A yeast trithorax-group complex is required for methylation of histone H3

Histone methylation has emerged as an important mechanism for regulating the transcriptional accessibility of chromatin. Several methyltransferases have been shown to target histone amino-terminal tails and mark nucleosomes associated with either euchromatic or heterochromatic states. However, the biochemical machinery responsible for regulating histone methylation and integrating it with other cellular events has not been well characterized. The purification, molecular identification, and genetic and biochemical characterization of the Set1 protein complex is reported that is necessary for methylation of histone H3 at lysine residue 4 in Saccharomyces cerevisiae. The seven-member 363-kDa complex contains homologs of Drosophila proteins Ash2 and Trithorax and C. elegans protein DPY-30, which are implicated in the maintenance of Hox gene expression and regulation of X chromosome dosage compensation, respectively. Mutations of Set1 protein comparable to those that disrupt developmental function of its Drosophila homolog Trithorax abrogate histone methylation in yeast. These studies suggest that epigenetic regulation of developmental and sex-specific gene expression are species-specific readouts for a common chromatin remodeling machinery associated mechanistically with histone methylation (Nagy, 2001).

A subset of methyltransferases contains a highly conserved motif known as the SET domain. This motif was originally discovered in Drosophila proteins Suppressor of variegation 3-9, Enhancer-of-zeste and Trithorax, which are involved in variegated or developmental gene expression. Although a functional role for the SET domain was not immediately evident, limited homology with plant methyltransferases prompted the discovery that Su(var)3-9 and its Schizosaccharomyces pombe homolog CLR4 are capable of transferring a methyl group onto Lys-9 of histone H3. This methyl tag has subsequently been shown to mark nucleosomes that are associated with transcriptionally silenced genes in the Sch. pombe mating type locus, and the association of Su(var)3-9 homologs with the Rb corepressor complex suggests that Lys-9 methylation may also play a role in transcriptional repression of euchromatic genes (Nagy, 2001 and references therein).

Human SET domain-containing proteins such as MLL, a homolog of Drosophila Trithorax, NSD2, and NSD3 are essential for normal development and are also directly implicated in the pathogenesis of cancer. However, it remains to be determined what role these proteins serve in establishing and maintaining chromatin states and whether malfunction of their chromatin-modifying activity directly contributes to alterations in gene expression profiles leading to cancer. These efforts are likely to be compromised by the large number of SET domain proteins in mammalian cells, their possible functional redundancy, and their interactions with a variety of heterologous partners. Conversely, Saccharomyces cerevisiae contains only six SET-domain proteins. One of these is Set1p, which appears to play a role in silencing at telomeres and mating type loci as well as in transcriptional activation of DNA repair genes. Its SET domain is 44% identical to that of Drosophila Trithorax, the founding member of the trxG transcriptional regulators required for the maintenance of homeotic gene expression in both mammals and Drosophila (Nagy, 2001 and references therein).

To characterize the role of Set1p as potentially part of a multiprotein complex, a clarified crude extract of yeast was passed through a Superose 6 sizing column. Western blot analysis revealed a Set1p peak at 440 kDa, which is considerably greater than the predicted 124-kDa molecular mass of monomeric Set1p, suggesting that it was associated with additional proteins in vivo (Nagy, 2001).

Denaturing gel electrophoretic analysis of the purified Set1p complex revealed the presence of seven polypeptides. These were identified by mass spectrometry as Set1p and six previously uncharacterized proteins encoded by YLR015W(BRE2), YAR003W, YPL138C, YKL018W, YBR175W, and YDR469W. These Set1p-associated factors are hereafter referred to as Bre2p, Saf49p, Saf41p, Saf37p, Saf35p, and Saf19p, respectively. Their predicted molecular masses totaled 363 kDa, which is compatible with the apparent mass of the Set1p complex determined by gel-filtration chromatography. Coomassie blue-stained gels of the purified complex suggested that each component was present in stoichiometric amounts, although one or more of the components may be present in nonequimolar ratios. Antisera generated against four of the six components showed that they cofractionated with Set1p in crude extract. Genomewide two-hybrid studies identified interactions between Saf49p and Saf35p as well as between Bre2p and Saf19p. Taken together, these observations establish that Set1p is a component of a previously undescribed multiprotein complex (Nagy, 2001).

Sequence alignment and motif analysis has shown that Bre2p contains an SPRY domain, a motif of unknown function that is present in a wide variety of proteins. Bre2p is most closely related to Drosophila Ash2, a trxG gene product required for imaginal disc pattern formation. However, Bre2p lacks a highly conserved PHD finger, which is found in Ash2 orthologs of D. melanogaster, C. elegans, and Sch. pombe. Interestingly, another component of the Set1p complex, Saf41p, contains a PHD finger similar to that of Yng2p, a component of the NuA4 histone acetyltransferase complex and its human homolog, the Ing1 tumor suppressor protein. Thus, it is possible that Bre2p and Saf41p together constitute a bipartite functional homolog of Ash2. The smallest member of the complex, Saf19p, is an apparent ortholog of C. elegans DPY-30, which is required for X-chromosome dosage compensation in hermaphrodite worms. Although DPY-30 itself has a diffuse nuclear localization, it has been shown to be essential for the sex-specific association of other dosage compensation factors with the X chromosome. The mechanism of DPY-30 activity is unknown. Finally, the Set1p complex also contains three WD-repeat-containing proteins (Saf49p, Saf37p, and Saf35p) that contain no other recognizable motifs (Nagy, 2001).

With the exception of the WD-repeat-containing Saf37p, all complex components are nonessential for vegetative growth in either the haploid or diploid state. Homozygous diploid mutants sporulate with the exception of the set1 strain. Yeast strains singly deficient for the nonessential components display similar flocculation phenotypes and comparable sensitivity to formamide, chlorpromazine, and rapamycin. Cells lacking Saf41p, the PHD finger protein, consistently displayed a less severe phenotype under these conditions. Strains deficient for Set1p complex components grow on nonglucose carbon sources and are able to activate the HO promoter, in contrast to yeast mutant for Swi2, another trxG-related protein. Synthetic effects on growth are not observed for compound mutants deficient for two or more nonessential components. The observed epistasis suggests that the components function in a common genetic pathway and is consistent with their participation in a biochemical complex (Nagy, 2001).

In addition to a potential role in activating transcription, Set1p has also been implicated in silencing at telomeres and mating-type loci. Consistent with previous studies of set1 strains, yeast deficient for other complex components, with the exception of bre2, show various levels of derepression of a telomeric URA3 marker as evidenced by 5-fluoroorotic acid (FOA) sensitivity and normal growth on Ura- plates. In contrast, the bre2 strain displays enhanced FOA resistance and partial uracil auxotrophy. This phenotype likely reflects that BRE2 shares its promoter with PPR1, which codes for the activator of URA3. Thus, full-length disruption of BRE2 may lead to alterations of the PPR1 promoter and reduced levels of Ppr1p. Based on these observations, it is suggested that the observed telomeric silencing deficiencies in strains lacking Set1p complex components might be URA3-specific events reflecting compensatory changes at the PPR1-BRE2 promoter. This theory is supported by an inability to detect silencing defects by using an ADE2 telomeric marker. Although other yeast orthologs of trxG proteins, such as Swi/Snf, have been implicated in both activating and repressive transcriptional processes, it remains to be determined whether a role for the Set1p complex in silencing is direct or indirect (Nagy, 2001).

Despite extensive genetic studies, the biochemical functions of most SET domain proteins are unknown. Recently, the SET domain protein SUV39H1 was shown to be a histone H3-specific methyltransferase whose methylation of Lys-9 recruits heterochromatin protein HP1 in heterochromatic silencing and euchromatic gene repression. Unlike higher eukaryotes and Sch. pombe, S. cerevisiae has no ortholog of SUV39H1 and lacks methylation of histone H3 Lys-9. However, lysine methylation is observed at residue 4 within the amino-terminal tail of histone H3 in S. cerevisiae. Therefore, whether yeast deficient for members of the Set1p complex displayed alterations in histone H3 Lys-4 methylation was evaluated. Histones were purified from various mutant strains and examined by Western blot analysis using an antiserum specific for methyllysine-4 of histone H3. Complete loss of H3 Lys-4 methylation is observed in the set1 strain as well as strains deficient for WD-repeat proteins Saf49p and Saf35p. Greater than 90% decrease in Lys-4-specific methylation is observed in the bre2 and saf19 strains, versus an approximate 50% decrease in the strain deficient for PHD protein Saf41p. Relative preservation of histone methylation in the latter correlates with its milder phenotypic readout in genetic assays. Loss of histone H3 lys4 methylation in the set1 strain is rescued by Set1p, but not by mutant Set1p lacking the carboxyl-terminal SET domain (set11-899). H3 lys4 methylation is restored by a C-terminal portion of Set1p containing the SET domain and flanking sequences (set1762-1080). However, methylation is not restored by comparable constructs harboring mutations in SET domain residues that are conserved in plant methyltransferases and essential for methyltransferase activity of SUV39H1 (set1H1117R) or the embryonic function of Drosophila Trithorax (set1G951S). These results are consistent with a role for Set1p as a histone methyltransferase; however, methylation of nucleosomes or histone tails by the purified Set1p complex or recombinant Set1p in vitro has not been seen. This observation suggests that its activity is constrained by undefined enzymatic requirements or, less likely, that it plays an indirect role in histone methylation. Nevertheless, the data indicate that the Set1p complex is essential for Lys-4 methylation of histone H3 in S. cerevisiae and implicate the SET domain of Set1p (and its MLL homolog) as likely histone methyltransferases (Nagy, 2001).

These observations suggest a model in which Trithorax-like SET domain proteins exert their influence on chromatin through histone methylation, whereas Ash2-like trxG proteins apparently modulate this activity by working in conjunction with DPY-30-like molecules. Based on the physical association of Bre2p (Ash2-related) and Saf19p (DPY-30-related), and the genetic similarities of their mutant phenotypes, it is proposed that maintenance of Hox gene expression in D. melanogaster (Ash2-dependent) and regulation of X chromosome dosage compensation in C. elegans (DPY-30-dependent) are species-specific readouts for a common chromatin remodeling machinery. Besides causing XX-specific lethality, dpy-30 mutations in XO animals cause developmental delay, small body size, an inability to mate, and abnormal tail morphology. These phenotypes suggest a broader role for DYP-30, and more detailed study of Hox gene expression patterns is warranted in dpy-30 mutants (Nagy, 2001).

WD-repeat-containing proteins of the Set1p complex are likely to target its methyltransferase activity to substrate histone H3. This suggestion is based on (1) the complete loss of histone H3 Lys-4 methylation in strains deficient for WD-repeat proteins Saf49p and Saf35p; (2) the fact that these proteins physically interact in a two-hybrid screen; and (3) the structural similarity of Saf35p to Cac3p and Hat2p, two other seven-WD-repeat-containing proteins that bind histones and are components of other chromatin-modifying complexes. The role of Saf37p, a WD-repeat protein that is the only essential member of the complex, is uncertain, but it is present exclusively in elution fractions from the sizing column that contains Set1p. This finding argues against its participation in other complexes, or in functions unrelated to Set1p (Nagy, 2001).

A potential role for histone H3 Lys-4 methylation effected by the Set1p machinery is suggested by recent studies in Sch. pombe that associate this modification with transcriptionally competent euchromatic regions. It has also been shown that histone H3 Lys-4 methylation is conserved from yeast to humans. In this context, the presence of Ash2 and Trithorax homologs in a single complex and their requirement for histone methylation in vivo provide a molecular basis for their genetic interaction in Drosophila and suggest a mechanism for maintenance of developmental gene expression (Nagy, 2001).

The set 1 methyltransferase opposes ipl 1 aurora kinase functions in chromosome segregation

A balance in the activities of the Ipl1 Aurora kinase (see Drosophila Aurora B) and the Glc7 phosphatase is essential for normal chromosome segregation in yeast. This balance is modulated by the Set1 methyltransferase. Deletion of SET1 suppresses chromosome loss in ipl1-2 cells. Conversely, combination of SET1 and GLC7 mutations is lethal. Strikingly, these effects are independent of previously defined functions for Set1 in transcription initiation and histone H3 methylation. Set1 is required for methylation of conserved lysines in a kinetochore protein, Dam1. Biochemical and genetic experiments indicate that Dam1 methylation inhibits Ipl1-mediated phosphorylation of flanking serines. These studies demonstrate that Set1 has important, unexpected functions in mitosis. Moreover, these findings suggest that antagonism between lysine methylation and serine phosphorylation is a fundamental mechanism for controlling protein function (Zhang, 2005).

The data reveal unexpected functional connections between the Set1 methyltransferase and phosphorylation events governed by the Ipl1 kinase and the Glc7 phosphatase. Loss of Set1 suppresses chromosome segregation defects caused by the ipl1-2 allele and is synthetic lethal with the glc7-127 allele. The mitotic functions of Set1 require Bre2, Swd1, and Sdc1, indicating that Set1 functions in the context of the COMPASS complex to modulate Ipl1-Glc7 functions in chromosome segregation (Zhang, 2005).

Previous studies have revealed a role for Set1 and COMPASS (Complex Proteins Associated with Set1) in gene transcription that requires Paf1 and ubiquitylation of histone H2B at K123. However, the data demonstrate that deletion of PAF1 or mutation of H2B K123 cannot suppress ipl1-2. Therefore, the suppression of ipl1-2 upon deletion of SET1 is independent of COMPASS functions in transcription initiation and early elongation (Zhang, 2005).

Prior to these studies, histone H3 K4 was the only known substrate of Set1. However, loss of H3 K4 methylation is not likely the molecular basis for the observed genetic interactions between SET1, IPL1, and GLC7: (1) mutations in SET1, PAF1, or histone H2B K123 all globally diminish H3 K4 methylation, yet only SET1 deletion suppresses ipl1-2; (2) no correlation was found between the effects of deletion of other COMPASS components on H3 K4 methylation and suppression of ipl1-2; (3) mutation of H3 K4 to R suppresses ipl1-2 more weakly than does deletion or mutation of SET1, and the H3 K4R mutation is not synthetic lethal with glc7-127; (4) chromatin immunoprecipitation results indicate that little or no H3 K4 methylation occurs at centromeres in S. cerevisiae, consistent with the replacement of H3 with Cse4 in centromeric nucleosomes (Zhang, 2005).

Unlike centromeres in S. pombe and most other organisms, centromeres in S. cerevisiae are not flanked by heterochromatic repeat elements, and this yeast does not contain HP1-like proteins or Suv39 methyltransferases. H3 K9 is not methylated in S. cerevisiae, and mutations in H3 S10 do not affect chromosome segregation. Moreover, no evidence was found of global changes in phosphorylation of S10 in the absence of Set1. Therefore, the effects of Set1 loss on Ipl1 functions do not likely reflect indirect effects on modifications at S10 or K9 in H3. Rather, the results indicate that these effects are mediated through Set1-mediated methylation of at least one nonhistone substrate, Dam1 (Zhang, 2005).

How might Dam1 methylation at histone H2B K233 or K194 contribute to proper chromosome segregation? By analogy to the effects of histone methylation on the occurrence of other posttranslational modifications of the histones, K233 methylation might directly affect phosphorylation of neighboring serines. This model is consistent with the observation that Ipl1-mediated phosphorylation of methylated Dam1 peptides is inhibited in vitro, as well as genetic data that reveal functional connections between K233 and S232, S234, and S235. The data indicate that prevention of K233 methylation by set1Δ allows improved phosphorylation of Dam1 by the cripples ipl1-2 kinase, as reflected by suppression of the ipl1-2 phenotype, but might allow too much or too persistent phosphorylation by wild-type Ipl1. Conversely, the suppression of the lethality of the DAM1 K233A allele by flanking S to A mutations or by the ipl1-2 mutation (but not by S to D mutations) strongly suggests that negative effects associated with loss of Dam1 methylation can be countered by decreased phosphorylation at these sites. Several previous findings that indicate a balance in the phosphorylation and dephosphorylation of IPL1 and GLC7 substrates is essential for normal cell growth and chromosome segregation. These strongly suggest that the region between K194 and S235 is a critical module in Dam1 that is regulated by both phosphorylation and methylation (Zhang, 2005).

Plant Trithorax homologs

The CURLEY LEAF gene of Arabidopsis is necessary for stable repression of a floral homeotic gene (AGAMOUS) required to specify stamen and carpel identity in whorls 3 and 4 respectively. AGAMOUS is a MADS box protein (see Drosophila Serum response factor and MEF2). The CURLEY LEAF protein shows extensive homology to Enhancer of zeste, with three regions conserved between the two proteins. First, the C-terminus contains a 115 amino-acid region, the SET domain, previously recognized as a conserved region in the products of E(z), Trithorax and Su(var)3-9. Second, residues 655-720 of CLF show 46% identity with the region of E(Z) (residues 538-603) that are rich in cysteine residues, but with an arrangement unlike that of zinc fingers. Third, residues 270-317 of CLF contain seven cysteines with a similar spacing to a region of E(Z) (residues 321-367) containing five cysteines, and there is a small region of sequence similarity at the N termini of these regions (Goodrich, 1997).

The genes of the trithorax (trxG) and Polycomb groups (PcG) are best known for their regulatory functions in Drosophila, where they control homeotic gene expression. Plants and animals are thought to have evolved multicellularity independently. Although homeotic genes control organ identity in both animals and plants, their homeotic genes are unrelated. Despite this fact, several plant homeotic genes are negatively regulated by plant genes similar to the repressors from the animal PcG. However, plant-activating regulators of the trxG have not been characterized. Genetic, molecular, functional, and biochemical evidence is provided that an Arabidopsis gene, ATX1, which is similar to the Drosophila trx, regulates floral organ development. The effects are specific: structurally and functionally related flower homeotic genes are under different control. ATX1 is an epigenetic regulator with histone H3K4 methyltransferase activity. This is the first example of this kind of enzyme activity reported in plants, and, in contrast to the Drosophila and the yeast trithorax homologs, ATX1 can methylate in the absence of additional proteins. In its ability to methylate H3K4 as a recombinant protein, ATX1 is similar to the human homolog. It is concluded that ATX1 functions as an activator of homeotic genes, like Trithorax in animal systems. The histone methylating activity of the ATX1-SET domain argues that the molecular basis of these effects is the ability of ATX1 to modify chromatin structure. These results suggest a conservation of trxG function between the animal and plant kingdoms despite the different structural nature of their targets (Alvarez-Venegas, 2003).

ATX1 contains a SET domain belonging to the Trithorax family. Like its animal counterpart, ATX1 acts as a master regulator involved in pleiotropic functions and in diverse developmental processes. Loss of organ identity in atx1-1 mutants, like sepal cells displaying meristematic characteristics, conversion of petals into stamenoid structures, conversion of petal cells into cells with anther characters, and carpeloid stamens, may result from the compromised expression of several flower homeotic genes. The observed phenotypes, supported by RT-PCR and in situ hybridizations, suggest that wild-type ATX1 regulates the expression of homeotic genes with counteracting activities. The fact that ATX1, which is highly expressed in the vegetative tissues of the plant, does not lead to ectopic expression of its homeotic target genes and makes it likely that ATX1 is involved in maintaining an established active state rather than creating it. Other factors, present in the inflorescence but not vegetative tissues, are needed to activate the homeotic genes, i.e., create an active state, while ATX1 is required to keep it active. The atx1-1 phenotypes and the fact that ATX1 belongs to the family of SET domain proteins with HTMase activity suggest that ATX1 is involved in epigenetic gene regulation by modifying chromatin structure. An important result of this study is that the effects of ATX1 are specific because structurally and functionally related (homeotic) genes are affected selectively (Alvarez-Venegas, 2003).

Mammalian Trithorax homologs

A gene has been identified at chromosome band 19q13.1 that is closely related to MLL. MLL is located in a region of chromosome 11q23 that has partial synteny with chromosome 19q. The gene at 19q13.1 has been named MLL2. MLL2 encodes a protein that exhibits a high level of similarity to MLL over several important protein domains. MLL2 is also ubiquitously expressed among adult human tissues, as is MLL. MLL is a homologue of the Drosophila gene trithorax, which encodes a regulator of homeotic gene expression. MLL is involved in chromosome rearrangements associated with leukemia in mammals. However, no MLL2 rearrangements associated with leukemia have been recorded (FitzGerald, 1999).

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).

The trithorax gene family contains members implicated in the control of transcription, development, chromosome structure, and human leukemia. A feature shared by some family members, and by other proteins that function in chromatin-mediated transcriptional regulation, is the presence of a 130- to 140-amino acid motif dubbed the SET or Tromo domain. Presented here is an analysis of SET1, a yeast member of the trithorax gene family that was identified by sequence inspection to encode a 1080-amino acid protein with a C-terminal SET domain. In addition to its SET domain, which is 40-50% identical to those previously characterized, SET1 also shares dispersed but significant similarity to Drosophila and human trithorax homologs. To understand SET1 function(s), a null mutant was created. Mutant strains, although viable, are defective in transcriptional silencing of the silent mating-type loci and telomeres. The telomeric silencing defect is rescued not only by full-length episomal SET1 but also by the conserved SET domain of SET1. set1 mutant strains display other phenotypes including morphological abnormalities, stationary phase defects, and growth and sporulation defects. Candidate genes that may interact with SET1 include those with functions in transcription, growth, and cell cycle control. These data suggest that yeast SET1, like its SET domain counterparts in other organisms, functions in diverse biological processes including transcription and chromatin structure (Nislow, 1997).

The MLL gene is interrupted and fused to a number of partner genes as a result of chromosomal translocations in human leukemias. MLL is a very large protein with a unique domain structure and large regions of homology to Drosophila trx. To define the key structural and functional domains of the MLL protein in vertebrates, the genomic region encoding an MLL-like gene has been cloned in the compact model vertebrate genome of Fugu rubripes. While the similarity between the mouse and human MLL proteins is very high, a lower overall similarity is present between the Fugu and mammalian proteins. Several new highly conserved regions have been identified in the portion of the protein included in the MLL leukemia-associated fusion proteins. The conserved nature of regions of similarity between vertebrate forms of MLL and the Drosophila Trx proteins, as well as other domains previously suggested to have a functional role in MLL (including the AT hooks and the DNA methyltransferase domain), is also observed. Therefore, strong evolutionary constraints limit sequence divergence within these domains. The information derived from this comparative analysis will form the basis for the functional study of the MLL protein, particularly as it relates to human leukemogenesis (Caldas, 1998).

The human ALL-1 gene is involved in acute leukemia through gene fusions, partial tandem duplications or a specific deletion. Several sequence motifs within the ALL-1 protein, such as the SET domain, PHD fingers and the region with homology to DNA methyl transferase are shared with other proteins involved in transcription regulation through chromatin alterations. However, the function of these motifs is still not clear. Studying ALL-1 presents an additional challenge because the gene is the human homolog of Drosophila Trithorax. Yeast two hybrid methodology, in vivo immunoprecipitation and in vitro 'pull down' techniques have been applied to show self association of the SET motifs of ALL-1, and the Drosophila Trithorax and Ash1 proteins. Point mutations in evolutionarily conserved residues of the Trithorax SET domain abolish the interaction. SET-SET interactions might act in integrating the activity of ALL-1 (TRX and ASH1) protein molecules, simultaneously positioned at different maintenance elements and directing expression of the same or different target genes (Rozovskaia, 2000).

During animal development, regions of the embryo become committed to position-specific identities, which are determined by spatially restricted expression of Hox/homeotic genes. This expression pattern is initially established by the activity of the segmentation genes and is subsequently maintained during the proliferative stage through the action of transcription factors encoded by the trithorax (trx) and Polycomb (Pc) groups of genes. trithorax and ash1 (absent, small, or homeotic 1) are members of the Drosophila trx group. Their products are associated with chromosomes and are believed to activate transcription of target genes through chromatin remodeling. Trx and Ash1 proteins act in concert to bind simultaneously to response elements located at close proximity within the same set of target genes. Extension of these and other studies to mammalian systems required identification and cloning of the mammalian homologue of ash1 (the mammalian homolog of trx, ALL-1, has been cloned previously). A human expressed sequence tag (EST) clone with similarity to the SET domain of Drosophila ASH1 has been identified, and it has been used to clone the human gene. huASH1 resides at chromosomal band 1q21. The gene is expressed in multiple tissues as an ~10.5-kb transcript and encodes a protein of 2962 residues. The protein contains a SET domain, a PHD finger, four AT hooks, and a region with homology to the bromodomain. The last region is not present in Drosophila ASH1, and as such might confer to the human protein a unique additional function. Using several anti-huASH1 Ab for immunostaining of cultured cells, it was found that the protein is distributed in intranuclear speckles, and unexpectedly also in intercellular junctions. There is no appreciable colocalization of the ALL-1 and huASH1 speckles. Double-immunofluorescence labeling of huASH1 and several junctional proteins localize the huASH1 protein into tight junctions. The significance of huASH1 dual location is discussed. In particular, the possibility is considered that translocation of the protein between the junctional membrane and the nucleus may be involved in adhesion-mediated signaling (Nakamura, 2000).

Translocations involving human chromosome band 11q23, found in acute lymphoid and myeloid leukemias, disrupt the MLL gene. This gene contains regions of homology to trithorax and the "AT-hook" DNA-binding motif of high mobility group proteins. The repression domain is located centromeric to the breakpoint region of MLL. The activation domain, located telomeric to the breakpoint region, activates transcription from a variety of promoters including ones containing only basal promoter elements. In translocations involving MLL, the protein produced includes the AT-hook domain and the repression domain. The MLL AT-hook domain can bind cruciform DNA, recognizing the structure rather than the sequence of the target DNA. The MLL AT-hook domain can bind to AT-rich scaffold attachment regions, but not to non-scaffold DNA fragments (Broeker, 1995)

Reciprocal chromosome translocations involving 11q23 are frequently associated with acute leukemias, with the t(4;11) translocation predominating among acute lymphoblastic leukemias, and the t(9;11), t(11;19) and t(6;11) translocations most common among acute myeloid leukemias. In each of these translocations the ALL-1 gene, located at 11q23 and constituting the human homolog of Drosophila trithorax, fuses to a specific gene on the partner chromosome to produce a chimeric protein. The partner gene from chromosome 6 (AF-6, a homolog of Drosophila Canoe) is expressed in a variety of cell types and encodes a protein of 1612 amino acids. The protein contains short stretches rich in prolines, charged amino acids, serines, or glutamines. In addition, the AF-6 protein contains the GLGF motif shared with several proteins of vertebrates and invertebrates thought to be involved in signal transduction at special cell-cell junctions (Prasad, 1993).

The ALL-1 protein distributes in cultured cells in a nuclear punctate pattern. Several chimeric ALL-1 proteins encoded by products of chromosome translocations and expressed in transfected cells show similar speckles. Dissection of the ALL-1 protein identifies within its approximately 1,100 N-terminal residues three polypeptides directing nuclear localization and at least two main domains conferring distribution in dots. The latter span two short sequences conserved in Drosophila Trithorax. Enforced nuclear expression of other domains of ALL-1, such as the PHD (zinc) fingers and the SET motif, results in uniform nonpunctate patterns. This indicates that positioning of the ALL-1 protein in subnuclear structures is mediated via interactions of ALL-1 N-terminal elements. It is suggested that the speckles represent protein complexes that contain multiple copies of the ALL-1 protein, positioned at ALL-1 target sites on the chromatin. Therefore, the role of the N-terminal portion of ALL-1 is to direct the protein to its target genes (Yano, 1997).

SET binding factor 1 (Sbf1) was originally discovered by virtue of its interaction with a highly conserved motif (the SET domain) of unknown function in Hrx, the protooncoprotein homolog of Drosophila Trithorax. Sbf1 shares extensive sequence similarity with myotubularin, a dual specificity phosphatase (dsPTPase) that is mutated in a subset of patients with inherited myopathies. Both Sbf1 and myotubularin interact with the SET domains of Hrx and other epigenetic regulatory proteins, but Sbf1 lacks phosphatase activity due to several evolutionarily conserved amino acid changes in its structurally preserved catalytic pocket. Thus, Sbf1 has features of an anti-phosphatase that could competitively antagonize dsPTPases; however, the in vivo role for such factors remains unknown. Given its ability to physically interact with Hrx, a developmental regulator subject to translocation-induced mutations in B cell precursor leukemias, the current studies were undertaken to assess the effects of Sbf1 on lymphopoiesis. After infection with recombinant Sbf1 retroviruses, bone marrow cells were plated under Whitlock-Witte conditions for long-term culture of B lineage cells. Sbf1-expressing cells rapidly dominated the cultures resulting in clonal outgrowths of B cell progenitors that retained a dependence on their primary bone marrow-derived stroma for continuous growth in vitro. Structure/function analyses demonstrate that the SET interaction domain of Sbf1 is necessary and sufficient for growth alterations of B cell progenitors. These observations support a model in which Sbf1 functions as a SET domain-dependent, positive regulator of growth-inducing, kinase signaling pathways that impinge on SET domain proteins. SET domain-dsPTPase interactions appear to be critically important for regulating the growth properties of B cell progenitors (De Vivo, 1998).

Several proteins that contribute to epigenetic mechanisms of gene regulation contain a characteristic motif of unknown function called the SET (Suvar3-9, Enhancer-of-zeste, Trithorax) domain. SET domains mediate highly conserved interactions with a specific family of proteins that display similarity with dual-specificity phosphatases (dsPTPases). These include myotubularin, which, as a gene, is mutated in a subset of patients with X-linked myotubular myopathy, and Sbf1, a newly isolated homolog of myotubularin. In contrast with myotubularin, Sbf1 lacks a functional catalytic domain that dephosphorylates phospho-tyrosine and serine-containing peptides in vitro. Competitive interference of endogenous SET domain-dsPTPase interactions by forced expression of Sbf1 induces oncogenic transformation of NIH 3T3 fibroblasts and impairs the in vitro differentiation of C2 myoblast cells. It is concluded that myotubularin-type phosphatases link SET-domain containing components of the epigenetic regulatory machinery with signalling pathways involved in growth and differentiation (Cui, 1998).

ALL1, the human homolog of Drosophila trithorax, is directly involved in human acute leukemias associated with abnormalities at 11q23. Using the differential display method, a gene was isolated that is down-regulated in All1 double-knockout mouse embryonic stem (ES) cells. The gene, designated ARP1 (also termed RIEG, Ptx2, or Otlx2), is a member of a family of homeotic genes containing a short motif shared with several homeobox genes. Using a bacterially synthesized All1 polypeptide encompassing the AT-hook motifs, a 0.5-kb ARP1 DNA fragment was identified that preferentially binds to the polypeptide. Within this DNA, a region of approximately 100 bp is protected by the All1 polypeptide from digestion by the enzymes ExoIII and DNase I. Whole-mount in situ hybridization to early mouse embryos of 9.5-10.5 days indicates a complex pattern of Arp1 expression spatially overlapping with the expression of All1. Although the ARP1 gene is expressed strongly in bone marrow cells, no transcripts were detected in six leukemia cell lines with 11q23 translocations. These results suggest that ARP1 is up-regulated by the All1 protein, possibly through direct interaction with an upstream DNA sequence of the former. The results are also consistent with the suggestion that ALL1 chimeric proteins resulting from 11q23 abnormalities act in a dominant negative fashion (Arakawa, 1998).

Control of cell identity during development is specified in large part by the unique expression patterns of multiple homeobox-containing (Hox) genes in specific segments of an embryo. Trithorax and Polycomb-group (Trx-G and Pc-G) proteins in Drosophila maintain Hox expression or repression, respectively. Mixed lineage leukemia (MLL) is frequently involved in chromosomal translocations associated with acute leukemia and is the one established mammalian homolog of Trx. Bmi-1 was first identified as a collaborator in c-myc-induced murine lymphomagenesis and is homologous to the Drosophila Pc-G member Posterior sex combs. The axial-skeletal transformations and altered Hox expression patterns of Mll-deficient mice and Bmi-1-deficient mice are normalized when both Mll and Bmi-1 are deleted, demonstrating their antagonistic role in determining segmental identity. Embryonic fibroblasts from Mll-deficient mice, compared with Bmi-1-deficient mice, demonstrate reciprocal regulation of Hox genes as well as an integrated Hoxc8-lacZ reporter construct. Reexpression of MLL is able to overcome repression, rescuing expression of Hoxc8-lacZ in Mll-deficient cells. Consistent with this, MLL and BMI-I display discrete subnuclear colocalization. Although Drosophila Pc-G and Trx-G members have been shown to maintain a previously established transcriptional pattern, MLL can also dynamically regulate a target Hox gene (Hanson, 1999).

Cotranscriptional histone methylations by Set1 and Set2 have been shown to affect histone acetylation at promoters and 3' regions of genes, respectively. While histone H3K4 trimethylation (H3K4me3) is thought to promote nucleosome acetylation and remodeling near promoters, this study shows that H3K4 dimethylation (H3K4me2) by Set1 leads to reduced histone acetylation levels near 5' ends of genes. H3K4me2 recruits the Set3 complex via the Set3 PHD finger, localizing the Hos2 and Hst1 subunits to deacetylate histones in 5' transcribed regions. Cells lacking the Set1-Set3 complex pathway are sensitive to mycophenolic acid and have reduced polymerase levels at a Set3 target gene, suggesting a positive role in transcription. It is proposed that Set1 establishes two distinct chromatin zones on genes: H3K4me3 leads to high levels of acetylation and low nucleosome density at promoters, while H3K4me2 just downstream recruits the Set3 complex to suppress nucleosome acetylation and remodeling (Kim, 2009).

Physical association and coordinate function of the H3 K4 methyltransferase MLL1 and the H4 K16 acetyltransferase MOF

A stable complex containing MLL1 (Drosophila homolog, Trx) and MOF has been immunoaffinity purified from a human cell line that stably expresses an epitope-tagged WDR5 subunit. Stable interactions between MLL1 and MOF were confirmed by reciprocal immunoprecipitation, cosedimentation, and cotransfection analyses, and interaction sites were mapped to MLL1 C-terminal and MOF zinc finger domains. The purified complex has a robust MLL1-mediated histone methyltransferase activity that can effect mono-, di-, and tri-methylation of H3 K4 and a MOF-mediated histone acetyltransferase activity that is specific for H4 K16. Importantly, both activities are required for optimal transcription activation on a chromatin template in vitro and on an endogenous MLL1 target gene, Hox a9, in vivo. These results indicate an activator-based mechanism for joint MLL1 and MOF recruitment and targeted methylation and acetylation and provide a molecular explanation for the closely correlated distribution of H3 K4 methylation and H4 K16 acetylation on active genes (Dou, 2005; full text of article).

Automethylation activities within the Mixed Lineage Leukemia-1 (MLL1) core complex reveal evidence supporting a 'two-active site' model for multiple histone H3 lysine 4 methylation

The Mixed Lineage Leukemia-1 (MLL1) core complex predominantly catalyzes mono- and dimethylation of histone H3 at lysine 4 (H3K4) and is frequently altered in aggressive acute leukemias. The molecular mechanisms that account for conversion of mono- to dimethyl H3K4 (H3K4me1,2) are not well understood. This paper reports that the SET domains from human MLL1 and Drosophila Trithorax undergo robust intramolecular automethylation reactions at an evolutionarily conserved cysteine residue in the active site, which is inhibited by unmodified histone H3. The location of the automethylation in the SET-I sub-domain indicates that the MLL1 SET domain possesses significantly more conformational plasticity in solution than suggested by its crystal structure. It is also reported that MLL1 methylates Ash2L in the absence of histone H3, but only when assembled within a complex including WDR5 and RbBP5, suggesting a restraint for the architectural arrangement of subunits within the complex. Using MLL1 and Ash2L automethylation reactions as probes for histone binding, it was observed that both automethylation reactions are significantly inhibited by stoichiometric amounts of unmethylated histone H3, but not by histones previously mono-, di- or trimethylated at H3K4. These results suggest that the H3K4me1 intermediate does not significantly bind to the MLL1 SET domain during the dimethylation reaction. Consistent with this hypothesis, it was demonstrated that the MLL1 core complex assembled with a catalytically inactive SET domain variant preferentially catalyzes H3K4 dimethylation using the H3K4me1 substrate. Taken together, these results are consistent with a 'two-active site' model for multiple H3K4 methylation by the MLL1 core complex (Patel, 2013).

Regulation of MLL1 H3K4 methyltransferase activity by its core components

Histone H3 Lys4 (H3K4) methylation is a prevalent mark associated with transcription activation. A common feature of several H3K4 methyltransferase complexes is the presence of three structural components (RbBP5, Ash2L and WDR5) and a catalytic subunit containing a SET domain. This study reports the first biochemical reconstitution of a functional four-component mixed-lineage leukemia protein-1 (MLL1) core complex. This reconstitution, combined with in vivo assays, allows direct analysis of the contribution of each component to MLL1 enzymatic activity and their roles in transcriptional regulation. Moreover, taking clues from a crystal structure analysis, it was demonstrated that WDR5 mediates interactions of the MLL1 catalytic unit both with the common structural platform and with the histone substrate. Mechanistic insights gained from this study can be generalized to the whole family of SET1-like histone methyltransferases in mammals (Dou, 2005).

MLL targets SET domain methyltransferase activity to hox gene promoters

MLL, the human homolog of Drosophila trithorax, maintains Hox gene expression in mammalian embryos and is rearranged in human leukemias resulting in Hox gene deregulation. How MLL or MLL fusion proteins regulate gene expression remains obscure. MLL regulates target Hox gene expression through direct binding to promoter sequences. The MLL SET domain is a histone H3 lysine 4-specific methyltransferase whose activity is stimulated with acetylated H3 peptides. This methylase activity is associated with Hox gene activation and H3 (Lys4) methylation at cis-regulatory sequences in vivo. A leukemogenic MLL fusion protein that activates Hox expression had no effect on histone methylation, suggesting a distinct mechanism for gene regulation by MLL and MLL fusion proteins (Milne, 2002).

How MLL regulates Hox gene expression is poorly understood. The domain structure of MLL is complex, making it difficult to unravel the key components of MLL function. Domains that may have a role in MLL function include the AT hooks, which bind DNA, a region homologous to DNA methyl transferases (DNMT), the cysteine-rich PHD domain, and a highly conserved SET domain. The SET domain is found in many proteins now demonstrated to mediate lysine-directed histone methylation. These findings suggest a possible role for MLL in chromatin remodeling mediated by histone methylation. However, early studies of this domain in MLL did not reveal evidence of enzymatic activity, leaving its function enigmatic. Furthermore, rearrangements of MLL that occur in leukemia consistently delete the PHD and SET domains and replace these sequences with one of over 30 different translocation partners that in general share little sequence homology (Milne, 2002).

Progress in understanding the mechanistic role of MLL in maintenance and gene regulation has also been slowed by a lack of known target binding sites for mammalian PcG or trxG homologs. To address these issues, attention was focused on how MLL regulates transcription of Hox c8. This target was chosen because it is tightly regulated by MLL and because it is the only Hox gene in which the sequences required for the correct initiation and maintenance of expression have been extensively mapped in vivo. Hox c8 is upregulated by MLL, supporting a transcriptional activating role for MLL. MLL binds directly to proximal promoter sequences but not to other regions of the Hox c8 locus, including the 5' and 3' enhancer sequences, suggesting that MLL-dependent regulatory elements in mammalian Hox genes are organized differently from those in Drosophila. The Hox c8 promoter is necessary and sufficient for MLL responsiveness and, along with the 5' enhancer, exhibits differential histone acetylation and H3 (Lys4) methylation in Mll+/+ as compared to Mll-/- cells. Reexpression of MLL in null cells results in methylation of H3 (Lys4) at the Hox c8 5' enhancer and promoter as well as at other Hox gene promoters. H3 (Lys4) methylation is dependent on an intact MLL SET domain and this methyltransferase activity is stimulated by H3 peptides that are acetylated at Lys9 or Lys14. Collectively, these experiments underscore the importance of a concerted series of histone and DNA modifications in the regulation and maintenance of target genes during mammalian development and provide a framework for comparing mechanisms of epigenetic forms of gene regulation by MLL and MLL fusion proteins (Milne, 2002).

ALL-1 is a member of the human trithorax/Polycomb gene family and is also involved in acute leukemia. ALL-1 is present within a stable, very large multiprotein supercomplex composed of ~29 proteins. The majority of the latter are components of the human transcription complexes TFIID (including TBP), SWI/SNF, NuRD, hSNF2H, and Sin3A. Other components are involved in RNA processing or in histone methylation. The complex remodels, acetylates, deacetylates, and methylates nucleosomes and/or free histones. The complex's H3-K4 methylation activity is conferred by the ALL-1 SET domain. Chromatin immunoprecipitations show that ALL-1 and other complex components examined are bound at the promoter of an active ALL-1-dependent Hox a9 gene. In parallel, H3-K4 is methylated, and histones H3 and H4 are acetylated at this promoter (Nakamura, 2002).

Strikingly, most ALL-1-associated proteins can be classified into well-known complexes involved in transcription. Of these, the SWI/SNF(BRM) and NuRD complexes and the hSNF2H protein are ATP-dependent chromatin remodelers: Sin3A and NuRD are histone deacetylases; two human homologs of components of the yeast Set1 complex (but not the Set1 protein) are involved in H3-K4 methylation, and TFIID acts in promoter recognition and in mediating activator responsiveness. The identification of TFIID components, including TBP, within the ALL-1 supercomplex is one of the most significant observations of this work. This finding indicates a direct connection between ALL-1 and the general transcription machinery. Several TFIID proteins have been identified as components of the Drosophila Polycomb multiprotein complex. Considering the known functions of the other complexes included within the ALL-1 supercomplex, SWI/SNF and hSNF2H may act both as activators and repressors, but Sin3A and NuRD complexes have been generally associated with transcriptional silencing. Since ALL-1 is an activator, the inclusion of these last two complexes within the ALL-1 supercomplex is surprising. However, HDAC1, a component of both Sin3A and NuRD complexes, has been found bound to active promoters of some Drosophila genes, including the trithorax-regulated Abd-B. Further, histone deacetylation might be required to enable H3-K4 methylation. Also, deacetylation might be applied to modulate the level of histone acetylation conferred by the ALL-1 complex and/or by acetyltransferases transiently associated with ALL-1. Moreover, the deacetylating complexes might target transcription factors regulated by acetylation. Finally, the inclusion within the ALL-1 supercomplex of CPSF and Symplekin involved in polyadenylation and of p116 associated with splicing provides support for the notion of direct connection between the promoter of a gene and how its transcript is processed (Nakamura, 2002 and references therein).

A major finding in this work is that the ALL-1 SET domain methylates H3-K4. Previous attempts to show methyltransferase activity of ALL-1 SET were unsuccessful, probably due to the low activity of this domain (at least 10-fold lower than the activity of SUV39H1 SET, which methylates H3-K9). Presently, two other proteins have been implicated in H3-K4 methylation. Human Set7/Set9 possess this intrinsic enzymatic activity conferred by the SET domain. A Saccharomyces cerevisiae complex containing the Set1 protein methylates H3-K4, and mutation analysis of the gene implicates it directly in that histone modification in yeast. Whereas human SET7/SET9 activates transcription, yeast Set1 represses transcription of ribosomal DNA. Nevertheless, diverse findings correlate H3-K4 methylation with an active state of transcription. Thus, this modification is specifically associated with transcriptionally active macronuclei but not with inactive micronuclei in Tetrahymena. Also, immunofluorescence studies of human female chromosomes show that H3-K4 methylation accumulates at transcribed regions of autosomes but is largely excluded from the inactive X chromosome. Moreover, ChIP experiments at the mating type locus of fission yeast have shown that, while histone H3-K4 methylation is localized to actively transcribed regions, H3-K9 methylation is detected in silent heterochromatin. Similar results have been observed in ChIP analysis of the beta-globin locus during erythropoiesis (Nakamura, 2002 and references therein).

Mammalian Polycomb-mediated repression of Hox genes requires the essential spliceosomal protein Sf3b1

Polycomb group (PcG) proteins are responsible for the stable repression of homeotic (Hox) genes by forming multimeric protein complexes. Physical interaction is shown between components of the U2 small nuclear ribonucleoprotein particle (U2 snRNP), including Sf3b1 and PcG proteins Zfp144 and Rnf2. Sf3b1 heterozygous mice exhibit skeletal transformations concomitant with ectopic Hox expressions. These alterations are enhanced by Zfp144 mutation but repressed by Mll mutation (a trithorax homolog). Importantly, the levels of Sf3b1 in PcG complexes are decreased in Sf3b1-heterozygous embryos. These findings suggest that Sf3b1-PcG protein interaction is essential for true PcG-mediated repression of Hox genes (Isono, 2005).

These results show a significant and novel mechanistic link between Sf3b1 (together probably with other U2 snRNP components) and PcG repressive complexes on Hox loci. This idea is strongly supported by the observation that heterozygous mutant mice for Sf3b2, another U2 snRNP component, exhibit skeletal abnormality similar to Sf3b1 phenotypes. However, although with respect to the PcG-mediated repression in the transcriptional-competent regions, these findings are in general accord with previous reports, nevertheless they indicate the presence a different gene silencing mechanism. Evidence that the level of Sf3b1 in PcG complexes affects the expressional boundary of Hox genes implies that Sf3b1 supports the activity of PcG complexes. The simplest explanation is that Sf3b1/U2 snRNP might be a PcG protein and could form repressive PcG complexes together with other PcG proteins. A more interesting hypothesis is that this interaction constitutes part of a mechanism that is designed to maintain the amount of Hox transcripts required to confer the appropriate positional identities. Regulation of Hox expressions in the vicinity of their boundaries is thought to be loose, because even wild type occasionally exhibits homeotic transformations. RNAs, mistranscribed beyond loose repression, may be tethered by Sf3b1/U2 snRNP bound to PcG complex, leading to the arrest of splicing and a normal Hox boundary as a consequence. However, in Sf3b1+/- cells, because of the decrease of PcG complex-bound Sf3b1, such mistranscribed RNAs become easily associated with splicing-active nucleoplasmic Sf3b1/U2 snRNP. This association leads to the achievement of a splicing reaction, which results in the anterior shift of Hox expression. In support of this model is the important evidence that the Mll mutation completely suppresses Sf3b1 phenotypes, indicating that the PcG-like function of Sf3b1 is very susceptible to levels of Mll; in other words the acting points of both proteins are spatially very close. Of further note is the fact that the human MLL supercomplex includes the 116-kDa protein specific to the U5 snRNP, which acts on pre-mRNA following the U2 snRNP. Finally, it appears that there are multiple interacting surfaces between PcG complexes and gene expression machineries. It might be that, through this interaction, PcG complexes act as a part of the modules that sense the transcriptional status in transcriptional competent regions of the Hox cluster (Isono, 2005).

The APC tumor suppressor counteracts beta-catenin activation and H3K4 methylation at Wnt target genes

The APC tumor suppressor controls the stability and nuclear export of β-catenin (β-cat), a transcriptional coactivator of LEF-1/TCF HMG proteins in the Wnt/Wg signaling pathway. β-cat and APC have opposing actions at Wnt target genes in vivo. The β-cat C-terminal activation domain associates with TRRAP/TIP60 and mixed-lineage-leukemia (MLL1/MLL2) SET1-type chromatin-modifying complexes in vitro, and β-cat promotes H3K4 trimethylation at the c-Myc gene in vivo. H3K4 trimethylation in vivo requires prior ubiquitination of H2B, and ubiquitin is found necessary for transcription initiation on chromatin but not nonchromatin templates in vitro. Chromatin immunoprecipitation experiments reveal that β-cat recruits Pygopus, Bcl-9/Legless, and MLL/SET1-type complexes to the c-Myc enhancer together with the negative Wnt regulators, APC, and βTrCP. Interestingly, APC-mediated repression of c-Myc transcription in HT29-APC colorectal cancer cells is initiated by the transient binding of APC, βTrCP, and the CtBP corepressor to the c-Myc enhancer, followed by stable binding of the TLE-1 and HDAC1 corepressors. Moreover, nuclear CtBP physically associates with full-length APC, but not with mutant SW480 or HT29 APC proteins. It is concluded that, in addition to regulating the stability of β-cat, APC facilitates CtBP-mediated repression of Wnt target genes in normal, but not in colorectal cancer cells (Sierra, 2006).

The data presented here support a model in which the APC tumor suppressor functions directly to counteract β-cat-mediated transcription at Wnt target genes in vivo. This possibility was first suggested by the finding that full-length APC cycles on and off the c-Myc enhancer in conjunction with β-cat and associated coactivators in LiCl-treated C2C12 cells. In contrast, the enhancer complex appears to be stable and does not cycle in HT29 CRC cells, which contain a Class II APC mutant protein that is unable to degrade β-cat. Most strikingly, the binding of the full-length APC protein to the c-Myc gene in HT29-APC cells correlates with the rapid disassembly of the Wnt enhancer complex in vivo and the subsequent decline in steady-state c-Myc mRNA levels, both of which significantly precede the drop in β-cat protein levels that occurs as a result of proteolytic degradation in the cytoplasm. Thus, the effect of APC on c-Myc transcription appears to be immediate and direct, and may serve to coordinate the switch between the β-cat coactivator and TLE1 corepressor complexes (Sierra, 2006).

The β-cat enhancer complex includes the Wnt coactivators Pygopus and Bcl-9/Lgs, which control the retention of β-cat in the nucleus and may also function directly in transcription. The observation that APC can also regulate nuclear transport of β-cat raises the possibility that these factors may reside within a larger regulatory complex that chaperones β-cat in and out of the nucleus and mediates its release from the DNA. Indeed, sequential ChIP (re-ChIP) data indicate that the mutant APC in HT29 colorectal cancer cells exists in a stable complex with β-cat and LEF-1 at the active c-Myc gene. This finding is unexpected because β-cat cannot bind simultaneously to APC and LEF-1, and thus, if the full-length APC is part of a larger β-cat:LEF enhancer complex, it may interact with other subunits. Alternatively, the full-length APC and β-cat may exist in different complexes that rapidly exchange at the enhancer. The current data indicate that targeting is mediated by the N-terminal half of the APC protein, and that CtBP and βTrCP appear only in conjunction with the full-length APC protein. How APC is recruited to Wnt enhancers remains an open and important question (Sierra, 2006).

The ChIP experiments also suggest that APC-mediated inhibition of c-Myc transcription in HT29 cells occurs in two steps, initiated by transient binding of APC, βTrCP, CtBP, and YY1 to the enhancer, and followed by stable binding of the TLE-1 and HDAC1 corepressors. The transient recruitment of APC and CtBP, at the time when β-cat, Bcl-9, Pygo, and other Wnt enhancer factors leave the DNA, strongly suggests a role for these factors in the exchange of Wnt coactivator and corepressor complexes. In this respect it is interesting that CtBP was shown recently to associate with APC, both in vivo and in vitro. The results confirm a high-affinity interaction between CtBP and the full-length APC protein induced in HT29-APC cells, as well as with the native (full-length) APC protein in 293 cells. Consequently, APC may function to recruit CtBP to Wnt enhancers. Although both CtBP and TLE-1 are well-established corepressors of Wnt target genes, the different functions of the two types of corepressors remain unclear, and the ChIP data suggest that they act at distinct steps. Together, these data suggest that APC counteracts β-cat function in the nucleus, as well as in the cytoplasm, and may facilitate turnover of the enhancer complex at responsive genes by recruiting βTrCP and CtBP (Sierra, 2006).

Methylation of histone H3R2 by PRMT6 and H3K4 by an MLL complex are mutually exclusive

Eukaryotic genomes are organized into active (euchromatic) and inactive (heterochromatic) chromatin domains. Post-translational modifications of histones (or 'marks') are key in defining these functional states, particularly in promoter regions. Mutual regulatory interactions between these marks -- and the enzymes that catalyse them -- contribute to the shaping of this epigenetic landscape, in a manner that remains to be fully elucidated. Asymmetric di-methylation of histone H3 arginine 2 (H3R2me2a) counter-correlates with di- and tri-methylation of H3 lysine 4 (H3K4me2, H3K4me3) on human promoters. This study shows that the arginine methyltransferase PRMT6 catalyses H3R2 di-methylation in vitro and controls global levels of H3R2me2a in vivo. H3R2 methylation by PRMT6 is prevented by the presence of H3K4me3 on the H3 tail. Conversely, the H3R2me2a mark prevents methylation of H3K4 as well as binding to the H3 tail by an ASH2/WDR5/MLL-family methyltransferase complex. Chromatin immunoprecipitation showed that H3R2me2a is distributed within the body and at the 3' end of human genes, regardless of their transcriptional state, whereas it is selectively and locally depleted from active promoters, coincident with the presence of H3K4me3. Hence, the mutual antagonism between H3R2 and H3K4 methylation, together with the association of MLL-family complexes with the basal transcription machinery, may contribute to the localized patterns of H3K4 tri-methylation characteristic of transcriptionally poised or active promoters in mammalian genomes (Guccione, 2007).

Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin (see Drosophila Menin-1) to regulate Hox gene expression

MLL (for mixed-lineage leukemia) is a proto-oncogene that is mutated in a variety of human leukemias. Its product, a homolog of Drosophila melanogaster trithorax, displays intrinsic histone methyltransferase activity and functions genetically to maintain embryonic Hox gene expression. This study reports the biochemical purification of MLL and demonstrates that it associates with a cohort of proteins shared with the yeast and human SET1 histone methyltransferase complexes, including a homolog of Ash2, another Trx-G group protein. Two other members of the novel MLL complex identified in this study are host cell factor 1 (HCF-1), a transcriptional coregulator, and the related HCF-2, both of which specifically interact with a conserved binding motif in the MLL(N) (p300) subunit of MLL and provide a potential mechanism for regulating its antagonistic transcriptional properties. Menin, a product of the MEN1 tumor suppressor gene, is also a component of the 1-MDa MLL complex. Abrogation of menin expression phenocopies loss of MLL and reveals a critical role for menin in the maintenance of Hox gene expression. Oncogenic mutant forms of MLL retain an ability to interact with menin but not other identified complex components. These studies link the menin tumor suppressor protein with the MLL histone methyltransferase machinery, with implications for Hox gene expression in development and leukemia pathogenesis (Yokoyama, 2007).

The BRCT-domain containing protein PTIP links PAX2 to a histone H3, lysine 4 methyltransferase complex

The MLL family of histone methyltransferases maintains active chromatin domains by methylating histone H3 on lysine 4 (H3K4). How MLL complexes recognize specific chromatin domains in a temporal and tissue-specific manner remains unclear. This study shows that the DNA-binding protein PAX2 promotes assembly of an H3K4 methyltransferase complex through the ubiquitously expressed nuclear factor PTIP (pax transcription activation domain interacting protein). PTIP copurifies with ALR, MLL3, and other components of a histone methyltransferase complex. PTIP promotes assembly of the ALR complex and H3K4 methylation at a PAX2-binding DNA element. Without PTIP, Pax2 binds to this element but does not assemble the ALR complex. Embryonic lethal ptip-null mutants and conditional mutants both show reduced levels of methylated H3K4. Thus, PTIP bridges DNA-binding developmental regulators to histone methyltransferase-dependent epigenetic regulation (Patel, 2007).

Bimodal degradation of MLL by SCFSkp2 and APCCdc20 assures cell cycle execution: a critical regulatory circuit lost in leukemogenic MLL fusions

Human chromosome 11q23 translocations disrupting MLL result in poor prognostic leukemias. It fuses the common MLL N-terminal ~1400 amino acids in-frame with >60 different partners without shared characteristics. In addition to the well-characterized activity of MLL in maintaining Hox gene expression, recent studies established an MLL-E2F axis in orchestrating core cell cycle gene expression including Cyclins. This study demonstrates a biphasic expression of MLL conferred by defined windows of degradation mediated by specialized cell cycle E3 ligases. Specifically, SCFSkp2 and APCCdc20 mark MLL for degradation at S phase and late M phase, respectively. Abolished peak expression of MLL incurs corresponding defects in G1/S transition and M-phase progression. Conversely, overexpression of MLL blocks S-phase progression. Remarkably, MLL degradation initiates at its N-terminal ~1400 amino acids, and tested prevalent MLL fusions are resistant to degradation. Thus, impaired degradation of MLL fusions likely constitutes the universal mechanism underlying all MLL leukemias. These data conclude an essential post-translational regulation of MLL by the cell cycle ubiquitin/proteasome system (UPS) assures the temporal necessity of MLL in coordinating cell cycle progression (Liu, 2007).

MLL, the mammalian homolog of Drosophila trithorax, is best known for its positive regulation of Hox gene expression. Homozygous disruption of MLL in mice results in early embryonic lethality, and heterozygous deficiency results in homeotic transformation due to impaired maintenance of Hox genes. The early lethality of MLL-/- mouse embryos precludes detailed investigation of its involvement in other signaling pathways. The demonstration that MLL is regulated by Taspase1-mediated proteolytic cleavage broadens the avenue in studying MLL in that noncleaved precursor MLL functions as a hypomorphic allele. Although the underlying mechanisms were not further investigated, initial characterizations on MLL-/- mice suggest a role of MLL in proliferation in addition to differentiation. For example, MLL-/- fetal liver or yolk sac hematopoietic cells grow more slowly and form smaller colonies in methyl cellulose assays. Studies on cells deficient for Taspase1 or bearing noncleavable alleles of MLL (MLLNC/NC) have recognized a participation of MLL through E2Fs in regulating cell proliferation (Takeda, 2006). The active involvement of MLL/trx in the cell cycle is conserved through evolution, in that genetic studies in Drosophila also highlighted a significant role of trx in cell proliferation (Muyrers-Chen, 2004). Recent studies have begin to elucidate the downstream targets for MLL in the cell cycle regulation that include Cyclins and CDKIs (Muyrers-Chen, 2004; Milne, 2005; Xia, 2005). A MLL-E2F axis has been defined in regulating Cyclin E/A/B expression for progressive cell cycle phase transition (Takeda, 2006). This study further investigated the participation of MLL in the cell cycle control and discovered a tightly controlled biphasic expression of MLL. This unique expression is conferred by defined windows of degradation mediated by specialized cell cycle E3 ligases: SCFSkp2 and APCCdc20. Importantly, individual peak expressions of MLL precede the induction of Cyclin E/A and Cyclin B to ensure proper G1/S transition and M-phase progression, respectively. Deregulation of this unique expression of MLL by shRNA-mediated knockdown causes corresponding defects in G1/S entry and M-phase progression. Furthermore, overexpression of MLL incurs specific S-phase defects, indicating the importance of down-regulating its activity in S phase. However, whether this resulted from sustained expression of Cyclin E/A and/or invoked not-yet-identified insults remains to be determined. These data highlight the significance of this biphasic expression of MLL in regulating cell proliferation and uncover a novel mechanism in regulating MLL through protein degradation -- another post-translational regulatory scheme in addition to Taspase1-mediated site-specific proteolysis. Since MLL directly activates the transcription of Cyclins that exhibit periodic expression during cell proliferation, it is necessary for a cell to incorporate MLL expression into the intricately assembled cell cycle circuitry to ensure correct transition of progressive phases. Current data not only consolidate the role of MLL in activating cell cycle but also discover a built-in program elegantly designed to turn off MLL with an impeccable temporal sequence (Liu, 2007).

Meis1 is an essential and rate-limiting regulator of MLL leukemia stem cell potential

Oncogenic mutations of the MLL histone methyltransferase confer an unusual ability to transform non-self-renewing myeloid progenitors into leukemia stem cells (LSCs) by mechanisms that remain poorly defined. Misregulation of Hox genes is likely to be critical for LSC induction and maintenance but alone it does not recapitulate the phenotype and biology of MLL leukemias, which are clinically heterogeneous -- presumably reflecting differences in LSC biology and/or frequency. TALE (three-amino-acid loop extension) class homeodomain proteins of the Pbx and Meis families are also misexpressed in this context, and thus knockout, knockdown, and dominant-negative genetic techniques were employed to investigate the requirements and contributions of these factors in MLL oncoprotein-induced acute myeloid leukemia. The studies show that induction and maintenance of MLL transformation requires Meis1 and is codependent on the redundant contributions of Pbx2 and Pbx3. Meis1 in particular serves a major role in establishing LSC potential, and determines LSC frequency by quantitatively regulating the extent of self-renewal, differentiation arrest, and cycling, as well as the rate of in vivo LSC generation from myeloid progenitors. Thus, TALE proteins are critical downstream effectors within an essential homeoprotein network that serves a rate-limiting regulatory role in MLL leukemogenesis (Wong, 2007).

In vitro hematopoietic differentiation of mouse embryonic stem cells requires the tumor suppressor menin and is mediated by Hoxa9

Inactivating mutations in the tumor suppressor gene MEN1 cause the inherited cancer syndrome multiple endocrine neoplasia type 1 (MEN1). The ubiquitously expressed MEN1 encoded protein, menin, interacts with MLL (mixed-lineage leukemia protein), and together they are essential components of a multiprotein complex with histone methyl transferase activity. MLL is also essential for hematopoiesis, and plays a critical role in leukemogenesis via epigenetic regulation of Hoxa9 expression that also requires menin. Therefore, the role of menin in hematopoiesis was investigated. Men1-/- embryonic stem (ES) cell lines were induced to differentiate in vitro. While these cells were able to form embryoid bodies (EBs) expressing the early markers Flk-1 and c-Kit, their ability to further differentiate into hematopoietic colonies was compromised. The Men1-/- ES cells show reduced expression of Hoxa9 that can be recovered by reexpression of Menin. The block in differentiation of Men1-/- ES cell lines can be rescued not only by the expression of menin but also that of Hoxa9. These results suggest that, similar to MLL, menin is required for hematopoiesis, and this requirement may be mediated through regulation of Hoxa9 expression (Novotny, 2009).

A vertebrate Polycomb response element governs segmentation of the posterior hindbrain

Chromatin remodeling by Polycomb group (PcG) and trithorax group (trxG) proteins regulates gene expression in all metazoans. Two major complexes, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2), are thought to mediate PcG-dependent repression in flies and mammals. In Drosophila, PcG/trxG protein complexes are recruited by PcG/trxG response elements (PREs). However, it has been unclear how PcG/trxG are recruited in vertebrates. This study identified a vertebrate PRE, PRE-kr, that regulates expression of the mouse MafB/Kreisler gene. PRE-kr recruits PcG proteins in flies and mouse F9 cells and represses gene expression in a PcG/trxG-dependent manner. PRC1 and 2 bind to a minimal PRE-kr region, which can recruit stable PRC1 binding but only weak PRC2 binding when introduced ectopically, suggesting that PRC1 and 2 have different binding requirements. Thus, evidence is provided that similar to invertebrates, PREs act as entry sites for PcG/trxG chromatin remodeling in vertebrates (Sing, 2009).

Analyses of the kr inversion suggest that PRE-kr directs PcG-protein-dependent repression in the anterior hindbrain and trxG-protein-dependent activation in the posterior hindbrain. A combination of factors might influence such position-specific function. (1) Analyses in Drosophila have shown that PREs associate robustly with promoters nearest to them. Thus, PRE-kr could interact with the regulatory elements or promoters from Nnat or MafB closest to it. (2) PRE-kr function could depend on the composition of the PcG protein complexes that bind it. Various studies suggest selective interactions of PREs with specific PcG subunits. For example, whereas redundancy of M33/Cbx2 with its homologs Cbx4, 6, 7, and 8 might contribute to the variable effects of M33 dosage on MafB, Cbx/Polycomb family members also have distinct roles in governing the cell cycle. Cbx4 does not affect replicative senescence of fibroblasts, a Cbx8-Bmi1 complex binds the INK4a-ARF locus to overcome senescence, and Cbx7-mediated bypass of senescence is Bmi1 independent. (3) Interactions of PcG proteins with other transcription factors could provide additional specificity. For example, Bmi1 interacts with the E2F6 transcription factor to repress Hox genes but acts independently of E2F6 to repress the Ink4a-Arf locus. (4) PRE-kr might be sensitive to signals governing anterior-posterior patterning since an extrinsic RARE can overcome PRE-kr-mediated repression in transgenic mice. Whether PRE-kr responds selectively to RA or also to other signaling pathways remains to be determined (Sing, 2009).

In Drosophila, trxG proteins interact selectively with PRE-kr. Only trxl/GAGA factor affected PRE-kr-directed repression in flies. Considering that vertebrates lack Trxl/GAGAF orthologs, the effect of Trxl on PRE-kr-dependent repression in flies suggests that a GAGAF-containing trxG complex, which also contains other trxG proteins, is conserved between flies and mice and promotes PRE-kr dependent activation. The SWI/SNF-related ATP-dependent Brahma chromatin-remodeling complex components, Brahma and Moira, did not affect PRE-kr-dependent repression in flies. This potentially reflects the selective action of SWI/SNF-containing complexes seen also in vertebrate neural development. Future studies will be needed to investigate if PRE-kr serves as a substrate for specific SWI/SNF-containing complexes that interact selectively with PREs governing early neural patterning genes (Sing, 2009).

In Drosophila, PcG complexes bind to discrete sequence platforms. Similarly, a distinct peak of SUZ12 and Bmi1 binding was observed within PRE-kr in F9 cells. By contrast, the H3K27 signature, which is thought to be laid down by Ezh1/2-Eed complex(es), covered the MafB locus. However, RA-induced decrease of the H3K27 mark and loss of PcG binding were highly localized. These observations suggest that two distinct H3K27 pools, only one of which depends directly on the presence of PRC1 and 2 binding, exist. Consistent with current observations, it has been proposed that distinct PRC2 complexes exist and only a subset specifically target PREs. Thus, other cues, such as nucleosomal modifications, could collaborate with the H3K27 mark to flag and re-enforce distinct SUZ12- and PRC1-binding platforms at PRE-kr (Sing, 2009).

A distinct difference was uncovered in sequence requirements for PRC1 and 2 binding. The minimal hcPRE-kr region could recruit Bmi1, but not SUZ12, binding as effectively in an exogenous context as in an endogenous context. Only stable binding of PRC1 appears to be required for PRE-kr to repress reporter gene expression at ectopic sites. Notably, in flies, recruitment of the PRC1 components Pc and Ph to PRE transgenic insertion sites has served as a criterion for validating ectopically introduced Drosophilid PREs, but binding of PRC2 components has not been similarly examined. Perhaps improved PRC2-specific antibodies will elucidate if the requirements for stable PRC1 and PRC2 occupancy differ in flies, as suggested by findings in F9 cells. Other studies in mammals have found that PRC1 is associated with repressive activity even in the absence of PRC2. CBX8 and Bmi1 exhibit similar levels of binding to many genes even in the absence of detectable H3K27 methylation in Suz12-/- ES cells. In Eed null cells, several PRC1 components are recruited to the inactive X, whereas maternally provided PRC1 components show Ezh2-independent targeting to paternal heterochromatin. Furthermore, PRC1 in vitro is able to repress transcription and inhibit ATP-dependent chromatin remodeling mediated by the human SWI/SNF complex -- a complex related to the Drosophila TrxG Brahma complex (Sing, 2009).

The observation that knockdown of SUZ12 affects Bmi1 binding to endogous and ectopic PRE-kr fits well with the 'hierarchical recruitment' model, which proposes sequential action of PRC2 and 1. Given that SUZ12 binding to ectopic hcPRE-kr was very low, it is proposed that a transient, unstable SUZ12 association with ectopic hcPRE-kr is sufficient to stabilize PRC1 binding -- perhaps by introducing the necessary methylation signature. An observation not directly in line with the 'hierarchical recruitment' model is that Bmi1 knockdown reduced SUZ12 binding to endogenous but not ectopic PRE-kr. This observation could be explained by a sequence-dependent role for PRC1 in supporting stable PRC2 binding or indirect effects of PRC1 on other PRC2 components, for example by reducing their levels. Thus, a possible interdependence of PRC1 and 2 binding and function still remains to be elucidated for PRE-kr and PREs (Sing, 2009).

These results have wide-ranging implications for PcG mechanisms, but also possibily for organization of transcriptional neighborhoods and human disorders. The finding that the kr inversion translocates a PRE from one rhombomere-specific gene to another might be a coincidence or could suggest that PREs are involved in long-range organization of transcriptional neighborhoods (Sing, 2009).

Histone H3 lysine 4 monomethylation modulates long-range chromatin interactions at enhancers
Long-range chromatin interactions between enhancers and promoters are essential for transcription of many developmentally controlled genes in mammals and other metazoans. Currently, the exact mechanisms that connect distal enhancers to their specific target promoters remain to be fully elucidated. This study shows that the enhancer-specific histone H3 lysine 4 monomethylation (H3K4me1) and the histone methyltransferases MLL3 and MLL4 (MLL3/4; see Drosophila Trithorax) play an active role in this process. This study demonstrates that in differentiating mouse embryonic stem cells, MLL3/4-dependent deposition of H3K4me1 at enhancers correlates with increased levels of chromatin interactions, whereas loss of this histone modification leads to reduced levels of chromatin interactions and defects in gene activation during differentiation. H3K4me1 facilitates recruitment of the Cohesin complex, a known regulator of chromatin organization, to chromatin in vitro and in vivo, providing a potential mechanism for MLL3/4 to promote chromatin interactions between enhancers and promoters. Taken together, these results support a role for MLL3/4-dependent H3K4me1 in orchestrating long-range chromatin interactions at enhancers in mammalian cells (Yan, 2018).

trithorax: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.