br Results br Discussion A key step in the regulation

Results
Discussion A key step in the regulation of gene AG 013736 is the methylation on H3K4, which is added by the multi-component MLL/COMPASS-like histone methyltransferases. Many studies have shown the requirement of shared core subunits for optimal methylation activity of catalytic SET protein. Here, we present a 4-Å resolution structure of the yeast COMPASS catalytic core complex. Together with systematic biochemical analyses in vitro and yeast genetics in vivo, the structure provides molecular insights into the assembly and function of COMPASS and COMPASS-like complexes. Cps50 provides the molecular scaffold for assembling the COMPASS complex. Earlier studies showed the importance of few short motifs at the Cps50/RbBP5 C-terminal unstructured region for methylation (Avdic et al., 2011a, Li et al., 2016, Zhang et al., 2015). In COMPASS, the winding Cps50 C-terminal tail positions nearly all the core subunits in contact with the catalytic SET domain. Abrogating the interaction between the tail and its partners affected the activity of COMPASS to different extents. Here, we also solved the crystal structure of Cps50 N-terminal WD40 domain, which shares the conserved seven β strand blades with Cps30 and other WD40 domain proteins. The Cps50 WD40 domain sandwiches its C-terminal end together with the Cps30 WD40 domain, potentially to orient the Cps30 protein in a strict geometry related to the catalytic SET protein. Moreover, the extensive contact between Cps50 WD40 domain and Cps40 likely anchors this helical protein for its function. The COMPASS structure reveals that Cps40 is a primarily α-helical protein, in agreement with secondary structure predictions. In addition, a chromatin-binding PHD domain is predicted to be located at its N terminus. Even though we could not model this region due to its flexibility, truncation of this N-terminal PHD domain affected the methylation activity of COMPASS in yeast (Figure 5E), which may reflect a reduced affinity with chromatin. Earlier studies have shown that the PHD domains can recognize methylated histone lysine residue—H3K4me3 (Li et al., 2006, Peña et al., 2006) and H3K9me3 (Iwase et al., 2007)—or unmethylated H3K4 (Lan et al., 2007). We note that the spacing between the Cps40 N-terminal region (PHD domain and helical-stack domain connected by a hinge to the two long helical rods) and Cps60/Cps25 base is about 10-nm wide, which is compatible with the dimensions of a nucleosome (Figure S7E). The difference in the two COMPASS complex conformations we observed mainly resides in the disposition of the Cps40 N-terminal half, including PHD domain and the Cps60/Cps25 base. It is plausible that such movement between Cps40 PHD domain and the Cps60/Cps25 module enables the complex to “flex” and adapt to the nucleosomal substrate where H3K4 is located (Figure S7E), although detailed COMPASS studies in complex with the nucleosome are required to delineate these events.
STAR★Methods
Acknowledgments This work was supported by NIH grant DK090165 (G.S.), an Outstanding Investigator Award R35CA197569 from the National Cancer Institute (A.S.), and a CIHR grant (J.-F.C.).
Histones are the small basic proteins found in eukaryotic cell nucleus, forming chromatin structures with DNA as the chief protein components. Covalent histone modifications such as phosphorylation, ubiquitination, acetylation and methylation play an important role in regulating chromatin dynamics and function. Enhancer of zeste homologue 2 (EZH2) is the core protein of Polycomb repressive complex 2 (PRC2) catalyzing primarily tri-methylation of histone H3 Lys-27 (H3K27). Tri-methylation of the lysine-27 has been suggested to cause the repression of specific genes, including many tumor suppressor genes. High EZH2 expression has been shown to be correlated with poor prognosis, high grade and high stage in several cancer types. In addition, several kinds of heterozygous mutations were found in ca. 7% of follicular lymphomas and ca. 22% of diffuse large B cell lymphomas (DLBCL). The DLBCL cell lines with EZH2 mutation at position of Tyr-641 or Ala-677 are known to be very sensitive to EZH2 inhibitors, indicating that the cell lines are dependent on EZH2 activity for cell growth. Thus, blocking its gene expression or enzyme activity is considered to be a promising therapeutic strategy to treat cancers. Recently, small molecule inhibitors of EZH2 have been reported to produce promising anti-tumor activity both in vitro and in vivo.