Genome-Wide CRISPR Screen Identifies an NF2-adheren junction mechanistic dependency for cardiac lineage

Cardiac differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear. Here, we performed a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells (hESCs) and identify NF2, a Moesin-Ezrin-Radixin Like (MERLIN) Tumor Suppressor, as an upstream driver of cardiomyocyte specification. Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of non-mesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anti-cardiac regulators CDX2, MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Since YAP is a known repressor of mesendoderm genes, we found that NF2 and AMOT cooperatively bind to YAP during mesendoderm formation, thereby preventing YAP activation independent on canonical MST-LATS1 kinase activity. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 Serine-518 phospho-mutants, but not NF2 FERM-domain blue-box mutants, showing that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for cardiomyocyte lineage differentiation. These results provide mechanistic insight into the essential role of NF2 for cardiomyocyte lineage specification by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.