BIOLOGICAL OVERVIEW

How do cells maintain or change gene expression during differentiation? Two genetically complex systems are involved: the Polycomb group (Pc-G) of proteins and the trithorax group (TRX-G). Each forms a large multiprotein complex to fulfill the functions of modifying gene expression. Molecular analysis of the components of these multiprotein complexes has resulted in an understanding of the role of chromatin structure in gene regulation. Furthering this understanding has been a comparison of homologies across species, from yeast to humans. Pc-G proteins cooperate to induce changes in chromatin that inactivate genes, while TRX-G proteins cooperate to modify chromatin structure to activate transcription.

Current understanding of the TRX-G stems from the intersection of classical genetics with the biochemistry of chromosomes. trithorax mutants mimic the homeotic transformations caused by mutations of homeotic genes such as Antennapedia and Ultrabithorax. trithorax mutants exhibit a transformation of posterior abdominal structures to more anterior structures. This interaction between trx and homeotic genes has lead to observations that trx is necessary to sustain homeotic gene expression past the gastrulation phase (Breen, 1991).

Chromatin is the constituent of nuclei and chromosomes that interacts with colorful dyes, when viewed with light microscopy. The main constituents of chromatin are DNA and histones, the proteins that organize the long DNA double helix into chromosomes. For many years, histologists have distinguished between two types of chromatin: the genetically active euchromatin and the genetically inactive heterochromatin. It is now clear that the Pc-G and TRX-G proteins are involved in the transition of DNA between activity and inactivity, and that this transition involves a change in the molecular makeup of chromatin. Various yeast proteins, part of a large protein complex, activate silenced yeast genes, in particular mating type genes.

Homology of structure implies homology of function. Discoveries of homology between organisms as diverse as yeast and man can be directly applied to understanding of function across all species. Many of the yeast proteins have both human and Drosophila counterparts. Just as the fly's TRX-G proteins activate transcription in Drosophila, the yeast and human proteins have homologous functions in their respective organisms. One protein of a large gene activating protein complex in yeast, the SWI/SNF complex (pronounced switch-sniff), is homologous to Drosophila brahma gene. The Brahma protein contains a bromo domain and a DNA-dependent ATPase/helicase domain. A second Drosophila protein, SNR1, has been found to be homologous to another subunit of the yeast SW1/SNF complex (Dingwall, 1995). Yet a third Drosophila protein (ISWI) is homologous to another subunit of SWI/SNF and known human member of the SWI/SNF family (Tsukiyama, 1995).

Nuclesomes constitute the principle histone multiprotein complex that make up the chromatin structure that organizes DNA into chromosomes. The biochemical action of the SWI/SNF complex (the disruption of nucleosomal structures) makes promoters available for activation by transcription factors. This chain of events has provided insight into the mechanisms by which TRX-G proteins activate transcription.

The Trithorax protein contains a zinc finger domain, homologous to many transcriptional activators, including a human homolog involved in oncogenic transformation in cases of acute leukemia. Polytene chromosomes are especially thick chromosomes in the fly's salivary glands in which the DNA has been duplicated a thousand times over. TRX protein can be immunolocalized on polytene chromosomes at 16 binding sites that overlap those occupied by Polycomb group proteins. This overlap suggests that Trithorax is involved in a multiprotein complex made up of both gene silencing (Polycomb) and activating (Trithorax) subunits. In the absence of certain members of the Pc-G (Enhancer of zeste or Posterior sex combs proteins), TRX protein becomes disassociated from its chromosomal binding site, providing further evidence of the likelihood that a large protein complex is involved in activating and repressing genes (Kuzin, 1994).

In these investigations, classical genetics and cell morphology have teamed with molecular genetics, cooperatively amassing information in an ongoing clarification of the roles of TRX-G genes. It has been established that there are two large protein complexes involving TRX-G genes, both of which activate genes, one activating inactive genes, and the other sustaining the activity of already activated genes. It is now also clear that transcription factors are not mere lumps of protein, sitting on promoters and activating transcripition by RNA polymerase, but rather are elements in a large array of proteins possessing enzymatic activity, able to use the energy of the cell to open chromatin structures or actively silence genes (Simon, 1995 and Orlando, 1995).

The COMPASS family of H3K4 Methylases in Drosophila

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 (Mohar, 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 (Mohar, 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 (Mohar, 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 (Mohar, 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 (Mohar, 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 (Mohar, 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 our understanding of the MLL1 and MLL2 complexes, specifically in their role in development (Mohar, 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 (Mohar, 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 (Mohar, 2011).

GENE STRUCTURE

The trithorax locus of Drosophila affects segment determination primarily in the thoracic region. Mutant flies show transformations of the third and, to a lesser extent, first thoracic segment toward the second thoracic segment; abdominal transformations also occur. Genetic evidence suggests that these effects are based on interactions between trx and genes of the bithorax complex and Antennapedia complex. Further, interactions between the maternal effect locus female sterile homeotic (fsh: coding for a combined pfkB family of carbohydrate kinase and Bromodomain protein) and trx have been observed. To aid in a molecular analysis of trx function, the locus has been cloned by a P-element transposon tagging approach. Five insertion mutations have been mapped within a region of about 10 kilobases; one of these mutations reverted coincident with the loss of the insertion. Transcription mapping suggests that two RNAs of about 12 and 15 kilobases are the major transcripts of the trx locus and that the transcription unit comprises a region of about 25 kilobases. Transcripts from the trx locus are distributed uniformly in early embryos, but at 14-16 hr after fertilization the ventral nerve cord contains a higher concentration of trx RNA than other regions of the embryo (Mozer, 1989).

The intron/exon structure of the trx gene and the large alternatively spliced TRX mRNAs are capable of encoding only two protein isoforms. TRX proteins differ only in a long Ser- and Gly-rich N-terminal region, encoded by exon III, present only in the larger trx isoform. Trithorax has a novel variant of the highly conserved nuclear receptor type of DNA binding domain (Stassen, 1995). Five different transcripts can be identified. Each one uses the same first exon. Transcript M has no second or third exon, and a short 3'UTR. Transcript ME also has no second or third exon, and a long 3'UTR. These two transcripts code for the low molecular weight protein isoform. Transcripts E1, E2 and L differ in their use of second and third exons as follows: E1 uses the second and not the third; E2 uses the third and not the second, and L uses both. E1and L, both using the third exon, code for the high molecular weight protein isoform, while E2 codes for the low (Sedkov, 1995 and Stassen, 1995).

Exons - eight, with alternative splicing of multiple 5' exons.

Bases in 3' UTR - There are two alternative 3'UTRs, one of 154 nucleotides and the second of 1578 nucleotides (Sedkov, 1994).

PROTEIN STRUCTURE

Amino Acids - 3358 and 3726 for the two isoforms (Stassen, 1995)

Structural Domains

The trithorax gene functions in segment determination in Drosophila through interaction with genes of the bithorax complex and Antennapedia complex. Genetic evidence suggests that trx may be considered a positive regulator of homeotic genes. Sequencing of cDNAs corresponding to the entire trx transcription unit reveal the existence of an unusually long open reading frame encoding 3759 amino acids. The main features of the predicted trx protein are several cysteine-rich regions that can be folded into zinc finger-like domains. Cysteine-rich portions expressed from trx cDNAs in Escherichia coli are capable of zinc binding in vitro, suggesting a possible function for the trx product as a metal-dependent DNA-binding protein. Analysis of trx mutant embryos with antibody to the Ultrabithorax gene product shows decreased staining in parasegment 6 of the ventral nerve cord of late embryos. However, expression of Ubx is not affected in embryos carrying the lethal mutation trxE3, in which one of the putative zinc finger-like domains of the Trx protein is deleted. This differential effect of the E3 mutation suggests that trx exhibits other function(s) besides those involved in the regulation of Ubx expression in the ventral nerve cord of the embryo (Mazo, 1990).

The smaller isoform of TRX lacks an N-terminal serine and glycine rich region of 368 residues. These residues are potential phosphorylation targets. There are four PEST residues found in proteins with short half lives. The DNA binding domain (DBD) found in residues 762-860 is a variant of that found in the nuclear receptor superfamily, and has high homology to Drosophila proteins Knirps, Knirps-like, Ecdysone receptor, Ultraspiracle, FtzF1 and Tailless, as well as retinoid-X receptors. The D box or second finger is involved in DNA binding-induced DBD dimerization The more N-terminal P Box (or first finger) contains three residues responsible for sequence specificity in DNA binding (Stassen, 1995).

The Trithorax protein has a C4HC3 zinc finger motif.

This C4HC3 motif has been identified in a variety of proteins including the Drosophila trithorax and its human homolog ALL-1 involved in oncogenic translocations in acute leukemias. This domain, whose proposed name is TTC (for Trithorax consensus), is more frequently referred to as PHD fingers (for plant homeodomain fingers). This domain is found in many transcriptional regulators or DNA-binding proteins. Interestingly, TTC/PHD was found in several bromo domain containing transcriptional adaptors including the E1A-binding p300 and the CREB-binding CBP proteins. In CBP, this domain does not appear to be involved in DNA, CREB or TFIIB binding. (Koken, 1995).

The cys-rich zinc finger central region is also present in Polycomb-like and RBP2, a retinoblastoma binding protein. TRX proteins terminate with another novel conserved domain. This tromo domain is shared with Drosophila Enhancer of zeste, Sup(var)3-9, and human and mouse ALL-1 proteins (Stassen, 1995).

The C-terminal tromodomain portion of the Trithorax protein (is part of a larger region previously shown to share extensive homology with a human protein (ALL-1/Hrx), implicated in acute leukemias. Molecular analysis of a Polycomb group protein, Enhancer of zeste, predicts a 760-amino-acid protein. A region of 116 amino acids near the E(z) carboxy terminus is 41.2% identical (68.4% similar) with a carboxy-terminal region of the Trithorax protein. Over this same 116 amino acids, E(z) and ALL-1/Hrx are 43.9% identical (68.4% similar). Otherwise, E(z) is not significantly similar to any previously described proteins. It is interesting that this region of sequence similarity is shared by two proteins with antagonistic functions; it may comprise a domain that interacts with a common target, either nucleic acid or protein. It is a region moderately enriched in both acidic and basic residues (Jones, 1993 and Stassen, 1995).

The C-terminal domain of Trx has been termed a tromodomain; the more generally accepted term for this is a SET domain (for Su(var)39, E(z) and trx, the three proteins in which domain was first identified). Trx has an atypical bromodomain between the third and fourth PHD Zn fingers; these Zn fingers are highly conserved domains in the middle of the protein. With regard to the antagonistic functions between Trx and E(z), some E(z) mutants enhance the trx phenotypes rather than repressing them (Manuel Diaz, personal communication to the editor of The Interactive Fly).

The D. virilis trithorax gene has been isolated and sequenced. It produces a set of transcripts similar to that of D. melanogaster, and it encodes a protein that shows sequence similarity in several domains which are also conserved in the human homolog, ALL-1/HRX (Tillib, 1995).

date revised: 2o September 2023

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