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Histone H2B Monoubiquitination Regulates Heart Development Via Epigenetic Control of Cilia Motility

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Histone H2B Monoubiquitination Regulates Heart Development Via Epigenetic Control of Cilia Motility Histone H2B monoubiquitination regulates heart development via epigenetic control of cilia motility Andrew Robsona,b,c,1,2, Svetlana Z. Makovab,1, Syndi Barishc, Samir Zaidic,3, Sameet Mehtac, Jeffrey Drozdb, Sheng Chih Jinc,4, Bruce D. Gelbd,e, Christine E. Seidmanf,g,h, Wendy K. Chungi,j, Richard P. Liftonc,k, Mustafa K. Khokhaa,b,c,5, and Martina Bruecknerb,c,5 aPediatric Genomics Discovery Program, Yale University School of Medicine, New Haven, CT 06510; bDepartment of Pediatrics, Yale University School of Medicine, New Haven, CT 06510; cDepartment of Genetics, Yale University School of Medicine, New Haven, CT 06510; dThe Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029; eDepartment of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY 10029; fDivision of Cardiovascular Medicine, Brigham and Women’s Hospital, Boston, MA 02115; gDepartment of Genetics, Harvard Medical School, Boston, MA 02115; hHoward Hughes Medical Institute, Chevy Chase, MD 20815; iDepartment of Pediatrics, Columbia University Medical Center, New York, NY 10032; jDepartment of Medicine, Columbia University Medical Center, New York, NY 10032; and kLaboratory of Human Genetics and Genomics, Rockefeller University, New York, NY 10065 Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved May 20, 2019 (received for review May 14, 2018) Genomic analyses of patients with congenital heart disease (CHD) development, the best understood role for cilia is establishing have identified significant contribution from mutations affecting cilia left–right (LR) asymmetry and determining the direction of genes and chromatin remodeling genes; however, the mechanism(s) heart looping. A highly conserved ciliated left–right organizer connecting chromatin remodeling to CHD is unknown. Histone H2B (LRO) utilizes motile and immotile cilia to generate and sense monoubiquitination (H2Bub1) is catalyzed by the RNF20 complex directional flow of extraembryonic fluid and transduce it to a consisting of RNF20, RNF40, and UBE2B. Here, we show significant calcium signal (13–16). Defects affecting cilia motility result in enrichment of loss-of-function mutations affecting H2Bub1 in CHD heterotaxy (Htx) (associated with CHD); 6.5% of patients with P = × −03 patients (enrichment 6.01, 1.67 10 ), some of whom had PCD (primary ciliary dyskinesia), a disorder defined by abnormal abnormal laterality associated with ciliary dysfunction. In Xenopus, rnf20 rnf40 cilia motility in the airway epithelia (17), also display Htx and BIOLOGY knockdown of and results in abnormal heart looping, CHD (18). defective development of left–right (LR) asymmetry, and impaired DEVELOPMENTAL cilia motility. Rnf20, Rnf40, and Ube2b affect LR patterning and cilia synergistically. Examination of global H2Bub1 level in Xenopus em- Significance bryos shows that H2Bub1 is developmentally regulated and requires Rnf20. To examine gene-specific H2Bub1, we performed ChIP-seq of Congenital heart disease affects 1% of infants, and variants mouse ciliated and nonciliated tissues and showed tissue-specific were identified affecting genes (RNF20, RNF40, and UBE2B) H2Bub1 marks significantly enriched at cilia genes including the tran- involved in a chromatin modification called histone 2B mono- scription factor Rfx3. Rnf20 knockdown results in decreased levels of ubiquitination (H2Bub1). How these variants lead to congenital rfx3 mRNA in Xenopus, and exogenous rfx3 can rescue the Rnf20 heart disease remains unknown. Here, we demonstrate how depletion phenotype. These data suggest that Rnf20 functions at the H2Bub1 pathway modulation during development leads to Rfx3 locus regulating cilia motility and cardiac situs and identify abnormal cardiac left–right patterning. In Xenopus, H2Bub1 H2Bub1 as an upstream transcriptional regulator controlling tissue- regulates cilia motility, and defects lead to abnormal inner specific expression of cilia genes. Our findings mechanistically link dynein arms, mirroring a patient with an RNF20 mutation. the two functional gene ontologies that have been implicated in Moreover, we show that H2Bub1 marks are enriched on the human CHD: chromatin remodeling and cilia function. cilia transcription factor Rfx3 in a tissue-specific manner; fur- thermore, addition of rfx3 mRNA to Rnf20-depleted embryos cilia | ubiquitination | congenital heart disease | RNF20 | histones restores cilia motility. Thus, our findings uncover a mechanism linking H2Bub1 with cilia and cardiac development. ongenital heart disease (CHD), a structural abnormality of ∼ Author contributions: A.R., S.Z.M., M.K.K., and M.B. designed research; A.R., S.Z.M., S.B., Cthe heart and/or great vessels, affects 1% of live born in- S.Z., J.D., S.C.J., M.K.K., and M.B. performed research; S.C.J., B.D.G., C.E.S., W.K.C., and fants. Despite survival of 90% of CHD patients to adulthood, M.B. contributed new reagents/analytic tools; A.R., S.Z.M., S.B., S.Z., S.M., S.C.J., R.P.L., CHD remains the leading cause of birth defect-related mortality M.K.K., and M.B. analyzed data; and A.R., S.Z.M., S.B., M.K.K., and M.B. wrote the paper. in the United States and Europe (1). Whole-exome sequencing The authors declare no conflict of interest. (WES) of CHD patients identified de novo mutations (DNMs) This article is a PNAS Direct Submission. affecting chromatin modifications (including H3K4 and H3K27 Published under the PNAS license. methylation and H2BK120 ubiquitination) contributing to 2–3% Data deposition: Final RNA-seq and processed data resulting from the ChIP-seq experi- of CHD (2–5). While the mechanism(s) by which H3K4 and ments reported in this paper have been deposited in the Gene Expression Omnibus (GEO) H3K27 methylation impact heart development is being in- database, https://www.ncbi.nlm.nih.gov/geo (accession nos. GSE132115–GSE132117, and GSM3844810–GSM3844844). vestigated (6, 7), the role of monoubiquitination of histone H2B 1 on K120 (H2Bub1) in heart development is unknown. H2Bub1 is A.R. and S.Z.M. contributed equally to this work. 2 linked to down-regulation of pluripotency factors in ES cell Present address: Sir William Dunn School of Pathology, Oxford OX3 9DU, United – Kingdom. differentiation (8 10). In mammals, H2Bub1 is catalyzed by the 3 RNF20–RNF40 complex together with the ubiquitin-conjugating Present address: Department of Medicine, Memorial Sloan Kettering Cancer Center, New SI Appendix A York, NY 10065. enzyme UBE2B (11) ( , Fig. S1 ). 4 Present address: Laboratory of Human Genetics and Genomics, Rockefeller University, In addition to mutations affecting chromatin-remodeling New York, NY 10065. genes, genomic analysis of CHD patients (3) and a genetic 5To whom correspondence may be addressed. Email: [email protected] or screen in mice (12) reveal a significant contribution from re- [email protected]. cessive mutations affecting cilia. Cilia are hair-like organelles This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. found on the surface of most vertebrate cells and serve functions 1073/pnas.1808341116/-/DCSupplemental. including signaling and extracellular fluid propulsion. In heart Published online June 24, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1808341116 PNAS | July 9, 2019 | vol. 116 | no. 28 | 14049–14054 Downloaded by guest on September 25, 2021 The structure of the cilium is tightly regulated in part by tran- of RNF20 and UBE2B (Fig. 1B) (25). The core complex inter- scription factors (TFs), such as FoxJ1, Rfx2, and Rfx3 (19–23). acts directly or indirectly with other proteins including the PAF1 Rfx3 binds to promoters of many cilia genes, including Foxj1, complex to regulate H2Bub1, and/or link H2Bub1 to other Dnah9,andDnah5 (20). Rfx2 functions in LR asymmetry devel- chromatin modifications. To evaluate the contribution to CHD opment in zebrafish (22) and Xenopus (23). Finally, FoxJ1 and of genes outside the RNF20 core complex affecting H2Bub1, we Rfx2 bind cooperatively at chromatin loops and promote gene identified 45 RNF20 core complex-interacting genes from expression in multiciliated cells (MCCs) (24). While our knowledge STRING (SI Appendix, Fig. S2A). We then used de novo ex- of the TFs that regulate ciliogenesis is emerging, the chromatin pectation analysis of the previous WES analysis of 2,645 CHD modifications that regulate these factors are mostly unexplored. proband–parent trios and 1,789 control–parent trios (unaffected Here, we demonstrate that the RNF20–RNF40 E3 ligase com- siblings of autism patients) (phs000571.v1.p1, phs000571.v2.p1, plex together with UBE2B (the RNF20 core complex) regulates and phs000571.v3.p2) (3, 26, 27) to test for enrichment in DNMs H2Bub1 during development and affects transcription of cilia genes comparing observed to expected DNMs. We identified signifi- required for LR patterning and cardiac development. Rnf20 de- cant enrichment in LoF and protein-altering and -damaging pletion in Xenopus leads to reduced expression of rfx3 at the left– (LoF + damaging missense, defined as MetaSVM-damaging) right organizer, cilia dysmotility, and abnormal LR development. DNMs affecting the RNF20-interactome genes in CHD patients H2Bub1 marks are enriched at cilia genes in mouse multiciliated but not in controls (Table 1). tissues, including at the Rfx3 locus. Together, our results identify The phenotypes of the patients with DNMs affecting H2Bub1 the RNF20 complex as a transcriptional regulator of cilia genes and encompassed the full range
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