Histone variant H3.3-specific readout of H3K36 trimethylation by ZMYND11 links transcription elongation control to tumour suppression. Homo sapiens

Recognition of modified histones by “reader” proteins plays a critical role in the regulation of transcription1. H3K36 trimethylation (H3K36me3) is deposited onto the nucleosomes in the transcribed regions following RNA polymerase II (Pol II) elongation. In yeast, this mark in turn recruits epigenetic regulators to reset the chromatin at an appropriate state to suppress cryptic transcription2,3. However, much less is known about the role of H3K36me3 in transcription regulation in mammals. This is further complicated by the transcription-coupled incorporation of the histone variant H3.3 in gene bodies4. Here we show that the candidate tumor suppressor ZMYND11 specifically recognizes H3K36me3 on H3.3 (H3.3K36me3) and regulates Pol II elongation. Structural studies reveal that in addition to the trimethyl-lysine binding by an aromatic cage within the PWWP domain, the H3.3-dependent recognition is mediated by the encapsulation of the H3.3-specific “Ser31” residue in a composite pocket formed by the tandem bromo-PWWP domains of ZMYND11. ChIP-sequencing analysis reveal a genome-wide colocalization of ZMYND11 with H3K36me3 and H3.3 in gene bodies, and its occupancy requires the pre-deposition of H3.3K36me3. Although ZMYND11 is associated with highly expressed genes, it functions as an unconventional transcription corepressor via modulating the transition of the promoter-proximal paused Pol II to elongation. ZMYND11 is critical for the repression of a transcriptional program that is essential for tumor cell growth; higher expression of ZMYND11 is observed in triple-negative breast cancer patients with better prognosis. Consistently, overexpression of ZMYND11 suppresses cancer cell growth and tumor formation in mice. Together, this study identifies ZMYND11 as an H3.3-specific reader of H3K36me3 that links the histone variant-mediated transcription elongation control to tumor suppression. Overall design: ChIP-seq analysis of ZMYND11, H3K36me3 in U2OS cells and ZMYND11 knockdown cells; ChIP-seq of H3.3 in Flag-H3.3 stable U2OS cells; RNA-seq of ZNYMD11 depleted U2OS cells.

“‘阅读器’蛋白识别修饰后的组蛋白，在转录调控中发挥关键作用¹。H3K36三甲基化（H3K36me3）会在RNA聚合酶II（Pol II）延伸后沉积于转录区域的核小体上。在酵母中，该修饰会进一步招募表观遗传调控因子，将染色质重置至适宜状态以抑制隐秘转录²,³。然而，目前对哺乳动物中H3K36me3在转录调控中的作用所知甚少，而基因体中伴随转录过程掺入的组蛋白变体H3.3更使这一问题复杂化⁴。
本研究表明，候选抑癌蛋白ZMYND11可特异性识别H3.3上的H3K36me3（即H3.3K36me3）并调控Pol II延伸。结构研究显示，除了通过PWWP结构域内的芳香笼结合三甲基赖氨酸残基外，H3.3的特异性识别还依赖于ZMYND11串联溴域-PWWP结构域形成的复合口袋对H3.3特有的‘Ser31’残基的包裹作用。
染色质免疫共沉淀测序（ChIP-seq）分析显示，ZMYND11与H3K36me3、H3.3在基因体上存在全基因组共定位，且其染色质富集依赖于H3.3K36me3的预先沉积。尽管ZMYND11与高表达基因相关，但它可通过调控启动子近端暂停的Pol II向延伸阶段的转变，发挥非常规转录共抑制因子的功能。
ZMYND11对于抑制肿瘤细胞生长必需的转录程序至关重要；三阴性乳腺癌患者中ZMYND11的高表达与更好的预后相关。与之一致的是，ZMYND11的过表达可抑制小鼠体内癌细胞生长及肿瘤形成。综上，本研究鉴定ZMYND11为H3.3特异性的H3K36me3阅读器蛋白，将组蛋白变体介导的转录延伸调控与肿瘤抑制联系起来。
整体实验设计：对U2OS细胞及ZMYND11敲低细胞中的ZMYND11、H3K36me3进行染色质免疫共沉淀测序（ChIP-seq）分析；对Flag-H3.3稳定表达的U2OS细胞中的H3.3进行ChIP-seq分析；对ZMYND11敲低的U2OS细胞进行RNA测序（RNA-seq）。