Pax6 as a key regulator of lens development

该项目的长期目标是阐明哺乳动物透镜的分子机制 发育和稳态直接由DNA结合转录因子Pax 6控制。 以往的研究表明Pax 6对建立透镜谱系和调节晶状体上皮细胞的生长和分化是必不可少的。 晶状体蛋白基因表达然而，Pax 6功能的完整频谱和范围以及 其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作为推定的直接Pax 6靶点。Mab 21 l1在透镜基板中高度表达， 启动子区含有Six 3的多个结合位点，Six 3是另一个重要的晶状体谱系特异性调节基因， 基因DNA酶Ⅱ在去核前的透镜纤维细胞中表达最高。证据存在 该基因的晶状体偏好表达受Pax 6、AP-2和Foxe 3的控制。与此相反， 在分化的透镜纤维细胞中，Hsf 4在DNase II的上调中起直接作用。我们还发现 Pax 6不仅促进了透镜的发展， 发育程序，如神经基因在透镜谱系中的表达。这些发现 表明Pax 6控制着表观遗传机制，该机制控制着个体细胞谱系的形成， 胚胎发育为了实现这一长期目标，提出了以下具体目标： (1)阐明透镜祖细胞存活所必需的基因Mab 21 l1的转录调控 细胞，通过Pax 6、Six 3等因子在转基因小鼠中的作用及细胞培养实验。(2)到 阐明DNA酶II？（一种透镜纤维细胞去核所需的酶）的转录调节， Pax 6、AP-2、Foxe 3和Hsf 4在转基因小鼠中通过蛋白质-DNA结合的组合 研究和基于细胞培养的报告基因测定。(3)为了鉴定那些新的直接Pax 6靶点， 通过远端5'和3'增强子调节，并产生Pax 6依赖性调节网络， 控制透镜和前脑的发展，使用染色质免疫沉淀分析， 平行DNA测序（ChIP-seq），正常和Pax 6突变组织中的RNA表达谱。

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 II¿ 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 II¿ 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-2¿ and Foxe3. In contrast, Hsf4 plays a direct role in the upregulation of DNase II¿ 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 II¿, an enzyme required for lens fiber cell denuclation, by Pax6, AP-2¿, 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.