Wonho Kim's research while affiliated with University of Pennsylvania and other places

The human genome functions as a three-dimensional chromatin polymer, driven by a complex collection of chromosome interactions1–3. Although the molecular rules governing these interactions are being quickly elucidated, relatively few proteins regulating this process have been identified. Here, to address this gap, we developed high-throughput DNA or RNA labelling with optimized Oligopaints (HiDRO)—an automated imaging pipeline that enables the quantitative measurement of chromatin interactions in single cells across thousands of samples. By screening the human druggable genome, we identified more than 300 factors that influence genome folding during interphase. Among these, 43 genes were validated as either increasing or decreasing interactions between topologically associating domains. Our findings show that genetic or chemical inhibition of the ubiquitous kinase GSK3A leads to increased long-range chromatin looping interactions in a genome-wide and cohesin-dependent manner. These results demonstrate the importance of GSK3A signalling in nuclear architecture and the use of HiDRO for identifying mechanisms of spatial genome organization.

Introduction: Cardiac fibrosis is mediated by the activation of fibroblasts to myofibroblasts, a cell state transition which involves the coordination of expression of hundreds of genes. Previous studies have demonstrated that inhibition of bromodomain and extra-terminal domain (BET) proteins attenuates fibrosis. We recently discovered a novel role of bromodomain-containing protein 4 (BRD4), a BET family member, in maintaining genome folding by stabilizing the cohesin complex (necessary for genome-genome interactions). We hypothesize that BRD4-mediated genome folding is critical for maintaining the enhancer-promoter interactions at genes required for fibroblast activation. Methods: A cardiac fibroblast cell line was generated in which a degron epitope tag was appended biallelically to Brd4 to enable acute BRD4 degradation. Fibroblasts were activated via addition of TGFβ. Meox1 , a critical transcription factor mediating a broad fibrotic gene program, was used as a model locus to investigate chromatin looping. The proximity between the Meox1 enhancer and promoter, Meox1 expression, and protein occupancy at the locus were determined using DNA fluorescence in situ hybridization, RT-qPCR, and chromatin immunoprecipitation (ChIP)-qPCR, respectively. Results: The proximity between the Meox1 enhancer and promoter and Meox1 expression increased upon TGFβ-induced activation. BRD4 occupancy was enriched at the enhancer of Meox1 upon activation, and BRD4 depletion reduced Meox1 expression and the co-localization of the Meox1 enhancer and promoter. BRD4 physically interacts with the cohesin agonist NIPBL, and we found that co-depletion of BRD4 and a cohesin antagonist normalizes the Meox1 enhancer and promoter proximity and Meox1 expression levels. Ongoing studies include genome-wide occupancy studies and expanding our findings to in vivo models of cardiac fibrosis. Conclusions: Our studies provide a mechanistic understanding of the functional relevance of genome organization during fibroblast activation. These studies will also expand our knowledge on how genome folding regulates cell plasticity and inform therapeutic approaches for targeting pathologic fibrotic remodeling.

Although the molecular rules governing genome organization are being quickly elucidated, relatively few proteins regulating this process have been identified. To address this gap, we developed a fully automated imaging pipeline, called HiDRO (high-throughput DNA or RNA labeling with optimized Oligopaints), that permits quantitative measurement of chromatin interactions across a large number of samples. Using HiDRO, we screened the human druggable genome and identified >300 factors that regulate chromatin folding during interphase, including 43 validated hits that either increase or decrease interactions between topological associating domains (TADs). We discovered that genetic or chemical inhibition of the ubiquitous kinase GSK3A enhances long-range interactions by dysregulating cohesin-mediated chromatin looping. Collectively, these results highlight a noncanonical role for GSK3A signaling in nuclear architecture and underscore the broader utility of HiDRO-based screening to identify novel mechanisms that drive the spatial organization of the genome.

Higher-order chromatin structure regulates gene expression, and mutations in proteins mediating genome folding underlie developmental disorders known as cohesinopathies. However, the relationship between three-dimensional genome organization and embryonic development remains unclear. Here we define a role for bromodomain-containing protein 4 (BRD4) in genome folding, and leverage it to understand the importance of genome folding in neural crest progenitor differentiation. Brd4 deletion in neural crest results in cohesinopathy-like phenotypes. BRD4 interacts with NIPBL, a cohesin agonist, and BRD4 depletion or loss of the BRD4–NIPBL interaction reduces NIPBL occupancy, suggesting that BRD4 stabilizes NIPBL on chromatin. Chromatin interaction mapping and imaging experiments demonstrate that BRD4 depletion results in compromised genome folding and loop extrusion. Finally, mutation of individual BRD4 amino acids that mediate an interaction with NIPBL impedes neural crest differentiation into smooth muscle. Remarkably, loss of WAPL, a cohesin antagonist, rescues attenuated smooth muscle differentiation resulting from BRD4 loss. Collectively, our data reveal that BRD4 choreographs genome folding and illustrates the relevance of balancing cohesin activity for progenitor differentiation.

Background: Gene regulatory networks control tissue homeostasis and disease progression in a cell-type specific manner. Ubiquitously expressed chromatin regulators modulate these networks, yet the mechanisms governing how tissue-specificity of their function is achieved are poorly understood. BRD4, a member of the BET (Bromo- and Extra-Terminal domain) family of ubiquitously expressed acetyl-lysine reader proteins, plays a pivotal role as a coactivator of enhancer signaling across diverse tissue types in both health and disease, and has been implicated as a pharmacologic target in heart failure. However, the cell-specific role of BRD4 in adult cardiomyocytes remains unknown. Methods: We combined conditional mouse genetics, unbiased transcriptomic and epigenomic analyses, and classical molecular biology and biochemical approaches to understand the role of BRD4 in adult cardiomyocyte homeostasis. Results: Here, we show that cardiomyocyte-specific deletion of Brd4 in adult mice leads to acute deterioration of cardiac contractile function with mutant animals demonstrating a transcriptomic signature enriched for decreased expression of genes critical for mitochondrial energy production. Genome-wide occupancy data show that BRD4 enriches at many downregulated genes (including the master co-activators Ppargc1a , Ppargc1b , and their downstream targets) and preferentially co-localizes with GATA4, a lineage determining cardiac transcription factor not previously implicated in regulation of adult cardiac metabolism. BRD4 and GATA4 form an endogenous complex in cardiomyocytes and interact in a bromodomainindependent manner, revealing a new functional interaction partner for BRD4 that can direct its locus and tissue specificity. Conclusions: These results highlight a novel role for a BRD4-GATA4 module in cooperative regulation of a cardiomyocyte specific gene program governing bioenergetic homeostasis in the adult heart.

Viable cells with reduced fitness are often eliminated by neighboring cells with greater fitness. This phenomenon, called cell competition, is an important mechanism for maintaining a high-quality population of cells in tissues. Foundational studies characterizing cellular competition and its molecular underpinnings were first carried out utilizing Drosophila as a model system. More recently, competitive behavior studies have extended into mammalian cell types. In this review, we highlight recent advances in the field, focusing on new insights into the molecular mechanisms regulating competitive behavior in various cellular contexts and in cancer. Throughout the review, we highlight new avenues to expand our understanding of the molecular underpinnings of cell competition and its role in tissue development and homeostasis.

Gene regulatory networks control tissue plasticity during basal homeostasis and disease in a cell-type specific manner. Ubiquitously expressed chromatin regulators modulate these networks, yet the mechanisms governing how tissue-specificity of their function is achieved are poorly understood. BRD4, a member of the BET (Bromo- and Extra-Terminal domain) family of ubiquitously expressed acetyl-lysine reader proteins, plays a pivotal role as a coactivator of enhancer signaling across diverse tissue types in both health and disease, and has been implicated as a pharmacologic target in heart failure. However, the cell-specific role of BRD4 in adult cardiomyocytes remains unknown. Here, we show that cardiomyocyte-specific deletion of BRD4 in adult mice leads to acute deterioration of cardiac contractile function with mutant animals demonstrating a transcriptomic signature enriched for decreased expression of genes critical for mitochondrial energy production. Genome-wide occupancy data show that BRD4 enriches at many downregulated genes and preferentially co-localizes with GATA4, a lineage determining cardiac transcription factor not previously implicated in regulation of adult cardiac metabolism. Co-immunoprecipitation assays demonstrate that BRD4 and GATA4 form a complex in a bromodomain-independent manner, revealing a new interaction partner for BRD4 that has functional consequences for target transactivation and may allow for locus and tissue specificity. These results highlight a novel role for a BRD4-GATA4 module in cooperative regulation of a cardiomyocyte specific gene program governing bioenergetic homeostasis in the adult heart.