PROJECT SUMMARY/ABSTRACT Epigenetic processes have been implicated in control of cardiac development and in the incidence and progression of disease. Heart failure in particular has been shown to involve the actions of chromatin remodeling enzymes and to proceed by temporal reorganization of histone modifications and gene expression. The scientific premise of this application is that understanding the principles of chromatin structure-function, including the roles of specific molecular targets such as the linker histone H1 family, is key to re-engineering healthy transcriptomes in the setting of disease. Based on preliminary data implicating its role in fibroblast phenotype, we will mechanistically investigate the role of linker histone H1.0 in chromatin organization, using gain- and loss-of- function approaches in cells and in vivo. We hypothesize that precise structural orientation of the genome is underpinned by cell type-specific molecular processes and that disease results from reorganization of global genome architecture, thereby enabling pathologic gene expression. To test this hypothesis, we will perform the first ever reconstruction of genome topology on distinct cell types—fibroblasts and myocytes—from the same organ. We will precisely measure differences in chromatin architecture in fibroblast nuclei from male and female mice, examining the role of sex differences in genome organization as a potential unexplored contributor to differences in gene expression and cardiovascular phenotype between the sexes. We will use dCas CLOuD9- based chromatin loop reorganization tools to definitively test the causative role of chromatin interactions in transcription and fibroblast activation. Based on preliminary data implicating a privileged role for linker histone H1.0 in cardiac fibroblasts, we will examine the molecular mechanisms whereby H1.0 controls fibroblast gene expression and locus specific chromatin accessibility. We will use gain- and loss-of-function approaches in isolated fibroblasts to examine the role of histone H1.0 to regulate nuclear condensation, fibroblast gel contraction, proliferation and myofibroblast protein expression. We will conclusively determine the in vivo role of histone H1.0 in cardiac fibroblast phenotype under basal conditions and in the setting of pressure overload, beta adrenergic stress by isoproterenol, or ischemic injury. This approach will allow us to test the role of histone H1.0 to regulate assembly of specific chromatin neighborhoods identified in our genome structure studies as well as to examine whether these neighborhoods are reorganized in a histone H1.0-dependent manner in vivo concomitant with development of disease. The proposed experiments will provide mechanistic insights into how histone H1.0 contributes to fibroblast activation and cardiac function in vivo, revealing molecular details underpinning epigenetic control of the heart’s response to injury.

PROJECT NARRATIVE How the genome is packaged within the nucleus and differentially utilized across different cells in the body is unknown. This project examines how changes in the structure of the genome during heart disease influences the progression of the symptoms. These investigations examine distinct cell types in the heart to identify new strategies to treat heart failure.