Pax6 as a key regulator of lens development

Pax6 作为晶状体发育的关键调节因子

基本信息

批准号：
8002004
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
项目类别：
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/8002004
关键词：
5&apos 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 public health relevance reconstruction research study transcription factor

项目摘要

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.

描述（由申请人提供）：该项目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稳态的分子机制。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至关重要。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。使用染色质免疫沉淀 (ChIP)，我们现已鉴定出大量新基因，包括 Mab21l1 和 DNase II2，作为假定的直接 Pax6 靶标。 Mab21l1 在晶状体基板中高度表达，其启动子区域包含另一个重要的晶状体谱系特异性调控基因 Six3 的多个结合位点。 DNase 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晶状体偏好表达受到 Pax6、AP-21 和 Foxe3 的控制。相比之下，Hsf4 在分化晶状体纤维细胞中 DNase II2 的上调中发挥直接作用。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，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，从而控制胚胎发育中个体细胞谱系的形成。为了实现这一长期目标，提出以下具体目标：（1）阐明转基因小鼠和晶状体祖细胞存活所必需的基因Mab21l1通过Pax6、Six3和其他因子的转录调控。通过细胞培养实验。 (2) 为了阐明转基因小鼠中 Pax6、AP-21、Foxe3 和 Hsf4 对 DNase II2（一种晶状体纤维细胞去核所需的酶）的转录调节，并通过蛋白质-DNA 结合研究和基于细胞培养的报告基因测定相结合。 (3) 鉴定那些通过远端 5' 和 3' 增强子调节的新型直接 Pax6 靶标，并使用大规模并行 DNA 测序 (ChIP-seq) 分析的染色质免疫沉淀来生成控制晶状体和前脑发育的 Pax6 依赖性调节网络），正常组织和 Pax6 突变组织中的 RNA 表达谱。公共卫生相关性：该应用与人类健康相关，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的必需基因，它控制晶状体谱系的形成和晶状体纤维细胞的终末分化。 PAX6 和此处研究的其他基因（FOXE3、HSF4 和 MAF）的突变已知会导致人类先天性眼病。 PAX6 突变还会引起多种神经系统疾病，并且 PAX6 还与 II 型糖尿病有关。