absent, small, or homeotic discs 2
EVOLUTIONARY HOMOLOGS

The SET protein complex in yeast

The trithorax genes encode an evolutionarily conserved family of proteins that function to maintain specific patterns of gene expression throughout cellular development. Members of this protein family contain a highly conserved 130- to 140-amino acid motif termed the SET domain. Subunits of a protein complex in the yeast Saccharomyces cerevisiae that includes the trithorax-related protein Set1 have been purified and molecularly identified. This protein complex, which has been named COMPASS (Complex Proteins Associated with Set1), consists of seven polypeptides ranging from 130 to 25 kDa. The same seven proteins were identified in COMPASS purified either by conventional biochemical chromatography or tandem-affinity tagging of the individual subunits of the complex. Null mutants missing any one of the six nonessential subunits of COMPASS grow more slowly than wild-type cells under normal conditions and demonstrate growth sensitivity to hydroxyurea. Furthermore, gene expression profiles of strains missing either of two nonessential subunits of COMPASS are altered in similar ways, suggesting these proteins have similar roles in gene expression in vivo. Molecular characterization of trithorax complexes will facilitate defining the role of this class of proteins in the regulation of gene expression and how their misregulation results in the development of human cancer (Miller, 2001).

The SET domain proteins, SUV39 and G9a are histone methyltransferases specific for lysines 9 and 27 (G9a only) of histone 3 (H3). The SET domains of the Saccharomyces cerevisiae Set1 and Drosophila trithorax proteins are closely related to each other but distinct from SUV39 and G9a. The complex associated with Set1 and Set1C has been characterized: it is comprised of eight members, one of which, Bre2, is homologous to the trithorax-group (trxG) protein, Ash2. Set1C requires Set1 for complex integrity and mutation of Set1 and Set1C components shortens telomeres. One Set1C member, Swd2/Cpf10 is also present in cleavage polyadenylation factor (CPF). Set1C methylates lysine 4 of H3, thus adding a new specificity and a new subclass of SET domain proteins known to methyltransferases. Since methylation of H3 lysine 4 is widespread in eukaryotes, databases were screened and other Set1 homologues were found. It is proposed that eukaryotic Set1Cs are H3 lysine 4 methyltransferases and are related to trxG action through association with Ash2 homologues (Roguev, 2001).

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 Set1 protein complex that is necessary for methylation of histone H3 at lysine residue 4 in Saccharomyces cerevisiae has been purified, molecularly identified, and genetically and biochemically characterized. The seven-member 363-kDa complex contains homologs of Drosophila melanogaster proteins Ash2 and Trithorax and Caenorhabditis 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, 2002).

Histone 3 lysine 4 (H3 Lys4) methylation in Saccharomyces cerevisiae is mediated by the Set1 complex (Set1C) and is dependent upon ubiquitinylation of H2B by Rad6. Mutually exclusive methylation of H3 at Lys4 or Lys9 is central to chromatin regulation; however, S. cerevisiae lacks Lys9 methylation. Furthermore, a different H3 Lys4 methylase, Set 7/9, has been identified in mammals, thereby questioning the relevance of the S. cerevisiae findings for eukaryotes in general. The majority of Lys4 methylation in Schizosaccharomyces pombe, like in S. cerevisiae, is mediated by Set1C and is Rad6-dependent. S. pombe Set1C mediates H3 Lys(4) methylation in vitro and contains the same eight subunits found in S. cerevisiae, including the homologue of the Drosophila trithorax Group protein, Ash2. Three additional features of S. pombe Set1C each involve PHD fingers. Notably, the Spp1 subunit is dispensable for H3 Lys(4) methylation in budding yeast but required in fission yeast, and Sp_Set1C has a novel proteomic hyperlink to a new complex that includes the homologue of another trithorax Group protein, Lid (Little imaginal discs). Thus, it is inferred that Set1C is highly conserved in eukaryotes but observed that its links to the proteome are not (Roguev, 2003).

Mammalian ash2 homologs

Drosophila ash2 belongs to the trithorax (trx) gene family. Ash2 positively regulates expression of homeotic selector genes, and is implicated in early development and formation of various disc patterns in the fruit fly. Through large-scale sequencing of human genomic DNA coupled with in silico gene trapping, a gene (Ash2L) has been identified on chromosome 8p11.2 whose predicted product is highly homologous to ash2. It has been characterized, and its mouse counterpart has been identified. The human ash2 cDNA is 2368 bp long, encoding 628 amino acids. The 16-exon gene spans more than 34 kb of genomic DNA between STS markers WI-9207 (centromere) and AFMA295ZD5 (telomere) on chromosome 8, with transcription oriented telomere to centromere. The ash2 genes are highly conserved among different species, including C. elegans and yeast. The presence of a conserved bipartite nuclear localization signal and a PHD finger motif in the human ash2 gene suggests that the gene product would function as a transcriptional regulator in humans, as its homologue does in Drosophila (Ikegawa, 1999).

Drosophila ash2 is a member of the trxG gene super family, some human homologues of which are involved in hematopoiesis and leukemia. The isolation of the human homologue of Drosophila ash2 and its alternative splicing isoforms, ASH2L1 and ASH2L2, is reported. ASH2L proteins are 60% homologous to Drosophila Ash2. ASH2L also has a zinc finger motif (C2C2) although it is not identical to that in ASH2. Expression profile analysis showed that the amount of ASH2L transcripts is extremely high in fetal liver, testis, and leukemia cell lines with erythroid and megakaryocytic potential such as K562, Hel, and Dami. These cells were treated with differentiation inducers phorbol ester and hemin. ASH2L is downregulated rapidly and dramatically in K562, Hel, and Dami cells during phorbol ester induced differentiation with megakaryocytic features. However, its expression is maintained at a high level during erythroid differentiation of K562 cells induced with hemin. These results suggest that ASH2L plays a role in hematopoiesis and is associated with some special kinds of leukemia (Wang, 2001).

SET protein complex in vertebrates methylates H3

The abundant and chromatin-associated protein HCF-1 is a critical player in mammalian cell proliferation as well as herpes simplex virus (HSV) transcription. Separate regions of HCF-1 critical for its role in cell proliferation associate with the Sin3 histone deacetylase (HDAC) and a previously uncharacterized human trithorax-related Set1/Ash2 histone methyltransferase (HMT). The Set1/Ash2 HMT methylates histone H3 at Lys 4 (K4), but not if the neighboring K9 residue is already methylated. HCF-1 tethers the Sin3 and Set1/Ash2 transcriptional regulatory complexes together even though they are generally associated with opposite transcriptional outcomes: repression and activation of transcription, respectively. Nevertheless, this tethering is context-dependent because the transcriptional activator VP16 selectively binds HCF-1 associated with the Set1/Ash2 HMT complex in the absence of the Sin3 HDAC complex. These results suggest that HCF-1 can broadly regulate transcription, both positively and negatively, through selective modulation of chromatin structure (Wysocka, 2003).

Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. These include activating signal cointegrator 2 (ASC-2), a recently isolated transcriptional coactivator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors and numerous other transcription factors. ASC-2 belongs to a steady-state complex of approximately 2 MDa (ASC-2 complex [ASCOM]) in HeLa nuclei. ASCOM contains retinoblastoma-binding protein RBQ-3, alpha/ß-tubulins, and trithorax group proteins ALR-1, ALR-2, HALR, and ASH2. In particular, ALR-1/2 and HALR contain a highly conserved 130- to 140-amino-acid motif termed the SET domain, which was recently implicated in histone H3 lysine-specific methylation activities. Indeed, recombinant ALR-1, HALR, and immunopurified ASCOM exhibit very weak but specific H3-lysine 4 methylation activities in vitro, and transactivation by retinoic acid receptor appears to involve ligand-dependent recruitment of ASCOM and subsequent transient H3-lysine 4 methylation of the promoter region in vivo. Thus, ASCOM may represent a distinct coactivator complex of nuclear receptors. Further characterization of ASCOM will lead to a better understanding of how nuclear receptors and other transcription factors mediate transcriptional activation (Goo, 2003).

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).

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 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 (see Drosophila Menin-1), 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 Trithorax group protein Ash2l and Saf-A are recruited to the inactive X chromosome at the onset of stable X inactivation

Mammals compensate X chromosome gene dosage between the sexes by silencing of one of the two female X chromosomes. X inactivation is initiated in the early embryo and requires the non-coding Xist RNA, which encompasses the inactive X chromosome (Xi) and triggers its silencing. In differentiated cells, several factors including the histone variant macroH2A and the scaffold attachment factor SAF-A are recruited to the Xi and maintain its repression. Consequently, in female somatic cells the Xi remains stably silenced independently of Xist. This study identified the Trithorax group protein Ash2l as a novel component of the Xi. Ash2l is recruited by Xist concomitantly with Saf-A and macroH2A at the transition to Xi maintenance. Recruitment of these factors characterizes a developmental transition point for the chromatin composition of the Xi. Surprisingly, expression of a mutant Xist RNA that does not cause gene repression can trigger recruitment of Ash2l, Saf-A and macroH2A to the X chromosome, and can cause chromosome-wide histone H4 hypoacetylation. This suggests that a chromatin configuration is established on non-genic chromatin on the Xi by Xist to provide a repressive compartment that could be used for maintaining gene silencing. Gene silencing is mechanistically separable from the formation of this repressive compartment and, thus, requires additional pathways. This observation highlights a crucial role for spatial organization of chromatin changes in the maintenance of X inactivation (Pullirsch, 2010).

Ash1l methylates Lys36 of histone H3 independently of transcriptional elongation to counteract polycomb silencing

Molecular mechanisms for the establishment of transcriptional memory are poorly understood. 5,6-dichloro-1-D-ribofuranosyl-benzimidazole (DRB) is a P-TEFb kinase inhibitor that artificially induces the poised RNA polymerase II (RNAPII), thereby manifesting intermediate steps for the establishment of transcriptional activation. In this study, using genetics and DRB, it was shown that mammalian Absent, small, or homeotic discs 1-like (Ash1l), a member of the trithorax group proteins, methylates Lys36 of histone H3 to promote the establishment of Hox gene expression by counteracting Polycomb silencing. Importantly, it was found that Ash1l-dependent Lys36 di-, tri-methylation of histone H3 in a coding region and exclusion of Polycomb group proteins occur independently of transcriptional elongation in embryonic stem (ES) cells, although both were previously thought to be consequences of transcription. Genome-wide analyses of histone H3 Lys36 methylation under DRB treatment have suggested that binding of the retinoic acid receptor (RAR) to a certain genomic region promotes trimethylation in the RAR-associated gene independent of its ongoing transcription. Moreover, DRB treatment unveils a parallel response between Lys36 methylation of histone H3 and occupancy of either Tip60 or Mof in a region-dependent manner. It was also found that Brg1 is another key player involved in the response. These results uncover a novel regulatory cascade orchestrated by Ash1l with RAR and provide insights into mechanisms underlying the establishment of the transcriptional activation that counteracts Polycomb silencing (Miyazaki, 2013).


absent, small, or homeotic discs 2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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