Epigenetic Regulation of the Hypoxic Response in the Mouse Heart

小鼠心脏缺氧反应的表观遗传调控

基本信息

批准号：
10460449
负责人：
Andrew Kekupa'a Knutson
金额：
$ 10万
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：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10460449
关键词：
Adenoviruses Affect Age Aging Alleles Architecture Cardiac Cardiac Myocytes Cardiac development Cardiovascular Diseases Cell Nucleus Cell Survival Cell physiology Cells ChIP-seq Chromatin DNA Deterioration Development Disease Progression Embryo Embryonic Heart Enhancers Enterobacteria phage P1 Cre recombinase Environment Enzymes Epigenetic Process Exposure to Female Fibrinogen Gene Expression Genes Genetic Genetic Transcription Heart Heart failure Higher Order Chromatin Structure Homeostasis Human Hypoxia Hypoxia Inducible Factor Hypoxia-Inducible Factor Pathway Immunofluorescence Immunologic In Situ Hybridization Knockout Mice Lamin Type A Lamins Lead Link Location Malignant Neoplasms Mass Spectrum Analysis Measures Mesoderm Messenger RNA Morphogenesis Morphology Motivation Mus Nuclear Nuclear Inner Membrane Nuclear Lamin Nuclear Lamina Organ Oxygen Pathology Pattern Phase Phenotype Predictive Factor Progeria Proteins RNA Regenerative capacity Regulation Research Role Sex Chromosomes Sex Differences Syndrome Techniques Testing Western Blotting X Chromosome Y Chromosome age related biological adaptation to stress cardiogenesis career career development chromatin remodeling conditional knockout epigenetic regulation experimental study heart cell heart function histone demethylase hypoxia inducible factor 1 insight male mouse genetics new therapeutic target next generation sequencing normoxia novel recruit response sex sexual dimorphism tool transcriptome transcriptome sequencing

项目摘要

PROJECT SUMMARY Cells adapt to low oxygen conditions by activating the Hypoxia Inducible Factor (HIF) pathway. Significant efforts are underway to better understand HIFs and the factors that regulate their activity in the heart, an organ requiring large amounts of oxygen to function properly. Central modulators of gene expression include chromatin factors and epigenetic mechanisms. However, the role of these factors and mechanisms in the heart and their relationship to oxygen sensing is only starting to be explored. The Shohet Lab recently identified a novel chromatin regulator of HIF activity called RACK7 that potently limits HIF action. It is unknown how RACK7 modulates HIF-regulated gene expression although previous studies point to a role in enhancer regulation through recruitment of specific histone demethylases. The experiments described in this proposal investigate the role of chromatin factors in regulating the hypoxic response in the mouse heart. Aim 1 will focus on understanding the role of RACK7 during heart development. The localization of Rack7 mRNA and protein will be assessed at different stages of heart morphogenesis using RNA in situ hybridization and immunofluorescence. The necessity of Rack7 in the developing heart will then be tested using a Rack7 conditional allele in combination with Cre recombinase lines that are expressed during early stages of heart development. Aim 2 will investigate the potential interaction between RACK7 and two histone demethylases encoded on the sex chromosomes, KDM5C and KDM5D. Previous studies have shown RACK7 interacts with these demethylases in human cancer, but it is unknown if this interaction occurs in mouse cardiomyocytes. Using IP-mass spectrometry and ChIP-seq experiments, the interaction between RACK7 and KDM5C and KDM5D will be assessed. In the second part of aim 2, conditional knockout lines of Kdm5c and Kdm5d will be created to delete these genes in cardiomyocytes. These cardiomyocytes will be exposed to normoxia or hypoxia and the changes in gene expression will be analyzed using RNA-seq, potentially revealing sex-differences in oxygen sensing. Finally, in aim 3, the relationship between oxygen sensing and the nuclear lamina will be explored. Nuclear integrity is a regulator of chromatin architecture and gene expression and deterioration of the nuclear lamina is associated with aging and progeria syndromes. Using ChIP-seq, Lamin Associated Domains (LADs) will be defined in normoxic and hypoxic cardiomyocytes. To test if lamins are important in regulating the hypoxic response in cardiomyocytes, RNA-seq will be performed on normal and Lmna-null cardiomyocytes exposed to normoxia or hypoxia. The localization of HIF-target genes to the nuclear periphery in hypoxic cardiomyocytes will also be assessed using DNA FISH. The central motivation of this proposal is to understand the chromatin factors that regulate the hypoxic response in the heart and investigate sex and age-dependent differences. This may lead to the identification of new therapeutic targets and will provide a better understanding of how HIF-1 is regulated in the heart.

项目概要细胞通过激活缺氧诱导因子 (HIF) 途径来适应低氧条件。重大努力正在进行中以更好地了解 HIF 以及调节其在心脏中活动的因素，心脏是一个需要大量的氧气才能正常运作。基因表达的中央调节剂包括染色质因子和表观遗传机制。然而，这些因素和机制在心脏中的作用及其作用与氧传感的关系才刚刚开始被探索。 Shohet 实验室最近发现了一本小说 HIF 活性的染色质调节因子称为 RACK7，可有效限制 HIF 的作用。尚不清楚RACK7如何调节 HIF 调节的基因表达，尽管之前的研究指出其在增强子调节中的作用通过招募特定的组蛋白去甲基化酶。本提案中描述的实验调查了染色质因子在调节小鼠心脏缺氧反应中的作用。目标 1 将重点关注了解 RACK7 在心脏发育过程中的作用。 Rack7 mRNA 和蛋白的定位将使用 RNA 原位杂交在心脏形态发生的不同阶段进行评估免疫荧光。然后将使用 Rack7 测试 Rack7 在发育中心脏的必要性条件等位基因与在心脏早期阶段表达的 Cre 重组酶系相结合发展。目标 2 将研究 RACK7 和两种组蛋白去甲基酶之间的潜在相互作用编码在性染色体 KDM5C 和 KDM5D 上。先前的研究表明 RACK7 与这些去甲基化酶在人类癌症中发挥作用，但尚不清楚这种相互作用是否发生在小鼠心肌细胞中。使用 IP-质谱和 ChIP-seq 实验，RACK7 与 KDM5C 和 KDM5D 之间的相互作用将进行评估。在目标 2 的第二部分中，将创建 Kdm5c 和 Kdm5d 的条件淘汰线，以删除心肌细胞中的这些基因。这些心肌细胞将暴露在常氧或缺氧的环境中，将使用RNA-seq分析基因表达的变化，可能揭示氧气的性别差异传感。最后，在目标 3 中，将探讨氧传感与核层之间的关系。核完整性是染色质结构和基因表达以及核退化的调节剂椎板与衰老和早衰综合症有关。使用 ChIP-seq、核纤层蛋白相关域 (LAD) 将在常氧和缺氧心肌细胞中定义。测试核纤层蛋白对于调节缺氧是否重要心肌细胞反应，RNA-seq 将在正常和 Lmna-null 心肌细胞上进行含氧量正常或缺氧。 HIF 靶基因在缺氧心肌细胞核周边的定位还将使用 DNA FISH 进行评估。该提案的核心动机是了解染色质调节心脏缺氧反应的因素并研究性别和年龄依赖性差异。这可能会导致新的治疗靶点的确定，并有助于更好地了解 HIF-1 的作用机制调节在心里。