Histone H2B monoubiquitination regulates heart development via epigenetic control of cilia motility [RNA-Seq]

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 are unknown. Histone H2B mono-ubiquitination (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=1.67x10-03), some of whom had abnormal laterality associated with cilia dysfunction. In Xenopus, knockdown of rnf20 and rnf40 results in abnormal heart looping, defective development of left-right asymmetry and impaired cilia motility. Rnf20, Rnf40 and Ube2b affect LR patterning and cilia synergistically. Examination of global H2Bub1 level in Xenopus embryos shows that H2Bub1 is developmentally regulated and requires Rnf20. To examine gene-specific H2Bub1, we performed ChIP-seq of mouse ciliated and non-ciliated tissues and showed tissue-specific H2Bub1 marks significantly enriched at cilia genes including the transcription factor Rfx3.  Rnf20 knockdown results in decreased levels of rfx3 mRNA in Xenopus, and exogenous rfx3 can rescue the Rnf20 depletion phenotype. These data suggest that Rnf20 functions at the Rfx3 locus 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. Xenopus embryo mRNA profiling in wt (in duplicate) and RNF20 morpholino (in triplicate) at 12 hpf, 13 hpf, 14 hpf, 15 hpf, and 16 hpf

针对先天性心脏病（congenital heart disease, CHD）患者的基因组分析已证实，影响纤毛基因与染色质重塑基因的突变对该病存在显著贡献，但目前仍未阐明染色质重塑与CHD之间的关联机制。组蛋白H2B单泛素化（histone H2B mono-ubiquitination, H2Bub1）由包含RNF20、RNF40与UBE2B的RNF20复合物催化完成。本研究发现，CHD患者中影响H2Bub1的功能丧失型突变存在显著富集（富集倍数=6.01，p=1.67×10^-3），其中部分患者伴有与纤毛功能异常相关的侧位发育异常。在爪蟾（Xenopus）模型中，敲低rnf20与rnf40会引发心脏环化异常、左右不对称发育缺陷以及纤毛运动能力受损。Rnf20、Rnf40与Ube2b可协同调控左右模式形成与纤毛功能。对爪蟾胚胎中整体H2Bub1水平的检测显示，H2Bub1的表达受发育进程调控，且其正常水平依赖于RNF20。为检测基因特异性的H2Bub1修饰，我们对小鼠的纤毛组织与非纤毛组织开展了染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）分析，结果表明组织特异性的H2Bub1标记在包括转录因子Rfx3在内的纤毛基因上存在显著富集。在爪蟾模型中敲低Rnf20会导致rfx3的mRNA水平下调，而外源性过表达rfx3可挽救Rnf20缺失引发的表型。上述结果表明，RNF20可作用于Rfx3基因座，调控纤毛运动与心脏位置，并证实H2Bub1作为上游转录调控因子，控制纤毛基因的组织特异性表达。本研究的发现从机制层面将与人类CHD相关的两类基因本体功能类别——染色质重塑与纤毛功能——建立了直接关联。本研究采集了受精后12、13、14、15及16小时（hours post fertilization, hpf）的野生型（wild type, wt，2次生物学重复）与RNF20吗啉寡聚核苷酸处理组（3次生物学重复）的爪蟾胚胎mRNA表达谱数据。