Chang Jie Mick Lee, Ph.D

The Human Genome Project marked a major milestone in the scientific community as it unravelled the  3 billion bases that are central to crucial aspects of human life. Despite this achievement, it only scratched the surface of understanding how each nucleotide matters, both individually and as part of a larger unit. Beyond the coding genome, which comprises only  2% of the whole genome, scientists have realized that large portions of the genome, not known to code for any protein, were crucial for regulating the coding genes. These large portions of the genome comprise the ’non-coding genome’. The history of gene regulation mediated by proteins that bind to the regulatory non-coding genome dates back many decades to the 1960s. However, the original definition of ’enhancers’ was first used in the early 1980s. In this Review, we summarize benchmark studies that have mapped the role of cardiac enhancers in disease and development. We highlight instances in which enhancer-localized genetic variants explain the missing link to cardiac pathogenesis. Finally, we inspire readers to consider the next phase of exploring enhancer-based gene therapy for cardiovascular disease.

The 3D chromatin architecture establishes a complex network of genes and regulatory elements necessary for transcriptomic regulation in development and disease. This network can be modeled by cis-regulatory hubs (CRH) which underscore the local functional interactions between enhancers and promoter regions and differ from other higher order chromatin structures such as topological associated domains (TAD). The Activity-by-contact (ABC) model of enhancer-promoter regulation has been recently used in the identification of these CRHs, but little is known about the role of CTCF on the ABC scores and the consequent impact on CRHs. Here we show that the loss of CTCF leads to a reorganization of the enhancer-promoter interactions resulting in a re-distribution of ABC scores of the putative enhancers in C57/bl6 mouse hearts. The loss of CTCF also leads to a global reduction of the total number of CRHs, and an increase in the size of the CRHs due to increase in the number of elements within each hub. In addition, CTCF loss led to more CRHs that cross TAD boundaries. These results provide another layer of evidence to support the importance of CTCF in the formation of regulatory networks necessary for gene regulation.Competing Interest StatementThe authors have declared no competing interest.ChIP-seqChromatin immunoprecipitation with sequencingATAC-seqAssay for Transposase Accessible Chromatin with sequencingHi-CHigh throughput chromatin conformation capture with sequencingABCActivity by contact algorithm

BACKGROUND: Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear. METHODS: We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 (MYH6)-GFP (green fluorescence protein) reporter system. After an independent secondary sgRNA validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal NF2 knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays. RESULTS: Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation. CONCLUSIONS: These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.

Over the past few decades, the rising burden of metabolic disease, including type 2 diabetes, prediabetes, obesity, and metabolic dysfunction-associated steatotic liver disease, has corresponded with fundamental shifts in the landscape of heart failure (HF) epidemiology, including the rising prevalence of HF with preserved ejection fraction. It has become increasingly important to understand the role of extracardiac contributors and interorgan communication in the pathophysiology and phenotypic heterogeneity of HF. Whereas traditional epidemiological strategies have separately examined individual contributions of specific comorbidities to HF risk, these approaches may not capture the shared mechanisms and more complex, bidirectional relationships between cardiac and noncardiac comorbidities. In this review, we highlight the cardiac, kidney, liver, and metabolism multiorgan interactions and pathways that complicate HF development and progression and propose research strategies to further understand HF in the context of multiple organ disease. This includes evolving epidemiological approaches such as multiomics and machine learning which may better capture common underlying mechanisms and interorgan crosstalk. We review existing preclinical models of HF and how they have enhanced our understanding of the role of multiorgan disease in the development of HF subtypes. We suggest recommendations as to how clinical practice across multiple specialties should screen for and manage multiorgan involvement in HF. Finally, recognizing the advent of novel combinatorial therapeutic agents that may have multiple indications across the cardiac-kidney-liver metabolism continuum, we review the current clinical trials landscape. We specifically highlight a pressing need for the design of more inclusive trials that examine the contributions of multimorbidity and incorporate multiorgan end points, which we propose may lead to outcomes that are evermore clinically relevant today.