Suppressor of variegation 3-9
In Schizosaccharomyces pombe the mating-type information is stored at two transcriptionally silent loci (mat2 and mat3). The region between these sites (K region) is inert for meiotic crossing over. The mating-type genes (M or P) are expressed only when present at a third, active locus (mat1). The positional regulation of P genes is based on repression at the silent site, caused by elements in the flanking DNA sequences. A sterile mat1 deleted strain has been mutagenized and cells that are able to conjugate were selected. Recessive mutations of this type should define genes encoding trans-acting factors involved in repression of the silent mating-type loci. Mutations in two genes, clr1 and swi6, have been shown to allow both expression of the silent loci and recombination in the K region. The sensitivity of the present selection is demonstrated by the isolation of new mutations that derepress one or both of the silent loci (M-mating or bi-mating). The frequency of M-mating mutants was almost two orders of magnitude higher than that of bi-mating mutants and in all mutants analyzed mat3-M expression was significantly higher than mat2-P expression. The mutations define three new genes, clr2, clr3 and clr4. In addition the rik1 mutant previously known to allow recombination in the K region also depresses the silent loci (Ekwall, 1994).
The ura4+ gene displays phenotypes consistent with variegated expression when inserted at 11 sites throughout fission yeast centromere 1. An abrupt transition occurs between the zone of centromeric repression and two adjacent expressed sites. Mutations in six genes alleviate repression of the silent-mating type loci and of ura4+ expressed from a site adjacent to the silent locus, mat3-M. Defects at all six loci affect repression of the ura4+ gene adjacent to telomeres and at the three centromeric sites tested. The clr4-S5 and rik1-304 mutations cause the most dramatic derepression at two out of three sites within cen1. All six mutations had only slight or intermediate effects on a third site in the center of cen1 or on telomeric repression. Strains with lesions at the clr4, rik1, and swi6 loci have highly elevated rates of chromosome loss. It is proposed that the products of these genes are integral in the assembly of a heterochromatin-like structure, with distinct domains, enclosing the entire centromeric region that reduces or excludes access to transcription factors. The formation of this heterochromatic structure may be an absolute requirement for the formation of a fully functional centromere (Allshire, 1995).
Transcriptional silencing is known to occur at centromeres, telomeres and the mating type region in the nucleus of fission yeast, Schizosaccharomyces pombe. Mating-type silencing factors also affect transcriptional repression within centromeres and to some extent at telomeres. Mutations in the clr4+, rik1+ and swi6+ genes dramatically reduce silencing at certain centromeric regions and cause elevated chromosome loss rates. Swi6p co-localizes with the three silent chromosomal regions. The involvement of clr4+, rik1+ and swi6+ in centromere function has been investigated in further detail. Fluorescence in situ hybridization (FISH) was used to show that, as in swi6 mutant cells, centromeres lag on late anaphase spindles in clr4 and rik1 mutant cells. This phenotype is consistent with a role for these three gene products in fission yeast centromere function. The Swi6 protein was found to be delocalized from all three silent chromosomal regions, and dispersed within the nucleus, in both clr4 and rik1 mutant cells. The phenotypic similarity observed in all three mutants is consistent with the products of both the clr4+ and rik1+ genes being required to recruit Swi6p to the centromere and other silent regions. Mutations in clr4, rik1 and swi6 also result in elevated sensitivity to reagents that destabilize microtubules and show a synergistic interaction with a mutation in the beta-tubulin gene (nda3). These observations suggest that clr4+ and rik1+ must play a role in the assembly of Swi6p into a transcriptionally silent, inaccessible chromatin structure at fission yeast centromeres that is required to facilitate interactions with spindle microtubules and to ensure normal chromosome segregation (Ekwall, 1996).
The clr4 gene, which is essential for silencing of centromeres and the mating-type loci in Schizosaccharomyces pombe, encodes a protein with high homology to the product of Su(var)3-9, a gene affecting PEV in Drosophila. Like Su(var)3-9p, Clr4p contains SET and chromo domains, motifs found in proteins that modulate chromatin structure. Site-directed mutations in the conserved residues of the chromo domain confirm that it is required for proper silencing and directional switching of the mating type, like SET domain. Surprisingly, RNA differential display experiments demonstrate that clr4+ can mediate transcriptional activation of certain other loci. These results show that clr4 plays a critical role in silencing at mating-type loci and centromeres through the organization of repressive chromatin structure and demonstrate a new, activator function for Clr4p (Ivanova, 1998).
Although DNA replication has been thought to play an important role in the silencing of mating type loci in Saccharomyces cerevisiae, recent studies indicate that silencing can be decoupled from replication. In Schizosaccharomyces pombe, mating type silencing is brought about by the trans-acting proteins, namely Swi6 (the yeast equivalent of Drosophila HP1), Clr1-Clr4, and Rhp6, in cooperation with the cis-acting silencers. The latter contain an autonomous replication sequence, suggesting that DNA replication may be critical for silencing in S. pombe. To investigate the connection between DNA replication and silencing in S. pombe, several temperature-sensitive mutants of DNA polymerase alpha were examined. One such mutant, swi7H4, exhibits silencing defects at mat, centromere, and telomere loci. This effect is independent of the checkpoint and replication defects of the mutant. Interestingly, the extent of the silencing defect in the swi7H4 mutant at the silent mat2 locus is further enhanced in absence of the cis-acting, centromere-proximal silencer. The chromodomain protein Swi6, which is required for silencing and is localized to mat and other heterochromatin loci, interacts with DNA polymerase alpha in vivo and in vitro in wild type cells. However, it does not interact with the mutant pol alpha and is delocalized away from the silent mat loci in the mutant. These results demonstrate a role of DNA polymerase alpha in the establishment of silencing. A recruitment model is proposed for the coupling of DNA replication with the establishment of silencing by the chromodomain protein Swi6, which may be applicable to higher eukaryotes (Ahmed, 2001).
The assembly of higher order chromatin structures has been linked to the covalent modifications of histone tails. In vivo evidence is provided that lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the Clr4 protein at heterochromatin-associated regions in fission yeast. Both the conserved chromo- and SET domains of Clr4 are required for H3 Lys9 methylation in vivo. Localization of Swi6, a homolog of Drosophila HP1, to heterochomatic regions is dependent on H3 Lys9 methylation. Moreover, an H3-specific deacetylase Clr3 and a beta-propeller domain protein Rik1 are required for H3 Lys9 methylation by Clr4 and Swi6 localization. These data define a conserved pathway wherein sequential histone modifications establish a 'histone code' essential for the epigenetic inheritance of heterochromatin assembly (Nakayama, 2001).
The encapsulation of otherwise transcribable loci within transcriptionally inactive heterochromatin is rapidly gaining recognition as an important mechanism of epigenetic gene regulation. In the fission yeast Schizosaccharomyces pombe, heterochromatinization of the mat2/mat3 loci silences the mating-type information encoded within these loci. The solution structure of the chromo domain from the cryptic loci regulator protein Clr4 is presented in this study. Clr4 is known to regulate silencing and switching at the mating-type loci and to affect chromatin structure at centromeres. Clr4 and its human and Drosophila homologs have been identified as histone H3-specific methyltransferases, further implicating this family of proteins in chromatin remodeling. The structure highlights a conserved surface that may be involved in chromo domain-ligand interactions. Two chromo domain mutants (W31G and W41G) that affect silencing and switching in full-length Clr4, were examined. Both mutants are significantly destabilized relative to wild-type (Horita, 2001).
In eukaryotes, histone H3 lysine 9 (H3K9) methylation is a common mechanism involved in gene silencing and the establishment of heterochromatin. The loci of the major heterochromatic H3K9 methyltransferase Su(var)3-9 and the functionally unrelated γ subunit of the translation initiation factor eIF2 are fused in Drosophila melanogaster. This study examined the phylogenetic distribution of this unusual gene fusion and the molecular evolution of the H3K9 HMTase Su(var)3-9. The gene fusion had taken place in the ancestral line of winged insects and silverfishs (Dicondylia) about 400 million years ago. Su(var)3-9 genes were cloned from a collembolan and a spider where both genes ancestrally exist as independent transcription units. In contrast, a Su(var)3-9-specific exon was found inside the conserved intron position 81-1 of the eIF2γ gene structure in species of eight different insect orders. Intriguinly, in the pea aphid Acyrthosiphon pisum, only sequence remains were detected of this Su(var)3-9 exon in the eIF2γ intron, along with an eIF2γ-independent Su(var)3-9 gene. This reveals an evolutionary re-fission of both genes in aphids. Su(var)3-9 chromo domains are similar to HP1 chromo domains, which points to a potential binding activity to methylated K9 of histone H3. SET domain comparisons suggest a weaker methyltransferase activity of Su(var)3-9 in comparison to other H3K9 HMTases. Astonishingly, 11 of 19 previously described, deleterious amino acid substitutions found in Drosophila Su(var)3-9 are seemingly compensable through accompanying substitutions during evolution. Therefore, examination of the Su(var)3-9 evolution revealed strong evidence for the establishment of the Su(var)3-9/eIF2γ gene fusion in an ancestor of dicondylic insects and a re-fission of this fusion during the evolution of aphids. The comparison of 65 selected chromo domains and 93 selected SET domains from Su(var)3-9 and related proteins offers functional predictions concerning both domains in Su(var)3-9 proteins (Krauss, 2006).
Fusions of two ancestrally independent genes with completely different functions similar to Su(var)3-9/eIF2γ have not been described so far. Other known gene fusions are supposed to be positively selected because the resulting gene products are fused players of the same cellular pathway, fused molecular interactors or perform at least one novel function using an acquired protein domain. How, then, was it possible that two proteins as different as Su(var)3-9 and eIF2γ in respect to sequence, structure, function, cellular localization and interactions were evolved to be derived from a single gene structure? Northern blots in Drosophila revealed that the eIF2γ mRNA is expressed strongly in each developmental stage, whereas the Su(var)3-9 mRNA is expressed weakly during the first nine hours of embryonal development and almost undetectable during later stages. Therefore, it was hypothesized that the Su(var)3-9-specific splice variant of the Su(var)3-9/eIF2γ gene 'parasitize' on the strong expression of the eIF2γ splice variant. The developmental changes of the Su(var)3-9 share in the Su(var)3-9/eIF2γ primary transcript are unable to influence the eIF2γ expression significantly because of the generally weak expression rate of Su(var)3-9. Under these conditions, it was possible that a Su(var)3-9 retrotransposition into the 81-1 exon of an ancient eIF2γ gene has taken place and that this event has immediately resulted in a functional, alternative spliced gene. The only additional prerequisite is an activation of a cryptic splice site at the 5'end of the Su(var)3-9-specific exon, which has to be sufficient weak to not disturb significantly the eIF2γ expression (Krauss, 2006).
To determine age and distribution of the Su(var)3-9/eIF2γ gene fusion, orthologues of both genes or of the gene fusion, respectively, were cloned in 19 selected genera of arthropods. The fusion is restricted to Ectognatha (Insecta) and, possibly, to Dicondylia (Pterygota + Zygentoma). According to palaeontological evidence with respect to the first true insect, the age of this unusual genomic assemblage can be estimated to about 400 million years. Irrespective of its long history, the gene fusion seems to impose a functional burden on the encoded gene products. In beetles and butterflies obvious splice artefacts, containing all exons of the fusion, are detectable. The coding potential of these artefacts comprises all eIF2γ exons under inclusion of the Su(var)3-9-specific exon, which renders the encoded protein functionally inactive, at least with regard to eIF2γ. Notably, the Su(var)3-9-specific part of the gene fusion consists in all analyzed 21 species of only one large exon (>1450 bp). Initially, this may have been caused by retrotransposition of Su(var)3-9 sequences into the eIF2γ gene. Afterwards, the establishment of internal Su(var)3-9 introns might have been suppressed by selection against abundant functionless or antimorphic splice artefacts, which would concomitantly decrease the expression of functional eIF2γ mRNAs. At the same time, the eIF2γ part of the gene fusion has acquired at least four novel introns, and the newly emerged Acyrthosiphon Su(var)3-9 gene has gained two novel introns (Krauss, 2006).
During this study, evidence was found for a reversion of the Su(var)3-9/eIF2γ gene fusion in aphids. The remnants of a Su(var)3-9-coding region in the eIF2γ intron 81-1 of Acyrthosiphon pisum reveal that these aphids descend from ancestors which harbored the gene fusion. Because the cicada Cercopis vulnerata possesses the fused gene, the fission of both gene parts has to be occurred during the evolution of the hemipterid group Sternorrhyncha (psyllids, whiteflies, aphids and coccids). It remains open whether a genomic duplication has happened, or a renewed retrotransposition of the Su(var)3-9 mRNA (Krauss, 2006).
The central role of the Su(var)3-9 histone H3K9 methyltransferase for the establishment of pericentromeric heterochromatin has been shown for mammals, Drosophila and Schizosaccharomyces. The observation of Su(var)3-9 orthologues in holocentric species of insects (butterflies, hemipterans, earwigs) argues for an important role of the protein also outside of the pericentromeric heterochromatin, possibly in euchromatic gene silencing, at telomeres and/or in chromosome segregation. Whether Su(var)3-9 proteins are involved in the establishment of heterochromatic regions in aphid chromosomes, which are mostly limited to telomeres and X chromosomes, and references therein), remains to be seen. Additionally, it would be interesting to evaluate function and nuclear distribution of a Su(var)3-9 ortholog in the coccid model system Planococcus citri, where H3K9 methylation is found exclusively in the paternally imprinted chromosome set (Krauss, 2006).
This examination of the evolution of the Su(var)3-9/eIF2γ gene fusion revealed strong evidence for the establishment of this fusion in a common ancestor of dicondylic insects. Because of the unrelatedness of Su(var)3-9 and eIF2γ and the demonstrated broad phylogenetic distribution of the fusion, this gene structure is a reliable synapomorphy, but appears not to invoke novel functions of the gene products. Therefore, this gene fusion is interpreted as an event of constructive neutral evolution. The identified re-fission of this fusion during the evolution of aphids shows the vulnerability of this structure to evolutionary decay, probably due to duplication and partial degeneration. Comparison of chromo domains and SET domains from Su(var)3-9 and related proteins offers functional predictions concerning both domains in Su(var)3-9 proteins. Su(var)3-9 chromo domains are similar to HP1 chromo domains, which points to a potential binding activity to methylated K9 of histone H3. SET domain comparisons suggest less enzymatic activity of Su(var)3-9 proteins in comparison to other H3K9 HMTases. Su(var)3-9 proteins combine two motifs in one molecule, which are typical for structural (chromo domain) or enzymatic components (SET domain) of chromatin. This raises an interesting question: Are evolutionary attenuations of the chromo domain histone H3 binding affinity and of the SET domain histone H3 methyltransferase activity necessary conditions to make Su(var)3-9 compatible to animal chromatin? Domain swapping experiments may give an answer (Krauss, 2006).
The chromo and SET domains are conserved sequence motifs present in chromosomal proteins that function in epigenetic control of gene expression, presumably by modulating higher order chromatin. Based on sequence information from the SET domain, human (SUV39H1) and mouse (Suv39h1) homologs of the dominant Drosophila modifier of position-effect-variegation (PEV) Su(var)3-9 have been isolated. Mammalian homologs contain, in addition to the SET domain, the characteristic chromo domain, a combination that is also preserved in the Schizosaccharyomyces pombe silencing factor clr4. Chromatin-dependent gene regulation is demonstrated by the potential of human SUV39H1 to increase repression of the pericentromeric white marker gene in transgenic flies. Immunodetection of endogenous Suv39h1/SUV39H1 proteins in a variety of mammalian cell lines reveals enriched distribution at heterochromatic foci during interphase and centromere-specific localization during metaphase. In addition, Suv39h1/SUV39H1 proteins associate with M31, currently the only other characterized mammalian SU(VAR) homolog. These data indicate the existence of a mammalian SU(VAR) complex and define Suv39h1/SUV39H1 as novel components of mammalian higher order chromatin (Aagaard, 1999).
The Su(var)3-9 gene is ubiquitously expressed and displays highest abundance from early to mid-embryogenesis in Drosophila (Tschiersch, 1994). To analyze the temporal and tissue-specific expression profile of Suv39h1 during mouse development, an RNase protection analysis was used with a 500 bp riboprobe specific for the SET domain of Suv39h1. Suv39h1-specific transcripts were detected throughout mouse embryogenesis, and their relative abundance reaches 2- to 3-fold higher levels between day E9.5 and day E13 of development. A similar increase was also observed after retinoic acid-induced in vitro differentiation of embryonic stem cells. In contrast, Suv39h1 transcripts remain at reduced levels during later stages of embryogenesis and in adult tissues (Aagaard, 1999).
The spatial expression profile of Suv39h1 was investigated using in situ hybridizations with a Suv39h1-specific riboprobe on sagittal sections of day E12.5 total mouse fetuses. The Suv39h1 antisense probe reveals a rather uniform expression throughout the entire fetus, with Suv39h1 transcripts being present in tissues derived from all three germ layers. In comparison with neuroectodermal structures, Suv39h1 expression is slightly elevated in the mesoderm-derived somites and reaches highest levels in fetal liver. Suv39h1 expression is also detected in heart, stomach and many other organs. Together with the RNase protection analysis shown above, these data indicate broad expression of Suv39h1 during embryonic and adult stages of mouse development (Aagaard, 1999).
To characterize the endogenous Suv39h1/SUV39H1 proteins in mammalian cells, a polyclonal rabbit antiserum was generated that was raised against a bacterially expressed, glutathione S-transferase (GST) fusion product comprising amino acids 82-412 from the murine Suv39h1 protein. Western blot analysis of in vitro translated SUV39H1 indicated that the alpha-Suv39h1 antiserum also recognizes the almost identical (95%) human protein, but not the related Suv39h2 gene product. Following affinity-purification, this polyclonal alpha-Suv39h1 antiserum was used to detect endogenous proteins in Ponceau S-adjusted nuclear extracts derived from a variety of human and mouse cell lines. In all eight cell lines tested, the alpha-Suv39h1 antiserum recognizes a specific endogenous protein of ~48 kDa that co-migrates with in vitro translated SUV39H1 and whose size is in good agreement with products predicted from the coding sequences of the respective mammalian cDNAs. In addition to the endogenous proteins, the alpha-Suv39h1 antiserum also detects ectopic (myc)3-SUV39H1 (~55 kDa) that is overexpressed in 'stably' transfected HeLa-B3 cells. No other proteins are visualized, demonstrating the specificity of this alpha-Suv39h1 antiserum, which is similarly efficient in detecting both mouse Suv39h1 and human SUV39H1. Protein abundance largely correlates (with the exception of NIH 3T3 cells) with the levels of endogenous Suv39h1/SUV39H1 mRNAs, indicating broad expression in mammalian cell lines (Aagaard, 1999).
To investigate the subnuclear localization of Suv39h1/SUV39H1 proteins, their distribution was characterized in interphase nuclei of several mouse and human cell lines. In contrast to the dispersed human interphase chromatin, mouse nuclei contain cytologically visible blocks of heterochromatin that can be highlighted with 4'-6'-diamidino-2-phenylindole (DAPI), which preferentially stains A/T-rich repeat sequences of constitutive heterochromatin. M31 is a mammalian homolog of Drosophila HP1. Co-localization analyses were performed with rat monoclonal alpha-M31 antibodies, which have been shown to define heterochromatic foci, and with human auto centromeric antibodies (hACA), which specifically decorate centromeric positions (Aagaard, 1999).
Indirect immunofluorescence of Triton X-100-extracted mouse Cop8 cells with the alpha-Suv39h1 antiserum indicates concentration of Suv39h1 protein at several (7-10) nuclear patches which overlap with the bright DAPI counterstaining and with the focal distribution of M31. The merged image of the Suv39h1 and M31 staining patterns demonstrate significant but not complete co-localization of these proteins. In addition to the prominent heterochromatic foci, some weakly staining areas are detected in which Suv39h1 and M31 may only partly coincide. This subnuclear distribution of Suv39h1 protein has been confirmed in other mouse cell lines (Aagaard, 1999).
In contrast to M31, the human auto centromeric antibodies (hACA) serum detects many discrete dots. However, the majority of these hACA positions appear enriched at the periphery of Suv39h1 foci, consistent with the clustering of centromeres around heterochromatic regions in interphase. It is concluded that endogenous Suv39h1 protein significantly co-localizes with M31 and preferably associates with heterochromatin in mouse interphase nuclei (Aagaard, 1999).
A possible association of the SUV39H1 protein with mitotic chromatin in several human cell lines was investigated. Logarithmically growing cells were treated with colcemid, resulting in metaphase arrest of ~20% of the cells. Distribution of endogenous SUV39H1 protein along unfixed metaphase chromosomes was then analysed by indirect immunofluorescence with the alpha-Suv39h1 antiserum and, as a comparison, with hACA antibodies (Aagaard, 1999).
Interestingly, endogenous SUV39H1 protein in HeLa metaphase spreads is detected at centromeric positions in a staining that resolves into the classical two-dotted pattern, which reflects the centromeres of sister chromatids. Higher magnification of the characteristic blocks of pericentromeric heterochromatin, which can be visualized by staining with distamycin A-DAPI (DA-DAPI) and which are prominent, for example, in human chromosome 1, demonstrates that SUV39H1 is specifically concentrated at the centromeres, but does not decorate the adjacent heterochromatic domain. Furthermore, co-localization analysis with the hACA serum indicates a very similar, yet distinct distribution between the SUV39H1 signals and the hACA staining. Indeed, higher magnification of the merged images illustrates that SUV39H1 is concentrated at the outer region of the centromeres, whereas hACA epitopes appear more internal. At this level of resolution, the extent of partial overlap between SUV39H1 and hACA epitopes in a common centromeric region is difficult to define (Aagaard, 1999).
Centromere-specific localization of SUV39H1 has been confirmed on metaphase spreads of other human cell lines, and is also observed at acrocentric mouse metaphase chromosomes. Together, these data classify endogenous SUV39H1/Suv39h1 as novel centromere-associated proteins in mammalian mitotic chromatin (Aagaard, 1999).
Centromeres of eukaryotes are frequently associated with constitutive heterochromatin and their activity appears to be coregulated by epigenetic modification of higher order chromatin. Mammalian Su(var)3-9 homologs encode novel centromeric proteins on metaphase-arrested chromosomes. A detailed analysis of the chromatin distribution of human SUV39H1 during the cell cycle is described in this study. Although there is significant heterochromatic overlap between SUV39H1 and M31 [HP1(beta)] during interphase, mitotic SUV39H1 displays a more restricted spatial and temporal association pattern with metaphase chromosomes than M31 [HP1(beta)], or the related HP1(alpha) gene product. SUV39H1 specifically accumulates at the centromere during prometaphase but dissociates from centromeric positions at the meta- to anaphase transition. In addition, SUV39H1 selectively associates with the active centromere of a dicentric chromosome and also with a neocentromere. Interestingly, SUV39H1 is shown to be a phosphoprotein with modifications at serine and, to a lesser degree, also at threonine residues. Whereas SUV39H1 steady-state protein levels appear constant during the cell cycle, two additional phosphorylated isoforms are detected in mitotic extracts. This intriguing localization and modification pattern would be consistent with a regulatory role(s) for SUV39H1 in participating in higher order chromatin organization at mammalian centromeres (Aagaard, 2000).
Higher-order chromatin has been implicated in epigenetic gene control and in the functional organization of chromosomes. Mouse (Suv39h1) and human (SUV39H1) histone H3 lysine 9-selective methyltransferases (Suv39h HMTases) modulate chromatin dynamics in somatic cells. A second murine gene, Suv39h2, has been characterized. Like Suv39h1, Suv39h2 encodes an H3 HMTase that shares 59% identity with Suv39h1 but which differs by the presence of a highly basic N terminus. Using fluorescent in situ hybridization and haplotype analysis, the Suv39h2 locus was mapped to the subcentromeric region of mouse chromosome 2, whereas the Suv39h1 locus resides at the tip of the mouse X chromosome. Notably, although both Suv39h loci display overlapping expression profiles during mouse embryogenesis, Suv39h2 transcripts remain specifically expressed in adult testes. Immunolocalization of Suv39h2 protein during spermatogenesis indicates enriched distribution at the heterochromatin from the leptotene to the round spermatid stage. Moreover, Suv39h2 specifically accumulates with chromatin of the sex chromosomes (XY body), which undergo transcriptional silencing during the first meiotic prophase. These data are consistent with redundant enzymatic roles for Suv39h1 and Suv39h2 during mouse development and suggest an additional function of the Suv39h2 HMTase in organizing meiotic heterochromatin that may even impart an epigenetic imprint to the male germ line (O'Carroll, 2000).
In contrast to stably repressive, constitutive heterochromatin and stably active, euchromatin, facultative heterochromatin has the capacity to alternate between repressive and activated states of transcription1. As such, it is an instructive source to understand the molecular basis for changes in chromatin structure that correlate with transcriptional status. Sirtuin 1 (SIRT1) and suppressor of variegation 3-9 homologue 1 (SUV39H1) are among the enzymes responsible for chromatin modulations associated with facultative heterochromatin formation. SUV39H1 is the principal enzyme responsible for the accumulation of histone H3 containing a tri-methyl group at its lysine 9 position (H3K9me3) in regions of heterochromatin. SIRT1 is an NAD+-dependent deacetylase that targets histone H4 at lysine 16, and through an unknown mechanism facilitates increased levels of H3K9me3. This study shows that the mammalian histone methyltransferase SUV39H1 is itself targeted by the histone deacetylase SIRT1 and that SUV39H1 activity is regulated by acetylation at lysine residue 266 in its catalytic SET domain. SIRT1 interacts directly with, recruits and deacetylates SUV39H1, and these activities independently contribute to elevated levels of SUV39H1 activity resulting in increased levels of the H3K9me3 modification. Loss of SIRT1 greatly affects SUV39H1-dependent H3K9me3 and impairs localization of heterochromatin protein 1. These findings demonstrate a functional link between the heterochromatin-related histone methyltransferase SUV39H1 and the histone deacetylase SIRT1 (Vaguero, 2007).
Mammalian SET domain-containing proteins define a distinctive class of chromatin-associated factors that are targets for growth control signals and oncogenic activation. SUV39H1, a mammalian ortholog of Drosophila Su(var)3-9, contains both SET and chromo domains, signature motifs for proteins that contribute to epigenetic control of gene expression through effects on the regional organization of chromatin structure. SUV39H1 represses transcription in a transient transcriptional assay when tethered to DNA through the GAL4 DNA binding domain. Under these conditions, SUV39H1 displays features of a long-range repressor capable of acting over several kilobases to silence basal promoters. A possible role in chromatin-mediated gene silencing is supported by the localization of exogenously expressed SUV39H1 to nuclear bodies with morphologic features suggestive of heterochromatin in interphase cells. In addition, SUV39H1 is phosphorylated specifically at the G(1)/S cell cycle transition and when forcibly expressed suppresses cell growth. Growth suppression as well as the ability of SUV39H1 to form nuclear bodies and silence transcription are antagonized by the oncogenic antiphosphatase Sbf1 that when hyperexpressed interacts with the SET domain and stabilizes the phosphorylated form of SUV39H1. These studies suggest a phosphorylation-dependent mechanism for regulating the chromatin organizing activity of a mammalian su(var) protein and implicate the SET domain as a gatekeeper motif that integrates upstream signaling pathways to epigenetic regulation and growth control (Firestein, 2000).
A novel histone methyltransferase, termed Set9, was isolated from human cells. Set9 contains a SET domain, but lacks the pre- and post-SET domains. Set9 methylates specifically lysine 4 (K4) of histone H3 (H3-K4) and potentiates transcription activation. The histone H3 tail interacts specifically with the histone deacetylase NuRD complex. Methylation of histone H3-K4 by Set9 precludes the association of NuRD with the H3 tail. Moreover, methylation of H3-K4 impairs Suv39h1-mediated methylation at K9 of H3 (H3-K9). The interplay between the Set9 and Suv39h1 histone methyltransferases is specific, since the methylation of H3-K9 by the histone methyltransferase G9a is not affected by Set9 methylation of H3-K4. These studies suggest that Set9-mediated methylation of H3-K4 functions in transcription activation by competing with histone deacetylases and by precluding H3-K9 methylation by Suv39h1. These results suggest that the methylation of histone tails can have distinct effects on transcription, depending on its chromosomal location, the combination of posttranslational modifications, and the enzyme (or protein complex) involved in the particular modification (Nishioka, 2002).
The organization of chromatin into higher-order structures influences chromosome function and epigenetic gene regulation. Higher-order chromatin has been proposed to be nucleated by the covalent modification of histone tails and the subsequent establishment of chromosomal subdomains by non-histone modifier factors. Human SUV39H1 and murine Suv39h1 -- mammalian homologs of Drosophila Su(var)3-9 and of Schizosaccharomyces pombe clr4 -- encode histone H3-specific methyltransferases that selectively methylate lysine 9 of the amino terminus of histone H3 in vitro. The catalytic motif has been mapped to the evolutionarily conserved SET domain, which requires adjacent cysteine-rich regions to confer histone methyltransferase activity. Methylation of lysine 9 interferes with phosphorylation of serine 10, but is also influenced by pre-existing modifications in the amino terminus of H3. In vivo, deregulated SUV39H1 or disrupted Suv39h activity modulates H3 serine 10 phosphorylation in native chromatin and induces aberrant mitotic divisions. These data reveal a functional interdependence of site-specific H3 tail modifications and suggest a dynamic mechanism for the regulation of higher-order chromatin (Rea, 2000).
Histone H3 lysine 9 methylation has been proposed to provide a major 'switch' for the functional organization of chromosomal subdomains. The murine Suv39h histone methyltransferases (HMTases) govern H3-K9 methylation at pericentric heterochromatin and induce a specialized histone methylation pattern that differs from the broad H3-K9 methylation present at other chromosomal regions. Suv39h-deficient mice display severely impaired viability and chromosomal instabilities that are associated with an increased tumor risk and perturbed chromosome interactions during male meiosis. These in vivo data assign a crucial role for pericentric H3-K9 methylation in protecting genome stability, and define the Suv39h HMTases as important epigenetic regulators for mammalian development (Peters, 2001).
This study provides evidence that H3-K9 methylation and DNA methylation systems can synergize to regulate silenced chromatin domains at major and minor satellite repeats in mammals. Histone H3 lysine 9 (H3-K9) methylation and DNA methylation are characteristic hallmarks of mammalian heterochromatin. H3-K9 methylation is a prerequisite for DNA methylation in Neurospora crassa and Arabidopsis thaliana. Currently, it is unknown whether a similar dependence exists in mammalian organisms. A physical and functional link is demonstrated between the Suv39h-HP1 histone methylation system and DNA methyltransferase 3b (Dnmt3b) in mammals. Whereas in wild-type cells Dnmt3b interacts with HP1alpha and is concentrated at heterochromatic foci, it fails to localize to these regions in Suv39h double null (dn) mouse embryonic stem (ES) cells. Consistently, the Suv39h dn ES cells display an altered DNA methylation profile at pericentric satellite repeats, but not at other repeat sequences. In contrast, H3-K9 trimethylation at pericentric heterochromatin is not impaired in Dnmt1 single- or Dnmt3a/Dnmt3b double-deficient ES cells. Pericentric heterochromatin is not transcriptionally inert and can give rise to transcripts spanning the major satellite repeats. In conclusion, these data demonstrate an evolutionarily conserved pathway between histone H3-K9 methylation and DNA methylation in mammals. While the Suv39h HMTases are required to direct H3-K9 trimethylation and Dnmt3b-dependent DNA methylation at pericentric repeats, DNA methylation at centromeric repeats occurs independent of Suv39h function. Thus, these data also indicate a more complex interrelatedness between histone and DNA methylation systems in mammals. Both methylation systems are likely to be important in reinforcing the stability of heterochromatic subdomains and thereby in protecting genome integrity (Lehnertz, 2003).
The use of immunofluorescence analyses and DNA methylation profiles in wt and mutant murine ES cells has demonstrated that Suv39h-mediated H3-K9 trimethylation can direct Dnmt3b to major satellite repeats present in pericentric heterochromatin. In addition, co-IP data suggest that Dnmt3b and Dnmt3a are part of a repressive complex that is targeted to methylated H3-K9 positions via HP1α and HP1β (Lehnertz, 2003).
The Suv39h-dependent DNA methylation defect at major satellites was only detectable upon digestion with MaeII and reflects a similar deficiency in heterochromatic DNA methylation as compared to Dnmt3a/Dnmt3b mutant ES cells. In contrast, genomic DNA prepared from Dnmt1-deficient ES cells displayed methylation defects that were observed after either MaeII or HpaII digestion. Sequence analyses identified no apparent HpaII sites within the 234 bp major satellite repeat unit, suggesting that they may be interspersed between satellite repeats or present at other repetitive sequences, which together comprise the large blocks of pericentric heterochromatin. It is currently unresolved whether DNA methylation at these HpaII sites is initiated by Dnmt3a/Dnmt3b in an Suv39h-dependent manner and then maintained by Dnmt1, or whether there may be differential target sensitivities of DNMTs to certain DNA sequences or even to chromosomal subdomains (Lehnertz, 2003).
In human embryonic carcinoma cell lines (Tera-1 and NCCIT), Dnmt3b also interacts with HP1α. Mutational inactivation of DNMT3b causes the rare ICF syndrome, which is in part characterized by extensive cytosine demethylation and chromosomal instabilities at pericentric heterochromatin containing satellite 2 and 3 repeats. Since human chromosomes display dense H3-K9 trimethylation at these satellites, it is anticipated that SUV39H-dependent histone methylation may also direct pericentric DNA methylation in humans (Lehnertz, 2003).
In contrast to the major satellites, Dnmt3b-dependent DNA methylation at minor satellites is not impaired in Suv39h dn ES cells. Recent immunofluorescence and chromatin immunoprecipitation analyses with highly specific antibodies that discriminate H3-K9 di- and H3-K9 trimethylation show that the histone methylation pattern differs between centromeric and pericentric heterochromatin. For example, centromeric minor satellites are enriched for H3-K9 dimethylation in both wt and Suv39h dn ES cells, whereas pericentric major satellites display selective H3-K9 trimethylation in an Suv39h-dependent manner. It is possible that Dnmt3b targeting to minor satellites could involve H3-K9 dimethylation, mediated by an HMTase that is distinct from the Suv39h enzymes and maintains a local concentration of HP1α or HP1β. This interpretation would be consistent with the robust HMTase activity associated with Dnmt3b in Suv39h-deficient nuclear extracts (Lehnertz, 2003).
In contrast to Dnmt3b, the pericentric localization of Dnmt1 and the more complete loss of DNA methylation at major satellites observed in Dnmt1 null versus Suv39h dn ES cells indicates that recruitment of Dnmt1 to pericentric regions also occurs independent of the function of the Suv39h HMTases. Indeed, Dnmt1 has been shown to be targeted via PCNA to major satellites during late replication. Similarly, in A. thaliana, maintenance of CpG methylation by the Dnmt1 homolog MET1 is not impaired in mutants of the KYP H3-K9 HMTase. These findings suggests that replication-coupled propagation of CpG methylation may be independent of H3-K9 methylation (Lehnertz, 2003).
Although H3-K9 methylation can be maintained at silent centromeric repeats in CpG (Dnmt1 homolog met1) or CpNpG (cmt3) DNA methylation-deficient mutants in A. thaliana, these studies also show that loss of DNA methylation can feed back on the persistence of H3-K9 methylation patterns if there is significant derepression of silenced loci, e.g., as observed with aberrant transcriptional activity of retro-transposons that had integrated into pericentric domains. Similarly, treatment of human cancer cell lines with the DNA-demethylating compound 5-aza-2'-deoxycytidine (5-aza-dC) results in transcriptional reactivation and reversal of repressive histone methyl marks at silenced tumor suppressor and cell cycle genes. In particular, 5-aza-dC induces a reduction in H3-K9 dimethylation while simultaneously increasing the levels for H3-K4 dimethylation and H3-K9 acetylation. RT-PCR analysis detects a weak upregulation of mouse major satellite transcripts in total RNA prepared from Suv39h dn ES cells, but not from the different DNMT-deficient cells However, pericentric heterochromatin remained underrepresented for H3-K4 dimethylation in wt and in all mutant ES cell lines examined. Together, these observations support a model in which reduced DNA methylation can alter histone methylation marks only if transcriptional reactivation is significantly induced. Since DNA methylation at major satellites is not fully lost in Dnmt1 null or Dnmt3a/Dnmt3b double-deficient ES cells, it remains possible that the complete absence of DNA methylation (e.g., in a triple-deficient Dnmt1/Dnmt3a/Dnmt3b ES cell line) would more drastically affect transcriptional activity and H3-K9 trimethylation patterns at the pericentric satellite repeats (Lehnertz, 2003).
Thus H3-K9 methylation and DNA methylation systems can synergize to regulate silenced chromatin domains at major and minor satellite repeats in mammals. Silencing is likely to be reinforced by binding of the methyl-CpG binding protein MeCP2 and associated histone deacetylases (HDACs) and HMTases. The selective impairment of Suv39h-dependent DNA methylation at the major satellites is intriguingly reminiscent of the recently discovered potential of DNA repeats to target H3-K9 methylation to a chromatin region via the generation of small double-stranded RNAs. Since transcripts spanning the mouse major satellites have been observed, it is conceivable that 'small heterochromatic' RNAs generated from these transcripts may guide recruitment of the Suv39h HMTases to direct H3-K9 trimethylation and, in turn, DNA methylation to pericentric heterochromatin. Ongoing studies are aimed to delineate the molecular mechanism(s) connecting these major epigenetic pathways (Lehnertz, 2003).
Heterochromatin protein 1 (HP1) is localized at heterochromatin sites where it mediates gene silencing. The chromo domain of HP1 is necessary for both targeting and transcriptional repression. In the fission yeast Schizosaccharomyces pombe, the correct localization of Swi6 (the HP1 equivalent) depends on Clr4, a homolog of the mammalian SUV39H1 histone methylase. Both Clr4 and SUV39H1 specifically methylate lysine 9 of histone H3. In this study it has been shown that mammalian HP1 can bind with high affinity to histone H3 methylated at lysine 9 but not at lysine 4. The chromo domain of HP1 is identified as its methyl-lysine-binding domain. A point mutation in the chromo domain, which destroys the gene silencing activity of HP1 in Drosophila, abolishes methyl-lysine-binding activity. Genetic and biochemical analysis in S. pombe shows that the methylase activity of Clr4 is necessary for the correct localization of Swi6 at centromeric heterochromatin and for gene silencing. These results provide a stepwise model for the formation of a transcriptionally silent heterochromatin: SUV39H1 places a 'methyl marker' on histone H3, which is then recognized by HP1 through its chromo domain. This model may also explain the stable inheritance of the heterochromatic state (Bannister, 2001).
Distinct modifications of histone amino termini, such as acetylation, phosphorylation and methylation, have been proposed to underlie a chromatin-based regulatory mechanism that modulates the accessibility of genetic information. In addition to histone modifications that facilitate gene activity, it is of similar importance to restrict inappropriate gene expression if cellular and developmental programs are to proceed unperturbed. Mammalian methyltransferases that selectively methylate histone H3 on lysine 9 (Suv39h HMTases) generate a binding site for HP1 proteins -- a family of heterochromatic adaptor molecules implicated in both gene silencing and supra-nucleosomal chromatin structure. High-affinity in vitro recognition of a methylated histone H3 peptide by HP1 requires a functional chromo domain; thus, the HP1 chromo domain is a specific interaction motif for the methyl epitope on lysine9 of histone H3. In vivo, heterochromatin association of HP1 proteins is lost in Suv39h double-null primary mouse fibroblasts but is restored after the re-introduction of a catalytically active SWUV39H1 HMTase. These data define a molecular mechanism through which the SUV39H-HP1 methylation system can contribute to the propagation of heterochromatic subdomains in native chromatin (Lachner, 2001).
The human ISWI-containing factor RSF (remodeling and spacing factor) mediates nucleosome deposition and, in the presence of ATP, generates regularly spaced nucleosome arrays. Using this system, recombinant chromatin was reconstituted with bacterially produced histones. Acetylation of the histone tails was found to play an important role in establishing regularly spaced nucleosome arrays. Recombinant chromatin lacking histone acetylation is impaired in directing transcription. Histone-tail modifications regulate transcription from the recombinant chromatin. Acetylation of the histone tails by p300 increases transcription. Methylation of the histone H3 tail by Suv39H1 represses transcription in an HP1-dependent manner. The effects of histone-tail modifications were observed in nuclear extracts. A highly reconstituted RNA polymerase II transcription system is refractory to the effect imposed by acetylation and methylation (Loyola, 2001).
Human (SUV39H1) and mouse (Suv39h1) histone methyltransferases (HMTases) have been isolated and have been shown to be important regulators for the organization of repressive chromatin domains. To investigate whether a SUV39H1-induced modulation of heterochromatin would affect mammalian development, transgenic mice were generated that over-express the SUV39H1 HMTase early during embryogenesis. SUV39H1 transgenic mice are growth retarded, display a weak penetrance of skeletal transformations and are largely characterized by impaired erythroid differentiation, consistent with highest transgene expression in fetal liver. Ex vivo transgenic fetal liver cultures initially contain reduced numbers of cells in G1 but progress to immortalized erythroblasts that are compromised in executing an erythroid differentiation program. The outgrowing SUV39H1-immortalized erythroblasts can maintain a diploid karyotype despite deregulation of several tumor suppressor proteins and dispersed distribution of the heterochromatin component HP1. Together, these data provide evidence of a role for the SUV39H1 HMTase during mammalian development and indicate a possible function for higher-order chromatin in contributing to the balance between proliferation and differentiation potentials of progenitor cells (Czvitkovich, 2001).
SUV39H1, a human homolog of the Drosophila position effect variegation modifier Su(var)3-9 and of the Schizosaccharomyces pombe silencing factor clr4, encodes a novel heterochromatic protein that transiently accumulates at centromeric positions during mitosis. Using a detailed structure-function analysis of SUV39H1 mutant proteins in transfected cells, it has been shown that deregulated SUV39H1 interferes at multiple levels with mammalian higher-order chromatin organization. (1) Forced expression of full-length SUV39H1 (412 amino acids) redistributes endogenous M31 (HP1beta) and induces abundant associations with inter- and meta-phase chromatin. These properties depend on the C-terminal SET domain, although the major portion of the SUV39H1 protein (amino acids 89 to 412) does not display affinity for nuclear chromatin. By contrast, the M31 interaction surface, which maps to the first 44 N-terminal amino acids, together with the immediately adjacent chromo domain, directs specific accumulation at heterochromatin. (2) Cells overexpressing full-length SUV39H1 display severe defects in mitotic progression and chromosome segregation. Surprisingly, whereas localization of centromere proteins is unaltered, the focal, G2-specific distribution of phosphorylated histone H3 at serine 10 (phosH3) is dispersed in these cells. This phosH3 shift is not observed with C-terminally truncated mutant SUV39H1 proteins or with deregulated M31. Together, these data reveal a dominant role(s) for the SET domain of SUV39H1 in the distribution of prominent heterochromatic proteins and suggest a possible link between a chromosomal SU(VAR) protein and histone H3 (Melcher, 2000).
In cultured mammalian cells the histone methylase SUV39H1 and the methyl-lysine binding protein HP1 functionally interact to repress transcription at heterochromatic sites. Lysine 9 of histone H3 is methylated by SUV39H1, creating a binding site for the chromo domain of HP1. SUV39H1 and HP1 are both involved in the repressive functions of the retinoblastoma (Rb) protein. Rb associates with SUV39H1 and HP1 in vivo by means of its pocket domain. SUV39H1 cooperates with Rb to repress the cyclin E promoter. In fibroblasts that are disrupted for SUV39, the activity of the cyclin E and cyclin A2 genes are specifically elevated. Chromatin immunoprecipitations show that Rb is necessary to direct methylation of histone H3, and is necessary for binding of HP1 to the cyclin E promoter. These results indicate that the SUV39H1-HP1 complex is not only involved in heterochromatic silencing but also has a role in repression of euchromatic genes by Rb and perhaps other co-repressor proteins (Nielsen, 2001).
The Rb protein functions as a repressor, at least partly, through the recruitment of histone deacetylase activity. Whether histone methylation might also be involved in Rb-mediated repression is considered in this study, since the SUV39H1 methylase has repressive potential. To establish whether Rb can associate with histone-methylase activity, a glutathione S-transferase (GST)-Rb fusion was incubated with nuclear extract, and any bound methylase activity was assayed on bulk histones as a substrate. GST-Rb can purify histone-methylase activity, whereas GST fusions to transcriptional activators such as P/CAF, E2F1, p53 and ATF2 do not. The Rb-associated methylase activity is specific for histone H3 and does not recognize the GAR substrate for arginine methylases (Nielsen, 2001).
An antibody directed against Rb can precipitate histone-methylase activity that is specific for histone H3. This methylase binds the pocket domain of Rb because tumor-derived mutations in the pocket (F706C), or truncations of the pocket (928 and 737), abolish binding to the methylase. The Rb-associated methylase has specificity for Lys 9 of histone H3 (Nielsen, 2001).
The SUV39H1 protein possesses lysine methylase activity, which resides within its conserved SET domain. Since this enzyme has specificity for Lys 9 of histone H3 an investigation was carried out to see whether SUV39H1 could be the methylase associated with Rb. A GST-Rb fusion can bind to transfected, hemagglutinin (HA)-tagged SUV39H1. Endogenous Rb also associates with endogenous SUV39H1, as shown by a co-immunoprecipitation analysis (Nielsen, 2001).
Whether SUV39H1 can act as co-repressor with Rb was investigated. SUV39H1 represses the activity of a promoter bearing GAL4 sites in a concentration-dependent manner in vivo, but only when Gal4-Rb is present at the promoter. The co-repressor functions of SUV39H1 can also be seen on the cyclin E promoter, a natural target for Rb-mediated repression. This promoter can be stimulated by E2F and is not affected by SUV39H1 alone. Under limiting conditions, where Rb represses E2F activity slightly, the SUV39H1 enzyme can further repress E2F activity in cooperation with Rb. When the methylase domain of SUV39H1 is removed, the resulting SUV39H1 SET domain is unable to mediate repression. These results suggest that SUV39H1 uses its methylase activity to repress the cyclin E promoter when it is targeted there by Rb (Nielsen, 2001).
SUV39H1 is known to form a complex with the HP1 protein. Recently, HP1 function has been placed downstream of SUV39H1 histone methylation, since HP1 recognizes specifically, and binds to, histone H3 methylated at Lys 9. This mechanistic link has prompted an investigation of the role of HP1 in Rb/SUV39H1-mediated repression. Rb and HP1 can interact in a two-hybrid screen in yeast, and it has been shown that there is an LXCXE motif (X is any amino acid) in HP1. It was therefore asked if HP1 binds to Rb in mammalian cells. A GST-HP1 fusion can bind Rb that is present in nuclear extracts; Rb and HP1 can associate in vivo, as determined by co-immunoprecipitation analysis. An LXCXE motif peptide can compete for the binding of histone H3 methylase activity to Rb, but does not affect the binding of H3 methylase activity to HP1, which is consistent with the finding that the methylase activity is associated with the Rb pocket (Nielsen, 2001).
Whether HP1 can recognize methylated lysines while associated with Rb was tested. To address this, a histone H3 peptide methylated at Lys 9 was used as an affinity resin. Recombinant Rb does not bind to this methylated peptide, but it can do so efficiently in the presence of recombinant HP1. This result confirms that HP1 can bind directly to Rb and that it can recognize Rb and methylated lysine simultaneously. A similar experiment was attempted using nuclear extracts as the source of protein. The H3 peptide methylated at Lys 9 binds to HP1, SUV39H1 and Rb, as detected by Western blotting (Nielsen, 2001).
These results suggest that an Rb-regulated promoter such as cyclin E should be associated with HP1. To test this chromatin immunoprecipitation analysis of the cyclin E promoter was performed. A nucleosome encompassing the cyclin E initiation site (cyclin Epr) that is known to be deacetylated is associated with HP1 in fibroblast cells of mouse embryos. Since the cyclin Epr nucleosome binds HP1, whether this nucleosome contains histone H3 that is methylated at Lys 9 was examined. To test this an antibody was produced that recognizes histone H3 when methylated at Lys 9. In Rb+/+ cells the cyclin Epr nucleosome contains methylated histone H3 and is associated with HP1. However, in Rb-/- cells histone H3 methylation and HP1 binding is significantly reduced. Thus, in the presence of Rb, methylase activity and HP1 are targeted to the cyclin E promoter (Nielsen, 2001).
Collectively, these results implicate each of the components of the SUV39H1-HP1 complex in the repression functions of the Rb protein. In this model Rb brings to the promoter the SUV39H1 enzyme (and possibly other members of this family) to methylate Lys 9 of histone H3 and provides a binding site for HP1. Methylation by SUV39H1 cannot take place on an already acetylated lysine. Thus the deacetylase activity associated with Rb may be a necessary preceding step to SUV39H1-mediated methylation. The precise function of HP1 in repression is unclear. HP1 may protect the methyl group on Lys 9 from attack from potential demethylases; it may bring in other repressive functions, or it may enhance the stability of the Rb-associated repressor complex (Nielsen, 2001).
HP1 is found associated with a number of transcriptional repressors, suggesting that it may have a role in repressing many other promoters. Thus, the results presented here extend the role of SUV39H1 and HP1 beyond heterochromatic gene silencing to a more general, genome-wide function in repressing gene transcription (Nielsen, 2001).
E2F-mediated gene repression plays a key role in regulation of neuron survival and death. However, the key molecules involved in such regulation and the mechanisms by which they respond to apoptotic stimuli are largely unknown. This study shows that p130 is the predominant Rb family member associated with E2F in neurons, that its major partner for repression of pro-apoptotic genes is E2F4, and that the p130-E2F4 complex recruits the chromatin modifiers HDAC1 and Suv39H1 to promote gene silencing and neuron survival. Apoptotic stimuli induce neuron death by sequentially causing p130 hyperphosphorylation, dissociation of p130-E2F4-Suv39H1-HDAC complexes, altered modification of H3 histone and gene derepression. Experimental suppression of such events blocks neuron death while interference with the synthesis of E2F4 or p130, or with the interaction of E2F4-p130 with chromatin modifiers, induces neuron death. Thus, neuron survival and death are dependent on the integrity of E2F4-p130-HDAC/Suv39H1 complexes (Liu, 2005).
Active repression of E2F-regulated genes by Rb family members is achieved by recruitment of chromatin-modifying proteins to complexes with E2F. Thus, a key finding here is that the mechanism by which p130-E2F4 complexes promote neuron survival is via gene repression that requires their interaction with the chromatin-modifying corepressors HDAC and Suv39H1. Moreover, apoptotic stimuli induce death by causing loss of such interactions. In support of this, p130 and p130-E2F4 fusion proteins mutated to abolish interaction with HDAC and Suv39H1 promote neuron death, while phosphorylation-resistant E2F4-p130 fusion proteins that do not lose association with HDAC or Suv39H1 under apoptotic conditions are protective. Although these studies identified HDAC1 as a partner for p130 in neuronal cells, preliminary findings indicate that other HDAC family members may also be involved in p130-mediated gene repression and neuron survival (Liu, 2005).
How might p130-tethered HDAC1 and Suv39H1 promote gene repression and neuron survival? One target for these enzymes is the N-terminal tail of histone H3. In the absence of corepressors, H3 is phosphorylated on Ser 10, and this facilitates acetylation of Lys 14. These modifications promote transcription and are essential for cell cycle progression in mitotically competent cells. When tethered to chromatin by Rb-E2F complexes, Suv39H1 methylates H3 residue Lys 9. This, in turn, inhibits phosphorylation of Ser 10 and, in concert with HDACs, favors deacetylation of Lys 14. Such changes lead to condensation of local chromatin and gene silencing. Consistent with this mechanism, it was observed that levels of Ac-p-H3 associated with the endogenous B-myb promoter are low in viable neuronal cells and greatly increase in response to an apoptotic stimulus. In support of the involvement of HDAC and Suv39H1, it was found that a death stimulus abolishes the association between p130 and HDAC and substantially diminishes levels of p130-associated HMT activity (Liu, 2005).
An issue raised by these studies is the target of E2F-mediated gene repression that regulates neuron survival and death. A variety of observations support the closely related B-myb and C-myb genes in such a role. The promoters for these genes contain E2F-binding sites, and their expression is subject to E2F-dependent repression. Apoptotic stimuli, including p130 down-regulation, induce Myb reporter activity in neuronal cells, and the findings in this study show that apoptotic stimuli lead to loss of repressive complexes containing E2F4-p130-HDAC-Suv39H1 from the endogenous B-myb promoter as well as changes in associated chromatin consistent with gene derepression. Moreover, B-myb and C-myb transcripts and proteins are significantly induced by apoptotic stimuli, and overexpression of B-myb and C-myb promotes neuron death. Finally, down-regulation of B-myb and C-myb with anti-sense and siRNA constructs protects neurons from apoptotic stimuli (Liu, 2005).
These findings strongly support a repression/derepression model for regulation of neuron survival/death by E2F4-p130 and associated chromatin modifiers. They identify both the molecules and mechanisms by which p130 promotes silencing of E2F-responsive genes in viable neurons and by which apoptotic stimuli lead to derepression of E2F-responsive genes and death (Liu, 2005).
p130 has been implicated as a promoter of quiescence in nonneuronal cells and may well contribute to the post-mitotic state of neurons. Thus, stimuli that lead to dissolution of p130 complexes and that thereby trigger the derepression of pro-apoptotic genes such as Mybs, might concomitantly stimulate neurons to attempt cell cycle re-entry. Such a situation may explain not only why a variety of cell cycle markers are observed in neurons affected by injury and neurodegenerative disorders, but also why treatments targeted at suppressing derepression of E2F-regulated genes in neurons may have a therapeutic benefit in preventing neuron degeneration. In this regard, the present findings provide several additional molecular targets for such an approach (Liu, 2005).
Pluripotent cells develop within the inner cell mass of blastocysts, a mosaic of cells surrounded by an extra-embryonic layer, the trophectoderm. This study shows that a set of somatic lineage regulators (including Hox, Gata and Sox factors) that carry bivalent chromatin enriched in H3K27me3 and H3K4me2 are selectively targeted by Suv39h1-mediated H3K9me3 and de novo DNA methylation in extra-embryonic versus embryonic (pluripotent) lineages, as assessed both in blastocyst-derived stem cells and in vivo. This stably repressed state is linked with a loss of gene priming for transcription through the exclusion of PRC1 (Ring1B) and RNA polymerase II complexes at bivalent, lineage-inappropriate genes upon trophoblast lineage commitment. Collectively, these results suggest a mutually exclusive role for Ring1B and Suv39h1 in regulating distinct chromatin states at key developmental genes and propose a novel mechanism by which lineage specification can be reinforced during early development (Alder, 2010).
Gene expression programmes must be tightly controlled to govern cell identity and lineage choice. An inherent challenge for developing organisms is to maintain a critical balance between stable and flexible gene regulation. This is most obvious in pluripotent embryonic stem (ES) cells, which are functionally characterised by their ability to self-renew and to generate all somatic lineages when induced. Accordingly, ES cells express genes that encode self-renewal factors, while repressing many lineage-specific regulators that are ultimately required during development. A series of recent reports have indicated how Polycomb-mediated gene repression might provide short-term, and therefore flexible, epigenetic silencing of such developmental genes in pluripotent cell lines. This contrasts with long-term repression mechanisms as conferred, for example, by DNA methylation at transposons, imprinted and pluripotency-associated genes in somatic cells (Alder, 2010).
Two distinct Polycomb Repressive Complexes (PRC), PRC1 and PRC2, are known to be important for the function of ES cells. PRC2 contains Ezh2, which catalyses histone H3 lysine 27 trimethylation (H3K27me3), as well as Eed and Suz12. PRC1 contains the E3 ubiquitin ligases Ring1A (also known as Ring1) and Ring1B (Rnf2 -- Mouse Genome Informatics) that mono-ubiquitylate histone H2A lysine 119. Other PRC1 components include Bmi1, Mel18 (Pcgf6 -- Mouse Genome Informatics) and proteins of the Cbx family with H3K27 methylation affinity. Candidate-based and genome-wide studies of histone methylation in ES cells led to the remarkable finding that many PRC2-target genes not only carry the repressive H3K27me3 mark, but are also enriched for conventional indicators of active chromatin, including methylated H3K4. These so-called bivalent chromatin domains are thought to silence key developmental regulators while keeping them primed for future activation (or repression), and thus generally resolve to monovalent configurations upon differentiation, in accordance with gene expression changes. Further work showed that multipotent stem cells and some differentiated cells also possess bivalent domains, albeit perhaps fewer than in ES cells, indicating that plasticity might be maintained at loci that are required for the function and differentiation of lineage-committed cells. Consistent with gene priming, bivalent genes assemble RNA polymerase II complexes preferentially phosphorylated on Ser5 residues (poised RNAP) and are transcribed at low levels. Productive expression is, however, prevented by the action of PRC1 with conditional deletion of Ring1A/B, resulting in an upregulation of target gene expression in ES cells (from primed to overt transcription) (Alder, 2010 and references therein).
Clearly, Polycomb repressors are functionally required to prevent premature expression of primed genes and thus to stably maintain a pluripotent ES cell identity in culture. Whether ES cell epigenetic signatures can also be seen in the developing embryo and when (and how) bivalent domains are established and subsequently resolved upon lineage commitment in vivo remains to be elucidated. This study focused on the earliest stages of mouse development to address whether poised chromatin structures are unique hallmarks of the founder (pluripotent) cells of the early embryo, and to investigate the kinetics of appearance and resolution of bivalent domains during the first lineage decision event (trophectoderm formation). In vivo evidence is provided that bivalent histone marking operates in the early mouse embryo from the eight-cell up to the blastocyst stage. Unexpectedly, it was shown that bivalent domains are retained at a subset of genes encoding key somatic lineage regulators in extra-embryonic restricted cells, as assessed both in vitro and in vivo. However, and in contrast to pluripotent cells, PRC1 (Ring1B) and poised RNAP are not engaged at these PRC2 (Suz12)-bound genes, consistent with a loss of gene priming. Instead, bivalent genes become selectively targeted by Suv39h1-mediated repression upon trophoblast lineage commitment. Collectively, these results suggest a mutually exclusive role for Ring1B and Suv39h1 in specifying the fate of bivalent genes in a lineage-specific manner upon blastocyst formation (Alder, 2010).
The JAK/STAT pathway has pleiotropic roles in animal development, and its aberrant activation is implicated in multiple human cancers. JAK/STAT signaling effects have been attributed largely to direct transcriptional regulation by STAT of specific target genes that promote tumor cell proliferation or survival. In a Drosophila hematopoietic tumor model, however, that JAK overactivation globally disrupts heterochromatic gene silencing, an epigenetic tumor suppressive mechanism. This disruption allows derepression of genes that are not direct targets of STAT, as evidenced by suppression of heterochromatin-mediated position effect variegation. Moreover, mutations in the genes encoding heterochromatin components heterochromatin protein 1 (HP1) and Su(var)3-9 enhance tumorigenesis induced by an oncogenic JAK kinase without affecting JAK/STAT signaling. Consistently, JAK loss of function enhances heterochromatic gene silencing, whereas overexpressing HP1 suppresses oncogenic JAK-induced tumors. These results demonstrate that the JAK/STAT pathway regulates cellular epigenetic status and that globally disrupting heterochromatin-mediated tumor suppression is essential for tumorigenesis induced by JAK overactivation (Shi, 2006).
Telomeres are capping structures at the ends of eukaryotic chromosomes composed of TTAGGG repeats bound to an array of specialized proteins. Telomeres are heterochromatic regions. Yeast and flies with defects in activities that modify the state of chromatin also have abnormal telomere function, but the putative role of chromatin-modifying activities in regulating telomeres in mammals is unknown. This study reports on telomere length and function in mice null with respect to both the histone methyltransferases (HMTases) Suv39h1 and Suv39h2 (called SUV39DN mice). Suv39h1 and Suv39h2 govern methylation of histone H3 Lys9 (H3-Lys9) in heterochromatic regions. Primary cells derived from SUV39DN mice have abnormally long telomeres relative to wild-type controls. Using chromatin immunoprecipitation (ChIP) analysis, it was found that telomeres are enriched in di- and trimethylated H3-Lys9 but that telomeres of SUV39DN cells have less dimethylated and trimethylated H3-Lys9 but more monomethylated H3-Lys9. Concomitant with the decrease in H3-Lys9 methylation, telomeres in SUV39DN cells have reduced binding of the chromobox proteins Cbx1, Cbx3 and Cbx5, homologs of Drosophila melanogaster heterochromatin protein 1 (HP1). These findings indicate substantial changes in the state of telomeric heterochromatin in SUV39DN cells that are associated with abnormal telomere elongation. Taken together, the results indicate epigenetic regulation of telomere length in mammals by Suv39h1 and Suv39h2 (Garcia-Cao, 2004).
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