Ectopic expression of the NtSET1 histone methyltransferase inhibits cell expansion, and affects cell division and differentiation in tobacco plants

The tobacco NtSET1 gene encodes a member of the SUV39H family of histone methyltransferases. Ectopic expression of NtSET1 causes an increase in methylated histone H3 lysine 9 and abnormal chromosome segregation in tobacco suspension cells, and inhibits tobacco plant growth. Here we show that the inhibition of plant growth was caused by reduced cell expansion as well as by abnormal cell division and differentiation. We found that deletion of the C-terminally located catalytic domain of the protein abolished the ectopic effects of NtSET1 on plant growth. Our results indicate that histone H3 lysine 9 methylation is a critical mark of epigenetic control for plant development.     

Insulation of the chicken beta-globin chromosomal domain from a chromatin-condensing protein,MENT

Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken beta-globin domain. We observed two sharp transitions of MENT concentration coinciding with the beta-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation.     

Multimerization and H3K9me3 Binding Are Required for CDYL1b Heterochromatin Association

Proteins containing defined recognition modules mediate readout and translation of histone modifications. These factors are thought to initiate downstream signaling events regulating chromatin structure and function. We identified CDYL1 as an interaction partner of histone H3 trimethylated on lysine 9 (H3K9me3). CDYL1 belongs to a family of chromodomain factors found in vertebrates. We show that three different splicing variants of CDYL1, a, b, and c, are differentially expressed in various tissues with CDYL1b being the most abundant variant. Although all three splicing variants share a common C-terminal enoyl-CoA hydratase-like domain, only CDYL1b contains a functional chromodomain implicated in H3K9me3 binding. A splicing event introducing an N-terminal extension right at the beginning of the chromodomain of CDYL1a inactivates its chromodomain. CDYL1c does not contain a chromodomain at all. Although CDYL1b displays binding affinity to methyl-lysine residues in different sequence context similar to chromodomains in other chromatin factors, we demonstrate that the CDYL1b chromodomain/H3K9me3 interaction is necessary but not sufficient for association of the factor with heterochromatin. Indeed, multimerization of the protein via the enoyl-CoA hydratase-like domain is essential for H3K9me3 chromatin binding in vitro and heterochromatin localization in vivo. In agreement, overexpression of CDYL1c that can multimerize, but does not interact with H3K9me3 can displace CDYL1b from heterochromatin. Our results imply that multimeric binding to H3K9me3 by CDYL1b homomeric complexes is essential for efficient chromatin targeting. We suggest that similar multivalent binding stably anchors other histone modification binding factors on their target chromatin regions.     

Structure-function analysis of SUV39H1 reveals a dominant role in heterochromatin organization, chromosome segregation, and mitotic progression

SUV39H1, a human homologue 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, we now show that deregulated SUV39H1 interferes at multiple levels with mammalian higher-order chromatin organization. First, forced expression of full-length SUV39H1 (412 amino acids) redistributes endogenous M31 (HP1beta) and induces abundant associations with inter- and metaphase 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 was mapped to the first 44 N-terminal amino acids, together with the immediately adjacent chromo domain, directs specific accumulation at heterochromatin. Second, cells overexpressing full-length SUV39H1 display severe defects in mitotic progression and chromosome segregation. Surprisingly, whereas localization of centromere proteins is unaltered, the focal, G(2)-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, our 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.     

SET3p monomethylates histone H3 on lysine 9 and is required for the silencing of tandemly repeated transgenes in Chlamydomonas

SET domain-containing proteins of the SU(VAR)3-9 class are major regulators of heterochromatin in several eukaryotes, including mammals, insects, plants and fungi. The function of these polypeptides is mediated, at least in part, by their ability to methylate histone H3 on lysine 9 (H3K9). Indeed, mutants defective in SU(VAR)3-9 proteins have implicated di- and/or trimethyl H3K9 in the formation and/or maintenance of heterochromatin across the eukaryotic spectrum. Yet, the biological significance of monomethyl H3K9 has remained unclear because of the lack of mutants exclusively defective in this modification. Interestingly, a SU(VAR)3-9 homolog in the unicellular green alga Chlamydomonas reinhardtii, SET3p, functions in vitro as a specific H3K9 monomethyltransferase. RNAi-mediated suppression of SET3 reactivated the expression of repetitive transgenic arrays and reduced global monomethyl H3K9 levels. Moreover, chromatin immunoprecipitation (ChIP) assays demonstrated that transgene reactivation correlated with the partial loss of monomethyl H3K9 from their chromatin. In contrast, the levels of trimethyl H3K9 or the repression of euchromatic sequences were not affected by SET3 downregulation; whereas dimethyl H3K9 was undetectable in Chlamydomonas. Thus, our observations are consistent with a role for monomethyl H3K9 as an epigenetic mark of repressed chromatin and raise questions as to the functional distinctiveness of different H3K9 methylation states.     

N-terminal phosphorylation of HP1{alpha} promotes its chromatin binding

The phosphorylation of heterochromatin protein 1 (HP1) has been previously described in studies of mammals, but the biological implications of this modification remain largely elusive. Here, we show that the N-terminal phosphorylation of HP1α plays a central role in its targeting to chromatin. Recombinant HP1α prepared from mammalian cultured cells exhibited a stronger binding affinity for K9-methylated histone H3 (H3K9me) than that produced in Escherichia coli. Biochemical analyses revealed that HP1α was multiply phosphorylated at N-terminal serine residues (S11-14) in human and mouse cells and that this phosphorylation enhanced HP1α's affinity for H3K9me. Importantly, the N-terminal phosphorylation appeared to facilitate the initial binding of HP1α to H3K9me by mediating the interaction between HP1α and a part of the H3 tail that was distinct from the methylated K9. Unphosphorylatable mutant HP1α exhibited severe heterochromatin localization defects in vivo, and its prolonged expression led to increased chromosomal instability. Our results suggest that HP1α's N-terminal phosphorylation is essential for its proper targeting to heterochromatin and that its binding to the methylated histone tail is achieved by the cooperative action of the chromodomain and neighboring posttranslational modifications.     

Catalytic properties and kinetic mechanism of human recombinant Lys-9 histone H3 methyltransferase SUV39H1: participation of the chromodomain in enzymatic catalysis

Histone H3 lysine 9 (H3K9) methylation is a major component of gene regulation and chromatin organization. SUV39H1 methylates H3K9 at the pericentric heterochromatin region and participates in the maintenance of genome stability. In this study, a recombinant purified SUV39H1 is used for substrate specificity and steady-state kinetic analysis with peptides representing the un- or dimethylated lysine 9 histone H3 tail or full-length human recombinant H3 (rH3). Recombinant SUV39H1 methylated its substrate via a nonprocessive mechanism. Binding of either peptide or AdoMet first to the enzyme made a catalytically competent binary complex. Product inhibition studies with SUV39H1 showed that S-adenosyl-l-homocysteine is a competitive inhibitor of S-adenosyl-l-methionine and a mixed inhibitor of substrate peptide. Similarly, the methylated peptide was a competitive inhibitor of the unmethylated peptide and a mixed inhibitor of AdoMet, suggesting a random mechanism in a bi-bi reaction for recombinant SUV39H1 in which either substrate can bind to the enzyme first and either product can release first. The turnover numbers (k(cat)) for the H3 tail peptide and rH3 were comparable (12 and 8 h(-)(1), respectively) compared to the value of 1.5 h(-)(1) for an identical dimethylated lysine 9 H3 tail peptide. The Michaelis constant for the methylated peptide (K(m)(pep)) was 13-fold lower compared to that of the unmethylated peptide. The Michaelis constants for AdoMet (K(m)(AdoMet)) were 12 and 6 microM for the unmethylated peptide substrate and rH3, respectively. A reduction in the level of methylation was observed at high concentrations of rH3, implying substrate inhibition. Deletion of the chromodomain or point mutation of the conserved amino acids, W64A or W67A, of SUV39H1 impaired enzyme activity despite the presence of an intact catalytic SET domain. Thus, SUV39H1 utilizes both the chromodomain and the SET domain for catalysis.     

The centromeric heterochromatin of Costus spiralis: poorly methylated and transiently acetylated during meiosis

The centromeric region of Costus spiralis is characteristically composed of a small heterochromatic DAPI(+) band flanked by a discrete decondensed region. High concentrations of serine 10 of histone H3 (H3S10ph) around the DAPI(+) band in pachytene chromosomes and the location of this heterochromatin at the chromosome region directed towards the poles during metaphase-anaphase I confirm its integration into the centromeric region. Antibodies against both typical components of euchromatin histones (histone H4 acetylated at lysine 5 (H4K5ac) and histone H3 dimethylated at lysine 4 (H3K4me2)) and heterochromatin (dimethylated lysine 9 of H3 (H3K9me2) and anti-5-methylcytosine (5-mC)) were used to characterize the centromeric chromatin of this species during meiosis. In pachytene chromosomes, the decondensed terminal euchromatin of the chromosome arms were seen to be richer in H4K5ac and H3K4me2 histones, while the more condensed proximal region was relatively stronger labeled with anti-H3K9me2 and anti-5-methylcytosine (5-mC). The centromeric region itself, including the DAPI(+) band, was poor in all of these chromatin modifications, but it was highly enriched in H4K5ac at pachytene. Before and after this stage, the centromeric region was poorly labeled with anti-H4K5ac. Hypomethylation and hyperacetylation of any kind of heterochromatin has rarely been reported, and it may be related to the dominant role of the centromere domain over the heterochromatin repeats.     

Multiple SET Methyltransferases Are Required to Maintain Normal Heterochromatin Domains in the Genome of Drosophila melanogaster

Methylation of histone H3 lysine 9 (H3K9) is a key feature of silent chromatin and plays an important role in stabilizing the interaction of heterochromatin protein 1 (HP1) with chromatin. Genomes of metazoans such as the fruit fly Drosophila melanogaster generally encode three types of H3K9-specific SET domain methyltransferases that contribute to chromatin homeostasis during the life cycle of the organism. SU(VAR)3-9, dG9a, and dSETDB1 all function in the generation of wild-type H3K9 methylation levels in the Drosophila genome. Two of these enzymes, dSETDB1 and SU(VAR)3-9, govern heterochromatin formation in distinct but overlapping patterns across the genome. H3K9 methylation in the small, heterochromatic fourth chromosome of D. melanogaster is governed mainly by dSETDB1, whereas dSETDB1 and SU(VAR)3-9 function in concert to methylate H3K9 in the pericentric heterochromatin of all chromosomes, with dG9a having little impact in these domains, as shown by monitoring position effect variegation. To understand how these distinct heterochromatin compartments may be differentiated, we examined the developmental timing of dSETDB1 function using a knockdown strategy. dSETDB1 acts to maintain heterochromatin during metamorphosis, at a later stage in development than the reported action of SU(VAR)3-9. Surprisingly, depletion of both of these enzymes has less deleterious effect than depletion of one. These results imply that dSETDB1 acts as a heterochromatin maintenance factor that may be required for the persistence of earlier developmental events normally governed by SU(VAR)3-9. In addition, the genetic interactions between dSETDB1 and Su(var)3-9 mutations emphasize the importance of maintaining the activities of these histone methyltransferases in balance for normal genome function.     

H1 family histones in the nucleus. Control of binding and localization by the C-terminal domain

H1 histones bind to DNA as they enter and exit the nucleosome. H1 histones have a tripartite structure consisting of a short N-terminal domain, a highly conserved central globular domain, and a lysine-and arginine-rich C-terminal domain. The C-terminal domain comprises approximately half of the total amino acid content of the protein, is essential for the formation of compact chromatin structures, and contains the majority of the amino acid variations that define the individual histone H1 family members. This region contains several cell cycle-regulated phosphorylation sites and is thought to function through a charge-neutralization process, neutralizing the DNA phosphate backbone to allow chromatin compaction. In this study, we use fluorescence microscopy and fluorescence recovery after photobleaching to define the behavior of the individual histone H1 subtypes in vivo. We find that there are dramatic differences in the binding affinity of the individual histone H1 subtypes in vivo and differences in their preference for euchromatin and heterochromatin. Further, we show that subtype-specific properties originate with the C terminus and that the differences in histone H1 binding are not consistent with the relatively small changes in the net charge of the C-terminal domains.     

Heterochromatin protein 1 binds to nucleosomes and DNA in vitro

Heterochromatin protein 1 (HP1) is a nonhistone chromosomal protein primarily associated with the pericentric heterochromatin and telomeres in Drosophila. The molecular mechanism by which HP1 specifically recognizes and binds to chromatin is unknown. The purpose of this study was to test whether HP1 can bind directly to nucleosomes. HP1 binds nucleosome core particles and naked DNA. HP1-DNA complex formation is length-dependent and cooperative but relatively sequence-independent. We show that histone H4 amino-terminal peptides bind to monomeric and dimeric HP1 in vitro. Acetylation of lysine residues had no significant effect on in vitro binding. The C-terminal chromo shadow domain of HP1 specifically binds H4 N-terminal peptide. Neither the chromo domain nor chromo shadow domain alone binds DNA; intact native HP1 is required for such interactions. Together, these observations suggest that HP1 may serve as a cross-linker in chromatin, linking nucleosomal DNA and nonhistone protein complexes to form higher order chromatin structures.     

Different domains control the localization and mobility of LIKE HETEROCHROMATIN PROTEIN1 in Arabidopsis nuclei

Plants possess a single gene for the structurally related HETEROCHROMATIN PROTEIN1 (HP1), termed LIKE-HP1 (LHP1). We investigated the subnuclear localization, binding properties, and dynamics of LHP1 proteins in Arabidopsis thaliana cells. Transient expression assays showed that tomato (Solanum lycopersicum) LHP1 fused to green fluorescent protein (GFP; Sl LHP1-GFP) and Arabidopsis LHP1 (At LHP1-GFP) localized to heterochromatic chromocenters and showed punctuated distribution within the nucleus; tomato but not Arabidopsis LHP1 was also localized within the nucleolus. Mutations of aromatic cage residues that recognize methyl K9 of histone H3 abolished their punctuated distribution and localization to chromocenters. Sl LHP1-GFP plants displayed cell type-dependent subnuclear localization. The diverse localization pattern of tomato LHP1 did not require the chromo shadow domain (CSD), whereas the chromodomain alone was insufficient for localization to chromocenters; a nucleolar localization signal was identified within the hinge region. Fluorescence recovery after photobleaching showed that Sl LHP1 is a highly mobile protein whose localization and retention are controlled by distinct domains; retention at the nucleolus and chromocenters is conferred by the CSD. Our results imply that LHP1 recruitment to chromatin is mediated, at least in part, through interaction with methyl K9 and that LHP1 controls different nuclear processes via transient binding to its nuclear sites.