Genome-Wide CRISPR Screen Identifies an NF2-Adherens Junction Mechanistic Dependency for Cardiac Lineage.

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.

Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells.

Selection of the target site is an inherent question for any project aiming for directed transgene integration. Genomic safe harbour (GSH) loci have been proposed as safe sites in the human genome for transgene integration. Although several sites have been characterised for transgene integration in the literature, most of these do not meet criteria set out for a GSH and the limited set that do have not been characterised extensively. Here, we conducted a computational analysis using publicly available data to identify 25 unique putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression and minimal disruption of the native transcriptome in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.

Systematic in vivo candidate evaluation uncovers therapeutic targets for LMNA dilated cardiomyopathy and risk of Lamin A toxicity.

BACKGROUND: Dilated cardiomyopathy (DCM) is a severe, non-ischemic heart disease which ultimately results in heart failure (HF). Decades of research on DCM have revealed diverse aetiologies. Among them, familial DCM is the major form of DCM, with pathogenic variants in LMNA being the second most common form of autosomal dominant DCM. LMNA DCM is a multifactorial and complex disease with no specific treatment thus far. Many studies have demonstrated that perturbing candidates related to various dysregulated pathways ameliorate LMNA DCM. However, it is unknown whether these candidates could serve as potential therapeutic targets especially in long term efficacy. METHODS: We evaluated 14 potential candidates including Lmna gene products (Lamin A and Lamin C), key signaling pathways (Tgfß/Smad, mTor and Fgf/Mapk), calcium handling, proliferation regulators and modifiers of LINC complex function in a cardiac specific Lmna DCM model. Positive candidates for improved cardiac function were further assessed by survival analysis. Suppressive roles and mechanisms of these candidates in ameliorating Lmna DCM were dissected by comparing marker gene expression, Tgfß signaling pathway activation, fibrosis, inflammation, proliferation and DNA damage. Furthermore, transcriptome profiling compared the differences between Lamin A and Lamin C treatment. RESULTS: Cardiac function was restored by several positive candidates (Smad3, Yy1, Bmp7, Ctgf, aYAP1, Sun1, Lamin A, and Lamin C), which significantly correlated with suppression of HF/fibrosis marker expression and cardiac fibrosis in Lmna DCM. Lamin C or Sun1 shRNA administration achieved consistent, prolonged survival which highly correlated with reduced heart inflammation and DNA damage. Importantly, Lamin A treatment improved but could not reproduce long term survival, and Lamin A administration to healthy hearts itself induced DCM. Mechanistically, we identified this lapse as caused by a dose-dependent toxicity of Lamin A, which was independent from its maturation. CONCLUSIONS: In vivo candidate evaluation revealed that supplementation of Lamin C or knockdown of Sun1 significantly suppressed Lmna DCM and achieve prolonged survival. Conversely, Lamin A supplementation did not rescue long term survival and may impart detrimental cardiotoxicity risk. This study highlights a potential of advancing Lamin C and Sun1 as therapeutic targets for the treatment of LMNA DCM.

BNIP-2 Activation of Cellular Contractility Inactivates YAP for H9c2 Cardiomyoblast Differentiation.

Rho GTPases and Hippo kinases are key regulators of cardiomyoblast differentiation. However, how these signaling axes are coordinated spatiotemporally remains unclear. Here, the central and multifaceted roles of the BCH domain containing protein, BNIP-2, in orchestrating the expression of two key cardiac genes (cardiac troponin T [cTnT] and cardiac myosin light chain [Myl2]) in H9c2 and human embryonic stem cell-derived cardiomyocytes are delineated. This study shows that BNIP-2 mRNA and protein expression increase with the onset of cTnT and Myl2 and promote the alignment of H9c2 cardiomyocytes. Mechanistically, BNIP-2 is required for the inactivation of YAP through YAP phosphorylation and its cytosolic retention. Turbo-ID proximity labeling corroborated by super-resolution analyses and biochemical pulldown data reveals a scaffolding role of BNIP-2 for LATS1 to phosphorylate and inactivate YAP in a process that requires BNIP-2 activation of cellular contractility. The findings identify BNIP-2 as a pivotal signaling scaffold that spatiotemporally integrates RhoA/Myosin II and LATS1/YAP mechanotransduction signaling to drive cardiomyoblast differentiation, by switching the genetic programming from YAP-dependent growth to YAP-silenced differentiation. These findings offer insights into the importance of scaffolding proteins in bridging the gap between mechanical and biochemical signals in cell growth and differentiation and the prospects in translational applications.

Epigenomes of Human Hearts Reveal New Genetic Variants Relevant for Cardiac Disease and Phenotype.

RATIONALE: Identifying genetic markers for heterogeneous complex diseases such as heart failure is challenging and requires prohibitively large cohort sizes in genome-wide association studies to meet the stringent threshold of genome-wide statistical significance. On the other hand, chromatin quantitative trait loci, elucidated by direct epigenetic profiling of specific human tissues, may contribute toward prioritizing subthreshold variants for disease association. OBJECTIVE: Here, we captured noncoding genetic variants by performing epigenetic profiling for enhancer H3K27ac chromatin immunoprecipitation followed by sequencing in 70 human control and end-stage failing hearts. METHODS AND RESULTS: We have mapped a comprehensive catalog of 47 321 putative human heart enhancers and promoters. Three thousand eight hundred ninety-seven differential acetylation peaks (FDR [false discovery rate], 5%) pointed to pathways altered in heart failure. To identify cardiac histone acetylation quantitative trait loci (haQTLs), we regressed out confounding factors including heart failure disease status and used the G-SCI (Genotype-independent Signal Correlation and Imbalance) test1 to call out 1680 haQTLs (FDR, 10%). RNA sequencing performed on the same heart samples proved a subset of haQTLs to have significant association also to gene expression (expression quantitative trait loci), either in cis (180) or through long-range interactions (81), identified by Hi-C (high-throughput chromatin conformation assay) and HiChIP (high-throughput protein centric chromatin) performed on a subset of hearts. Furthermore, a concordant relationship between the gain or disruption of TF (transcription factor)-binding motifs, inferred from alternative alleles at the haQTLs, implied a surprising direct association between these specific TF and local histone acetylation in human hearts. Finally, 62 unique loci were identified by colocalization of haQTLs with the subthreshold loci of heart-related genome-wide association studies datasets. CONCLUSIONS: Disease and phenotype association for 62 unique loci are now implicated. These loci may indeed mediate their effect through modification of enhancer H3K27 acetylation enrichment and their corresponding gene expression differences (bioRxiv: https://doi.org/10.1101/536763). Graphical Abstract: A graphical abstract is available for this article.

Robust CTCF-Based Chromatin Architecture Underpins Epigenetic Changes in the Heart Failure Stress-Gene Response.

BACKGROUND: The human genome folds in 3 dimensions to form thousands of chromatin loops inside the nucleus, encasing genes and cis-regulatory elements for accurate gene expression control. Physical tethers of loops are anchored by the DNA-binding protein CTCF and the cohesin ring complex. Because heart failure is characterized by hallmark gene expression changes, it was recently reported that substantial CTCF-related chromatin reorganization underpins the myocardial stress-gene response, paralleled by chromatin domain boundary changes observed in CTCF knockout. METHODS: We undertook an independent and orthogonal analysis of chromatin organization with mouse pressure-overload model of myocardial stress (transverse aortic constriction) and cardiomyocyte-specific knockout of Ctcf. We also downloaded published data sets of similar cardiac mouse models and subjected them to independent reanalysis. RESULTS: We found that the cardiomyocyte chromatin architecture remains broadly stable in transverse aortic constriction hearts, whereas Ctcf knockout resulted in ≈99% abolition of global chromatin loops. Disease gene expression changes correlated instead with differential histone H3K27-acetylation enrichment at their respective proximal and distal interacting genomic enhancers confined within these static chromatin structures. Moreover, coregulated genes were mapped out as interconnected gene sets on the basis of their multigene 3D interactions. CONCLUSIONS: This work reveals a more stable genome-wide chromatin framework than previously described. Myocardial stress-gene transcription responds instead through H3K27-acetylation enhancer enrichment dynamics and gene networks of coregulation. Robust and intact CTCF looping is required for the induction of a rapid and accurate stress response.