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.Histone H3 lysine 4 methylation (H3K4me) is associated with the transcriptionally active regions of the genome in yeast, flies, and mammals (3,23,35). Set1 was identified as a component of a macromolecular protein complex named COMPASS (complex of proteins associated with Set 1), as the first H3K4 methylase, and it is responsible for all mono-, di-, and trimethylation of H3K4 in yeast (22,31,40,52). In Drosophila melanogaster, four SET domain-containing proteins, namely, Trithorax (Trx), Trithorax-related (Trr), dSet1, and Ash1, have been reported to implement H3K4 methylation (10). All but Ash1, which has subsequently been demonstrated to be an H3K36 methyltransferase (49,59), are related to subunits of the six COMPASS and COMPASS-like complexes in mammals. trx was originally characterized as a gene that when mutated caused homeotic transformations (6, 18). Detailed genetic and molecular analyses showed that Trx is required to maintain activation states of its target genes throughout development and counteracts the repressive effects of the Polycomb group proteins (PcG) (39, 41). Trr was identified based on sequence similarity to Trx but was shown to function in the regulation of hormone-responsive gene expression (42). dSet1 was identified based on sequence homology to the Saccharomyces cerevisiae and mammalian Set1 proteins (53, 58).In mammals, there are at least six SET1-related proteins that form COMPASS-like complexes, namely, SET1A, SET1B, and MLL1 to MLL4. SET1A and SET1B are orthologous to dSet1; MLL1 and MLL2 are orthologous to Drosophila Trx; MLL3 and MLL4 (also known as ALR) are orthologous to Drosophila Trr (33,43,45). All of the m...

Drosophila O-GlcNAcase Deletion Globally Perturbs Chromatin O-GlcNAcylation

“…Primary antibodies and dilutions used for staining were: rabbit anti-ASH1 (Novus number 50100002), anti-RNA Pol II Ser2P (Abcam, ab5095), anti-Polycomb (Santa Cruz, number sc-25762), and mouse HGAC-85 anti-O-GlcNAc (Thermo Scientific, number MA1-076). Rabbit anti-SET1 and anti-TRX antibodies were a kind gift from Dr. Shilatifard (38). All secondary antibodies were Alexa Fluor 488-or Alexa Fluor 568-conjugated (Invitrogen) and used at 1/250 dilution.…”

“…300 g of protein was used for immunoprecipitation in 500 l of PBS, Triton X-100. ASH1 (Novus, number 50100002), BRE1 (Novus, number 40280002), SET1, and TRX antibodies were described (38). All primary antibodies were used at 1/100 dilution.…”

Genome-wide activities of Polycomb complexes control pervasive transcription

“…Recent studies have attributed H3K4me1 to TRR (Ardehali et al 2011;Herz et al 2012), although it has also been assigned to Trithorax (Tie et al 2014). Significantly, CBP and TRR each form a complex containing UTX (Mohan et al 2011;Tie et al 2012), suggesting that these complexes may work in tandem demethylating H3K27 and writing H3K27ac and H3K4me1. To test whether CBP and TRR are involved in intergenic derepression, we knocked each down at 31°C and found that the derepression levels were reduced ( Fig.…”

“…RNAi knockdown experiments show that CBP and TRR contribute to transcriptional derepression but to various extents, depending on the site tested. How H3K27ac and H3K4me1 are mechanistically connected is still not well understood, but both CBP and TRR complexes are reported to include UTX (Mohan et al 2011;Tie et al 2012), which is associated with active transcription (Smith et al 2008). In vitro, mammalian and fly UTX demethylases act preferentially on H3K27me2/3 (Lan et al 2007;Smith et al 2008).…”

Paternal H3K4 methylation is required for minor zygotic gene activation and early mouse embryonic development

“…Mll2 also displayed a high FPKM score (31.451), whereas Mll4, Setd1a, and Setd1b were expressed to much lower degrees, and Mll1 was barely detectable (Supplementary Table S1A). Interestingly, among these six enzymes, only Mll3 and Mll4, both of which are orthologs of Drosophila Trr mediating H3K4me1 at enhancer regions rather than promoter regions [26][27][28], are categorized as "minor ZGA" depending on their expression patterns during the preimplantation stages (Supplementary Table S1A). Because these six methyltransferases are composed of three distinct protein complexes (Supplementary Table S1A) [29], we further examined the expression of molecules unique to each complex.…”