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  • Suz is required for methyltransferase

    2022-05-17

    Suz12 is required for methyltransferase activity, silencing function, and PRC2 protein stabilization in somatic and human ESCs (Cao and Zhang, 2004, Pasini et al., 2004, Collinson et al., 2016). Our experiments indicate that, in addition to associating with the H3K27 demethylases KDM6A (UTX) (Wang et al., 2013), KDM6B (JMJD3), and KDM7A (KIAA1718), the H3K36 methyltransferase SETD2 (Chen et al., 2012) and Ser2P-PolII (Yoh et al., 2007), Spt6 also interact with Suz12 in ESCs. Interaction of Spt6 with Eed, another core PRC2 subunit, has been previously reported (Cao et al., 2014), even though we were unable to consistently detect it. The methyltransferase SET domain of Ezh2 makes direct contact with HO-3867 located within the ZnF motif of Suz12, and such interactions are involved in establishing an architecture suitable for PRC2 allosteric regulation (Ciferri et al., 2012). A region spanning the ZnF domain and including additional Suz12 amino acids (406–545) directly interacted with Spt6. In vivo and in vitro titration of Spt6 reduced Suz12-Ezh2 association, indicating competition between Spt6 and Ezh2 for Suz12 binding. Consistently, Spt6 inhibits Ezh2 recruitment and H3K27me3 and mitigates transcriptional repression induced by heterologous Gal4-Suz12 in mammalian cells. Nucleoplasmic Spt6-Suz12 interaction may regulate the stoichiometry of Spt6 available for chromatin recruitment, and limiting Spt6 would preferentially affect regions particularly vulnerable to decreased Spt6 accretion, such as SEs. Recent work indicates that PRC2 binds by default to non-transcribed CGI genes and that blocking transcription or transcriptional elongation is sufficient to recruit PRC2. Interestingly, preventing transcriptional elongation via Ser2P-PolII inhibition was more efficient in inducing Suz12 recruitment than causing PolII degradation (Riising et al., 2014). While the difference in Suz12 recruitment might be an intrinsic effect of the inhibitors employed, Ser2P-PolII inhibition is expected to hinder Spt6 recruitment, which would result in increased PRC2 accretion. An additional mechanism involved in increased H3K27me3 following Spt6 depletion may be related to RNA production. RNA binding has been shown to negatively modulate PRC2 chromatin recruitment and methyltransferase activity (Davidovich et al., 2013, Cifuentes-Rojas et al., 2014, Davidovich et al., 2015). In addition to interacting with Suz12, Spt6 may also indirectly counteract PRC2 recruitment and activity at transcribed regions by sustaining RNA production. Recombinant Spt6 did not inhibit in vitro methyltransferase activity of preformed PRC2 complex (data not shown), suggesting that Spt6 may prevent PRC2 assembly rather than promoting its disassembly. In Drosophila melanogaster, deletion of the Suz12 region involved in Ezh2 and Spt6 interactions results in reduced PRC2 binding to chromatin targets and strong loss of function in genetic rescue tests (Rai et al., 2013). In summary, our findings indicate that, in addition to behaving as a histone chaperone and transcriptional elongation factor (Kwak and Lis, 2013), Spt6 has the potential of partitioning the genome in transcribed and repressed regions by counteracting formation and recruitment of PRC2 and H3K27me3 at defined regulatory regions.
    STAR★Methods
    Author Contributions
    Acknowledgments We thank Gustavo Gutierrez-Cruz (NIAMS Sequencing Facility) for library preparation and sequencing, Elif Eren (Laboratory of Structural Biology Research, NIAMS) for help with interpreting the Spt6/Suz12 interaction results, and Danny Reinberg (New York University) for providing reagents. This work was supported by the Intramural Research Program of the NIAMS at the NIH (AR041126-17).