Highlights d HP1, SUV39H1, and TRIM28 constitute heterochromatic H3K9me3 recognition complexes d These complexes contain multiple H3K9me3 reader chromodomains d Multivalent H3K9me3-chromodomain engagement triggers liquid-liquid phase separation d Histone modifications regulate chromatin compartmentalization via phase separation

Histone acetylation, including acetylated H3K14 (H3K14ac), is generally linked to gene activation. Monomethylated histone H3 lysine 4 (H3K4me1), together with other gene-activating marks, denotes active genes. In contrast to usual gene-activating can function of H3K14ac and H3K4me1, we here show that the dual histone modification mark H3K4me1-H3K14ac is recognized by ZMYND8 (also called RACK7) and can function to counteract gene expression. We identified ZMYND8 as a transcriptional corepressor of the H3K4 demethylase JARID1D. ZMYND8 antagonized the expression of metastasis-linked genes, and its knockdown increased the cellular invasiveness in vitro and in vivo. The plant homeodomain (PHD) and Bromodomain cassette in ZMYND8 mediated the combinatorial recognition of H3K4me1-H3K14ac and H3K4me0-H3K14ac by ZMYND8. These findings uncover an unexpected role for the signature H3K4me1-H3K14ac in attenuating gene expression and reveal a previously unknown metastasis-suppressive epigenetic mechanism in which ZMYND8’s PHD-Bromo cassette couples H3K4me1-H3K14ac with downregulation of metastasis-linked genes.

High-frequency point mutations of genes encoding histones have been identified recently as novel drivers in a number of tumors. Specifically, the H3K36M/I mutations were shown to be oncogenic in chondroblastomas and undifferentiated sarcomas by inhibiting H3K36 methyltransferases, including SETD2. Here we report the crystal structures of the SETD2 catalytic domain bound to H3K36M or H3K36I peptides with SAH (S-adenosylhomocysteine). In the complex structure, the catalytic domain adopts an open conformation, with the K36M/I peptide snuggly positioned in a newly formed substrate channel. Our structural and biochemical data reveal the molecular basis underying oncohistone recognition by and inhibition of SETD2.Supplemental material is available for this article.Received May 16, 2016; revised version accepted June 20, 2016. Histone post-translational modifications (PTMs) are linked to tumorigenesis, mostly via dysfunction of their regulators (e.g., readers, writers, and erasers) that are frequently mutated in tumors (Dawson and Kouzarides 2012). Recently, high-frequency mutations in genes encoding histones themselves, rather than the histone regulators, were identified in a number of cancer types. Exome sequencing studies have identified recurrent hot spot missense mutations in histone H3. Notably, these mutations are located at or adjacent to H3 lysine residues that undergo acetylation and/or methylation. For example, the H3K27M mutation was identified in the majority of pediatric diffuse intrinsic pontine gliomas (Schwartzentruber et al. 2012;Wu et al. 2012), and the H3K36M mutation was found to occur predominantly in chondroblastomas (Behjati et al. 2013) and rarely in other cancer types such as head and neck squamous cell carcinoma and colorectal cancer (Shah et al. 2014). In addition, the H3K36-neighboring G34 mutations, such as G34R/V and G34W/L, have been detected in pediatric non-brain stem gliomas (Schwartzentruber et al. 2012;Wu et al. 2012) and giant cell tumors of the bone (Behjati et al. 2013), respectively.Biochemical and cellular studies showed that H3K27M reduced global H3K27 methylation in vitro and in vivo by inhibiting the methyltransferase activity of polycombrepressive complex 2 (PRC2) (Chan et al. 2013;Lewis et al. 2013;Justin et al. 2016). Recently, we and others identified a similar "poisoning" mechanism underlying H3K36M-driven tumorigenesis involving the inactivation of H3K36 methyltransferases (Fang et al. 2016;Lu et al. 2016). H3K36 can be methylated by enzymes such as NSD1/2/3, ASH1L, and SETD2 (Wagner and Carpenter 2012). Among them, SETD2 serves as the major H3K36 methyltransferase that is able to generate the trimethylated H3K36 from the unmethylated, monomethylated, or dimethylated states in vitro and in cells (Edmunds et al. 2008;Hu et al. 2010). SETD2 plays important roles in cellular processes such as transcription elongation (Yoh et al. 2008), RNA splicing (de Almeida et al. 2011;Kim et al. 2011), and DNA damage repair (Li et al. 2013;Pai et al. 2014). However, due to the lack...