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

基本信息

批准号：
10629421
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
项目类别：
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/10629421
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 Diffusion 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 LoxP-flanked allele Mesenchyme Methods Microphthalmos Morphogenesis Morphology Mus Mutation NCOR1 gene Neural Crest Nuclear Nuclear Receptors Optic vesicle Optics Repression Retinoic Acid Binding Retinoic Acid Receptor Retinoic Acid Response Element Retinol dehydrogenase Signal Pathway Testing Tissues Transcriptional Regulation Transgenes Tretinoin Untranslated RNA Vitamin A candidate identification conditional knockout eye formation gene function gene regulatory network gene repression genetic corepressor improved in vivo insight interest mutant novel strategies optic cup posttranscriptional recruit retinaldehyde dehydrogenase transcriptome sequencing

项目摘要

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.

项目摘要维生素A（视黄醇）代谢物视网膜酸（RA）的最关键功能之一是控制眼睛发育。在小鼠中，RA合成发生在视野的早期，视黄醇的表达脱氢酶10（RDH10）和所有三种视网膜脱氢酶（aldh1a1，aldh1a2， Aldh1a3）将视黄醇转换为RA。 RA在整个视线座上的组织中扩散到组织囊泡和相邻的间质刺激视力囊泡的折叠，以形成视杯。 E10.5。在E12.5-E14.5处，需要RA才能进一步形态发生视觉杯和周围毛骨间质； RA的丧失导致微观恐惧症。 RA通过与核RA结合而起作用 RA响应元件（RARES）的受体激活或抑制钥匙的转录基因。 RA与RA受体的结合调节转录核心调节剂的募集，例如核受体共激活因子（NCOA）和核受体Corepressor（NCOR），又通用共激活器p300和通用核心prc2的控制结合。但是，主要未解决的问题是RA在眼睛发育过程中控制的关键基因是什么？仅知道两个候选直接RA靶基因（PITX2和FOXC1）。作为RA的损失或收益活性改变了数千个基因的表达（也许是由于转录后效应引起的），确定是RA的直接转录靶标的基因仍然很难。在我们的初步中我们的研究通过比较了来自 Aldh1a2 - / - 缺乏RA合成的胚胎，因此鉴定出RA时表达改变的基因缺少附近的H3K27AC（基因激活标记）的RA调节沉积或 H3K27me3（基因抑制标记）与稀有物相关。如此罕见的增强剂/消音器是被鉴定出已知胚胎发育所需的近基因，从而验证了我们的方法。 CRIS敲除几个预测的新直接RA目标基因验证了他们的开发要求。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）和用于野生型与RA缺陷的视力囊泡和眼睛的RNA-Seq，以及野生型与Pitx2敲除眼。我们还将在眼中确定RA调节的增强剂和消音器以发现机制 RA通过其中调节PITX2，FOXC1或其他基因。我们的研究将提供重要信息关于RA和PITX2使用的机制来控制眼睛中的转录，并将识别眼睛形成过程中的基因调节网络。这些知识将有助于确定眼睛发生缺陷，识别可能是引起突变靶标的新基因或增强子/消音器人眼缺陷并改善治疗眼缺陷的策略。