| Summary: | <p>In this thesis, I leveraged multi-omics, computational and functional analysis to shed light on the 3D genome organization and its impact on transcriptional regulation in pancreatic ductal adenocarcinoma (PDAC) and dilated cardiomyopathy (DCM), which are two of the world's deadliest diseases. </p>
<p>I generated a compendium of high-resolution 3D-chromatin contact maps (~1.2x1010 reads) from human DCM/non-failing hearts and uncovered a DCM-specific 3D chromatin architecture. I discovered that DCM-specific H3K27ac HiChIP loops largely reside in pre-established high-order chromatin architectures and pre-accessible chromatin sites bound by reactivated HAND1. Forced overexpression of HAND1 in human iPSC-derived cardiomyocytes (hiPSC-CM) induces a distinct gain of enhancer/promoter connectivity and increases the expression of their associated genes implicated for DCM etiology, recapitulating the transcriptional signature in human DCM hearts. In line with this, electrophysiology analysis demonstrated that overexpression of HAND1 in hiPSC-CM induces defects in calcium handling. Furthermore, cardiomyocyte-specific overexpression of Hand1 resulted in cardiac hypertrophy/dilated left ventricle and cardiac dysfunction in mouse. These data provided unique insights into topological mechanisms of DCM and demonstrated that HAND1 rewires enhancer/promoter connectome and thereby drives DCM pathogenesis.</p>
<p>Recent studies demonstrated that the dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current therapeutics. I performed a comprehensive 3D-epigenomic analyses in A13A_Org (primary) and A13D_Org (metastatic) PDAC CSC spheres from the same patient using integrative approaches: HiChIP /ChIA-PET for enhancer connectome/chromatin topology, ChIP-seq for annotation of regulatory elements, ATAC-seq for chromatin accessibility and RNA-seq for transcriptional output to reveal potential topological mechanisms that regulate metastasis-specific transcription in PDAC. By comparing A13D_Org to A13A_Org, I found that H3K27ac-associated enhancer connectome significantly contributes to A13D_Org-specific gene transcription, especially to those prognostic molecular markers in PDAC (e.g. NRP1) and cancer stem cell markers (e.g. BMI1). In addition, we demonstrated that chromatin accessibility is associated with the formation of A13D_Org-specific H3K27ac HiChIP loop anchors. Moreover, by transcription factor motif scan analysis, we showed that FOSL1 is enriched on A13D_Org-specific H3K27ac HiChIP loop anchors, the expression of which is upregulated in A13D_Org and associated with poor prognosis in PDAC patients. To further dissect the topological mechanism of enhancer-promoter interactions, we performed CTCF ChIA-PET analysis and uncovered that CTCF binding on the promoters but not alteration of topological associated domains (TADs) is linked to a specific enhancer-promoter connectome. In sum, the comprehensive 3D-epigenomic analyses suggest integrated epigenomic and topological mechanisms that regulate PDAC-specific transcription, and provide novel insights into the interplay of epigenetics, and 3D chromatin architecture in PDAC progression. </p>
<p>By performing small-molecule compound screening (142 epigenetic enzymes), my colleague and I uncovered that BRD9 is a key chromatin regulator to orchestrate the self-renewal/stemness of pancreatic CSCs. Perturbation of BRD9 (chemical inhibitor I-BRD9 or genetic ablation) can block the self-renewal and spheroid formation of pancreatic CSCs. Single-cell RNA-seq further revealed that perturbation of BRD9, particularly combining with gemcitabine, dramatically reduces the CSC population within patient tumor cells. In line with this, perturbation of BRD9 combined with gemcitabine, dramatically ameliorates tumorigenesis in mice. Mechanistically, Inhibition of BRD9 reprograms the transcriptional landscape related to stemness and TGF-beta pathway via disruption of enhancer-promoter connectome. Collectively, this study identified the important roles of BRD9 in orchestrating PDAC stemness and tumorigenesis, highlighting BRD9 perturbation as a potential avenue for PDAC treatment.</p>
<p>In summary, I provided reliable and extensive 3D-epigenomic data to interrogate the underlying 3D-chromatin mechanisms that control gene transcription in two human lethal diseases (i.e. DCM and PDAC). These datasets represent a significant advance in our understanding of the etiology of human diseases from a new perspective (chromatin architecture). In addition, the disease-relevant chromatin loops and their regulators (e.g. HAND1, BRD9) identified in these studies could be developed as novel biomarkers for disease diagnosis and be targeted for disease intervention. </p>
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