Genomic analyses of patients with congenital heart disease (CHD) have identified significant contribution from mutations affecting cilia genes and chromatin remodeling genes; however, the mechanism(s) connecting chromatin remodeling to CHD is unknown. Histone H2B monoubiquitination (H2Bub1) is catalyzed by the RNF20 complex consisting of RNF20, RNF40, and UBE2B. Here, we show significant enrichment of loss-of-function mutations affecting H2Bub1 in CHD patients (enrichment 6.01,P= 1.67 × 10−03), some of whom had abnormal laterality associated with ciliary dysfunction. InXenopus, knockdown ofrnf20andrnf40results in abnormal heart looping, defective development of left–right (LR) asymmetry, and impaired cilia motility. Rnf20, Rnf40, and Ube2b affect LR patterning and cilia synergistically. Examination of global H2Bub1 level inXenopusembryos shows that H2Bub1 is developmentally regulated and requires Rnf20. To examine gene-specific H2Bub1, we performed ChIP-seq of mouse ciliated and nonciliated tissues and showed tissue-specific H2Bub1 marks significantly enriched at cilia genes including the transcription factorRfx3. Rnf20 knockdown results in decreased levels ofrfx3mRNA inXenopus, and exogenousrfx3can rescue the Rnf20 depletion phenotype. These data suggest that Rnf20 functions at theRfx3locus regulating cilia motility and cardiac situs and identify H2Bub1 as an upstream transcriptional regulator controlling tissue-specific expression of cilia genes. Our findings mechanistically link the two functional gene ontologies that have been implicated in human CHD: chromatin remodeling and cilia function.

De novo variants affecting the core complex required for monoubiquitination of histone H2B (H2Bub1) are enriched in human congenital heart disease. H2Bub1 is an enigmatic chromatin modification required in stem cell differentiation, cilia function, post-natal cardiomyocyte maturation and transcriptional elongation. However, it is still unknown how H2Bub1 affects cardiogenesis (heart structure formation), which is distinct from cardiomyocyte maturation and underlies congenital heart disease. Here we show that the RNF20-core complex (RNF20-RNF40-UBE2B) is required for cardiogenesis in mouse embryos and is essential for differentiation of human iPSCs into cardiomyocytes. Mice with cardiac-specific deletion of Rnf20 are e12.5 lethal, have thinned myocardium, a deficient ventricular septum, and abnormal cardiac sarcomere organization. We analyzed H2Bub1 marks during the time course of differentiation of human iPSCs into cardiomyocytes, and demonstrated that H2Bub1 marks are erased from a majority of genes at the transition from cardiac mesoderm to cardiac progenitor cells, but are preserved on a subset of long cardiac-specific genes. Sarcomeric gene expression is dependent on normal H2Bub1 both in mice and in human iPSC-derived cardiomyocytes. Finally, we identify an accumulation of H2Bub1 near the center of tissue-specific genes in human cardiomyocytes, mouse embryonic fibroblasts, and human fetal osteoblasts associated with transcriptional elongation efficiency that is absent in UBE2B knock-out H2Bub1-deficient cardiomyocytes. In summary, normal H2Bub1 distribution is required for cardiac morphogenesis and cardiomyocyte differentiation, and we propose that H2Bub1 regulates tissue-specific gene expression by increasing the efficiency of transcriptional elongation.

De-novo mutations affecting Histone2B-K120 monoubiquitination (H2Bub1) are enriched in congenital heart disease; however, how H2Bub1 affects heart development beyond its function in establishing left-right asymmetry remains unknown. Here we show that the RNF20-core complex (RNF20-RNF40-UBE2B), which catalyzes H2Bub1, is required for cardiac development in-vivo in mice and in-vitro in iPSC-derived cardiomyocytes. Mice with cardiac-specific deletion of Rnf20 have abnormal myocardium and ventricular septal defects; iPSCs with mutations affecting H2Bub1 cannot efficiently differentiate into cardiomyocytes. Sarcomeric gene expression is dependent on normal H2Bub1 both in mice and in iPSC-derived cardiomyocytes. Finally, we identify an accumulation of H2Bub1 near the center of tissue-specific genes in cardiomyocytes and MEFs associated with transcriptional efficiency that is reduced in UBE2B-/- cardiomyocytes. In summary, normal H2Bub1 distribution is required in cardiac development, and H2Bub1 accumulation acts as a general mechanism for tissue-specific regulation of transcriptional elongation efficiency.