Structural analysis of the core COMPASS family of histone H3K4 methylases from yeast to human

Abstract

Histone H3 lysine 4 (H3K4) methylation is catalyzed by the highly evolutionarily conserved multiprotein complex known as Set1/COMPASS or MLL/COMPASS-like complexes from yeast to human, respectively. Here we have reconstituted fully functional yeast Set1/COMPASS and human MLL/COMPASS-like complex in vitro and have identified the minimum subunit composition required for histone H3K4 methylation. These subunits include the methyltransferase C-terminal SET domain of Set1/MLL, Cps60/Ash2L, Cps50/RbBP5, Cps30/WDR5, and Cps25/Dpy30, which are all common components of the COMPASS family from yeast to human. Three-dimensional (3D) cryo-EM reconstructions of the core yeast complex, combined with immunolabeling and two-dimensional (2D) EM analysis of the individual subcomplexes reveal a Y-shaped architecture with Cps50 and Cps30 localizing on the top two adjacent lobes and Cps60-Cps25 forming the base at the bottom. EM analysis of the human complex reveals a striking similarity to its yeast counterpart, suggesting a common subunit organization. The SET domain of Set1 is located at the juncture of Cps50, Cps30, and the Cps60-Cps25 module, lining the walls of a central channel that may act as the platform for catalysis and regulative processing of various degrees of H3K4 methylation. This structural arrangement suggested that COMPASS family members function as exo-methylases, which we have confirmed by in vitro and in vivo studies.

Fig. 1.

Recombinant Set1/COMPASS complexes and in vitro histone methyltransferase activities. (A) Purifications of recombinant COMPASS consisting of full-length Set1 with various combinations of Cps60, Cps50, Cps40, Cps35, Cps30, and Cps25. Subunit composition of purified COMPASS was confirmed by Western blotting using anti-FLAG (top box) and anti-Set1 (middle box) antibodies. In vitro H3K4 methyltransferase activities toward free histone H3 of various recombinant COMPASS complexes were examined by Western blotting using anti-H3K4me1, me2, and me3 antibodies (bottom box). (B) Recombinant COMPASS complexes were prepared using Set1 (938–1,080) instead of full-length Set1. COMPASS composition and in vitro H3K4 methyltransferase activities were analyzed in the same way as (A).

Fig. 2.

EM mapping of core COMPASS. (A) Purification of Core Set1/COMPASS by size exclusion chromatography and analysis of the active fractions by HMTase assay. Core Set1/COMPASS consisting of Cps25, Cps60, Cps50, Set1 (938–1,080) was reconstituted and fractionated by size exclusion chromatography over a Superose-6 PC 3.2/30 (GE Healthcare). The H3K4 methylase activities of the resulting fractions were tested as shown. (B) Representative 2D averages of negative stained core COMPASS (fraction 18 in A) including Cps25, Cps60, Cps50, Set1 (938–1,080) which is denoted as SET938 for simplicity in Figs. 3 and 4, and Cps30. The inset shows the 2D projection profile of a WD40 domain for comparison (Scale bar, 10 nm). (C) Representative 2D averages of the Cps25-Cps60 assembly. (D) Representative 2D averages of negative stained Cps25-Cps60-Cps50-SET938 complex. (E) 2D average of Cps25-Cps60-Cps50-SET938 complex labeled with a Fab fragment against SET938. (F) Representative 2D class averages of negative stained human core MLL1/COMPASS-like complex. Schematic representations: Cps25/DPY30-Cps60/Ash2L (gray), SET938/MLL1 (blue), Cps50/RbBP5 (orange), and Cps30/WDR5 (green).

Fig. 4.

COMPASS family members are exomethylases. (A) Zoom-in view of the central channel formed in the complex, with the histone peptide of the MLL1 (as cocrystallized) shown in the red space-filling model. (B) Schematic model of core COMPASS family architecture. The red star indicates the histone peptide-binding region of SET938. (C) The N-terminally extended version histone H3 was constructed by inserting FLAG sequence (DYKDDDDK) between the Met start codon and the second Ala codon. COMPASS shows no H3K4 methylase activity with the FLAG-extended H3 tails in vivo (this figure) and in vitro (SI Appendix, Fig. S15).

Fig. 3.

3D cryo-EM reconstruction and modeling of core COMPASS. Different views of the cryo-EM 3D map for the Cps25-Cps60-Cps50-SET938-Cps30 COMPASS complex. Each view shows the solid rendered map accompanied by a transparent map with modeled crystal structures of the WD40 domain of Cps50 (orange), Cps30 (green), and MLL1/SET938 (cyan). The red arrow indicates the expected position of the Cps50 arm that bridges with the Cps25-Cps60 module.