Pax6 as a key regulator of lens development

Pax6 作为晶状体发育的关键调节因子

• 批准号: 8204917
• 负责人: Ales Cvekl
• 金额: $ 51.51万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8204917
• 关键词: 5' Flanking Region; Ablation; Affect; Aniridia; Apoptotic; Binding; Binding Sites; Biological Assay; Biology; Blindness; Cataract; Cell Culture Techniques; Cell Lineage; Cell Survival; Cells; Chromatin; Computer Analysis; Crystallins; DNA; DNA Binding; DNA Sequence; DNA-Binding Proteins; Deoxyribonucleases; Development; Distal; Embryo; Embryonic Development; Enhancers; Enzymes; Epigenetic Process; Essential Genes; Eye Abnormalities; Eye Development; Forebrain Development; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Transcription; Goals; Health; Homeostasis; Human; Individual; Lens Diseases; Lens Fiber; Lens Placodes; Light; Link; Modeling; Molecular; Molecular Profiling; Mus; Mutate; Mutation; Neurons; Non-Insulin-Dependent Diabetes Mellitus; Nucleic Acid Regulatory Sequences; Play; Process; Promoter Regions; Regulation; Regulator Genes; Reporter; Role; Signal Transduction Pathway; Stem cells; TFAP2A gene; Testing; Tissues; Transcriptional Regulation; Transgenic Mice; Up-Regulation; Validation; base; chromatin immunoprecipitation; chromatin remodeling; combinatorial; fiber cell; histone modification; in vivo; insight; lens; nervous system disorder; novel; nuclease; programs; promoter; reconstruction; research study; transcription factor

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.

这个项目的长期目标是阐明哺乳动物晶状体的分子机制。 由DNA结合转录因子Pax6直接控制的发育和动态平衡。 以前的研究表明，Pax6对于建立晶状体谱系和调节 晶状体蛋白基因的表达。然而，Pax6‘S函数的完整谱和范围以及 其影响晶状体发育的机制尚不清楚。使用染色质 免疫沉淀(CHIP)，我们现在已经发现了包括Mab21l1和Mab21l1在内的大量新基因 DNaseII？被认为是Pax6的直接靶标。Mab21l1在晶状体胎盘中高表达，其 启动子区域包含Six3的多个结合位点，Six3是另一种重要的晶状体特异性调控基因 吉恩。脱氧核糖核酸酶II在晶状体纤维细胞去核前高表达。证据是存在的 该基因的镜头偏好表达受Pax6、AP-2和Foxe3的调控。相比之下， HSF4在晶状体纤维细胞分化过程中直接上调DNA酶II？的表达。我们还发现 Pax6不仅促进了晶状体的发育，同时也抑制了另一种选择 发育计划，如神经基因在晶状体谱系中的表达。这些发现 提示Pax6控制表观遗传机制，控制单个细胞谱系的形成 胚胎发育。为实现这一长远目标，现提出以下具体目标： (1)阐明晶状体前体细胞生存所必需的基因Mab21l1的转录调控 细胞，通过Pax6、Six3等因子在转基因小鼠体内进行细胞培养实验。(2)至 阐明DNA酶II？，晶状体纤维细胞去核所需的一种酶的转录调控。 Pax6、AP-2β、Foxe3和HSF4在转基因小鼠中的表达及其与蛋白质和DNA的结合 以研究和细胞培养为基础的记者分析。(3)识别那些新的直接Pax6-靶标 通过远端5‘和3’增强子调节，并产生依赖于Pax6的调节网络，该网络 通过大量分析的染色质免疫沉淀控制晶状体和前脑的发育 并行DNA测序(CHIP-SEQ)，正常组织和Pax6突变组织的RNA表达谱。