InteractiveFly: GeneBrief

Trithorax-related: Biological Overview | References

BIOLOGICAL OVERVIEW

Methylation of histone H3 lysine 4 (H3K4) in Saccharomyces cerevisiae is implemented by Set1/COMPASS (Complex Proteins Associated with Set1), which was originally purified based on the similarity of yeast Set1 to human MLL1 and Drosophila (Trx). While humans have six COMPASS family members, Drosophila has a representative of the three subclasses within COMPASS-like complexes: dSet1 (human SET1A/SET1B), Trx (human MLL1/2), and Trr (human MLL3/4), the subject of this web-site. This study reports the biochemical purification and molecular characterization of the Drosophila COMPASS family. A one-to-one similarity occurs in subunit composition with their mammalian counterparts, with the exception of (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).

Histone H3 lysine 4 methylation (H3K4me) is associated with the transcriptionally active regions of the genome in yeast, flies, and mammals. 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. In Drosophila, four SET domain-containing proteins, namely, Trithorax (Trx), Trithorax-related (Trr), dSet1, and Ash1, have been reported to implement H3K4 methylation. All but Ash1, which has subsequently been demonstrated to be an H3K36 methyltransferase, 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. 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). Trr was identified based on sequence similarity to Trx but was shown to function in the regulation of hormone-responsive gene expression (Sedkov, 2003). dSet1 was identified based on sequence homology to the Saccharomyces cerevisiae and mammalian Set1 proteins (Mohan, 2011).

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 (Mohan, 2010; Shilatifard, 2008; Smith, 2010). All of the mammalian COMPASS family of H3K4 methylases share ASH2L, RBBP5, DPY30, and WDR5 as common components. Analysis of the mammalian complexes allows classification into three classes based on unique components within each class: COMPASS, represented by SET1A and SET1B, contains WDR82 and CXXC1, proteins implicated in regulating trimethylation by yeast COMPASS; the MLL1/2 complexes contain Menin, implicated in targeting MLL1 to the Hox genes; the MLL3/4 complexes contain PTIP, PA-1, and NCOA6 (Cho, 2007), which are important for the gene-specific targeting of these complexes, and UTX, a histone H3K27 demethylase thought to be involved in counteracting PcG-mediated gene silencing (Eissenberg, 2010: Hughes, 2005; Lee, 2007; Mohan, 2011 and references therein).

This study purified and characterized the dSet1, Trx, and Trr complexes. In contrast to a previous report that Trx formed a heterotrimeric complex with CBP and SBF1, this study found instead that Trx forms a COMPASS-like complex containing orthologs of all known components of the MLL1 complex in mammals. These studies also demonstrate that Drosophila Set1 is the major contributor to the bulk in vivo dimethylation and trimethylation of H3K4 and that this depends on a conserved form of histone cross talk, where monoubiquitinated H2B is required for H3K4 trimethylation by dSet1. It was also found that mammalian MLL3/4 are represented in flies by two genes, Trr and LPT, and that the encoded proteins exist together in a COMPASS-like Trr complex. Taken together, this evidence for the existence of one representative complex in Drosophila for each of the three classes of the six COMPASS family proteins in mammals provides a unique opportunity to discover the differences in the targeting and function of H3K4 methylation by these complexes (Mohan, 2011).

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).

Histone recognition and nuclear receptor co-activator functions of Drosophila Cara Mitad, a homolog of the N-terminal portion of mammalian MLL2 and MLL3

MLL2 and MLL3 histone lysine methyltransferases are conserved components of COMPASS-like co-activator complexes. In vertebrates, the paralogous MLL2 and MLL3 contain multiple domains required for epigenetic reading and writing of the histone code involved in hormone-stimulated gene programming, including receptor-binding motifs, SET methyltransferase, HMG and PHD domains. The genes encoding MLL2 and MLL3 arose from a common ancestor. Phylogenetic analyses reveal that the ancestral gene underwent a fission event in some Brachycera dipterans, including Drosophila species, creating two independent genes corresponding to the N- and C-terminal portions. In Drosophila, the C-terminal SET domain is encoded by trithorax-related (trr), which is required for hormone-dependent gene activation. This study identified the cara mitad (cmi, Lost PHDs of trr) gene, which encodes the previously undiscovered N-terminal region consisting of PHD and HMG domains and receptor-binding motifs. The cmi gene is essential and its functions are dosage sensitive. CMI associates with TRR, as well as the EcR-USP receptor, and is required for hormone-dependent transcription. Unexpectedly, although the CMI and MLL2 PHDf3 domains could bind histone H3, neither showed preference for trimethylated lysine 4. Genetic tests reveal that cmi is required for proper global trimethylation of H3K4 and that hormone-stimulated transcription requires chromatin binding by CMI, methylation of H3K4 by TRR and demethylation of H3K27 by the demethylase UTX. The evolutionary split of MLL2 into two distinct genes in Drosophila provides important insight into distinct epigenetic functions of conserved readers and writers of the histone code (Chauhan, 2012).

Nuclear receptors (NRs) function as transcription factors that respond to cellular signals to initiate new gene expression programs and have essential roles in embryonic development, growth and differentiation. NRs collaborate with greater than 300 co-factors that provide important enzymatic and regulatory functions. Co-factors can be activators or repressors and are typically recruited to gene promoters through associations with receptors (Bulynko, 2011). Some co-factors direct changes in the epigenetic environment of target genes by direct covalent chromatin modification or nucleosome remodeling. Co-activators are recruited in a ligand-dependent manner, whereas unliganded receptors often associate with co-repressors. Co-activators exist in large complexes required for the transcription of genes that are regulated by at least 48 vertebrate NRs, including retinoic acid receptor (RAR), liver-X-receptor (LXR), farnesoid-X-receptor (FXR), as well as a co-activator for p53. Disruptions of both NRs and their co-regulators have been linked to many cancers and developmental disorders (Chauhan, 2012).

Hormone signaling pathways in Drosophila melanogaster rely on two primary hormones, the steroid hormone 20-hydroxyecdysone (20HE) and sesquiterpenoid juvenile hormone (JH), and 18 receptors representing all major conserved nuclear receptor subfamilies. Drosophila Ecdysone Receptor (EcR) is an FXR/LXR ortholog, whereas its heterodimeric partner Ultraspiracle (USP) is an RXR ortholog (Chauhan, 2012).

Drosophila Trithorax-related (TRR) is a co-activator of EcR-USP. TRR is a histone lysine methyltransferase (HMT) that trimethylates histone 3 on lysine 4 (H3K4me3) and TRR functions are essential for activating ecdysone-regulated genes (Sedkov, 2003). TRR is closely related to another Drosophila protein, Trithorax (TRX), which regulates homeotic (Hox) gene expression through similar methyltransferase activity. The mammalian counterparts of TRR are MLL2 (also known as ALR or MLL4) and MLL3 (also known as HALR). MLL2 and MLL3 are enormous (5537 aa and 4911 aa, respectively), with multiple conserved domains, including histone methyltransferase (SET domain), five plant homeodomain (PHD) zinc fingers, an HMG-I binding motif, LXXLL NR binding motifs and FY-rich regions. Through the SET domain, both MLL2 and MLL3 directly methylate histone H3 to mediate transcription activation (Chauhan, 2012).

MLL2 and MLL3 are components of large SET1/COMPASS-like co-activator complexes that are required for NR-directed gene regulation. These complexes have important human disease connections, including developmental disorders and cancers. MLL2 and MLL3 are mutated in many Kabuki syndrome patients. MLL2 is frequently mutated in childhood medulloblastomas (14%), follicular lymphoma (89%) and diffuse large B-cell lymphoma (32%) (the two most common forms of non-Hodgkin lymphoma), suggesting that MLL2 and MLL3 COMPASS-like complex activities have important epigenetic gene regulatory roles that normally function to inhibit cancer progression (Chauhan, 2012).

Proteins that co-purify with the MLL2 include ASH2, RBBP5 (RBQ3), DPY30, WDR5, adaptor protein ASC2, PTIP, PA1 and histone demethylase UTX. Recently, Trithorax-related was found in Drosophila COMPASS-like complexes (Mohan, 2011). Despite functional similarities, TRR is much smaller than MLL2 or MLL3 with homology limited to the C-terminal SET domain portion (Sedkov, 2003). TRR lacks the N-terminal PHD and HMG domains that might contribute to chromatin binding. MLL2-related family members are always encoded by large single genes in species other than Brachycera dipterans. To further studies on epigenetic regulation of ecdysone target genes, Drosophila genes were sought that could encode a protein highly related to the N-terminal half of MLL2, and a single open reading frame (CG5591) was identified. The gene was named cara mitad (cmi; translated as 'dear half'). Although cmi is unlinked to trr in the genome, genetic studies using null mutants, in vivo depletion and overexpression revealed functions for cmi as a nuclear receptor co-factor necessary for hormone-regulated gene expression. Unexpectedly, the CMI type 3 PHD finger (PHDf3) was found to accommodate non-methylated, mono- and dimethylated H3K4, rather than trimethylated H3K4. Moreover, CMI-dependent activation also required demethylation functions of UTX, suggesting that NR-stimulated transcription involved at least three steps: binding of H3K4me1/2 by CMI, trimethylation of H3K4 by TRR and demethylation of H3K27 by UTX. The intriguing possibility that COMPASS-like functions in NR-directed transcription are associated with two independent proteins in flies suggests that recognition and binding to modified histones is a distinct step, separate from the epigenetic modification associated with other enzymes in the complex. This presents a unique opportunity to examine functions of histone recognition/binding and covalent histone tail lysine modifications as separate and essential features of NR-directed activation (Chauhan, 2012).

Although the precise roles of proteins directly participating in nuclear receptor signaling remain largely speculative, many are thought to regulate transcription through effects on chromatin. The MLL2 and MLL3 co-activators function to epigenetically decode or modify histone lysine residues and provide activation functions for NR signaling at target genes. In Drosophila, CMI and TRR together have a single MLL family homolog. This is the first example of an evolutionary 'splitting' of an epigenetic regulator involved in nuclear receptor signaling, whereby the essential gene regulatory functions of one protein have been parsed into two distinct proteins. CMI forms complexes with TRR, associates directly with hormone receptors and interacts with other putative COMPASS-like components, suggesting that Drosophila contains a functional counterpart to the mammalian ASCOM-MLL2 nuclear receptor co-activator complex (Chauhan, 2012).

The MLL histone lysine methyltransferases (KMTs) can be divided into two conserved groups, the MLL1-MLL4(2) and MLL2(4)/ALR-MLL3/HALR subfamilies. Each MLL member is capable of forming related discrete complexes with several common components. The MLL-based complexes activate transcription in part through methyltransferase activity on histone H3 Lys4 residues within promoter-associated nucleosomes. There might be partial functional overlap between MLL2 and MLL3; however, they are not redundant with the MLL1-MLL4 subfamily. The SET-domain methyltransferase activity of the MLL proteins is essential for transcription activation through histone lysine methylation, but the precise biological role of PHD fingers remains somewhat elusive. Closely related PHDf3 fingers bind H3K4me3/2, the product of the methyltransferase activity. Within the context of a single protein, such as MLL1, the PHDf3 recognition and binding of H3K4me3 is required for transcription activation of target genes (Chang, 2010; Chauhan, 2012).

The findings that CMI and TRR function coordinately in a COMPASS-like complex suggest that cmi and trr probably split from a common ancestor. Gene-protein fusions are four times more common than fissions, perhaps reflecting a simpler genetic event. In cases in which fissions occur, it has been suggested that many involve subunits of multimeric complexes in which the two independent proteins interact physically. The process of splitting into two independent genes might involve gene duplication with subsequent partial degeneration, as has been observed in the monkey king (mkg) gene family in Drosophila (Chauhan, 2012).

The notion that a large protein contains domains that function both together and independently is not without precedent. TRX and MLL1 are cleaved by a specific protease, taspase-1. The two 'halves' interact with each other in a functional complex, but there is evidence that the N-terminal TRX peptide (TRX-N) binds chromatin without its TRX-C partner in transcribed regions of Hox genes (Schuettengruber, 2009; Schwartz, 2010). Transcription factor TFIIA and herpes simplex virus host cell factor (HCF1) are cleaved during maturation, with both halves necessary for a functional product. There is presently no evidence that MLL2 or MLL3 are cleaved or processed (Chauhan, 2012).

An important question is whether both the chromatin-binding and methyltransferase functions of the MLL family are required for transcription activation. The data indicate that depletion of trr can suppress the effects of overexpressing cmi, suggesting that the activation potential of CMI depends on TRR methyltransferase activity. Similarly, simultaneous depletion of cmi and trr produces stronger phenotypes than depletion of either alone, indicative of cooperation on similar gene targets. Moreover, in vivo depletion of cmi results in reduced global H3 trimethylation, despite a functional trr gene (Chauhan, 2012).

Phenotypes associated with changes in CMI levels reveal important functions in hormone-regulated development. The larval defects in molting, morphogenetic furrow progression and necrosis associated with a cmi null allele, similar to trr, are consistent with impaired hormone signaling. Similarly, depletion of MLL2 in HeLa cells using siRNA led to reduced expression of genes known to be important for development and trimethylation of H3K4 was reduced at some promoters. Knockdown of MLL2 in MCF-7 cells impaired estrogen receptor (ERα) transcription activity and inhibited estrogen-dependent growth. Inactivation of the murine Mll3 resulted in stunted growth and reduced PPARγ-dependent adipogenesis with increased insulin sensitivity (Lee, 2008). Perhaps reflecting synonymous functions in Drosophila, cmi/CG5591 was found to be important for regulating muscle triglyceride levels, suggesting conserved adipogenic functions (Pospisilik, 2010). Furthermore, CG5591 (cmi) is involved in phagocytosis (Stroschein-Stevenson, 2006) and regulation of caspase functions in response to cellular stress (Yi, 2007), implicating cmi in immune-cell regulation. The increased hemocyte number associated with elevated CMI suggests functions in hemocyte development, perhaps as an effector of chromatin remodeling or signaling (JAK/STAT, Hedgehog, Notch) pathways. It was previously shown that trr was important for Hedgehog (HH)-dependent signaling during eye development (Sedkov, 2003) and cmi overexpression and depletion data are consistent with that possibility. However, the dosage-dependent cmi wing phenotypes are not consistent with changes in HH signaling, raising the possibility that cmi and trr are important for other growth and signaling pathways in wing development, including Decapentaplegic (DPP/TGFβ) and Wingless (WG/WNT) pathways (Chauhan, 2012).

Several steps are involved in activation of hormone-responsive target genes, including methylation of H3K4 by the MLL2-MLL3 COMPASS-like complex and displacement of demethylases (Vicent, 2011). Reduced cmi function resulted in lower hormone-responsive enhancer activation and genetic interactions between cmi, trr and Utx revealed that chromatin binding by CMI was important for gene activation in vivo. Furthermore, RNAi depletion of Utx suppressed HA-cmi overexpression wing phenotypes, suggesting that demethylation of H3K27 is a pre-requisite for activation of some hormone target genes. This is supported by genetic evidence from C. elegans that indicated both histone H3K4 methylation by SET-16 (MLL2/MLL3 ortholog) and H3K27 demethylation by UTX-1 were required for attenuation of RAS signaling in the vulva (Fisher, 2010; Li, 2011) and MLL2-MLL3 complex-related components were required for proper germ line development (Li, 2011). Genetic epistasis data reveals that Utx, trr and cmi functions are all required for activation in Drosophila (Chauhan, 2012).

Unexpectedly, the CMI PHDf3.b showed binding to mono- and dimethylated H3K4, rather than trimethylated H3K4 (Sanchez, 2011). Although CMI contains two PHDf3 domains in two clusters similar to MLL3, MLL2 contains one PHDf3 most closely related to the CMI and MLL3 PHDf3.b domains. The second cluster appears in all isoforms of MLL3, whereas the N-terminal 'a' cluster is optional. Additionally, the 'b' cluster is more closely related to the PHD cluster found in other MLL family proteins. PHD modules are thought to bind histones and present tail residues to the modifying enzyme subunits or stabilize those enzymes with their substrates. Recently, RNAi knockdown of trr in S2 cells was shown to affect H3K4 mono-, di-, and trimethylation, revealing widespread functions in regulating methylation in vivo (Ardehali, 2011) and suggesting that loss of TRR might destabilize the co-activator complex leading to de-protection of H3K4 methylation. One possibility is that CMI binds mono- and dimethylated H3K4 to prevent demethylation and stabilize TRR to allow for hormone-stimulated methylation and gene activation. CMI might disengage to allow for removal of methylation marks as hormone levels decrease and gene transcription is reduced. In contrast to MLL1-TRX function in maintenance of active gene transcription, CMI and TRR might be required for NR-targeted gene activation in response to temporally restricted hormone-dependent genome reprogramming (Chauhan, 2012).

The MLL3/MLL4 branch of the COMPASS family is a major H3K4 monomethylase at enhancers

Histone H3 lysine 4 (H3K4) can be mono-, di-, and trimethylated by members of the COMPASS (COMplex of Proteins ASsociated with Set1) family from yeast to human and these modifications can be found at distinct regions of the genome. Monomethylation of histone H3K4 (H3K4me1) is relatively more enriched at metazoan enhancer regions compared to trimethylated histone H3K4 (H3K4me3), which are found at transcription start sites in all eukaryotes. Recent studies in Drosophila demonstrated that the Trithorax-related (Trr) branch of the COMPASS family regulates enhancer activity and is responsible for the implementation of H3K4me1 at these regions. There are six COMPASS family members in mammals, two of which, MLL3 and MLL4, are most closely related to Drosophila Trr. This study used ChIP-seq of this class of COMPASS family members in both human HCT116 cells and mouse embryonic stem cells and found that MLL4 is preferentially found at enhancer regions. MLL3 and MLL4 are frequently mutated in cancer, and indeed, the widely used HCT116 cancer cell line contains inactivating mutations in the MLL3 gene. Using HCT116 cells in which MLL4 has also been knocked out, it was demonstrated that MLL3 and MLL4 are major regulators of H3K4me1 in these cells, with the greatest loss of monomethylation at enhancer regions. Moreover, a redundant role was found between Mll3 and Mll4 in enhancer H3K4 monomethylation in mouse embryonic fibroblast (MEF) cells. These findings suggest that mammalian MLL3/MLL4 function in the regulation of enhancer activity and enhancer-promoter communication during gene expression and that mutations of MLL3 and MLL4 found in cancer could exert their properties through enhancer malfunction (Hu, 2013).

Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing

Trithorax (Trx) antagonizes epigenetic silencing by Polycomb group (PcG) proteins, stimulates enhancer-dependent transcription, and establishes a 'cellular memory' of active transcription of PcG-regulated genes. The mechanisms underlying these Trx functions remain largely unknown, but are presumed to involve its histone H3K4 methyltransferase activity. This study report that the SET domains of Trx and Trx-related (Trr) have robust histone H3K4 monomethyltransferase activity in vitro and that Tyr3701 of Trx and Tyr2404 of Trr prevent them from being trimethyltransferases. The trxZ11 missense mutation (G3601S), which abolishes H3K4 methyltransferase activity in vitro, reduces the H3 H3K4me1 but not the H3K4me3 level in vivo. trxZ11 also suppresses the impaired silencing phenotypes of the Pc3 mutant, suggesting that H3K4me1 is involved in antagonizing Polycomb silencing. Polycomb silencing is also antagonized by Trx-dependent H3K27 acetylation by CREB-binding protein (CBP). Perturbation of Polycomb silencing by Trx overexpression requires CBP. It was also shown that Trx and Trr are each physically associated with CBP in vivo, that Trx binds directly to the CBP KIX domain, and that the chromatin binding patterns of Trx and Trr are highly correlated with CBP and H3K4me1 genome-wide. In vitro acetylation of H3K27 by CBP is enhanced on K4me1-containing H3 substrates, and independently altering the H3K4me1 level in vivo, via the H3K4 demethylase LSD1, produces concordant changes in H3K27ac. These data indicate that the catalytic activities of Trx and CBP are physically coupled and suggest that both activities play roles in antagonizing Polycomb silencing, stimulating enhancer activity and cellular memory (Tie, 2014).

The major findings presented in this study are: (1) TRX and TRR are monomethyltransferases and together account for the bulk of the H3K4me1 in vivo; (2) the catalytic activities of both TRX and CBP are required to antagonize PcG silencing; (3) TRX and TRR are physically associated with CBP in vivo and TRX binds directly to the CBP KIX domain via a region that contains multiple KIX-binding motifs; (4) TRX and TRR colocalize genome-wide with H3K4me1 and CBP at PREs and enhancers; and (5) H3K4me1 enhances histone acetylation by CBP. Together, these data suggest that the primary target of TRX monomethyltransferase activity is not promoters but PREs and neighboring enhancers. They suggest a new model for how TRX antagonizes Polycomb silencing, stimulates active enhancers, and establishes a cellular memory of active transcription. This differs significantly from the previous view that TRX trimethylates H3K4 (Tie, 2014).

The evidence presented in this study indicates that the SET domains of TRX, TRR and their human orthologs possess intrinsic H3K4 monomethyltransferase activities and are prevented from being trimethyltransferases by the presence of the bulkier Tyr residue at their respective F/Y switch positions, as previously shown for MLL1. Although these data do not rule out the possibility that TRX and TRR complexes might have some H3K4 trimethylation activity in vivo in some chromatin contexts, the reduced H3K4me1 and apparently normal H3K4me3 levels in the catalytically inactive trx^Z11 and trr³ mutants strongly suggest that H3K4 monomethylation is the predominant activity of TRX and TRR in vivo. Moreover, the absence of detectable H3K4me1 in trr³; trx^Z11 double-mutant embryos suggests that they are the principal H3K4 monomethyltransferases in vivo, consistent with their genome-wide colocalization with H3K4me1 at PREs and enhancers. Suppression of Pc³ mutant phenotypes by trx^Z11 further suggests that the monomethyltransferase activity of TRX plays a role in antagonizing Polycomb silencing (Tie, 2014).

This study found that TRX and TRR are physically associated with CBP in embryo extracts, confirming a previous report for TRX. The direct binding of TRX to the CBP KIX domain and the genome-wide correlation of H3K27ac with H3K4me1 on active genes suggests that their activities are coupled in vivo. Consistent with this, TRX-CBP complexes pulled down from embryo extracts have both H3K4 monomethyltransferase and H3K27 acetyltransferase activities. Moreover, the impaired Polycomb silencing caused by TRX overexpression in vivo (which elevates both H3K4me1 and H3K27ac levels) requires CBP and presumably the TRX-CBP interaction. Mutating the CID will be required to show this conclusively. No direct interaction between CBP and the TRR C-terminus was found, but it has been previously reported that CBP interacts directly with the H3K27 demethylase UTX, which is another subunit of the TRR complex. Together, these data suggest that these direct interactions are required for TRX- and TRR-dependent H3K27 acetylation and further suggest that TRX and TRR complexes function by fundamentally similar mechanisms (Tie, 2014).

The enhanced in vitro acetylation of H3K27 on K4me1-containing recombinant H3 substrates suggests that H3K4me1 might be a preferred CBP substrate in vivo. Consistent with this, altering the H3K4me1 level in vivo by manipulating LSD1 causes concordant changes in H3K27ac in adults. Moreover, a genome-wide analysis of hundreds of bona fide enhancers in purified mesodermal cells from Drosophila embryos revealed that H3K27ac is not present on enhancers without H3K4me1, whereas H3K4me1 is present without H3K27ac prior to enhancer 'activation'. This suggests that the presence of H3K4me1 might be a prerequisite for the deposition of H3K27ac at enhancers. Interestingly, some of the catalytically inactive trx^Z11 mutants survive until the late pupal period and exhibit strong homeotic transformations. This suggests that TRX catalytic activity might be more important for stimulating enhancers that drive robust homeotic gene expression, whereas the physical association of TRX with CBP, which is intact in trx^Z11, is more important for preventing silencing of normally active PcG-regulated genes in the embryo (Tie, 2014).

H3K4me1 and CBP are part of a conserved chromatin 'signature' of enhancers and H3K27ac marks 'active' enhancers. The data strongly suggest that TRX and TRR are responsible both for the H3K4me1 on enhancers and, via their physical association with CBP, for the H3K27ac on active enhancers. Determining which H3K4me1 is TRX dependent will require ChIP-seq analysis of trx^Z11 mutant cells (Tie, 2014).

Like TRX, H3K4me1 and CBP are also present at PRE/TREs of both active and inactive genes, suggesting that PRE/TREs have a functional connection to enhancers. Functional analyses of the strong bxd PRE/TRE in vivo suggest that PRE/TREs are distinct from enhancers, do not possess enhancer activity, but can boost enhancer-dependent transcription in a TRX-dependent manner. A GAL4-TRX fusion protein tethered to a transgene reporter exhibits these same properties (Tie, 2014).

TRR was recently shown to occupy many presumed enhancers. This study has found that TRR binds more sites than TRX and also co-occupies most TRX binding sites genome-wide, including PRE/TREs. This raises the possibility that both TRX and TRR regulate many PcG-regulated genes, perhaps in different contexts or in response to different signals. The presence of UTX in the TRR complex suggests that TRR can facilitate switching of PcG-regulated genes from silent to active, whereas TRX might only be capable of maintaining the expression of genes activated prior to the onset of Polycomb silencing in the early embryo, or genes subsequently derepressed by the UTX activity associated with the TRR complex. This might explain the previously reported critical requirement for TRX in early embryogenesis (0-4 hours; i.e. prior to the onset of Polycomb silencing) for later robust expression of the homeotic genes in imaginal discs. Absence of TRX in 0- to 4-hour embryos cannot be compensated by its subsequent restoration. Further investigation will be required to determine whether and in what contexts there is functional collaboration or division of labor between TRX and TRR (Tie, 2014).

Although it is required continuously, the critical early requirement for TRX might provide an important clue to its function. This suggests that TRX and CBP, bound to PRE/TREs, might be required for de novo 'priming' of surrounding enhancers with H3K4me1 and H3K27ac in the early embryo, prior to the onset of Polycomb silencing and perhaps even prior to transcriptional activation of the zygotic genome (Tie, 2014).

There is little detectable H3K27me3 in 0- to 4-hour embryos, whereas the H3K27ac level is already high relative to later embryonic stages. H3K4me1 is already present during syncytial stages. It is speculated that before zygotic genome activation, TRX and CBP are constitutively bound to PRE/TREs and deposition of H3K4me1 and H3K27ac might initially be restricted to nucleosomes adjacent to PRE/TREs. Binding of activators to early-acting enhancers promotes spreading of H3K4me1 and H3K27ac from PRE/TREs across adjacent cis-regulatory regions to form broad domains, perhaps facilitated by interactions between activators and TRX/CBP complexes. Spreading of H3K27ac initially proceeds unchecked by H3K27me3, encompassing all surrounding enhancers, including those that will be 'activated' later (e.g. the imaginal disc enhancers) and protects them from subsequent deposition of H3K27me3 by PRC2 at the onset of Polycomb silencing. PcG-regulated genes that are not activated in the early embryo become subject to deposition/spreading of H3K27me3 in similar broad domains, blocking subsequent H3K27 acetylation. There might also be some active removal of pre-existing H3K27ac by PRC1/PRC2-associated RPD3. Subsequent activation requires removal of H3K27me3 by UTX, and thus might require TRR, which is also present at PRE/TREs and so is poised to respond to the binding of TRR-dependent activators, such as EcR (Tie, 2014).

Other functions of H3K4me1 and H3K27ac at PREs and enhancers are not yet understood, but they might (1) recruit H3K4me1 and H3K27ac 'readers' that further stimulate/maintain the active transcriptional state, (2) facilitate the targeting of enhancers to promoters and (3) perpetuate the broad domains of H3K4me1 and H3K27ac by enhancing their own deposition by TRX and CBP, as suggested by the enhancing effect of H3K4me1 on H3K27 acetylation in vitro. Perpetuation of the broad domains of H3K4me1 and possibly H3K27ac through replication and mitosis could also constitute the elusive cellular memory of past transcriptional activity (Tie, 2014).

Methylation at lysine 4 of histone H3 in ecdysone-dependent development of Drosophila

Steroid hormones fulfill important functions in animal development. In Drosophila, ecdysone triggers moulting and metamorphosis through its effects on gene expression. Ecdysone works by binding to a nuclear receptor, EcR, which heterodimerizes with the retinoid X receptor homologue Ultraspiracle. Both partners are required for binding to ligand or DNA. Like most DNA-binding transcription factors, nuclear receptors activate or repress gene expression by recruiting co-regulators, some of which function as chromatin-modifying complexes. For example, p160 class coactivators associate with histone acetyltransferases and arginine histone methyltransferases. The Trithorax-related gene of Drosophila encodes the SET domain protein TRR. This study reports that TRR is a histone methyltransferase capable of trimethylating lysine 4 of histone H3 (H3-K4). trr acts upstream of hedgehog (hh) in progression of the morphogenetic furrow, and is required for retinal differentiation. Mutations in trr interact in eye development with EcR, and EcR and TRR can be co-immunoprecipitated on ecdysone treatment. TRR, EcR and trimethylated H3-K4 are detected at the ecdysone-inducible promoters of hh and BR-C in cultured cells, and H3-K4 trimethylation at these promoters is decreased in embryos lacking a functional copy of trr. It is proposed that TRR functions as a coactivator of EcR by altering the chromatin structure at ecdysone-responsive promoters (Sedkov, 2003).

Molecular genetic analysis of the Drosophila trithorax-related gene which encodes a novel SET domain protein

The products of the trithorax and Polycomb groups genes maintain the activity and silence, respectively, of many developmental genes including genes of the homeotic complexes. This transcriptional regulation is likely to involve modification of chromatin structure. The cloning and characterization of a new gene, trithorax-related (trr), shares sequence similarities with members of both the trithorax and Polycomb groups. The trr transcript is 9.6 kb in length and is present throughout development. The Trr protein, as predicted from the nucleotide sequence of the open reading frame, is 2431 amino acids in length and contains a PHD finger-like domain and a SET domain, two highly conserved protein motifs found in several trithorax and Polycomb group proteins, and in modifiers of position effect variegation. Trr is most similar in sequence to the human ALR protein, suggesting that trr is a Drosophila homolog of the ALR. Trr is also highly homologous to Drosophila Trithorax protein and to its human homolog, ALL-1/HRX. However, preliminary genetic analysis of a trr null allele suggests that TRR protein may not be involved in regulation of homeotic genes (i.e. not a member of the trithorax or Polycomb groups) or in position effect variegation. Lack of zygotic trr activity, combined with a 50% reduction of maternal trr+ activity, results in embryonic lethality, with dead embryos displaying wild-type cuticle morphology (Sedkov, 1999).

Trr contains two stretches of basic residues that are putatitive nuclear localization sequences, consistent with the proposed Trr role as a nuclear protein involved in transcriptional regulation. There are several regions with an unusually high fraction of proline, glutamic acid, serine, aspartic acid and threonine residues. Such PEST sequences are characteristic of short-lived protein. The striking feature of the TRR protein is that it shares several domains of sequence similarity with ALR, Trithorax, and ALL-1, the vertebrate homolog of Trithorax. In addition to the C-terminal SET domain, Trr also contains a PHD finger-like domain termed ZNF. This Cys-His-rich cluster is located in the C-terminal portion of both Trr and its homolog ALR, and in the central region of Trithorax and Trithorax's homolog ALL-1. In fact, it is the differing positions of ZNF between [Trr and ALR] and [Trx and ALL-1] that place these two pairs in separate branches of the trx-G family. The TNF extends over 108 residues and shows 55% and 47% identity, respectively, to ALR and TRX. ALR, but not Trr, contains a second ZNF domain located at the N-terminus of the protein. In addition, database searches have identified a C. elegans protein of unknown function that also contains this novel N-terminal ALR domain (Sedkov, 1999).

Overall, Trr is most similar to ALR. The Trr and Alr SET domains (C-terminal in all these proteins) is 70% identical to ALR SET, as compared to only 45% identity with Trithorax SET. In addition to the SET domain and the ZNF domain, all four proteins (Trr, ALR, Trx and ALL-1) contain two relatively short homologous domains, which have previously been called ATT1 and ATT2. Trr and ALR also show a relatively higher degree of conservation in these two domains. Trr is substantially smaller than ALR, and does not contain conventional PHD fingers, which are found at the N-terminus of ALR (Sedkov, 1999).

TRR mRNA is uniformly distributed at the preblastoderm stage, suggesting the transcript is maternal. TRR transcripts are not expressed in ovarian stem cells, oogonia, or early cysts and are first detectable at stage 8 in the cytoplasm of nurse cells, which is consistent with the maternally provided RNAs. At stage 10 of oogenesis, TRR mRNA is seen in the anterior end of the oocyte, and is uniformally distributed later on. The maternal TRR mRNA is quite stable. At the germband extended stage, TRR mRNA is enriched in the mesoderm. During germband retraction, it is strongly expressed in the anterior and posterior midgut. At the germband retracted stage, the TRR transcript becomes less abundant and is mainly localized to the ventral nerve cord and the brain. In third instar larvae, TRR is strongly and almost ubiquitously expressed in all imaginal discs. A low level of TRR is expressed in salivary glands, but there is not detectable expression in larval brain and gut tissues (Sedkov, 1999).

Search PubMed for articles about Drosophila Trithorax-related

Ardehali M. B., et al. (2011). Drosophila Set1 is the major histone H3 lysine 4 trimethyltransferase with role in transcription. EMBO J. 30: 2817-2828. PubMed ID: 21694722

Bulynko Y. A. and O'Malley B. W. (2011). Nuclear receptor coactivators: Structural and functional biochemistry. Biochemistry 50: 313-328. PubMed ID: 21141906

Chang, P. Y., et al. (2010). Binding of the MLL PHD3 finger to histone H3K4me3 is required for MLL-dependent gene transcription. J. Mol. Biol. 400: 137-144. PubMed ID: 20452361

Chauhan, C., Zraly, C. B., Parilla, M., Diaz, M. O. and Dingwall. A. K. (2012). Histone recognition and nuclear receptor co-activator functions of Drosophila Cara Mitad, a homolog of the N-terminal portion of mammalian MLL2 and MLL3. Development 139(11): 1997-2008. PubMed ID: 22569554

Cho, Y. W., et al. (2007). PTIP associates with MLL3- and MLL4-containing histone H3 lysine 4 methyltransferase complex. J. Biol. Chem. 282: 20395-20406. PubMed ID: 17500065

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

Fisher K., et al. (2010). Methylation and demethylation activities of a C. elegans MLL-like complex attenuate RAS signalling. Dev. Biol. 341: 142153. PubMed ID: 20188723

Hu, D., Gao, X., Morgan, M. A., Herz, H. M., Smith, E. R. and Shilatifard, A. (2013). The MLL3/MLL4 branch of the COMPASS family is a major H3K4 monomethylase at enhancers. Mol Cell Biol. 33(23): 4745-54. PubMed ID: 24081332

Hughes, C. M., et al. (2004). Menin associates with a trithorax family histone methyltransferase complex and with the hoxc8 locus. Mol. Cell 13: 587-597. PubMed ID: 14992727

Lee S., et al. (2008). Activating signal cointegrator-2 is an essential adaptor to recruit histone H3 lysine 4 methyltransferases MLL3 and MLL4 to the liver X receptors. Mol. Endocrinol. 22: 1312-1319. PubMed ID: 18372346

Li T. and Kelly W. G. (2011). A role for Set1/MLL-related components in epigenetic regulation of the Caenorhabditis elegans germ line. PLoS Genet. 7: e1001349. PubMed ID: 21455483

Mohan, M., Lin C., Guest, E. and Shilatifard, A. (2010). Licensed to elongate: a molecular mechanism for MLL-based leukaemogenesis. Nat. Rev. Cancer 10: 721-728. PubMed ID: 20844554

Mohan M., et al. (2011). The COMPASS family of H3K4 methylases in Drosophila. Mol. Cell. Biol. 31: 4310-4318. PubMed ID: 21875999

Pospisilik J. A., et al. (2010). Drosophila genome-wide obesity screen reveals hedgehog as a determinant of brown versus white adipose cell fate. Cell 140: 148-160. PubMed ID: 20074523

Sanchez, R. and Zhou, M. M. (2011). The PHD finger: a versatile epigenome reader. Trends Biochem. Sci. 36: 364-372. PubMed ID: 21514168

Schuettengruber B., et al. (2009). Functional anatomy of polycomb and trithorax chromatin landscapes in Drosophila embryos. PLoS Biol. 7: e13. PubMed ID: 19143474

Schwartz Y. B., (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(1-2): 171-9. PubMed ID: 1035448

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

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

Stroschein-Stevenson S. L., et al. (2006). Identification of Drosophila gene products required for phagocytosis of Candida albicans. PLoS Biol. 4: e4. PubMed ID: 16336044

Tie, F., Banerjee, R., Saiakhova, A. R., Howard, B., Monteith, K. E., Scacheri, P. C., Cosgrove, M. S. and Harte, P. J. (2014). Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing. Development 141: 1129-1139. PubMed ID: 24550119

Vicent G. P., et al. (2011). Four enzymes cooperate to displace histone H1 during the first minute of hormonal gene activation. Genes Dev. 25: 845-862. PubMed ID: 21447625

Yi, C. H., et al. (2007). A genome-wide RNAi screen reveals multiple regulators of caspase activation. J. Cell Biol. 179: 619-626. PubMed ID: 17998402

date revised: 2 December 2022

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