Dual histone H3 methylation marks at lysines 9 and 27 required for interaction with CHROMOMETHYLASE3

Both DNA methylation and post-translational histone modifications contribute to gene silencing, but the mechanistic relationship between these epigenetic marks is unclear. Mutations in two Arabidopsis genes, the KRYPTONITE (KYP) histone H3 lysine 9 (H3K9) methyltransferase and the CHROMOMETHYLASE3 (CMT3) DNA methyltransferase, cause a reduction of CNG DNA methylation, suggesting that H3K9 methylation controls CNG DNA methylation. Here we show that the chromodomain of CMT3 can directly interact with the N-terminal tail of histone H3, but only when it is simultaneously methylated at both the H3K9 and H3K27 positions. Furthermore, using chromatin immunoprecipitation analysis and immunohistolocalization experiments, we found that H3K27 methylation colocalizes with H3K9 methylation at CMT3-controlled loci. The H3K27 methylation present at heterochromatin was not affected by mutations in KYP or in several Arabidopsis PcG related genes including the Enhancer of Zeste homologs, suggesting that a novel pathway controls heterochromatic H3K27 methylation. Our results suggest a model in which H3K9 methylation by KYP, and H3K27 methylation by an unknown enzyme provide a combinatorial histone code for the recruitment of CMT3 to silent loci.     

DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis

We propose a model for heterochromatin assembly that links DNA methylation with histone methylation and DNA replication. The hypomethylated Arabidopsis mutants ddm1 and met1 were used to investigate the relationship between DNA methylation and chromatin organization. Both mutants show a reduction of heterochromatin due to dispersion of pericentromeric low-copy sequences away from heterochromatic chromocenters. DDM1 and MET1 control heterochromatin assembly at chromocenters by their influence on DNA maintenance (CpG) methylation and subsequent methylation of histone H3 lysine 9. In addition, DDM1 is required for deacetylation of histone H4 lysine 16. Analysis of F(1) hybrids between wild-type and hypomethylated mutants revealed that DNA methylation is epigenetically inherited and represents the genomic imprint that is required to maintain pericentromeric heterochromatin.     

The Arabidopsis SDG4 contributes to the regulation of pollen tube growth by methylation of histone H3 lysines 4 and 36 in mature pollen

Plant SET domain proteins are known to be involved in the epigenetic control of gene expression during plant development. Here, we report that the Arabidopsis SET domain protein, SDG4, contributes to the epigenetic regulation of pollen tube growth, thus affecting fertilization. Using an SDG4-GFP fusion construct, the chromosomal localization of SDG4 was established in tobacco BY-2 cells. In Arabidopsis, sdg4 knockout showed reproductive defects. Tissue-specific expression analyses indicated that SDG4 is the major ASH1-related gene expressed in the pollen. Immunological analyses demonstrated that SDG4 was involved in the methylation of histone H3 in the inflorescence and pollen grains. The significant reduction in the amount of methylated histone H3 K4 and K36 in sdg4 pollen vegetative nuclei resulted in suppression of pollen tube growth. Our results indicate that SDG4 is capable of modulating the expression of genes that function in the growth of pollen tube by methylation of specific lysine residues of the histone H3 in the vegetative nuclei.     

ING2 recruits histone methyltransferase activity with methylation site specificity distinct from histone H3 lysines 4 and 9

p33ING2 belongs to the ING-gene family that is involved in tumor suppression, DNA repair, cell cycle regulation, and cellular senescence. Most functions are dependent on the tumor suppressor p53. p33ING2 was also shown to bind to trimethylated lysine 4 of histone H3. Here, we show that p33ING2 contains a transferable silencing function, which is independent of p53. p33ING2-mediated gene silencing is resistant to the HDAC-inhibitor trichostatin A indicating that p33ING2 uses a non-HDAC class I or II pathway for gene repression in reporter assays. In line with that we show that p33ING2 is associated with histone methyltransferase (HMT) activity in vitro and in vivo, methylating specifically histone H3. Interestingly, the specificity is distinct from the MeCP2-recruited HMT. Mutation or methylation of lysine 9, a mark well known for repression, abrogates histone methylation by MeCP2 but not by the p33ING2 complex. Instead, the ING2-associated HMT shows an increased methylation activity if lysine 9 is methylated. In contrast, mutation or methylation of lysine 4, a methylation preferentially detected at active genes, led to a reduction of the ING2-associated HMT. Notably, also p33ING1 recruits HMT activity suggesting a more general biochemical interaction between members of p33ING family and HMT activity. Deletion analyses revealed that the ING2 C-terminus recruits HMT activity, which correlates with silencing function.     

Partitioning of the maize epigenome by the number of methyl groups on histone H3 lysines 9 and 27

We report a detailed analysis of maize chromosome structure with respect to seven histone H3 methylation states (dimethylation at lysine 4 and mono-, di-, and trimethylation at lysines 9 and 27). Three-dimensional light microscopy and the fine cytological resolution of maize pachytene chromosomes made it possible to compare the distribution of individual histone methylation events to each other and to DNA staining intensity. Major conclusions are that (1) H3K27me2 marks classical heterochromatin; (2) H3K4me2 is limited to areas between and around H3K27me2-marked chromomeres, clearly demarcating the euchromatic gene space; (3) H3K9me2 is restricted to the euchromatic gene space; (4) H3K27me3 occurs in a few (roughly seven) focused euchromatic domains; (5) centromeres and CENP-C are closely associated with H3K9me2 and H3K9me3; and (6) histone H4K20 di- and trimethylation are nearly or completely absent in maize. Each methylation state identifies different regions of the epigenome. We discuss the evolutionary lability of histone methylation profiles and draw a distinction between H3K9me2-mediated gene silencing and heterochromatin formation.     

Loss of CpG methylation is strongly correlated with loss of histone H3 lysine 9 methylation at DMR-LIT1 in patients with Beckwith-Wiedemann syndrome

To clarify the chromatin-based imprinting mechanism of the p57(KIP2)/LIT1 subdomain at chromosome 11p15.5 and the mouse ortholog at chromosome 7F5, we investigated the histone-modification status at a differentially CpG methylated region of Lit1/LIT1 (DMR-Lit1/LIT1), which is an imprinting control region for the subdomain and is demethylated in half of patients with Beckwith-Wiedemann syndrome (BWS). Chromatin-immunoprecipitation assays revealed that, in both species, DMR-Lit1/LIT1 with the CpG-methylated, maternally derived inactive allele showed histone H3 Lys9 methylation, whereas the CpG-unmethylated, paternally active allele was acetylated on histone H3/H4 and methylated on H3 Lys4. We have also investigated the relationship between CpG methylation and histone H3 Lys9 methylation at DMR-LIT1 in patients with BWS. In a normal individual and in patients with BWS with normal DMR-LIT1 methylation, histone H3 Lys9 methylation was detected on the maternal allele; however, it disappeared completely in the patients with the DMR-LIT1 imprinting defect. These findings suggest that the histone-modification status at DMR-Lit1/LIT1 plays an important role in imprinting control within the subdomain and that loss of histone H3 Lys9 methylation, together with CpG demethylation on the maternal allele, may lead to the BWS phenotype.     

Differential histone H3 Lys-9 and Lys-27 methylation profiles on the X chromosome

Histone H3 tail modifications are among the earliest chromatin changes in the X-chromosome inactivation process. In this study we investigated the relative profiles of two important repressive marks on the X chromosome: methylation of H3 lysine 9 (K9) and 27 (K27). We found that both H3K9 dimethylation and K27 trimethylation characterize the inactive X in somatic cells and that their relative kinetics of enrichment on the X chromosome as it undergoes inactivation are similar. However, dynamic changes of H3K9 and H3K27 methylation on the inactivating X chromosome compared to the rest of the genome are distinct, suggesting that these two modifications play complementary and perhaps nonredundant roles in the establishment and/or maintenance of X inactivation. Furthermore, we show that a hotspot of H3K9 dimethylation 5' to Xist also displays high levels of H3 tri-meK27. However, analysis of this region in G9a mutant embryonic stem cells shows that these two methyl marks are dependent on different histone methyltransferases.     

Distinct methylation patterns in histone H3 at Lys-4 and Lys-9 correlate with up- & down-regulation of genes by ethanol in hepatocytes

Ethanol induced liver injury is associated with a global change in gene expression but its mechanisms are not known. We studied whether alcohol-induced gene expression is associated with post-translational methylations of histone H3. Primary culture of rat hepatocytes was treated with ethanol (50 or 100 mM) for 24 h and the status of methylation of H3 at lys 4 (H3dimeK4) or lys 9 (H3dimeK9) was monitored by Western blotting using antibodies to dimethylated histone H3 at lys 4 or lys 9. The cells exposed to ethanol showed strikingly opposing behaviors in methylation patterns; H3dimeK9 methylation was decreased whereas H3dimeK4 increased. Similar results were obtained in the interphase nuclei. Their binding on the metaphase chromosomes exhibits distinct site specific pattern of accumulation. Next, chromatin immunoprecipitation of the ethanol treated samples with antibodies for methylated lys 4 or lys 9 histone H3 followed by amplification of the immunoprecipitated DNA, was used to determine their association with the promoters of genes up- or downregulated by ethanol. Lys4 methylation was associated with ethanol upregulated genes (Adh, GST-yc2) whereas lys 9 methylation with downregulated genes (Lsdh, cytP4502c11) demonstrating a difference between these two methylations. These results suggest that exposure of hepatocytes to ethanol changes the expression of several susceptible genes which are associated with site specific modification of dimethylated forms of histone H3 amino termini at their regulatory regions.     

Trimethylation of histone H3 lysine 4 impairs methylation of histone H3 lysine 9

Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4^me3) while heterochromatin contains the H3K9^me3 marks. The H3K9^me3 modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4^me3. In addition, we show that SETDB1 fails to methylate substrates containing the H3K4^me3 mark. Likewise, the functionally related H3K9 KMTs G9A, GLP, and SUV39H1 also fail to bind and to methylate H3K4^me3 substrates. Accordingly, we provide in vivo evidence that H3K9^me2-enriched histones are devoid of H3K4^me2/3 and that histones depleted of H3K4^me2/3 have elevated H3K9^me2/3. The correlation between the loss of interaction of these KMTs with H3K4^me3 and concomitant methylation impairment leads to the postulate that, at least these four KMTs, require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1α. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4^me3 and H3K9^me3. Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners.     

Heterochromatin,HP1 and methylation at lysine 9 of histone H3 in animals

We show that methylated lysine 9 of histone H3 (Me9H3) is a marker of heterochromatin in divergent animal species. It localises to both constitutive and facultative heterochromatin and replicates late in S-phase of the cell cycle. Significantly, Me9H3 is enriched in the inactive mammalian X chromosome (Xi) in female cells, as well as in the XY body during meiosis in the male, and forms a G-band pattern along the arms of the autosomes. Me9H3 is a constituent of imprinted chromosomes that are repressed. The paternal and maternal pronuclei in one-cell mouse embryos show a striking non-equivalence in Me9H3: the paternal pronucleus contains no immunocytologically detectable Me9H3. The levels of Me9H3 on the parental chromosomes only become equivalent after the two-cell stage. Finally, we provide evidence that Me9H3 is neither necessary nor sufficient for localisation of heterochromatin protein 1 (HP1) to chromosomal DNA.     

Whole-genome analysis of histone H3 lysine 27 trimethylation in Arabidopsis

Trimethylation of histone H3 lysine 27 (H3K27me3) plays critical roles in regulating animal development, and in several cases, H3K27me3 is also required for the proper expression of developmentally important genes in plants. However, the extent to which H3K27me3 regulates plant genes on a genome-wide scale remains unknown. In addition, it is not clear whether the establishment and spreading of H3K27me3 occur through the same mechanisms in plants and animals. We identified regions containing H3K27me3 in the genome of the flowering plant Arabidopsis thaliana using a high-density whole-genome tiling microarray. The results suggest that H3K27me3 is a major silencing mechanism in plants that regulates an unexpectedly large number of genes in Arabidopsis (~4,400), and that the maintenance of H3K27me3 is largely independent of other epigenetic pathways, such as DNA methylation or RNA interference. Unlike in animals, where H3K27m3 occupies large genomic regions, in Arabidopsis, we found that H3K27m3 domains were largely restricted to the transcribed regions of single genes. Furthermore, unlike in animals systems, H3K27m3 domains were not preferentially associated with low–nucleosome density regions. The results suggest that different mechanisms may underlie the establishment and spreading of H3K27me3 in plants and animals.     

Enrichment for histone H3 lysine 9 methylation at Alu repeats in human cells

The aim of this study was to identify in human cells common targets of histone H3 lysine 9 (H3-Lys9) methylation, a modification that is generally associated with gene silencing. After chromatin immunoprecipitation using an H3-Lys9 methylated antibody, we cloned the recovered DNA and sequenced 47 independent clones. Of these, 38 clones (81%) contained repetitive elements, either short interspersed transposable element (SINE or Alu elements), long terminal repeat (LTR), long interspersed transposable element (LINE), or satellite region (ALR/Alpha) DNA, and three additional clones were near Alu elements. Further characterization of these repetitive elements revealed that 32 clones (68%) were Alu repeats, corresponding to both old Alu (23 clones) and young Alu (9 clones) subfamilies. Association of H3-Lys9 methylation was confirmed by chromatin immunoprecipitation-PCR using conserved Alu primers. In addition, we randomly selected 5 Alu repeats from the recovered clones and confirmed association with H3-Lys9 by PCR using primer sets flanking the Alu elements. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine rapidly decreased the level of H3-Lys9 methylation in the Alu elements, suggesting that H3-Lys9 methylation may be related to the suppression of Alu elements through DNA methylation. Thus H3-Lys9 methylation is enriched at human repetitive elements, particularly Alu elements, and may play a role in the suppression of recombination by these elements.