Regulation of the hypoxic response by the RACK7 chromatin factor in the mouse heart

RACK7染色质因子对小鼠心脏缺氧反应的调节

基本信息

批准号：
10023171
负责人：
Andrew Kekupa'a Knutson
金额：
$ 6.93万
依托单位：
UNIVERSITY OF HAWAII AT MANOA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10023171
关键词：
ATAC-seq Adult Anterior Area Arteries Attention B-Lymphocytes Binding Binding Proteins Biochemical Biological Breast Cancer Cell Cardiac Cardiac Myocytes Cardiovascular Diseases Cell Survival Cells ChIP-seq Chromatin Coronary Data Development Disease Disease Progression Enhancers Enterobacteria phage P1 Cre recombinase Event Excision Exposure to Feedback Foundations Future Gene Expression Genes Genetic Enhancer Element Glycolysis Heart Histones Homeostasis Human Hypoxia Hypoxia Inducible Factor Hypoxia-Inducible Factor Pathway Infarction Inherited Ischemia Lead Left Ligation LoxP-flanked allele Malignant Neoplasms Measures Messenger RNA Molecular Molecular Analysis Mus Myocardial Infarction Myocardial Ischemia Neonatal Oxygen Pathway interactions Phenotype Protein Kinase C Proteins RNA Regulation Resources Response Elements Role Signal Transduction Technology Testing Therapeutic Tissues Transgenic Mice Triage Up-Regulation Vascularization Ventricular Function Work angiogenesis attenuation biological adaptation to stress chromatin immunoprecipitation chromatin modification chromatin remodeling conditional knockout deprivation experimental study genome-wide heart dimension/size heart function histone demethylase histone modification hypoxia inducible factor 1 inhibitor/antagonist insight malignant breast neoplasm myocardial hypoxia new therapeutic target novel overexpression programs promoter prostate cancer cell line protective effect recruit response single molecule stress tolerance targeted treatment therapeutic target therapy development transcription factor transcriptome transcriptome sequencing

项目摘要

PROJECT SUMMARY Oxygen deprivation in the heart induces the expression of hundreds of genes involved in angiogenesis, glycolysis, and cell survival. At the core of this response are the Hypoxia Inducible Factor (HIF) transcription factors with the most potent HIF factor being HIF-1. Significant efforts are underway to develop therapies that target the HIF pathway in hopes of treating various conditions including cardiovascular disease. To understand the molecular activities of active HIF-1 in the mouse heart, the Shohet Lab created a transgenic mouse expressing the constitutively active form of human HIF-1a. As expected, majority of mice showed increased heart size and vascularization due to activation of HIF-1-target genes. A fraction of transgenic mice, however, did not present these phenotypes, suggesting a potential inhibitory mechanism. Chromatin immunoprecipitation analysis of “unresponsive” hearts revealed significant enrichment of HIF-1a at the promoter of the Rack7 gene and upregulation of RACK7. RACK7 is a chromatin factor shown to recruit specific histone demethylases to loci, including cis-regulatory enhancer elements, and cause gene expression changes in human breast and prostate cancer cell lines and mouse B cells. The role of RACK7 in the heart is unknown and its relationship to the hypoxia machinery is only starting to be revealed. The experiments described in this proposal investigate the role of RACK7 and its ability to inhibit the HIF-1-dependent hypoxic response through chromatin remodeling. Aim 1 will test the sufficiency of RACK7 to inhibit HIF-1 activity in cultured neonatal cardiomyocytes. RACK7 will be overexpressed using a lentiviral strategy, cells will be exposed to normoxic or hypoxic conditions, and RNAs will be profiled using RNA-seq. To investigate possible inhibitory mechanisms used by RACK7, genome-wide HIF- 1a occupancy will be probed using ChIP-seq. Enhancer activity will also be profiled using ATAC-seq and ChIP- seq for the histone modifications H3K27ac and H3K4me1. Aim 2 will investigate the effect of removing Rack7 from the adult, ischemic heart. Rack7 will be deleted in the heart using an established floxed-allele combined with a cardiac-specific CRE recombinase. Myocardial infarctions will be induced in Rack7cKO and control adult mice by ligating the left anterior descending artery and infarct size and heart function will be measured. The localization of mRNAs transcribed from HIF-1-target genes will also be assessed in infarcted Rack7cKO and control hearts using single molecule FISH. While the biochemical mechanisms controlling HIF-1 activity are known, its regulation at the chromatin level is less understood. The data resulting from successful completion of these experiments will reveal unexplored molecular and cellular events that occur in the hypoxic mammalian heart and will provide insight into chromatin factors that may be involved in cardiovascular disease progression. Evidence of RACK7 as a potent inhibitor of HIF action may lead to the development of new therapies that target RACK7 and will provide a better understanding of how HIF-1 activity is regulated in the heart.

项目概要心脏缺氧会诱导数百种参与血管生成的基因表达，该反应的核心是缺氧诱导因子 (HIF) 转录。最有效的 HIF 因子是 HIF-1，目前正在大力开发治疗方法。以 HIF 通路为目标，希望能够治疗包括心血管疾病在内的各种疾病。为了研究小鼠心脏中活性 HIF-1 的分子活性，Shohet 实验室创建了转基因小鼠正如预期的那样，大多数小鼠表现出人 HIF-1a 的组成型活性形式的表达增加。然而，由于 HIF-1 靶基因的激活，一小部分转基因小鼠的心脏大小和血管形成发生了变化。没有呈现这些表型，表明存在潜在的染色质免疫沉淀机制。对“无反应”心脏的分析揭示了 Rack7 基因启动子处 HIF-1a 的显着富集 RACK7 的上调是一种染色质因子，可招募特定的组蛋白去甲基酶至位点，包括顺式调节增强子元件，并引起人类乳腺和前列腺的基因表达变化癌细胞系和小鼠 B 细胞 RACK7 在心脏中的作用及其与缺氧的关系尚不清楚。该提案中描述的实验才刚刚开始揭示其作用。 RACK7 及其通过染色质重塑抑制 HIF-1 低依赖性反应的能力将达到目的 1。测试 RACK7 是否足以抑制培养的新生儿心肌细胞中的 HIF-1 活性。使用慢病毒策略过度表达，细胞将暴露于常氧或缺氧条件下，RNA将使用 RNA-seq 进行分析以研究 RACK7（全基因组 HIF-）使用的可能抑制机制。 1a 占用率将使用 ChIP-seq 进行探测，也将使用 ATAC-seq 和 ChIP- 进行分析。组蛋白修饰 H3K27ac 和 H3K4me1 的 seq 目标 2 将研究删除 Rack7 的效果。使用已建立的 floxed 等位基因组合，将在心脏中删除来自成人缺血心脏的 Rack7。心脏特异性 CRE 重组酶将在 Rack7cKO 和对照成人中诱导心肌梗塞。通过结扎小鼠左前降支，测量梗塞面积和心脏功能。从 HIF-1 靶基因转录的 mRNA 的定位也将在梗死的 Rack7cKO 和使用单分子 FISH 控制心脏，而控制 HIF-1 活性的生化机制是众所周知，其在染色质水平上的调节知之甚少。这些实验将揭示缺氧哺乳动物中发生的未探索的分子和细胞事件心脏并将提供对可能参与心血管疾病进展的染色质因素的深入了解。 RACK7 作为 HIF 作用的有效抑制剂的证据可能会导致针对 HIF 的新疗法的开发 RACK7 将使人们更好地了解 HIF-1 活性如何在心脏中受到调节。