Epigenetic Regulation of the Hypoxic Response in the Mouse Heart

项目总结 细胞通过激活缺氧诱导因子(HIF)途径适应低氧环境。重大努力 正在进行中，以更好地了解HIF及其在心脏中调节其活动的因素，心脏是一个需要 大量的氧气才能正常运作。基因表达的中枢调节因子包括染色质因子 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 与氧气感应的关系才刚刚开始探索。Shohet实验室最近发现了一本小说 HIF活性的染色质调节因子RACK7可以有效地限制HIF的作用。尚不清楚RACK7是如何 调节HIF调节的基因表达，尽管先前的研究指出在增强子调节中起作用 通过招募特定的组蛋白去甲基酶。本提案中描述的实验研究 染色质因子在调节小鼠心脏缺氧反应中的作用。目标1将专注于 了解RACK7在心脏发育中的作用。Rack7mRNA和蛋白Will的定位 在心脏形态发生的不同阶段用RNA原位杂交和 免疫荧光。然后将使用Rack7测试Rack7在发育心脏中的必要性 在心脏早期表达的条件等位基因与Cre重组酶系的结合 发展。目的2将研究RACK7和两个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。此前的研究表明，RACK7与 这些去甲基酶在人类癌症中的作用，但尚不清楚这种相互作用是否发生在小鼠心肌细胞中。vbl.使用 IP-MS和CHIP-SEQ实验，RACK7与KDM5C和KDM5D的相互作用将 被评估。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低氧中， 基因表达的变化将使用rna-seq进行分析，可能揭示氧气中的性别差异。 感官。最后，在目标3中，我们将探讨氧感应与核层的关系。 核的完整性是染色质结构和基因表达以及核退化的调节因素 椎板与衰老和早衰症有关。使用CHIP-SEQ、LAMIN关联域(LAAD) 将在常氧和低氧心肌细胞中被定义。测试Lamins是否在调节低氧中起重要作用 在心肌细胞中的反应，RNA-seq将在正常和Lmna缺失的心肌细胞上进行 常氧或低氧。缺氧心肌细胞HIF靶基因在核周的定位 也将使用DNA FISH进行评估。这一提议的中心动机是理解染色质 调节心脏低氧反应的因素，并研究性别和年龄的差异。这 可能导致新的治疗靶点的确定，并将提供更好的了解HIF-1是如何 在心脏中起调节作用。

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.