Set1: Biological Overview | References
Gene name - Set1
Cytological map position - 3L
Function - protein lysine methyltransferase enzyme
Keywords - component of a conserved H3K4 trimethyltransferase complex
Symbol - Set1
FlyBase ID: FBgn0040022
Genetic map position - 3LHet:593,050..600,636 [+]
Classification - COMPASS complex Set1 subunit, N-SET domain; Histone-lysine N-methyltransferase; Nucleotide-binding, alpha-beta plait; Post-SET domain; RNA recognition motif domain; SET domain (SET domains are protein lysine methyltransferase enzymes)
Cellular location - nuclear
|Recent literature||Ford, D. J., Zraly, C. B., Perez, J. H. and Dingwall, A. K. (2020). The Drosophila MLR COMPASS-like complex regulates bantam miRNA expression differentially in the context of cell fate. Dev Biol 468(1-2): 41-53. PubMed ID: 32946789
The conserved MLR COMPASS-like complexes (Complex of Proteins Associated with Set1) are histone modifiers that are recruited by a variety of transcription factors to enhancer regions where they act as necessary epigenetic tools for enhancer establishment and function. A critical in vivo target of the Drosophila MLR complex is the bantam miRNA that regulates cell survival and functions in feedback regulation of cellular signaling pathways during development. Loss of Drosophila MLR complex function in developing wing and eye imaginal discs results in growth and patterning defects that are sensitive to bantam levels. Consistent with an essential regulatory role in modulating bantam transcription, the MLR complex binds to tissue-specific bantam enhancers and contributes to fine-tuning expression levels during larval tissue development. In wing imaginal discs, the MLR complex attenuates bantam enhancer activity by negatively regulating expression; whereas, in differentiating eye discs, the complex exerts either positive or negative regulatory activity on bantam transcription depending on cell fate. Furthermore, while the MLR complex is not required to control bantam levels in undifferentiated eye cells anterior to the morphogenetic furrow, it serves to prepare critical enhancer control of bantam transcription for later regulation upon differentiation. This investigation into the transcriptional regulation of a single target in a developmental context has provided novel insights as to how the MLR complex contributes to the precise timing of gene expression, and how the complex functions to help orchestrate the regulatory output of conserved signaling pathways during animal development.
Histone H3 lysine 4 trimethylation (H3K4me3) is a major hallmark of promoter-proximal histones at transcribed genes. This study reports that a previously uncharacterized Drosophila H3K4 methyltransferase, dSet1, and not the other putative histone H3K4 methyltransferases (Trithorax; Trithorax-related protein), is predominantly responsible for histone H3K4 trimethylation. Functional and proteomics studies reveal that dSet1 is a component of a conserved H3K4 trimethyltransferase complex and polytene staining and live cell imaging assays show widespread association of dSet1 with transcriptionally active genes. dSet1 is present at the promoter region of all tested genes, including activated Hsp70 and Hsp26 heat shock genes and is required for optimal mRNA accumulation from the tested genes. In the case of Hsp70, the mRNA production defect in dSet1 RNAi-treated cells is accompanied by retention of Pol II at promoters. These data suggest that dSet1-dependent H3K4me3 is responsible for the generation of a chromatin structure at active promoters that ensures optimal Pol II release into productive elongation (Ardehali, 2011).
Genomic DNA of eukaryotic cells is organized into a nucleoprotein structure called chromatin. The fundamental building block of chromatin is the nucleosome core particle, which consists of 147 base pairs of DNA wrapped around an octamer of the histones H2A, H2B, H3, and H4. Nucleosomes can form an obstacle to processes requiring access to the DNA such as transcription. Covalent modifications of histones at particular residues can alter the properties of nucleosomes by both changing chromatin's compactness and accessibility and by specifying new interactions of histones with transcription factors. One of these modifications, histone H3 lysine 4 trimethylation (H3K4me3), has been shown to be a major conserved mark of chromatin at nucleosomes immediately downstream of promoters of transcribed genes in yeast, Drosophila, and mammals. Although nucleosomes carrying this modification are targeted by a number of transcription and chromatin regulators, its contribution to transcription remains mysterious. In baker's yeast, the mutation in the gene coding for the H3K4 methyltransferase, SET1, does not cause obvious transcription defects during vegetative growth, while in human cells, the functional study of this mark is hampered by the existence of several genes encoding H3K4 trimethyltransferases (Eissenberg, 2010; Ardehali, 2011 and references therein).
H3K4 methylation is introduced into nucleosomes by the type-2 histone lysine methyltransferases (KMT2s; Allis, 2007). The prototypic KMTs from Drosophila are Suppressor of variegation 3–9, Enhancer of zeste, and Trithorax (Trx), and they share similarity in their catalytic SET domain. Of these, only Trx is H3K4-specific, and has been proposed to be the major KMT2 in flies (Shilatifard, 2008; Eissenberg, 2010). Flies possess a second, Trithorax-related (Trr) protein, which functions in the regulation of hormone-response gene expression (Sedkov, 1999; Sedkov, 2003). The two Drosophila Trx relatives are related to the mammalian Mixed lineage leukaemia (Mll)1–4 KMT2s (Eissenberg, 2010). While some Mll complexes contain subunits found in the yeast Set1 complex (COMPASS), Trx/Mll relatives have also been shown to assemble into complexes that are unrelated to COMPASS and contain histone acetyltransferases. These findings suggest a functional diversification of Trx/Mll complexes. Surprisingly, genome-wide studies in flies and mammals revealed that Trx/Mll relatives do not localize to over 97% of H3K4me3 domains, including those adjacent to transcription start sites (TSSs) of most expressed genes. Recent studies led to the identification of two Set1 orthologues in humans and showed that they assemble into complexes similar to yCOMPASS; however, their contribution to overall H3K4me3 is still obscure. Since a Set1 homologue was not found in Drosophila, the role of Set1 versus Trx/Mll relatives in global, transcription-linked H3K4 trimethylation at promoters is still unclear in multicellular organisms (Ardehali, 2011).
This study reports the identification and characterization of a Set1 homologue from Drosophila, CG40351/dSet1. Proteomics and functional studies revealed that the dSet1 complex is identical in its composition to its human counterpart and has strong H3K4 trimethyltransferase activity towards recombinant nucleosomes. RNAi-mediated knockdown (KD) studies demonstrated that dSet1 is responsible for bulk H3K4 di- and trimethylation, while the KD of Trx or Trr had less pronounced effects on H3K4me2/3. dSet1 co-localizes with H3K4me3 and transcribing Pol II on polytene chromosome, and the loss of the dSet1-complex subunit, dCfp1, diminishes dSet1 and H3K4me3 at transcription puffs. The KD of dSet1 causes reduced mRNA levels at all tested genes, including heat shock (HS) genes. Live cell imaging studies also revealed that EGFP–dSet1 is rapidly recruited to the Hsp70 HS loci upon activation. Photobleaching/recovery assays demonstrated that EGFP–dSet1 is continuously exchanged at the activated hsp70 loci. Moreover, time course HS experiments showed that KD of dSet1 caused increased Pol II levels at the hsp70 promoter during extended HS periods. These data supports a model in which dSet1-dependent H3K4me3 regulates chromatin changes at promoter-proximal nucleosomes that positively influence the release of Pol II into productive elongation, thereby contributing to optimal mRNA levels (Ardehali, 2011).
The Drosophila dSet1 complex is a component of a conserved complex responsible for bulk H3K4me3. dSet1 is required for H3K4me3 at all transcribed genes tested and its loss leads to a reduction of transcription levels accompanied by the accumulation of Pol II at promoters, which occur during phases of maximal transcription up-regulation. These findings support a model in which dSet1-dependent H3K4me3 generates a chromatin architecture facilitating the passage of Pol II through promoter-proximal nucleosomes during multiple rounds of transcription (Ardehali, 2011).
To this date, the role of Trx/Mll versus Set1 orthologues in transcription-linked H3K4 methylation is not fully understood. These studies support that dSet1 and Trx/Mll relatives form distinct subfamilies that most likely have non-overlapping functions in H3K4 methylation. While it has been proposed that Trx functions in complexes similar to Set1 in flies (Eissenberg, 2010), this study along with other reports support that Set1 homologues have a broader role in transcription-linked H3K4 methylation (Petruk, 2001; Schwartz, 2010; Ardehali, 2011 and references therein).
Recent studies showed that Trx might even have a role in development independent of its KMT activity (Schwartz, 2010). A C-terminal cleavage product of Trx (Trx-Cter) containing the SET domain was found at sites lacking H3K4me3. Trx-Nter distributed over large domains of active PcG-target genes, which also lacked H3K4me3, but were acetylated at H3K27 (H3K27ac). H3K27ac is introduced by CBP, and its KAT activity depends on its association with Trx-Nter. This acetylation protects H3K27 from methylation by PcG complexes, suggesting that Trx can function in positive gene regulation independent of its KMT activity. Consistent with this model, the loss of the elongation factor, Kismet, abolished the association of Trx with chromatin, while global H3K4me3 levels were unaffected. In these studies, chromosomal H3K4me3 was also unchanged in trx mutants. These findings are consistent with observations that H3K4me3 nearly fully overlaps with dSet1 and depended at most sites on the dSet1 complex (Ardehali, 2011).
These studies and previous reports support that Trr has a more widespread role in H3K4 methylation compared with Trx (see Sedkov, 2003). The SET domain of Trr had robust monomethyltransferase activity in vitro, and it was observed that the KD of Trr affected H3K4me1. Since isolated SET domains of KMT2s are only capable of monomethylating H3K4 (Cosgrove, 2010), further functional studies on purified Trr complexes will be necessary to address whether this factor has limited H3K4 KMT activity. In summary, dSet1 complexes are likely to regulate H3K4me3 in most chromatin regions, including the promoters of the majority of active genes, while Trx and Trr might function in diverse complexes with primary roles in developmental gene regulation (Ardehali, 2011).
The characterization of the yeast and human Set1 complexes supported a functional and structural conservation between these complexes (Lee, 2005; Eissenberg, 2010). Proteomics studies indicate that this is also the case for the Drosophila Set1 complex, which is identical in its composition to the human complex. Functional similarities were observed between the Cfp1 subunit of Set1 complexes in metazoans. In dCfp1 mutants, dSet1 dissociated from chromatin and H3K4me3 levels on polytene chromosomes were severely reduced. In humans, Cfp1 is essential for the targeting of hCOMPASS to euchromatin (Tate, 2010), while yeast yCfp1p/Spp1p is required for the trimethyltransferase activity of yCOMPASS (Takahashi, 2009a, Chandrasekharan, 2010; Murton, 2010; Ardehali, 2011).
in vitro methyltransferase assays revealed that dSet1 complexes trimethylate H3K4 in recombinant nucleosomes. In vivo, H2B ubiquitination stimulates nucleosomal H3K4 trimethylation by Set1 (Takahashi, 2009; Eissenberg, 2010). The data suggests that H2Bub has no direct regulatory role on the dSet1 complex, which is consistent with studies reporting that H2Bub prevents the loss of H2A/H2B heterodimers by Pol II-dependent transcription in vivo (Takahashi, 2009a; Chandrasekharan, 2010). This might explain why the stable association of Set1 complexes with nucleosomes is less dependent on H2Bub in a transcription-independent context. In conclusion, the data supports that dCOMPASS and its counterparts in other eukaryotes are highly conserved at the structural and functional level (Ardehali, 2011).
The role of Set1 in transcription initiation versus elongation is still under debate (Eissenberg, 2010). It is well established that the 5′ end of the hsp genes are occupied by paused Pol II. The current data indicates that promoter occupancy by Pol II at the HS and many other loci does not depend on dSet1. Pol II levels were also mostly unchanged at transcription puffs in dCfp1 mutants, and the KD of dSet1 did not cause a drop in Pol II levels at the activated Hsp70 gene. Furthermore, dSet1 and H3K4me3 levels increased at hsp loci after their activation, further supporting that dSet1 accumulation positively correlates with transcriptional activity. In fact, dSet1-dependent H3K4me3 appears to directly correlate with gene expression levels, and the KD of dSet1 has stronger impact on highly expressed genes such as α-tubulin (84B), sda, R, or the hsp loci. Interestingly, the hsp26, hsp70, and hsp83 mRNA accumulation defects occurred in dSet1-depleted cells during phases of maximal up-regulation between 10 and 20 min post-HS. Recent genome-wide studies on the role of Ash2, a subunit of the dSet1 complex, revealed that Ash2-dependent H3K4me3 was most critical for the expression of strongly expressed genes (Perez-Lluch, 2011), which is fully consistent with these studies (Ardehali, 2011).
The retention of Pol II at the hsp70 promoter in dSet1-depleted cells is accompanied with a decrease in Pol II density in the body of the gene. This suggests that dSet1 might have a role in the release of Pol II into productive elongation, which has been shown to be the rate-limiting step in transcription. This appears to be different from yeast, in which the promoter occupancy of Pol II was reduced at the MET16 locus in cells lacking SET1. Given the relatively modest effect of dSet1 KD on the Pol II density across the body of Hsp70 gene, it is likely that dSet1-dependent H3K4me3 has other functions in transcription; however, these roles must be restricted to promoter-proximal nucleosomes due to the accumulation of dSet1 in the 5′ regions of active genes. Considering that H3K4me3 is required for the recruitment of pre-mRNA processing and elongation factors to the 5′ regions of genes, it cannot be excluded that the observed hsp mRNA accumulation defects are due in part to the degradation of improperly processed pre-mRNAs. No major changes were observed in the phosphorylation states of Pol IIo along the axis of the activated hsp70 gene. This was not unexpected, since serine 5 phosphorylation and Pol II pausing at the hsp70 promoters precedes H3K4me3, supporting that this process is independent of H3K4me3 (Ardehali, 2011).
The data support a model in which dSet1-dependent H3K4me3 successively leads to the generation of a chromatin structure facilitating the repeated passage of Pol II through promoter-proximal chromatin. Since the release of Pol II into productive elongation is rate limiting, H3K4me3 might form a docking platform for other chromatin modifiers, which generate nucleosomes that are less refractive to Pol II passage. It has been shown that the nucleosomes at promoters of highly expressed genes are enriched for H3.3 and H2A.Z in hyperacetylated form. The candidate H3.3-exchange factor, CHD1, was shown to bind to H3K4-trimethylated nucleosomes, and H3.3 is the main target for H3K4 trimethylation at active genes. It is tempting to speculate that H3K4me3 might contribute to the generation of unstable nucleosomes containing H3.3 and H2A.Z, which would be consistent with the observed continuous exchange of dSet1 at the activated hsp70 puffs. On the other hand, it cannot rule out that histone H3K4me3-independent functions of the dSet1 complex are critical for its role in transcription (Ardehali, 2011).
Finally, dSet1-dependent H3K4me3 is likely to recruit factors that generate a chromatin architecture at promoters that is critical for optimal transcription levels. In fact, a number of transcription and chromatin regulators were confirmed to interact with nucleosomes trimethylated at H3K4 (Eissenberg, 2010); however, repressive complexes like the HDAC1 complexes were also found to be recruited by this mark. Future studies are necessary to identify other key regulators that interact with dSet1 complexes in the regulation of maximal transcription levels (Ardehali, 2011).
In eukaryotes, the post-translational addition of methyl groups to histone H3 lysine 4 (H3K4) plays key roles in maintenance and establishment of appropriate gene expression patterns and chromatin states. This study describes an essential locus within chromosome 3L centric heterochromatin that encodes the previously uncharacterized Drosophila ortholog (dSet1, CG40351) of the Set1 H3K4 histone methyltransferase (HMT). The results suggest that dSet1 acts as a 'global' or general H3K4 di- and trimethyl HMT in Drosophila. Levels of H3K4 di- and trimethylation are significantly reduced in dSet1 mutants during late larval and post-larval stages, but not in animals carrying mutations in genes encoding other well-characterized H3K4 HMTs such as trr, trx, and ash1. The latter results suggest that Trr, Trx, and Ash1 may play more specific roles in regulating key cellular targets and pathways and/or act as global H3K4 HMTs earlier in development. In yeast and mammalian cells, the HMT activity of Set1 proteins is mediated through an evolutionarily conserved protein complex known as Complex of Proteins Associated with Set1 (COMPASS). Biochemical evidence is presented that dSet1 interacts with members of a putative Drosophila COMPASS complex and genetic evidence is presented that these members are functionally required for H3K4 methylation. Taken together, these results suggest that dSet1 is responsible for the bulk of H3K4 di- and trimethylation throughout Drosophila development, thus providing a model system for better understanding the requirements for and functions of these modifications in metazoans (Hallson, 2012).
Post-translational modification of histones can alter the local chromatin environment and affect the recruitment of transcriptional regulatory machinery. These modifications can play diverse roles in transcriptional activation or silencing, and cross talk between different activating and silencing modifications may fine-tune levels of transcription (Hallson, 2012).
The post-translational addition of up to three methyl groups to histone H3 lysine 4 (H3K4) residues (H3K4me1, H3K4me2, and H3K4me3) correlates with active transcription. H3K4 di- and trimethylation is often enriched at the promoter and 5' coding regions of active genes, whereas H3K4 monomethylation is commonly found near the 3' ends of active genes and within enhancer elements. Although the mechanisms of methyl-H3K4-mediated transcriptional activation are not fully elucidated, trimethyl-H3K4 is thought to act as a docking scaffold for the recruitment of the transcription pre-initiation complex and transcriptionally activating chromatin-remodelling complexes (Hallson, 2012).
In the budding yeast, Saccharomyces cerevisiae, all mono-, di-, and trimethylation of H3K4 is catalyzed by the Set1 enzyme, and the enzymatic activity of Set1 is modulated through a multi-subunit protein complex known as the Complex of Proteins Associated with Set1 (COMPASS) (Miller, 2001; Roguev, 2001; Nagy, 2002; Dehe, 2005). COMPASS is evolutionarily conserved, with functional orthologs of Set1 acting as major H3K4 histone methyltransferases (HMTs) in metazoans (Lee, 2005; Lee, 2007; Simonet, 2007; Hallson, 2012 and references therein).
Higher metazoans possess additional H3K4 methylases, the mixed lineage leukemia (MLL) class of proteins, which act through distinct complexes similar to COMPASS (reviewed by Eissenberg, 2010). The MLL proteins (MLL1-5) are required at limited but important subsets of gene targets, such as homeotic and hormone response genes. H3K4 methylases identified in Drosophila melanogaster to date include Trx (homologous to MLL1-2), Trr (homologous to MLL3-4), and Ash1. Ash1 and Trx are members of the Trithorax group of proteins that antagonize Polycomb group-mediated gene silencing. In addition, Trx methylates H3K4 at heat-shock loci upon induction and appears to be required for mediating stress responses to heat stimuli. Trr is recruited to and required for H3K4 methylation at gene targets responsive to the insect nuclear hormone ecdysone. Although these HMTs are known to catalyze H3K4 methylation and are widely believed to act as the main global H3K4 methylases in Drosophila, the functional roles of the Drosophila ortholog of Set1 (dSet1) have remained undefined, largely because its location within centric heterochromatin makes genetic and molecular analysis particularly challenging (Hallson, 2012).
In efforts to functionally annotate essential heterochromatic genes in Drosophila, this study has linked dSet1/CG40351, the Drosophila set1 ortholog, to an essential genetic locus previously known as lethal 5 or l(3L)h5, residing in chromosome 3L centric heterochromatin (Marchant, 1988; Fitzpatrick, 2005). Surprisingly, dSet1, and not Trx, Trr, or Ash1, was shown to act as the main global H3K4 di- and trimethylase throughout Drosophila development. Genetic and molecular evidence is provided that Drosophila orthologs of other COMPASS members are required for H3K4 methylation and physically interact with dSet1. These findings establish a foundation for examining transcriptional regulatory mechanisms underlying this key post-translational modification (Hallson, 2012).
These results indicate that dSet1 acts as the main global H3K4 methylase throughout Drosophila development and is required for completion of late developmental stages. Although developmental roles of Set1 have been reported in Caenorhabditis elegans (Simonet, 2007), this is the first report that a Set1 ortholog is essential for the somatic development of a multicellular organism (Hallson, 2012).
A persisting maternal contribution of dSet1 mRNA or protein to the catalysis of bulk H3K4me2/me3 during early developmental stages cannot be ruled out. Indeed, RNA sequencing data available on FlyBase indicate that dSet1 is present in embryos aged 0-2 hr post egg lay, suggesting significant maternal loading of dSet1 transcripts. However, knocking down this maternal contribution during embryogenesis by expressing dSet1 RNAi in a dSet1 mutant background only slightly increases lethality at the L3 larval stage relative to dSet1 mutants. It is possible that, in addition to dSet1, other H3K4 methylases are responsible for 'early' bulk H3K4 methylation. Consistent with this, mutations in trr result in major losses of H3K4me2 and H3K4me3 levels during Drosophila embryogenesis (Sedkov, 2003). Moreover, bulk H3K4 dimethylation during C. elegans embryogenesis depends mostly on ASH-2, and not on SET-2 (the C. elegans dSet1 ortholog), suggesting that other H3K4 HMT players are involved (Xiao, 2011; Hallson, 2012 and references therein).
Trx and Trr, while apparently not required for bulk H3K4 methylation, may be important for transcriptional regulation of a subset of specific gene targets later in development. This would be consistent with their proposed role in human cells, where the Trr and Trx homologs MLL1-2 and MLL3-4 are thought to methylate H3K4 at a limited number of non-overlapping gene targets (Wang, 2009; Ansari, 2010; Eissenberg, 2010). Recent findings suggest that the predominant function of Ash1 is the catalysis of H3K36, and not H3K4 methylation (Hallson, 2012).
This study has shown that dSet1 interacts within the Drosophila COMPASS and has demonstrated the requirement of other Drosophila COMPASS members for H3K4 methylation, facilitating dissection of the functional roles of individual COMPASS members. Upon hcf and dWdr82 RNAi knockdown, only levels of H3K4me3 are reduced, an effect differing from that associated with loss of dSet1 function and suggesting specialized roles for Hcf and dWdr82 within the COMPASS. A nearly complete loss of H3K4me2 and H3K4me3 in ash2 mutants and dRbbp5 and wds RNAi knockdown animals suggests that these members are critical for COMPASS function; a loss of H3K4 monomethylation in these same animals is an effect not observed in dSet1 mutants and suggests distinct roles of dRbbp5, Wds, and Ash2 aside from their roles within the COMPASS (Hallson, 2012).
Although Set1 has been reported to target H3K4me1 in Saccharomyces cerevisiae, there have been no reports indicating that Set1 plays a similar role in metazoans, and no reductions were observed in monomethyl H3K4 in dSet1 mutants. The results also rule out the individual contributions of Ash1, Trr, or Trx to general H3K4 monomethylation. It has been reported that the human MLL/COMPASS complex subunits WDR5, Rbbp5, ASH2L, and DPY-30 form a complex (known as WRAD) that monomethylates recombinant histone H3 at lysine 4 in vitro (Patel, 2009; Patel, 2011). The involvement of a Drosophila form of WRAD in H3K4me1 seems plausible as ash2 mutations as well as wds and dRbbp5 RNAi knockdown result in decreased levels of H3K4me1. Alternatively, bulk H3K4 monomethylation may be catalyzed or targeted by an as-yet-uncharacterized H3K4 HMT complex containing these members or by combinatorial effects of Trr- and Trx-containing complexes (Hallson, 2012).
In summary, these results indicate that dSet1 acts through the COMPASS to promote global H3K4 di- and trimethylation and appears to be indispensable during Drosophila development. Another study (Ardehali, 2011) has reported that dSet1 is associated within a COMPASS complex and is responsible for the majority of H3K4 methylation in Drosophila S2 tissue culture cells. Mohan (2011) has also recently reported on the central role of dSet1; moreover, that study has comprehensively characterized all three COMPASS complexes in Drosophila containing dSet1, Trx, or Trr and associated proteins (Mohan, 2011). This work provides a complementary analysis of dSet1 function in the context of whole-organism development and includes data on functional roles for other COMPASS members. These results lay the groundwork for studying mechanisms and functional roles of H3K4 methylation by the COMPASS and other HMTs in metazoans (Hallson, 2012).
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 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). This study reports the biochemical purification and molecular characterization of the Drosophila COMPASS family. A one-to-one similarity in subunit composition with their mammalian counterparts was observed, 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. This 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 (Mohan, 2011).
Modifications of histones and the protein machinery for the generation and removal of such modifications are highly conserved and are associated with processes such as transcription, replication, recombination, repair, and RNA processing. Histone H3K4 methylation, particularly trimethylation, has been mapped to transcription start sites in all eukaryotes tested and is generally believed to be a hallmark of active transcription. The H3K4 methylation machinery was first identified in yeast and named Set1/COMPASS. Six H3K4 methyltransferase complexes have been identified in humans, including SET1A/B, which are subunits of human COMPASS, and MLL1 to MLL4, which are found in COMPASS-like complexes (Mohan, 2011).
Although Trx and Trr were identified quite some time ago, their relative contributions to different states of overall H3K4 methylation were not known. Studies of human cells and Drosophila cells has shown that SET1 is the major contributor of H3K4 trimethylation levels in cell. During the preparation of the manuscript, a study of Drosophila also showed that dSet1, as a part of COMPASS, is responsible for the majority of H3K4 di- and trimethylation (Ardehali, 2011), which is in line with the findings presented in this study. These findings suggest that dSet1 could be responsible for the deposition of H3K4 trimethylation at the transcription start sites of the most actively transcribed genes as a consequence of postinitiation recruitment via the PAF complex (Smith, 2010: see Recruitment of histone-modifying activities by RNA Pol II). Trx and Trr both show extensive distribution along polytene chromosomes, although neither protein is required for bulk levels of H3K4me3. Perhaps Trx and Trr implement H3K4 methylation in a more gene-specific manner, at distinct stages of transcriptional regulation, or alternatively, have other substrates or functions (Mohan, 2011).
These biochemical studies have demonstrated that the Drosophila complexes are very similar to their mammalian counterparts in subunit composition. These studies have also demonstrated the utility of a baculovirus superinfection system for expressing proteins in Drosophila cells. Large-scale transient transfections offer several potential advantages over generating clonal stable cell lines, one of which is that the overexpression of some proteins could be toxic to cells. This can be a problem even when using inducible promoters, such as the Mtn promoter, due to leaky expression under uninduced conditions. Moreover, the baculovirus infection and expression strategy took about 3 weeks from the cloning of the cDNA into the viral vector, generating the virus, infection of S2 cells, and purification of the complexes from nuclear extracts. In contrast, conventional cloning took 4 months from cloning the cDNA into the vector to generating and characterizing the clonal cell lines. FLAG-HA-dWDR82 was purified from both stably transfected S2 cells and from the superinfection system and both strategies yielded a strikingly similar enrichment of target proteins (Mohan, 2011).
All of the COMPASS family members in Drosophila have several common subunits, namely, Ash2, Rbbp5, Wdr5, and Dpy30, which are homologs of CPS60, CPS50, CPS30, and CPS25, respectively, as well as each having complex-specific subunits. Many of these subunits have established, conserved roles in both the yeast and mammalian complexes: ASH2L is required for proper H3K4 trimethylation, as is CPS60 in yeast; both WDR5 in humans and CPS30 in yeast are required for the mono-, di-, and trimethylation of H3K4, and each is required for proper formation of the COMPASS and MLL complexes. Conservation of this degree in the H3K4 methylation machinery suggests that Drosophila might have similar machinery. However, it had previously been reported that Trx forms a complex with CBP and SBF, but no corresponding complexes have been found in mammals (Mohan, 2011).
The demonstration of the presence of shared components between COMPASS and COMPASS-like complexes in Drosophila supports the findings that these proteins are required for the proper functional architecture critical for the methylation of H3K4. The complex-specific components found in association with the dSet1, Trx, and Trr complexes further demonstrate a one-to-one correspondence of subunits between the Drosophila and human COMPASS family members that will allow the use of Drosophila as a model system for understanding the function of the human complexes. For example, while Set1/COMPASS is conserved from yeast to humans, it is possible that the metazoan complexes have additional functions needed for development. As the subunit compositions of both the SET1A and SET1B complexes are identical, it is likely that their functional analysis would be hindered by redundancy between the two complexes. The presence of a single dSet1 complex in flies may serve as an excellent starting point to dissect the metazoan-specific functions of the SET1 complexes (Mohan, 2011).
MLL-related proteins are multidomain proteins with the capacity to bind to many other proteins that may modulate their function. For example, Menin binds to the extreme N terminus of MLL1/2 and is required for proper targeting of the MLL1/2 complex to chromatin. Owing to its conserved components and interactions, but nonredundant nature, investigation of the Drosophila Trx complex promises to aid in understanding of the MLL1 and MLL2 complexes, specifically in their role in development (Mohan, 2011).
Currently there is very limited understanding of the functions of the various domains within the MLL3/4 proteins. The identification of LPT, which is homologous to the N terminus of MLL3/4, as a component of the Trr complex indicates the importance of PHD fingers residing in the LPT protein for the proper functioning and/or targeting of the Trr complex to chromatin. This separation of the MLL3/4 protein in Drosophila as Trr and LPT could allow dissection of the functions of N and C termini. Various studies have identified mutations in MLL3, MLL4, and UTX in a variety of cancers. Therefore, studies of the LPT-Trr complex could improve understanding of the targeting and regulation of these complexes with relevance to human disease (Mohan, 2011).
Importantly, Drosophila has a single representative of each class of COMPASS family members found in mammals, in which two representatives of each complex exist. In contrast, nematodes, such as the genetically tractable C. elegans, contain only a Set1 and MLL3/4-related protein, but no MLL1/2 representative. Given the power of genetic manipulation, the identification of the COMPASS, Trx, and Trr complexes in Drosophila that share similar subunits with their mammalian counterparts will greatly facilitate an understanding of the biological functions of the H3K4 methylation machinery in development and differentiation (Mohan, 2011).
The stimulation of trimethylation of histone H3 Lys4 (H3K4) by H2B monoubiquitination (H2Bub) has been widely studied, with multiple mechanisms having been proposed for this form of histone cross-talk. Cps35/Swd2 within COMPASS (complex of proteins associated with Set1) is considered to bridge these different processes. However, a truncated form of Set1 (762-Set1) is reported to function in H3K4 trimethylation (H3K4me3) without interacting with Cps35/Swd2, and such cross-talk is attributed to the n-SET domain of Set1 and its interaction with the Cps40/Spp1 subunit of COMPASS. This study used biochemical, structural, in vivo, and chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) approaches to demonstrate that Cps40/Spp1 and the n-SET domain of Set1 are required for the stability of Set1 and not the cross-talk. Furthermore, the apparent wild-type levels of H3K4me3 in the 762-Set1 strain are due to the rogue methylase activity of this mutant, resulting in the mislocalization of H3K4me3 from the promoter-proximal regions to the gene bodies and intergenic regions. Detailed screens were performed, and yeast strains were identified lacking H2Bub but containing intact H2Bub enzymes that have normal levels of H3K4me3, suggesting that monoubiquitination may not directly stimulate COMPASS but rather works in the context of the PAF and Rad6/Bre1 complexes. This study demonstrates that the monoubiquitination machinery and Cps35/Swd2 function to focus COMPASS's H3K4me3 activity at promoter-proximal regions in a context-dependent manner (Thorton, 2014).
Search PubMed for articles about Drosophila Set1
Allis, C. D., et al. (2007) New nomenclature for chromatin-modifying enzymes. Cell 131: 633-636. PubMed ID: 18022353
Ansari, K. I. and Mandal, S. S. (2010) Mixed lineage leukemia: roles in gene expression, hormone signalling and mRNA processing. FEBS J. 277: 1790-1804. PubMed ID: 20236313
Ardehali, M. B., et al. (2011). Drosophila Set1 is the major histone H3 lysine 4 trimethyltransferase with role in transcription. EMBO J. 30: 2817-28. PubMed ID: 21694722
Chandrasekharan, M. B., et al. (2010). Histone H2B C-terminal helix mediates trans-histone H3K4 methylation independent of H2B ubiquitination. Mol. Cell Biol. 30(13): 3216-32. PubMed ID: 20439497
Cosgrove, M. S. and Patel, A. (2010). Mixed lineage leukemia: a structure-function perspective of the MLL1 protein. FEBS J. 277: 1832-1842. PubMed ID: 20236310
Dehe, P.-M., et al. (2005). Histone H3 lysine 4 mono-methylation does not require ubiquination of histone H2B. J. Mol. Biol. 353(3): 477-484. PubMed ID: 16185711
Eissenberg, J. C., et al. (2007). The trithorax-group gene in Drosophila little imaginal discs encodes a trimethylated histone H3 Lys4 demethylase. Nat. Struct. Mol. Biol. 14: 344-346. PubMed ID: 17351630
Eissenberg, J. C. and Shilatifard, A. (2010). Histone H3 lysine 4 (H3K4) methylation in development and differentiation. Dev. Biol. 339: 240-249. PubMed ID: 19703438
Fitzpatrick K. A., et al. (2005). A genetic and molecular profile of third chromosome centric heterochromatin in Drosophila melanogaster. Genome 48(4): 571-584. PubMed ID: 16094423
Hallson, G., et al. (2012). dSet1 is the main H3K4 di- and tri-methyltransferase throughout Drosophila development. Genetics 190(1): 91-100. PubMed ID: 22048023
Lee, J. H., and Skalnik, D. G. (2005). CpG-binding protein (CXXC finger protein 1) is a component of the mammalian Set1 histone H3-Lys4 methyltransferase complex, the analogue of the yeast Set1/COMPASS complex. J. Biol. Chem. 280: 41725-41731. PubMed ID: 16253997
Lee, J. H., Tate, C. M., You, J. S. and Skalnik, D. G. (2007). Identification and characterization of the human Set1B histone H3-Lys4 methyltransferase complex. J. Biol. Chem. 282(18): 13419-13428. PubMed ID: 17355966
Marchant G. E. and Holm D. G. (1988) Genetic analysis of the heterochromatin of chromosome 3 in Drosophila melanogaster. II. Vital loci identified through EMS mutagenesis. Genetics 120: 519-532. PubMed ID: 17246481
Miller, T., et al. (2001). COMPASS: a complex of proteins associated with a trithorax-related SET domain protein. Proc. Natl. Acad. Sci. 98(23): 12902-12907. PubMed ID: 11687631
Mohan, M., et al. (2011). The COMPASS family of H3K4 methylases in Drosophila. Mol. Cell. Biol. 31(21): 4310-8. PubMed ID: 21875999
Nagy, P. L., Griesenbeck, J., Kornberg, R. D. and Cleary, M. L. (2002). A trithorax-group complex purified from Sacchromyces cerevisiae is required for methylation of histone H3. Proc. Natl. Acad. Sci. 99(1): 90-94. PubMed ID: 11752412
Patel, A., Dharmarjan, V., Vought, V. E. and Cosgrove M. S. (2009). On the mechanism of multiple lysine methylation by the human mixed lineage leukemia protein-(MLL1) complex. J. Biol. Chem. 284(36): 24242-24256. PubMed ID: 19556245
Patel, A., Vought, V. E., Dharmarjan, V. and Cosgrove, M. S. (2011). A novel non-SET domain multi-subunit methyltransferase required for the sequential nucleosomal histone H3 methylation by the mixed lineage leukemia protein-1 (MLL1) core complex. J. Biol. Chem. 286(5): 3359-3369. PubMed ID: 21106533
Perez-Lluch, S., et al. (2011). Genome-wide chromatin occupancy analysis reveals a role for ASH2 in transcriptional pausing. Nucleic Acids Res. 39(11): 4628-39. PubMed ID: 21310711
Petruk, S., et al. (2001). Trithorax and dCBP acting in a complex to maintain expression of a homeotic gene. Science 294: 1331-1334. PubMed ID: 11701926
Roguev, A., et al. (2001). The Sacchromyces cerevisiae Set1 complex includes an Ash2 homologue and methylates histone H3 lysine 4. EMBO J. 20(24): 7137-7148. PubMed ID: 11742990
Schwartz, Y. B., et al.(2010) Alternative epigenetic chromatin states of polycomb target genes. PLoS Genet 6: e1000805. PubMed ID: 20062800
Sedkov, Y., et al. (1999). Molecular genetic analysis of the Drosophila trithorax-related gene which encodes a novel SET domain protein. Mech. Dev. 82: 171-179. PubMed ID: 10354481
Sedkov, Y., et al. (2003). Methylation at lysine 4 of histone H3 in ecdysone-dependent development of Drosophila. Nature 426: 78-83. PubMed ID: 14603321
Shilatifard, A. (2008). Molecular implementation and physiological roles for histone H3 lysine 4 (H3K4) methylation. Curr. Opin. Cell Biol. 20: 341-348. PubMed ID: 18508253
Simonet, T., Dulermo, R., Schott, S. and Palladino, F. (2007). Antagonistic functions of SET-2/SET1 and HPL/HP1 proteins in C. elegans. Dev. Biol. 312(1): 367-383. PubMed ID: 17967446
Smith E. and Shilatifard, A. (2010). The chromatin signaling pathway: diverse mechanisms of recruitment of histone-modifying enzymes and varied biological outcomes. Mol. Cell 40: 689-701. PubMed ID: 21145479
Takahashi, Y. H., et al. (2009a). Regulation of H3K4 trimethylation via Cps40 (Spp1) of COMPASS is monoubiquitination independent: implication for a Phe/Tyr switch by the catalytic domain of Set1. Mol. Cell Biol. 29: 3478-3486. PubMed ID: 19398585
Takahashi, Y. H. and Shilatifard, A. (2009b). Structural basis for H3K4 trimethylation by yeast Set1/COMPASS. Adv. Enzyme Regul. 50: 104-110. PubMed ID: 20005892
Tate, C. M., Lee, J. H. and Skalnik, D. G. (2010). CXXC finger protein 1 restricts the Setd1A histone H3K4 methyltransferase complex to euchromatin. FEBS J. 277: 210-223. PubMed ID: 19951360
Thornton, J. L., Westfield, G. H., Takahashi, Y. H., Cook, M., Gao, X., Woodfin, A. R., Lee, J. S., Morgan, M. A., Jackson, J., Smith, E. R., Couture, J. F., Skiniotis, G. and Shilatifard, A. (2014). Context dependency of Set1/COMPASS-mediated histone H3 Lys4 trimethylation. Genes Dev 28: 115-120. PubMed ID: 24402317
Wang, P., et al. (2009). Global analysis of H3K4 methylation defines MLL family member targets and points to a role for MLL1-mediated H3K4 methylation in the regulation of transcriptional initiation by RNA polymerase II. Mol. Cell. Biol. 29(22): 6074-6085. PubMed ID: 19703992
Xiao, Y., et al. (2011). Caenorhabditis elegans chromatin-associated proteins SET-2 and ASH-2 are differentially required for histone H3 Lys 4 methylation in embryos and adult germ cells. Proc. Natl. Acad. Sci. 108(20): 8305-8310. PubMed ID: 21527717
date revised: 10 February 2014
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