male-specific lethal 3

MSL-3 homologs from yeast to humans

The seven member, human MORF4 related gene (MRG) family has been identified based on the ability of Mortality factor on chromosome 4 (MORF4) to induce replicative senescence in immortal cell lines assigned to complementation group B. Initial computer based similarity searches identified human retinoblastoma binding protein 1 (RBP-1), Drosophila melanogaster male specific lethal-3 (Msl-3), S. pombe altered polarity-13 (Alp13) and S. cerevisiae Eaf3p, a component of the yeast NuA4 HAT complex, as having similarity to the human MRG protein family. This suggested that the MRG family might be found in multiple species, and analysis of other homologs would provide functional and evolutionary insights into this gene family. This study reports that MRG family members are present in twenty-three species based on molecular assays and sequence similarity searches. The new family members were divided into two groups based on similarity to the predominant human MRG family members, MRG15 and MRGX. The family members similar to MRG15 define a new, highly conserved subsection of the chromo domain superfamily. Additionally, conservation in the C-terminal two thirds of all the MRG family members and the Drosophila and human MSL-3 proteins defines a new protein domain, the MRG domain. These results indicate a highly conserved role for the MRG family in transcriptional regulation via chromatin remodeling by histone acetylation (Bertram, 2001).

In Drosophila, the MSL complex is required for the dosage compensation of X-linked genes in males and contains a histone acetyltransferase, MOF. A point mutation in the MOF acetyl-CoA-binding site results in male-specific lethality. Yeast Esa1p, a MOF homolog, is essential for cell cycle progression and is the catalytic subunit of the NuA4 acetyltransferase complex. NuA4 purified from yeast with a point mutation in the acetyl-CoA-binding domain of Esa1p exhibits a strong decrease in histone acetyltransferase activity, yet has no effect on growth. Eaf3p (Esa1p-associated factor-3 protein), a yeast protein homologous to the Drosophila dosage compensation protein MSL3, is also a stable component of the NuA4 complex. Unlike other subunits of the complex, it is not essential, and the deletion mutant has no growth phenotype. NuA4 purified from the mutant strain has a decreased apparent molecular mass, but retains wild-type levels of histone H4 acetyltransferase activity. The EAF3 deletion and the ESA1 mutation lead to a decrease in PHO5 gene expression; the EAF3 deletion also significantly reduces HIS4 and TRP4 expressions. These results, together with those previously obtained with both the MSL and NuA4 complexes, underscore the importance of targeted histone H4 acetylation for the gene-specific activation of transcription (Eisen, 2001).

Mammalian MSL-3 homologs

The Drosophila male-specific lethal (MSL) genes regulate transcription from the male X chromosome in a dosage compensation pathway that equalizes X-linked gene expression in males and females. The members of this gene family, including msl-1, msl-2, msl-3, mle, and mof, encode proteins with no sequence homology. However, mutations in each of these genes produce a similar phenotype: sex-specific lethality of male embryos caused by the failure of mutants to increase transcription from the single male X chromosome. The MSL gene products assemble into a multiprotein transcriptional activation complex at hundreds of sites along the chromatin of the X chromosome. A human gene, named MSL3L1, has been isolated an characterized that encodes a protein with significant homology to Drosophila MSL-3 in three distinct regions, including two putative chromo domains. MSL3L1 was identified by database queries with genomic sequence from BAC GS-590J6 (GenBank AC0004554) in Xp22.3 and was evaluated as a candidate gene for several developmental disorders mapping to this region, including OFD1 and Spondyloepiphyseal Dysplasia Tarda (SED tarda), as well as Aicardi syndrome and Goltz syndrome (Prakash, 1999)

A multisubunit complex in mammals homologous to the Drosophila dosage compensation complex

It is well known that histone acetylases are important chromatin modifiers and that they play a central role in chromatin transcription. Evidence is presented for novel roles of histone acetylases. The TIP60 histone acetylase purifies as a multimeric protein complex. Besides histone acetylase activity on chromatin, the TIP60 complex possesses ATPase, DNA helicase, and structural DNA binding activities. Ectopic expression of mutated TIP60 lacking histone acetylase activity results in cells with defective double-strand DNA break repair. Importantly, the resulting cells lose their apoptotic competence, suggesting a defect in the cells' ability to signal the existence of DNA damage to the apoptotic machinery. These results indicate that the histone acetylase TIP60-containing complex plays a role in DNA repair and apoptosis (Ikura, 2000).

The NuA4 histone acetyltransferase (HAT) multisubunit complex is responsible for acetylation of histone H4 and H2A N-terminal tails in yeast. Its catalytic component, Esa1, is essential for cell cycle progression, gene-specific regulation and has been implicated in DNA repair. Almost all NuA4 subunits have clear homologues in higher eukaryotes, suggesting that the complex is conserved throughout evolution to metazoans. NuA4 complexes are indeed present in human cells. Tip60 and its splice variant Tip60b/PLIP were purified as stable HAT complexes associated with identical polypeptides, with 11 of the 12 proteins being homologs of yeast NuA4 subunits. This indicates a highly conserved subunit composition and the identified human proteins underline the role of NuA4 in the control of mammalian cell proliferation. ING3, a member of the ING family of growth regulators, links NuA4 to p53 function which has been confirmed in vivo. Proteins specific to the human NuA4 complexes include ruvB-like helicases and a bromodomain-containing subunit linked to ligand-dependent transcription activation by the thyroid hormone receptor. Subunits MRG15 and DMAP1 are present in distinct protein complexes harboring histone deacetylase and SWI2-related ATPase activities, respectively. Finally, analogous to yeast, a recombinant trimeric complex formed by Tip60, EPC1, and ING3 is sufficient to reconstitute robust nucleosomal HAT activity in vitro. In conclusion, the NuA4 HAT complex is highly conserved in eukaryotes, in which it plays primary roles in transcription, cellular response to DNA damage, and cell cycle control (Doyon, 2004).

A stable, multisubunit human histone acetyltransferase complex (hMSL) is described that contains homologs of the Drosophila dosage compensation proteins MOF, MSL1, MSL2, and MSL3. This complex shows strong specificity for histone H4 lysine 16 in chromatin in vitro, and RNA interference-mediated knockdown experiments reveal that it is responsible for the majority of H4 acetylation at lysine 16 in the cell. hMOF is a component of additional complexes, forming associations with host cell factor 1 and a protein distantly related to MSL1 (hMSL1v1). Two versions of hMSL3 were found in the hMSL complex that differ by the presence of the chromodomain. Lastly, reduction in the levels of hMSLs and acetylation of H4 at lysine 16 were found to be correlated with reduced transcription of some genes and with a G(2)/M cell cycle arrest. This is of particular interest given the recent correlation of global loss of acetylation of lysine 16 in histone H4 with tumorigenesis (Smith, 2005).

The longer form of hMSL3 most likely corresponds to the previously characterized hMSL3L1 protein, based on its migration on SDS-PAGE, while the shorter form is consistent with the predicted sizes of the hMSL3L1 isoform 'c' protein identified from cDNA sequencing projects and hMSL3L2, an expressed retrogene of the hMSL3L1c isoform. hMSL3L1c and hMSL3L2 lack an amino-terminal chromodomain characteristic of MSL3 and its related proteins human MRG15 and yeast Eaf3p (EMM protein family). Although both long and short forms of hMSL3 protein are consistently observed, the ratios varied from cell line to cell line and even in different preparations from the same cell line. Additionally, RT-PCR measurements indicate that the ratios of the chromodomain containing hMSL3L1, the shorter splice isoform, and the product of its retrogene vary widely among different cell lines and tissues. Together, these results indicate that the presence or absence of the chromodomain in hMSL3 is a highly regulated phenomenon most likely implicated in controlling the association of the hMSL proteins to specific chromatin regions. Interestingly, the related human protein MRG15 has also been shown to have splice variants lacking the chromodomain region as well as two shorter paralogs with the same feature. hMRG15 and chromodomain-less MRGX proteins have both been found associated with a distinct human HAT protein, Tip60 (Smith, 2005).

Affinity purification of hMOF and hMSL3L1 identifies a stable human MSL HAT complex. To determine if the hMSL proteins are components of a stable HAT complex and if there are additional proteins associated with them, transduced cell lines carrying either TAP-tagged hMOF or hMSL3L1 were used. Both purified hMOF and hMSL3 proteins were found stably associated with the other hMSLs as shown by Western analysis. Furthermore, both preparations had HAT activity with specificity toward nucleosomal histone H4. Proteins copurifying with hMSL3L1 through the two affinity steps were separated by SDS-PAGE and stained with SYPRO Ruby, and gel slices were subjected to in-gel digestion and tandem mass spectrometry. Strikingly, the three major bands detected by staining were identified by mass spectrometry as hMSL1, hMSL2, and hMOF, in addition to hMSL3L1. A previous report suggested that hMOF was present in a complex with another MSL3-related protein, MRG15, a known component of the TIP60 HAT complex. This suggests that MSL3 family proteins could interact with more than one MYST family histone acetyltransferase. To determine if hMOF was the only histone acetyltransferase associated with hMSL3L1, partially purified hMSL3L1 preparations were subjected to immunoprecipitation with hMOF antibodies. HAT activity was observed in the partially purified hMSL3L1 fractions and in the sequential hMOF immunoprecipitate but not in the unbound fraction, indicating that hMOF is the only HAT protein associated with hMSL3L1 (Smith, 2005).

TAP-tagged hMOF was also subjected to TAP purification and identification of associated proteins by Western blotting and tandem mass spectrometry. In contrast to purified hMSL3L1, the pattern of bands obtained on stained gels was less clear, with hMOF-TAP being by far the most prominent band. This result suggests that hMOF can associate with polypeptides other than hMSLs, for example, with HCF-1. HCF-1 was originally identified as a cellular protein that associates with VP16 to activate immediate early genes during the lytic cycle of herpes simplex virus infection. In addition, HCF-1 associates with a Sin3 histone deacetylase complex as well as with ASH1 and MLL1 histone methyltransferase complexes, including an MLL1 complex containing MOF. It was confirmed that HCF-1 associates specifically with hMOF by performing Western analysis with anti-HCF antibodies on hMOF-TAP, hMSL3L1-TAP, and mock TAP-purified samples. These data clearly indicate that hMOF associates with HCF-1 in a complex distinct from the hMSL complex obtained with hMSL3L1 (Smith, 2005).

Another protein specifically identified in the hMOF-TAP preparation, but not with hMSL3L1-TAP, is distantly related to hMSL1: LOC284058 (which will be referred to as hMSL1v1. hMSL1v1 and the closely related hMSL1v2 (FLJ23861) share similarity with hMSL1 at their C termini. The MSL1 C terminus was shown to mediate binding to MOF and MSL3 in Drosophila. The interactions between MSL1 with MOF and MSL3 were extensively mapped, and distinct regions of the C-terminal domain were shown to be responsible for binding to MOF or MSL3. While the region of similarity between the MSL1 variants and the true MSL1 orthologs does not extend to the region of MSL1 that is implicated in binding to MSL3, significant homology is found within the putative MOF-interacting region. Importantly, hMSL1v1 was not found in TAP-purified hMSL3L1 preparations, reflecting at least one functional difference between hMSL1 and hMSL1v (Smith, 2005).

Nucleosomal HAT activity of the human MOF complexes is specific for histone H4 lysine 16. A notable feature of the Drosophila MSL complex is its specificity for acetylation of H4 at lysine 16. hMSL3L1-TAP and hMOF-TAP complexes were assayed with free histones and nucleosomes. hMSL3-TAP and hMOF-TAP complexes specifically label histone H4 in the context of chromatin, while also acetylating histone H3 when presented with a mixture of free histones. The site specificity of the hMOF-TAP complexes was tested on recombinant histone H4, and acetylation was detected with site-specific antisera. While purified yeast NuA4 complex acetylates both lysine 12 and 16 of H4 as expected, the hMOF-TAP complexes have a strong preference for lysine 16. To confirm this specificity for lysine 16 in the context of nucleosomes, hMOF immunoprecipitates were assayed with mononucleosomes and 3H-labeled acetyl-CoA. The histones were separated, and the band corresponding to histone H4 was subjected to Edman degradation. Incorporation of acetate was deduced by scintillation counting of the product from each cycle of Edman degradation. The majority of the released [3H]acetate can be attributed to acetylation of lysine 16. The post-lysine 16 trailing pattern is likely due to sequencing lag, a result previously observed when sequencing of H4 was attempted from membrane supports (Smith, 2005).

male-specific lethal 3: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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