The COMPASS family of H3K4 methylases in Drosophila

Abstract

Methylation of histone H3 lysine 4 (H3K4) in Saccharomyces cerevisiae is implemented by Set1/COMPASS, which was originally purified based on the similarity of yeast Set1 to human MLL1 and Drosophila melanogaster Trithorax (Trx). While humans have six COMPASS family members, Drosophila possesses a representative of the three subclasses within COMPASS-like complexes: dSet1 (human SET1A/SET1B), Trx (human MLL1/2), and Trr (human MLL3/4). Here, we report the biochemical purification and molecular characterization of the Drosophila COMPASS family. We observed a one-to-one similarity in subunit composition with their mammalian counterparts, with the exception of LPT (lost plant homeodomains [PHDs] of Trr), which copurifies with the Trr complex. LPT is a previously uncharacterized protein that is homologous to the multiple PHD fingers found in the N-terminal regions of mammalian MLL3/4 but not Drosophila Trr, indicating that Trr and LPT constitute a split gene of an MLL3/4 ancestor. Our study demonstrates that all three complexes in Drosophila are H3K4 methyltransferases; however, dSet1/COMPASS is the major monoubiquitination-dependent H3K4 di- and trimethylase in Drosophila. Taken together, this study provides a springboard for the functional dissection of the COMPASS family members and their role in the regulation of histone H3K4 methylation throughout development in Drosophila.

Fig. 1.

dSet1 is the major H3K4 methyltransferase, while Trx and Trr are minor contributors of global H3K4 methylation. Wing imaginal discs from Drosophila third-instar larvae were stained with antibodies specific to H3K4 methyltransferases. With the UAS-GAL4 system, enGAL4 was used to drive the expression of UAS-hairpin constructs targeting dSet1, Trx, and Trr; the domain of knockdown was marked by GFP. Loss of signal in the posterior half demonstrates the knockdown of dSet1 (a and a′), Trx (d and d′), and Trr (i and i′) and the specificity of the antibodies to their respective proteins. dSet1 knockdown results in near total loss of both H3K4me2 (b and b′) and H3K4me3 (c and c′) from the discs, whereas Trx (e and f′) and Trr (g and h′) knockdown does not result in any significant reduction of either H3K4me2 or H3K4me3.

Fig. 2.

Distributions of dSet1, Trx, Trr, and LPT on polytene chromosomes. (A) Immunostaining of dSet1, Trx, Trr, and LPT on polytene chromosome squashes from third-instar larval salivary glands. Labels in panel a denote the cytological positions of landmark puffs. (B) Details of the distal X chromosome stained for of dSet1, Trx, Trr, and LPT. Labels denote the cytological positions of the indicated stained sites. Trx-CT, antibody raised against the C-terminal domain of Trithorax protein. (C and D) Colocalization of dSet1 (red) with RNA Pol II (S2P; green) (C) and with RNA Pol II (S5P; green) (D). The significant amounts of yellow signals in the merge channels (C, panel c) and (D, panel c) indicate colocalization of dSet1 with active regions of the genome.

Fig. 3.

Baculovirus superinfection for the expression of epitope-tagged proteins in Drosophila S2 cells. (A) Immunohistochemistry against the HA epitope in S2 cells infected with either pBacEGFP/MT-FLAG-HA (empty BacPAK8) or pBacEGFP/MT-FLAG-HA-dWdr82, showing the expression of tagged protein. (B) Western blot analysis with HA antibody demonstrated the expression of dWdr82 and dPA1 in S2 cells infected with baculovirus (pBacEGFP-MT-FLAG-HA-dWdr82 and pBacEGFP-MT-FLAG-HA-dPA1).

Fig. 4.

Composition of COMPASS and COMPASS-like complexes in Drosophila. (A) Proteins purified and identified by MudPIT with FLAG-HA-dWdr82, Ash2, dPA1, and dUTX are shown in the table. For Ash2, a clonal S2 cell line expressing FLAG-HA-Ash2 was used. For dWdr82 and dPA1, baculovirus infection systems were used (see Fig. 3). Ubiquitously driven (Act5C-Gal4) UAS-dUTX-HA-2×FLAG fly embryos were used to purify dUTX complexes, and yw embryos were used as a control. Each box contains the number of peptides identified followed by the normalized spectral abundance factor (dNSAF) (60). Red boxes indicate components identified in yeast and mammalian complexes; blue boxes indicate subunits of Drosophila complexes identified in this study; green shows components expected due to homology. (B) HMTase assays with Ash2, dWdr82, dPA-1 (CG11750), dRbbp5 (CG5585), and wild-type S2 FLAG eluates on free recombinant histones were performed overnight in the presence of SAM and analyzed by Western blotting using antibodies specific to H3K4me1. The H3K4me1 signal demonstrated the histone methyltransferase activity of purified complexes. Total H3 levels were assayed as a loading control.

Fig. 5.

Ortholog analysis of COMPASS and COMPASS-like subunits between humans and Drosophila. (A) Proteins identified with purifications of shared and unique components of dCOMPASS and COMPASS-like complexes were domain aligned with human orthologs using SMART (http://smart.embl-heidelberg.de). WD40 repeat-containing proteins, such as dWdr82, and dRbbp5, were found to have similar domain organization with their human counterparts. (B) LPT is the missing link between MLL3/MLL4 and Trithorax-related (Trr) in Drosophila. The schematic alignment indicates that two proteins in Drosophila, LPT and Trr, likely resulted from a split into two genes of an ancestral MLL3/4 gene. Shown are LPT from Drosophila melanogaster (GI:24762433) and parasitic wasp (Nasonia vitripennis; GI:156551804), MLL3-related proteins from human, body louse (Pediculus humanus; GI:242016925), flour beetle (Tribolium castaneum; GI:270001730), and Trithorax-related protein (Trr) from Drosophila (GI:24639197) and Nasonia (GI:156551806). LPT together with the Trithorax-related proteins contain the overall domain architecture of MLL3/4 proteins from other insects and other animals, including vertebrates. (C) Schematic view of the phylogenetic relationship (30) between the insect orders represented in panel B. Bold text highlights the genus name for the protein represented in panel B, with a second representative of the order listed if we were able to determine that it also had a split or intact MLL3/4 gene. (D) RNAi-mediated knockdown of LPT using enGal4 resulted in significant loss of signal in the posterior half of the wing disc, demonstrating the knockdown of LPT and the specificity of the antibody. (E) Whole-cell lysates from S2 cells were immunoprecipitated with LPT or Trr antibody or rabbit IgG, and the blots were probed with either Trr or dUTX antibody. Input was 1% of the immunoprecipitated nuclear extracts.

Fig. 6.

Requirement of monoubiquitinated H2B by COMPASS for H3K4 trimethylation is conserved in flies. RNAi-mediated knockdown of Bre1, the homolog of yeast BRE1 and human RNF20/40 in wing imaginal discs, led to significantly reduced levels of H3K4 trimethylation (a and a′) and H3K4 dimethylation (b and b′). RNAi-mediated knockdown of dWdr82 in wing imaginal discs led to a similar reduction in H3K4 trimethylation (c and c′), but not H3K4 dimethylation (d and d′), demonstrating the mechanistic conservation between yeast, Drosophila, and mammalian SET1 complexes in generating H3K4 trimethylation.

Fig. 7.

Subunit composition for the histone H3K4 methyltransferase COMPASS and COMPASS-like complexes in yeast and Drosophila. Yeast Set1/COMPASS, the founding member of the H3K4 methylases, is represented as a direct descendant, reflecting the similarity of its protein composition to that of Drosophila Set1/dCOMPASS. Two other COMPASS-like complexes are the Trithorax-containing complex and the LPT/Trr complex. LPT and Trr together constitute the ortholog of the MLL3/4 proteins in mammals.