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

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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.