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.

该计划的长期目标是阐明哺乳动物晶状体的分子机制 DNA 结合转录因子 Pax6 直接控制发育和稳态。 先前的研究表明 Pax6 对于建立晶状体谱系和调节 晶状体蛋白基因表达。然而，Pax6 功能的完整谱系和范围以及 它影响晶状体发育的机制仍有待确定。使用染色质 通过免疫沉淀（ChIP），我们现已鉴定出大量新基因，包括 Mab21l1 和 DNase II 作为假定的直接 Pax6 靶标。 Mab21l1 在晶状体基板及其中高度表达 启动子区域包含 Six3 的多个结合位点，Six3 是另一个重要的晶状体谱系特异性调节因子 基因。 DNase II 在晶状体纤维细胞去核前表达最高。有证据存在 该基因的晶状体偏好表达受 Pax6、AP-2¿ 和 Foxe3 的控制。相比之下， Hsf4 在分化晶状体纤维细胞中 DNase II 的上调中发挥直接作用。我们还发现 Pax6不仅促进了镜片的发展，同时也抑制了替代品 发育程序，例如晶状体谱系中神经源基因的表达。这些发现 表明 Pax6 控制表观遗传机制，从而控制单个细胞谱系的形成 胚胎发育。为实现这一长期目标，提出以下具体目标： (1) 阐明Mab21l1的转录调控，Mab21l1是晶状体祖细胞生存所必需的基因 细胞，通过转基因小鼠中的 Pax6、Six3 和其他因子以及通过细胞培养实验。 (2) 至 阐明 DNase II 的转录调控，DNase II 是一种晶状体纤维细胞去核所需的酶，通过 Pax6、AP-2¿、Foxe3 和 Hsf4，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分析。 (3) 识别那些新颖的直接 Pax6 靶标 通过远端 5' 和 3' 增强子进行调节，并生成 Pax6 依赖性调节网络 使用大规模分析的染色质免疫沉淀控制晶状体和前脑发育 平行 DNA 测序 (ChIP-seq)、正常组织和 Pax6 突变组织中的 RNA 表达谱分析。