3D Genome Reorganization and Epigenome Dynamics of Clonal Hematopoiesis

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PROJECT SUMMARY Clonal hematopoiesis (CH) commonly occurs with aging and is associated with increased mortality, a higher risk of leukemia, and cardiovascular disease. As the population of older adults expands in the US, there is an urgent unmet need to mitigate CH. Age-related CH is the abnormal expansion of hematopoietic cells that have somatic mutations, mostly in genes encoding the DNA methyltransferase DNMT3A and the demethylase TET2. These 'CH-mutations' change the hematopoietic stem cell (HSC) epigenome (i.e., DNA methylation and chromatin accessibility) at promoters and enhancers. To better understand how these changes impact gene regulation to confer a competitive advantage to a subset of HSCs and contribute to CH development, we propose a multi- omics approach that considers the effects of epigenomic changes on the physical interactions between enhancers and promoters, which are critical to activating transcription. By mapping the genome's 3D chromatin interactions, we will identify the specific enhancer-promoter pairs that control key HSC cellular functions such as self-renewal and response to inflammation. Although it is known that the epigenome organization of HSCs changes with age, it remains unclear how aging and CH-mutations jointly remodel the '3D epigenome' (3D chromatin organization and epigenome) of HSCs and contribute to CH. Our central hypothesis is that the 3D epigenome is remodeled with age and impacts transcriptional programs critical for HSC function. Thus, to identify therapeutic targets for elongating healthspan and delaying or preventing the occurrence of CH-associated disease in the aging US population, this proposal seeks to systematically uncover the 3D epigenomic configurations of cellular competition in CH and to prioritize candidate gene targets that emerge in early aging. Using mouse models of CH and human primary cells, we will test this hypothesis via bulk and single-cell multi- omics mapping (including RNA-seq, chromatin accessibility, and chromatin interactions) of normal and mutant HSCs to systematically determine their 3D epigenome remodeling during early aging. In Aim 1, we will determine whether 3D epigenome organization is linked to an aging transcriptional program in mouse and human HSCs by identifying and integrating HSC-specific 3D genomic, epigenomic, and transcriptomic signatures from young, middle-age, and old individuals of both species and validate the roles of top candidate genes in HSC aging in vivo. In Aim 2, we will define age-dependent 3D epigenome remodeling in HSCs of CH mouse models and validate the roles of top candidate genes in clonal expansion of mutant HSCs in vivo. Aging signatures that emerge at middle age will likely be the initial cause of functional changes in aged HSCs. Thus, the study will reveal primary and modifiable targets for therapeutic intervention. In Aim 3, we will determine the response of CH-associated chromatin reorganization in aged HSCs to epigenetic therapy and anti-inflammatory drugs in vivo. We anticipate that the findings from this study will accelerate the development of early interventions to preempt CH-associated disease in aging individuals.