Pax6 as a key regulator of lens development

描述（由申请人提供）： 该项目的长期目标是阐明哺乳动物透镜发育和稳态的分子机制，这些机制直接由DNA结合转录因子Pax 6控制。以往的研究表明Pax 6在透镜谱系的建立和晶状体蛋白基因表达的调控中起重要作用。然而，Pax 6功能的完整光谱和范围以及它影响透镜发育的机制仍有待确定。使用染色质免疫沉淀（ChIP），我们现在已经确定了大量的新基因，包括Mab 21 l1和DNase II 2作为推定的直接Pax 6目标。Mab 21 l1在透镜基板中高度表达，其启动子区含有另一个重要的晶状体谱系特异性调控基因Six 3的多个结合位点。DNA酶Ⅱ 2在去核前的透镜纤维细胞中表达最高。有证据表明，该基因的晶状体偏好性表达受Pax 6、AP-21和Foxe 3的控制。相反，Hsf 4在分化透镜纤维细胞中的DNase II 2的上调中起直接作用。我们还发现Pax 6不仅促进透镜的发育，而且还有害地抑制替代性发育程序，例如透镜谱系中神经源性基因的表达。这些发现表明Pax 6控制着胚胎发育中控制单个细胞谱系形成的表观遗传机制。为了实现这一长期目标，本研究提出了以下具体目标：（1）通过转基因小鼠和细胞培养实验，阐明Pax 6、Six 3等因子对透镜祖细胞存活所必需的基因Mab 21 l1的转录调控。(2)通过蛋白质-DNA结合研究和基于细胞培养的报告基因分析，阐明Pax 6、AP-21、Foxe 3和Hsf 4对透镜纤维细胞去核所需的DNA酶II 2的转录调控。(3)为了鉴定通过远端5'和3'增强子调控的新的直接Pax 6靶点，并使用通过大规模平行DNA测序（ChIP-seq）分析的染色质免疫沉淀，在正常和Pax 6突变组织中的RNA表达谱，产生控制透镜和前脑发育的Pax 6依赖性调控网络。 公共卫生相关性： 由于透镜白内障是世界范围内失明的主要原因，因此该应用与人类健康相关。Pax 6基因是哺乳动物眼发育的必需基因，控制透镜谱系的形成和透镜纤维细胞的终末分化。已知PAX 6和本文研究的其他基因（FOXE 3，HSF 4和MAF）的突变会导致人类先天性眼病。PAX 6的突变也会导致各种神经系统疾病，PAX 6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.