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

Keywords: MLL1, single particle analysis, negative stain EM

Reconstitution of Core COMPASS.

To obtain stable COMPASS for biochemical characterization, we reconstituted the yeast and human complex through baculovirus-mediated cotransfection and overexpression of different subunit combinations in insect cells. Based on affinity purification of the recombinant Set1 and Cps proteins, we obtained nearly pure preparations of reconstituted COMPASS complexes (SI Appendix, Figs. S1–S4) that are enzymatically active in in vitro H3K4 methylation assays with full-length histone H3 (Fig. 1A). This approach enabled us to prepare any combination of wild-type (WT) or modified COMPASS components, including truncated forms of Set1, for studies that have been previously hampered by the absence of subunits such as Cps50 or Cps30 (8). Accordingly, COMPASS containing full-length Set1, truncated Set1 (762–1,080), Set1 (780–1,080), Set1 (938–1,080), or without Set1 were successfully prepared (SI Appendix, Figs. S1–S2). Curiously, full-length Set1 purified from yeast COMPASS consistently runs on SDS/PAGE as a doublet, although the reasons behind this property of Set1 or its biological significance remain unclear.

As a first step, we investigated the effects of specific Cps subunits on in vitro H3K4 methylation by preparing full-length Set1 (1–1,080) with various Cps combinations (Fig. 1A). Each introduced component was purified through FLAG affinity purification as shown by anti-FLAG or anti-Set1 Western blots (Fig. 1A, top and middle boxes, respectively). Consistent with our previous reports of in vivo and in vitro H3K4 methylation using cps60Δ and cps25Δ strains (8), the absence of Cps60 and Cps25 significantly impaired the catalytic activity of Set1 with the total loss of H3K4 trimethylation, and more than an 80% reduction of H3K4 mono- and dimethylation (Fig. 1A, lane 7). In contrast to earlier in vivo studies (8, 12–14), almost no monomethylation or very marginal di- and trimethylation changes of H3K4 were observed in the absence of Cps40 and Cps35 under conditions where Set1 levels are not a limiting reagent in our in vitro experiments (Fig. 1A). This study suggests that full-length Set1 stability in vivo is reduced in either cps40Δ or cps35Δ strains, compared to the strain expressing all five subunits. To this end, we found that Cps40 coeluted with other COMPASS components in the presence of the N-terminally extended region of Set1, including full-length Set1, Set1 (762–1,080), Set1 (780–1,080), but not Set1 (938–1,080) (SI Appendix, Fig. S2). Cps35 was also shown to require a region beyond 762–1,080 of Set1, because it did not coelute in the macromolecular fractions with any other truncated Set1 variants (SI Appendix, Fig. S2).

Next, we evaluated the intrinsic enzymatic activity of soluble Set1 protein in isolation. We did not detect any H3K4 methylation by Set1 alone or in the presence of putative Set1-stabilizing components Cps50 and Cps30 (Fig. 1A, lanes 2 and 3), thereby further confirming that Cps60 and Cps25 are required for H3K4 methylation activity by Set1. In the reconstituted system, Set1 (938–1,080) was sufficient in replacing the full-length protein in terms of stability and enzymatic activity (Fig. 1 A and B), except in the absence of Cps60 and Cps25 (Fig. 1 A, lane 7 and B, lane 7). Whereas full-length Set1-containing COMPASS supported low levels of residual in vitro H3K4 mono- and dimethylation in the absence of Cps60 and Cps25, this activity was completely abolished in Set1 (938–1,080)-containing COMPASS.

Recent studies with H3 peptide substrates have suggested that WDR5 RbBP5, Ash2L, and Dpy30 (also known as the WRAD complex), the human homologs of Cps30, Cps50, Cps60, and Cps25, respectively, display histone H3K4 methyltransferase activity independent of the Set1 domain of MLL1 (15–17). Despite the highly conserved components between the yeast COMPASS and the human MLL/COMPASS-like complexes, we could not detect any H3K4 methylation signals in a reconstituted Cps30, Cps60, Cps50, and Cps25 complex in the absence of Set1 (Fig. 1 A and B; see lane 10 in both figures). This observation is consistent with our previous in vivo studies where we were unable to detect any histone H3K4 methylation in strains with SET1 deletions (8), suggesting that Set1 is the only histone H3K4 methylase in yeast, and that the yeast WRAD complex does not demonstrate substantial HMTase activity associated with it in vivo or in vitro. Indeed, our enzymatic analyses (SI Appendix, Fig. S5) demonstrated the presence of less than 1% of H3K4me1 and virtually undetectable levels of H3K4me2-3 when using WRAD as compared to wild-type Set1/COMPASS or MLL/COMPASS-like complexes. Given the extremely weak catalytic rates reported for the reconstituted WRAD complex as a SET-domain independent histone H3K4 methylase (15–17), the biological relevance of such activity needs to be further demonstrated.

Insect cell-expressed core COMPASS also enabled us to investigate the regulation of histone H3K4 methylation by Cps50 and Cps30. Systematic analysis for in vitro H3K4 methyltransferase activity (SI Appendix, Fig. S3) shows that core COMPASS is rather insensitive to the lack of Cps30 with comparable H3K4 mono- and dimethylation, but partially defective trimethylation activity (SI Appendix, Fig. S3D, lanes 2 and 10). This finding suggests that COMPASS is enzymatically active in the absence of Cps30, but the processivity of the SET domain from H3K4 di- to trimethylation is regulated by Cps30. In contrast, reconstituted core COMPASS lacking either Set1 (938–1,080), Cps60, Cps50, or Cps25 completely lacked enzymatic activities (SI Appendix, Fig. S3D, lanes 1, 6, 11, and 12; note that the low levels of Set1 in lane 6 may also contribute to the absence of activity observed in that condition). Thus, we have identified Set1 (938–1,080), Cps60, Cps50, Cps30, and Cps25 as the necessary core COMPASS components for high levels of in vitro H3K4 mono-, di- and trimethylation using histone H3 as a substrate. Both Cps60 and Cps25 are essential for in vitro enzymatic activity regardless of the combination of other core COMPASS subunits (Fig. 1A lanes 3, 4, and 7, SI Appendix, Fig. S3D, lanes 3–8, 11–14). It is noted that the Cps60-Cps25 subcomplex (6) coeluted with Set1 (938–1,080) upon size exclusion chromatography when reconstituted together (fractions 21 to 23; SI Appendix, Fig. S4B). This result demonstrates a direct interaction between the SET domain and the Cps60-Cps25 module. In contrast, in the absence of Cps60 and Cps25, soluble Set1 (938–1,080) elutes broadly on the same size exclusion chromatography system (fractions 13 to 29; SI Appendix, Fig. S4A). Given that coexpression of neither Cps50 nor Cps30 had any effects on Set1 (938–1,080) elution profile (SI Appendix, Fig. S4C), Cps60 and Cps25 may play an additional role in the correct assembly of COMPASS through interactions with Set1.

Electron Microscopic Mapping of Core COMPASS.

To gain insight into the architecture of yeast core COMPASS, we employed electron microscopy to visualize preparations consisting of purified Set1 (938–1,080), Cps60, Cps50, Cps30, and Cps25 (Fig. 2A, fraction 18; SI Appendix, Fig. S1B). Raw images of negative stained specimen revealed a monodisperse population of complexes with similar sizes (SI Appendix, Fig. 6A). To analyze these particles, 35,527 projections were interactively selected and grouped into 100 classes by reference-free alignment and classification (SI Appendix, Fig. S7A). The two-dimensional (2D) class averages revealed flexible conformers of two major populations of particle projections: one displays a Y-shaped particle with a triangular base connecting through an extension to two adjacent oval lobes, one of which is in closer proximity to the base (Fig. 2B, right; SI Appendix, Fig. S7A); the second population reveals a similar triangular base and an arm extension connecting to a single circular doughnut-like domain with a distinct stain accumulation region in the center (Fig. 2B, left). The doughnut-shaped domain appears identical to the projection profile of a WD40 domain (inset in Fig. 2B), which defines both Cps50 and Cps30, further suggesting that a subpopulation of the reconstituted COMPASS complexes might be missing one of the two WD40 domain-containing subunits.

In order to identify each domain within the core COMPASS, we visualized individual subcomplexes by negative stain EM. We first examined the structure of the Cps60-Cps25 subcomplex, where classification and averaging (Fig. 2C; SI Appendix, Fig. S8 A and C) revealed a triangular structure with similar dimensions and shape to the triangular base found in the images of core COMPASS (Fig. 2B). This result suggests that Cps60 and Cps25 form the base of core COMPASS. We next analyzed EM images of reconstituted core COMPASS lacking the Cps30 subunit (core COMPASS/-Cps30) (SI Appendix, Fig. S8 B and D). 2D averages from this analysis show the well defined triangular base, with an arm extension and an additional oval lobe near the top of the complex (Fig. 2D). Missing, however, is the second, more distant oval lobe that is present in core COMPASS. This observation indicates that the Cps30 subunit is the more terminal of the two lobes away from the triangular base, while the lobe closer to the middle of the complex belongs to the WD40 domain of Cps50. In addition, a few averages reveal a small density extending from the connection between the Cps60-Cps25 base and the Cps50 domain, which might be attributed to the SET domain (Fig. 2D, right box). To test this hypothesis, we incubated core COMPASS/-Cps30 with a Fab fragment conjugated to SET and analyzed the complexes by single-particle EM. 2D projection averages of Fab-labeled complexes clearly reveal the extra Fab density at the level of the region connecting the Cps60-Cps25 base and Cps50. (Fig. 2E). Next, we examined the architecture of the reconstituted human core COMPASS consisting of MLL SET domain, RbBp5, Ash2L, WDR5, and Dpy30. Negative stain 2D averages from this preparation revealed an architecture with striking resemblance to the yeast complex (Fig. 2F). Thus, both the structure and function and COMPASS-like complexes appear to be highly conserved from yeast to human.

3D Architecture of Core COMPASS.

To further characterize the architecture of the core COMPASS, we sought to examine the three-dimensional (3D) structure of the yeast assembly. In the first step, we calculated several 3D models from negative stained particles belonging to the individual groups produced by classification (SI Appendix, Fig. S9). For this approach, we used the corresponding 60° tilted projections and the random conical tilt method to calculate initial reconstructions, which were further refined after the inclusion of 0° particle views. The 3D reconstructions of particles belonging to classes with only one globular domain connected to the Cps60-Cps25 base reveal that these particles have indeed only one WD40-like domain. In contrast, 3D reconstructions from Y-shaped particles reveal the Cps60-Cps25 module having two globular domains connected to it at different distances (SI Appendix, Fig. S9). In these 3D reconstructions, we notice variability in the disposition of the Cps60-Cps25 base as well as in the proximity between the two WD40-like lobes of Cps50 and Cps30. We reasoned that the various conformations might be the result of inherent complex flexibility, resulting in deformations due to the negative stain preparation on the carbon support of the grids. Therefore, we focused our efforts on cryo-EM of the complex in holey carbon grids, and successfully obtained particle images from specimen suspended in thin vitreous ice (SI Appendix, Fig. S6B). Because our negative stain analysis of core COMPASS indicated the presence of two major populations (+/- one WD40 domain), we subjected 21,583 cryo-EM images to multiple reference-supervised alignment (18, 19) using two initial 3D references (SI Appendix, Fig. S10A): a 3D volume representing only the Cps60-Cps25 base and one WD40-like domain (SI Appendix, Fig. S10A, left), and a 3D volume that included the base and both WD40-like domains (SI Appendix, Fig. S10A, right). This approach allowed us to effectively separate the two major populations within the cryo-EM dataset, resulting in stable particle assignments after several cycles of multireference alignment. We then employed single reference alignment and reconstruction to produce a final cryo-EM map of the core COMPASS at a resolution of 24 Å (FSC = 0.5) (SI Appendix, Fig. S10B). The cryo-EM 3D map displayed features that are clearly distinct from those of the starting reference 3D map from negative stain EM. To validate our reconstruction, we also employed different initial references for cryo-EM projection alignment, thereby, producing very similar final maps (SI Appendix, Fig. S11). Furthermore, 2D classification of the cryo-EM projections (SI Appendix, Fig. S12A) revealed averages that are in very good agreement with reprojections of the 3D map (SI Appendix, Fig. S12B).

Molecular Modeling of Core COMPASS.

To obtain deeper insights to the architecture of the core COMPASS, we generated a model by docking the available or homologous crystal structures into their corresponding positions within the cryo-EM map and according to our assignment from the 2D projection analysis (Fig. 3). Due to the limits in resolution, we performed rigid body manual docking based on visual inspection of the fit. The two neighboring globular domains of the 3D map represent the two WD40 domains, with the lobe more distal to the Cps60-Cps25 base belonging to Cps30 (Figs. 3 and 4B, green) and the other to Cps50 (orange). To model Cps50, which contains a β-propeller fold and a C-terminal tail of ∼80 amino acids, we docked a homologous WD40 domain (orange; PDBID:2XL2) in its corresponding position. A recent crystal structure of WDR5, a Cps30 homolog, has been obtained with peptides of both the C-terminal tail of RbBP5 (Cps50 homolog) and the Win motif that is N-terminally adjacent to the SET domain of MLL1 (Set1 homolog) bound onto the opposite faces of WDR5 (20, 21). We thus docked the cocrystal structure (PDBID:3P4F) of the MLL1 Win peptide-WDR5-RbBP5 tail peptide in the position of Cps30 with an orientation that places the RbBP5 tail peptide close to the Cps50 position on the cryo-EM map. Our Fab labeling experiments position the SET domain in the region connecting the two WD40 domains and the Cps60-Cps25 base. Accordingly, the Set1/SET domain from the homologous crystal structure (11, 22) was docked in this position. Although yeast Set1 does not have the canonical Win motif upstream of the SET domain, we identified a similar Ala-Arg-Ser motif at positions 943–945 of the Set1 primary sequence, and found that core COMPASS harboring either Set1 (R944A) or Set1 (S945A) substitutions almost completely loses its H3K4 methyltransferase activity (SI Appendix, Fig. S13). This observation suggests that the Ala-Arg-Ser motif is important in complex formation like the Win motif in the MLL1 core complex. Based on this result, we oriented the N terminus of Set1 close to Cps30 and opposite to Cps50 (SI Appendix, Fig. S14). According to these interactions, and given the spatial constraints of this region, we could only dock the Set1 domain in a single orientation. In this model, one side of Set1 (938–1,080) appears to extensively interact with Cps30, while another side is forming a bridge with density stemming from the Cps50 WD40 domain (Fig. 4A). Thus, our modeling places the active site of Set1 in the middle of a central channel that runs through the complex, starting from the connection between Cps60-25 and Cps50, and running adjacent to Set1, exiting behind the interface of Cps30 with Cps50 (Fig. 4 A and B). In this configuration, the peptide bound to the active site of the SET domain, as shown in the crystal structure (PDBID:2W5Z), would reside directly in the middle of the COMPASS channel (shown in red in Fig. 4A). Thus, this configuration may limit the substrates that COMPASS can recognize and methylate.

COMPASS Family Members Are Exo-Methylases.

Our structural analysis and modeling suggest that the centrally located active site within the COMPASS channel may only be reached by flexible peptide terminals, indicating that COMPASS family may function primarily as exo- and not endo-methylases. To test this hypothesis, we engineered a Flag sequence to the N terminus of the only copy of histone H3 in our yeast strain and tested H3K4 methylation by Set1/COMPASS. As shown in Fig. 4C (lanes 1, 3), WT H3 can be methylated on K4 by Set1/COMPASS in vivo. However, H3 bearing a single Flag sequence on its N terminus (making the H3K4 site an internal site) is no longer methylated, although this H3 can be fully methylated on K36 and K79 (Fig. 4C lanes 5, 6). This finding illustrates that Set1/COMPASS specifically methylates the N-terminal tail of histone H3 and that the addition of a short heterologous sequence to the substrate’s N terminus, making the site of methylation an internal site, blocks methylation by Set1/COMPASS in yeast cells. To further confirm this observation in a reconstituted system, we also tested whether N-terminally 10-His-tagged H3 can be methylated either by Set1/COMPASS or MLL/COMPASS-like complex. Unlike WT H3, an internally engineered H3K4 site can no longer serve as a substrate for COMPASS (SI Appendix, Fig. S15, lanes 1–14) further suggesting that the COMPASS family members preferentially methylate N-terminal and not internal lysine sites.

Plasmids and Yeast Strains.

Full-length Set1, Set1 (762–1,080), Set1 (780–1,080), Set1 (938–1,080), and the Cps subunits of COMPASS (Cps60, Cps50, Cps40, Cps35, Cps30, Cps25) except for Cps15 were fused with the FLAG epitope tag on their N termini and cloned into the transfer vector pBacPAK8 (Clontech). The MLL1 core complex components including MLL1-Win-SET(3,745–3,969), full-length WDR5, Ash2L, RbBP5, and Dpy30 were tagged with FLAG epitope tag on their N termini and cloned into the pBacPAK8 vector in the same manner. Yeast shuffle strain YBL574 was transformed with modified pWZ414-F12 plasmid, which encodes an N-terminally FLAG-tagged hht2 gene, and used for the analysis of exo-methylation by COMPASS.

Protein Preparation.

Recombinant COMPASS and MLL1 complexes were prepared through the BacPAK Baculovirus Expression System (Clontech). To prepare COMPASS complexes of various combinations of the Set1 and Cps subunits, exponentially growing Sf9 insect cell cultures were cotransfected with a mixture of viruses expressing specific combinations of COMPASS components and subsequently FLAG-purified. The MLL1 core complex was prepared in the same manner. For further details refer to SI Appendix.

In Vitro and In Vivo H3K4 Methylation Analysis.

Recombinant COMPASS complexes were incubated with 0.5 μg of free histone H3 and 200 μM S-adenosylmethionine in methyltransferase reaction buffer (50 mM Tris-HCl [pH 8.8], 20 mM KCl, 5 mM MgCl₂, 0.5 mM dithiothreitol) for at least 2 h at 30 °C. The methylation of histone H3 was examined by Western analysis using anti-H3K4me1, me2, and me3 specific antibodies. Histone methylation in vivo was determined by Western analysis of cleared cell lysates using anti-H3K4me1, me2, me3, H3K79me2, me3, and H3K36me3 specific antibodes.

Specimen Preparation, EM Imaging, 2D Classification, and 3D Reconstruction of Negative Stained COMPASS Complexes.

Core COMPASS and subcomplexes were prepared for electron microscopy using the conventional negative staining protocol (30), and imaged at room temperature with a Tecnai T12 electron microscope operated at 120 kV using low-dose procedures. 2D reference-free alignment and classification of particle projections was performed using SPIDER (31). The random conical tilt technique (32) was used to calculate a first back projection map from individual classes using the images of the tilted specimen. FREALIGN (33) was then used for further refinement of the orientation parameters and for correction of the contrast transfer function to produce final 3D reconstructions. For further details refer to SI Appendix.

Cryo-EM Specimen Preparation, Imaging, and 3D Reconstruction.

Vitrified specimen was visualized on a Tecnai F20 electron microscope (FEI) equipped with a field emission electron source operated at 200 kV. Particles from cryo-EM images were excised using Boxer [part of the EMAN 1.9 software suite] (34). A total of 21,583 particles were initially subjected to multiple reference-supervised alignment with EMAN (18, 19), and the separated particle projections were then subsequently submitted to single reference refinement and 3D reconstruction. For further details refer to SI Appendix.

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Supplementary Materials