权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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）代谢产物视黄酸（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，并表达视黄醇脱氢酶-10（RDH 10）和所有三种视黄醇脱氢酶（ALDH 1A 1，ALDH 1A 2， ALDH 1A 3），将视黄醇转化为RA。RA扩散至整个视基板、视神经囊泡和邻近的间充质，以刺激视泡折叠，从而通过 E10.5。在E12.5-E14.5，视杯和周围的进一步形态发生需要RA 视周间充质; RA缺失导致小眼球。RA通过与核RA结合发挥功能 RA反应元件（RARE）上的受体，其激活或抑制关键转录因子的转录。基因. RA与RA受体的结合调节转录辅助调节因子的募集，例如核受体辅激活子（NCOA）和核受体辅阻遏子（NCOR），控制通用共激活因子p300和通用共阻遏因子PRC 2的结合。但目前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; 只有两个候选的直接RA靶基因是已知的（Pitx 2和Foxc 1）。作为RA的损失或增益活性改变了数千个基因的表达（可能许多是由于转录后效应），仍然难以鉴定作为RA的直接转录靶的基因。在我们的初步我们通过比较ChIP-seq和RNA-seq的组织来解决这个问题，缺乏RA合成的Aldh 1a 2-/-胚胎，从而鉴定RA时表达改变的基因缺失也具有附近RA调节的H3 K27 ac沉积（基因激活标记）的基因，或 H3 K27 me 3（基因阻遏标记）与RARE相关。这种RARE增强子/沉默子是确定了已知胚胎发育所需的近基因，从而验证了我们的研究结果。 approach.几个预测的新的直接RA靶基因的CRISPR敲除验证了它们的有效性。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，通过比较ChIP-seq（H3 K27 ac & H3 K27 me 3）和用于野生型与RA缺陷视泡和眼以及野生型与Pitx 2敲除眼的RNA-seq。我们还将确定眼睛中RA调节的增强子和沉默子，以揭示其机制。 RA通过其调节Pitx 2、Foxc 1或其他基因。我们的研究将提供重要的信息 RA和PITX 2控制眼睛转录的机制，并将确定眼睛形成过程中的基因调控网络。这些知识将有助于确定眼睛缺陷发生时，识别可能是突变靶点的新基因或增强子/沉默子，人类眼睛缺陷和治疗眼睛缺陷改进策略。