Retinoic acid target genes and transcriptional mechanisms during eye development

眼睛发育过程中视黄酸靶基因和转录机制

• 批准号: 10201360
• 负责人: GREGG L DUESTER
• 金额: $ 39万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10201360
• 关键词: ALDH1A2 gene; ATAC-seq; Address; All-Trans-Retinol; Binding; Biological Assay; Candidate Disease Gene; ChIP-seq; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Defect; Deposition; Development; Diffuse; Disease; EP300 gene; Embryo; Embryonic Development; Enhancers; Epigenetic Process; Exhibits; Eye; Eye Development; FOXC1 gene; Failure; Gene Activation; Gene Silencing; Genes; Genetic Enhancer Element; Genetic Transcription; Genome; Goals; Growth; Human; Knock-out; Knowledge; Learning; Mesenchyme; Methods; Microphthalmos; Morphogenesis; Morphology; Mus; Mutation; Neural Crest; Nuclear; Nuclear Receptors; Optic vesicle; Optics; Regulator Genes; Repression; Response Elements; Retinoic Acid Binding; Retinoic Acid Receptor; Retinoic Acid Response Element; Retinol dehydrogenase; Signal Pathway; Testing; Tissues; Transcriptional Regulation; Transgenes; Tretinoin; Untranslated RNA; Vitamin A; conditional knockout; eye formation; gene function; gene repression; genetic corepressor; improved; in vivo; insight; interest; mutant; novel strategies; optic cup; receptor; recruit; retinaldehyde dehydrogenase; transcriptome sequencing

PROJECT SUMMARY One of the most critical functions of the vitamin A (retinol) metabolite retinoic acid (RA) is control of eye development. In mouse, RA synthesis occurs early in the optic field with expression of retinol dehydrogenase-10 (RDH10) and all three retinaldehyde dehydrogenases (ALDH1A1, ALDH1A2, ALDH1A3) that convert retinol to RA. RA diffuses to tissues throughout the optic placode, optic vesicle, and adjacent mesenchyme to stimulate folding of the optic vesicle to form the optic cup by E10.5. At E12.5-E14.5, RA is needed for further morphogenesis of the optic cup and surrounding perioptic mesenchyme; loss of RA leads to microphthalmia. RA functions by binding to nuclear RA receptors at RA response elements (RAREs) that either activate or repress transcription of key genes. Binding of RA to RA receptors regulates recruitment of transcriptional coregulators such as nuclear receptor coactivator (NCOA) and nuclear receptor corepressor (NCOR), which in turn control binding of the generic coactivator p300 and the generic corepressor PRC2. However, a major unsolved problem is what are the key genes controlled by RA during development of the eye; only two candidate direct RA target genes are known (Pitx2 and Foxc1). As loss or gain of RA activity alters expression of thousands of genes (perhaps many due to post-transcriptional effects), it remains difficult to identify genes that are direct transcriptional targets of RA. In our Preliminary Studies we addressed this question by comparing ChIP-seq and RNA-seq for tissues from Aldh1a2-/- embryos lacking RA synthesis, thus identifying genes with altered expression when RA is missing that also have nearby RA-regulated deposition of H3K27ac (gene activation mark) or H3K27me3 (gene repression mark) associated with RAREs. Such RARE enhancers/silencers were identified near genes already known to be required for embryonic development, thus validating our approach. CRISPR knockouts for several predicted new direct RA target genes verified their requirements for development. Here, we plan to use this approach to identify RA target genes and PITX2 target genes during eye development by comparing ChIP-seq (H3K27ac & H3K27me3) and RNA-seq for wild-type vs RA-deficient optic vesicle and eye, and wild-type vs Pitx2 knockout eye. We will also identify RA-regulated enhancers and silencers in the eye to uncover the mechanisms through which RA regulates Pitx2, Foxc1, or other genes. Our studies will provide vital information on the mechanisms utilized by RA and PITX2 to control transcription in the eye and will identify gene regulatory networks during eye formation. This knowledge will help determine how eye defects occur, identify new genes or enhancers/silencers that may be mutational targets causing human eye defects, and improve strategies to treat eye defects.

项目总结 维生素A(视黄醇)代谢物维甲酸(RA)最关键的功能之一是控制 眼睛发育。在小鼠，视黄酸合成发生在视场的早期，视黄醇的表达 脱氢酶-10(RDH10)和所有三种视黄醛脱氢酶(ALDH1A1，ALDH1A2， ALDH1A3)将视黄醇转化为视黄酸。RA扩散到整个视盘组织，视盘 囊泡和邻近的间质刺激视泡的折叠以形成视杯 E10.5。在E12.5-E14.5，视杯及其周围的进一步形态发生需要RA 视神经周围间充质；RA缺失导致小眼炎。RA通过与核RA结合发挥作用 激活或抑制KEY转录的RA反应元件受体(RARE) 基因。RA与RA受体的结合调节转录辅助调节因子的招募，如 核受体辅活化子(NCOA)和核受体辅阻遏子(NCoR)，进而 控制通用辅活化子p300和通用辅抑制子PrC2的结合。然而，a 尚未解决的主要问题是，在眼睛发育过程中，RA控制的关键基因是什么； 目前只有两个候选的直接RA靶基因(Pitx2和Foxc1)已知。作为RA的损失或收益 活动改变了数千个基因的表达(可能许多是由于转录后效应)， 目前还很难确定哪些基因是RA的直接转录靶点。在我们的预赛中 我们通过比较CHIP-SEQ和RNA-SEQ来解决这个问题 Aldh1a2-/-胚胎缺乏RA合成，从而识别RA时表达改变的基因 附近也有RA调节的H3K27ac(基因激活标记)沉积的缺失或 H3K27me3(基因抑制标记)与RES相关。这种罕见的增强剂/消音剂是 发现了已知的胚胎发育所需的基因附近，从而验证了我们的 接近。几个预测的新的直接RA靶基因的CRISPR敲除验证了它们的 发展的要求。在这里，我们计划使用这种方法来识别RA靶基因和 通过比较CHIP-seq(H3K27ac和H3K27me3)和PITX2在眼睛发育过程中的靶基因 野生型与RA缺陷型视泡和眼以及野生型与Pitx2基因敲除眼的RNA-seq。 我们还将在眼睛中识别RA调节的增强剂和消音器，以揭示其机制 RA通过该基因调控Pitx2、Foxc1或其他基因。我们的研究将提供重要的信息 关于RA和PITX2控制眼睛转录的机制，并将识别 眼睛形成过程中的基因调控网络。这一知识将有助于确定眼睛 缺陷发生，识别可能是突变靶标的新基因或增强子/沉默子 人类眼睛缺陷，并改进治疗眼睛缺陷的策略。