱分析，建立依赖Pax6的调控网络，控制晶状体和前脑的发育。 公共卫生相关性： 这一应用关系到人类的健康，因为晶状体白内障是全球失明的主要原因。Pax6是哺乳动物眼睛发育所必需的基因，它控制晶状体系谱的形成和晶状体纤维细胞的终末分化。PAX6和这里研究的其他基因(FOXE3、HSF4和MAF)的突变是已知的导致人类先天性眼病的原因。PAX6的突变还会导致各种神经疾病，PAX6也与II型糖尿病有关。

DESCRIPTION (provided by applicant): The long-term goal of this program is to elucidate those molecular mechanisms of mammalian lens development and homeostasis that are directly controlled by DNA-binding transcription factor Pax6. Previous studies have shown that Pax6 is essential for establishing lens lineage and regulation of crystallin gene expression. However, the complete spectrum and range of Pax6's function and the mechanism by which it affects lens development remain to be determined. Using chromatin immunoprecipitation (ChIP), we have now identified a large number of novel genes including Mab21l1 and DNase II2 as putative direct Pax6 targets. Mab21l1 is highly expressed in the lens placode and its promoter region contains multiple binding sites for Six3, another important lens-lineage specific regulatory gene. DNase II2 is most highly expresed in lens fiber cells just prior to their denucleation. Evidence exists that lens-preferred expression of this gene is under the control of Pax6, AP-21 and Foxe3. In contrast, Hsf4 plays a direct role in the upregulation of DNase II2 in differentiating lens fiber cells. We also found that Pax6 not only promotes lens development but it also simultanously suppresses alternative developmental programs such as the expression of neurogenic genes in lens lineage. These findings suggest that Pax6 controls epigenetic mechanims that control individual cell lineage formation in embryonic development. In order to carry out this long-term goal, the following specific aims are proposed: (1) To elucidate transcriptional regulation of Mab21l1, a gene essential for the survival of lens progenitor cells, by Pax6, Six3 and other factors in transgenic mouse and via cell culture experiments. (2) To elucidate transcriptional regulation of DNase II2, an enzyme required for lens fiber cell denuclation, by Pax6, AP-21, Foxe3 and Hsf4, in transgenic mouse and through a combination of protein-DNA binding studies and cell culture based reporter assays. (3) To identify those novel direct Pax6-targets that are regulated via distal 5' and 3' enhancers and to generate a Pax6-dependent regulatory network that controls lens and forebrain development using chromatin immunoprecipitations analyzed by massively parallel DNA sequencing (ChIP-seq), RNA expression profiling in normal and Pax6 mutated tissues. PUBLIC HEALTH RELEVANCE: This application is relevant to human health as lens cataract is a major cause of worldwide blindness. Pax6 is an essential gene for mammalian eye development, it controls lens lineage formation and terminal differentiation of lens fiber cells. Mutations in PAX6 and other genes studied here (FOXE3, HSF4 and MAF) are known to cause human congenital eye diseases. Mutations in PAX6 also cause a variety of neurological disorders and PAX6 has also been implicated in type II diabetes.