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

项目摘要 心脏缺氧会诱导数百种参与血管生成的基因表达， 糖酵解和细胞存活。这种反应的核心是缺氧诱导因子（HIF）转录 最强的HIF因子是HIF-1。正在进行重大努力以开发 靶向HIF通路，希望治疗包括心血管疾病在内的各种疾病。了解 活性HIF-1在小鼠心脏中的分子活性，Shohet实验室创造了一种转基因小鼠， 表达人HIF-1 α的组成型活性形式。正如预期的那样，大多数小鼠表现出增加 心脏大小和血管化由于HIF-1-靶基因的激活。然而，一小部分转基因小鼠， 没有呈现这些表型，提示潜在的抑制机制。染色质免疫沉淀 对“无反应”心脏的分析显示，Rack 7基因启动子处的HIF-1a显著富集 和RACK 7的上调。RACK 7是一种染色质因子，显示其将特异性组蛋白脱甲基酶募集到基因座， 包括顺式调节增强子元件，并引起人乳腺和前列腺中的基因表达变化 癌细胞系和小鼠B细胞。RACK 7在心脏中的作用尚不清楚，其与缺氧的关系 机器才刚刚开始被发现。本提案中描述的实验研究了 RACK 7及其通过染色质重塑抑制HIF-1依赖性缺氧反应的能力。目标1将 测试RACK 7抑制培养的新生心肌细胞中HIF-1活性的充分性。RACK 7将是 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，而RNA将 使用RNA-seq进行分析。为了研究RACK 7可能的抑制机制，全基因组HIF-1 α基因表达下调。 将使用ChIP-seq探测1a占用。还将使用ATAC-seq和ChIP分析增强子活性。 seq用于组蛋白修饰H3 K27 ac和H3 K4 me 1。目标2将研究删除Rack 7的影响 从成人缺血心脏中取出。Rack 7将在心脏中使用已建立的等位基因组合缺失 心脏特异性CRE重组酶将在Rack 7 cKO和对照成人中诱导心肌梗死 通过结扎小鼠的左前降支动脉，测量梗死面积和心脏功能。的 从HIF-1-靶基因转录的mRNA的定位也将在梗死的Rack 7 cKO中进行评估， 使用单分子FISH控制心脏。虽然控制HIF-1活性的生化机制是 已知，其在染色质水平的调节尚不清楚。成功完成的数据 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， 心脏和将提供深入了解染色质因素，可能参与心血管疾病的进展。 RACK 7作为HIF作用的有效抑制剂的证据可能导致靶向HIF的新疗法的开发。 RACK 7，并将提供一个更好地了解如何HIF-1活性在心脏调节。

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.