Epigenetic Regulation of the Hypoxic Response in the Mouse Heart

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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.