Lys Methylation Research Articles

Binding of the CHD4 PHD2 finger to histone H3 is modulated by covalent modifications

CHD4 (chromodomain helicase DNA-binding protein 4) ATPase is a major subunit of the repressive NuRD (nucleosome remodelling and deacetylase) complex, which is involved in transcriptional regulation and development. CHD4 contains two PHD (plant homeodomain) fingers of unknown function. Here we show that the second PHD finger (PHD2) of CHD4 recognizes the N-terminus of histone H3 and that this interaction is facilitated by acetylation or methylation of Lys9 (H3K9ac and H3K9me respectively) but is inhibited by methylation of Lys4 (H3K4me) or acetylation of Ala1 (H3A1ac). An 18 microM binding affinity toward unmodified H3 rises to 0.6 microM for H3K9ac and to 0.9 microM for H3K9me3, whereas it drops to 2.0 mM for H3K4me3, as measured by tryptophan fluorescence and NMR. A peptide library screen further shows that phosphorylation of Thr3, Thr6 or Ser10 abolishes this interaction. A model of the PHD2-H3 complex, generated using a combination of NMR, data-driven docking and mutagenesis data, reveals an elongated site on the PHD2 surface where the H3 peptide is bound. Together our findings suggest that the PHD2 finger plays a role in targeting of the CHD4/NuRD complex to chromatin.

Mandatory fortification with folic acid in the United States appears to have adverse effects on histone methylation in women with pre-cancer but not in women free of pre-cancer

To evaluate whether mandatory fortification of grain products with folic acid in the US is associated with changes in histone methylation in cells involved in cervical carcinogenesis. Cervical specimens obtained before (1990 to 1992) and after mandatory folic acid fortification (2000 to 2002) were used to examine the degree of histone methylation (H3 Lys-9) by immunohistochemistry. 91 women (51 before and 40 after fortification) were diagnosed with cervical intraepithelial neoplasia (CIN) grade 3 or carcinoma in situ (CIS) and sections utilized in the study also contained normal, reactive or metaplastic cervical epithelium, CIN 1 or CIN 2. 64 women (34 before and 30 after fortification) were free of CIN and these sections contained only normal or reactive cervical epithelium. Immunohistochemical staining for H3 Lys-9, its assessment in different cell or lesion types and data entry were blinded for fortification status. For each cell type or lesion category we used PROC MIXED in SAS with the specimen identifier as a random effect and the robust variance estimator to estimate age- and race-adjusted intensity score for H3 Lys-9 in the pre- and post-fortification periods. Degree of H3 Lys-9 methylation was significantly higher (P < 0.0001) in ≥CIN 2 lesions (CIN 2, CIN 3 and CIS) than in ≤CIN 1 lesions (CIN 1, normal, reactive and metaplastic), in both pre- and post-fortification CIN 3/CIS specimens. Age- and race-adjusted mean H3 Lys-9 score was significantly higher in all cell or lesion types in CIN 3/CIS specimens obtained in the post-fortification period compared to pre-fortification period (P < 0.05, all comparisons). In contrast, in specimens obtained from women free of CIN, Lys-9 methylation in normal/reactive cervical epithelium was significantly lower in post-fortification specimens than in pre-fortification specimens (P = 0.03). Higher levels of Lys-9 methylation in ≥CIN 2 compared to ≤CIN 1 lesions suggest that higher Lys-9 methylation is associated with progression of lower grade CIN to higher grade CIN. Higher Lys-9 methylation in cervical tissues of women diagnosed with CIN 3 in the post-fortification period than in pre-fortification period suggest that fortification may adversely affect histone methylation in already initiated cells. Lower Lys-9 methylation in normal/reactive cervical cells of women free of CIN in the post-fortification period than pre-fortification on the other hand suggests that fortification is likely to protect against initiation of carcinogenic process in the cervix. These results suggest that mandatory fortification with folic acid in the US seems to have different effects on cancer depending on the stage of carcinogenesis. Because this is the first study to report folic acid fortification-associated differences in histone methylation and because of the limitations inherent to the approach we have taken to demonstrate these differences, validation of the results in other study populations or with other techniques for assessing histone methylation is necessary.

Mandatory fortification with folic acid in the United States appear to have adverse effects on histone methylation in women with pre‐cancer but not in women free of pre‐cancer

The objective of this study was to evaluate whether mandatory fortification of grain products with folic acid in the USA is associated with changes in histone methylation in cells involved in cervical carcinogenesis. Cervical specimens (free of pre‐cancer or diagnosed with pre‐cancer cervical intraepithelial neoplasia grade 3 [CIN 3]) obtained before (1990‐92) or after mandatory folic acid fortification (2000‐02) were used to examine the degree of histone methylation (H3 Lys‐9) by immunohistochemistry. Age‐ and race‐adjusted mean H3 Lys‐9 score was significantly higher in CIN 3 specimens obtained in the post‐fortification period compared to pre‐fortification period (P &lt; 0.05). In contrast, in specimens obtained from women free of CIN, H3 Lys‐9 scores were significantly lower in post‐fortification specimens compared to pre‐fortification specimens (P &lt; 0.05). Altered histone methylation has emerged as key to DNA methylation‐related gene silencing and this type of gene silencing in cancer is accompanied by an increase in H3 Lys9 methylation. Therefore, higher Lys‐9 methylation in cervical tissues of women diagnosed with CIN 3 in the post‐fortification period compared to pre‐fortification period suggest that fortification may have adverse effects on histone methylation in already initiated cells. Lower Lys‐9 methylation in cervical cells of women free of CIN in the post fortification period compared to pre fortification on the other hand suggest that fortification is likely to protect against the initiation of carcinogenic process in the cervix.Supported by NCI R03 CA 102893

Distinct transcriptional outputs associated with mono- and dimethylated histone H3 arginine 2

Dimethylation of histone H3 Arg2 (H3R2me2) maintains transcriptional silencing by inhibiting Set1 mediated trimethylation of H3K4. Here we demonstrate that Arg2 is also monomethylated (H3R2me1) in yeast but that its functional characteristics are distinct from H3R2me2: (i) H3R2me1 does not inhibit histone H3 Lys4 (H3K4) methylation; (ii) it is present throughout the coding region of genes; and (iii) it correlates with active transcription. Collectively, these results indicate that different H3R2 methylation states have defined roles in gene expression.

Histone H3 Lys79 methylation is required for efficient nucleotide excision repair in a silenced locus of Saccharomyces cerevisiae

Methylation of specific histone lysine residues regulates gene expression and heterochromatin function, but little is known about its role in DNA repair. To examine how changes in conserved methylated residues of histone H3 affect nucleotide excision repair (NER), viable H3K4R and H3K79R mutants were generated in Saccharomyces cerevisiae. These mutants show decreased UV survival and impaired NER at the transcriptionally silent HML locus, while maintaining normal NER in the constitutively expressed RPB2 gene and transcriptionally repressed, nucleosome loaded GAL10 gene. Moreover, the HML chromatin in these mutants has reduced accessibility to Micrococcal nuclease (MNase). Importantly, chromatin immunoprecipitation analysis demonstrates there is enhanced recruitment of the Sir complex at the HML locus of these mutants, and deletion of the SIR2 or SIR3 genes restores the MNase accessibility and DNA repair efficiency at this locus. Furthermore, following UV irradiation expression of NER genes in these mutants remains at wild type levels, with the exception of RAD16 which decreases by more than 2-fold. These results indicate that impaired NER occurs in the silenced chromatin of H3K79R and H3K4,79R mutants as a result of increased binding of Sir complexes, which may reduce DNA lesion accessibility to repair enzymes.

The Rtt106 Histone Chaperone Is Functionally Linked to Transcription Elongation and Is Involved in the Regulation of Spurious Transcription from Cryptic Promoters in Yeast*♦

Rtt106 is a histone chaperone that has been suggested to play a role in heterochromatin-mediated silencing in Saccharomyces cerevisiae. It interacts physically and functionally with the chromatin assembly factor-1 (CAF-1), which is associated with replication-coupled nucleosomal deposition. In this work, we have taken several approaches to study Rtt106 in greater detail and have identified a previously unknown function of Rtt106. We found genetic interactions between rtt106Delta and mutations in genes encoding transcription elongation factors, including Spt6, TFIIS, and members of the PAF and yeast DSIF complexes. In addition, chromatin immunoprecipitation analysis indicates that Rtt106 is associated with transcribed regions of active genes. Furthermore, our results show that Rtt106 is required for the repression of transcription from a cryptic promoter within a coding region. This observation strongly suggests that Rtt106 is involved in the regulation of chromatin structure of transcribed regions. Finally, we provide evidence that Rtt106 plays a role in regulating the levels of histone H3 transcription-coupled deposition over transcribed regions. Taken together, our results indicate a direct link for Rtt106 with transcription elongation and the chromatin dynamics associated with RNA polymerase II passage.

Age-related difference of site-specific histone modifications in rat liver

Aging is associated with decrease in activities of the transcription, replication and DNA repair that can result in deterioration of cellular and tissue functions. Changes of chromatin structures with age are likely major underling mechanisms for the functional decline. Chromatin consists of DNA and histones as well as non-histone proteins. While age-associated change of DNA methylation is well documented, little information is available on site-specific histone modifications in aging. We studied here age-related change of selected modifications of rat liver histone, i.e., histone H3 Lys9 acetylation (H3K9ac), H3 Lys9 methylation (H3K9me), H3 Ser10 phosphorylation (H3S10ph) and H3 Lys14 acetylation (H3K14ac). H3K9ac was decreased and H3S10ph was increased with age significantly. In view of reports indicating that decrease in acetylation and increase in phosphorylation of H3 histones can suppress gene activity, our findings suggest that a mechanism of decreased chromatin functions with age is due to such epigenetic changes.

Ers1, a Rapidly Diverging Protein Essential for RNA Interference-dependent Heterochromatic Silencing in Schizosaccharomyces pombe

Centromeric silencing and heterochromatin formation in Schizosaccharomyces pombe require the RNA interference (RNAi) machinery. Three factors that mediate this mechanism have been identified: 1) the RNA-dependent RNA polymerase complex RdRC, 2) the Argonaute-containing RITS (RNA-induced initiation of transcriptional silencing) complex, and 3) the endoribonuclease Dicer ortholog Dcr1. S. pombe mutants lacking a new factor described here, Ers1, are completely defective in RNAi-dependent silencing of centromeric regions but, importantly, not in RNAi-independent silencing at the mat3M or tel2R loci. ers1Delta cells likewise fail to convert centromeric pre-small interfering RNA transcripts into small interfering RNAs, are defective in histone H3 Lys(9) methylation, and are unable to recruit the RITS complex to centromeric sequences. Surprisingly, Ers1 lacks obvious orthologs outside of the genus Schizosaccharomyces. Within this group, it is diverging rapidly, raising the possibility that it is coevolving with target RNA elements.

Epigenetic Control of rDNA Loci in Response to Intracellular Energy Status

Intracellular energy balance is important for cell survival. In eukaryotic cells, the most energy-consuming process is ribosome biosynthesis, which adapts to changes in intracellular energy status. However, the mechanism that links energy status and ribosome biosynthesis is largely unknown. Here, we describe eNoSC, a protein complex that senses energy status and controls rRNA transcription. eNoSC contains Nucleomethylin, which binds histone H3 dimethylated Lys9 in the rDNA locus, in a complex with SIRT1 and SUV39H1. Both SIRT1 and SUV39H1 are required for energy-dependent transcriptional repression, suggesting that a change in the NAD(+)/NADH ratio induced by reduction of energy status could activate SIRT1, leading to deacetylation of histone H3 and dimethylation at Lys9 by SUV39H1, thus establishing silent chromatin in the rDNA locus. Furthermore, eNoSC promotes restoration of energy balance by limiting rRNA transcription, thus protecting cells from energy deprivation-dependent apoptosis. These findings provide key insight into the mechanisms of energy homeostasis in cells.

A Kinetic Study of Human Protein Arginine N-Methyltransferase 6 Reveals a Distributive Mechanism

Human protein arginine N-methyltransferase 6 (PRMT6) transfers methyl groups from the co-substrate S-adenosyl-L-methionine to arginine residues within proteins, forming S-adenosyl-L-homocysteine as well as omega-N(G)-monomethylarginine (MMA) and asymmetric dimethylarginine (aDMA) residues in the process. We have characterized the kinetic mechanism of recombinant His-tagged PRMT6 using a mass spectrometry method for monitoring the methylation of a series of peptides bearing a single arginine, MMA, or aDMA residue. We find that PRMT6 follows an ordered sequential mechanism in which S-adenosyl-L-methionine binds to the enzyme first and the methylated product is the first to dissociate. Furthermore, we find that the enzyme displays a preference for the monomethylated peptide substrate, exhibiting both lower K(m) and higher V(max) values than what are observed for the unmethylated peptide. This difference in substrate K(m) and V(max), as well as the lack of detectable aDMA-containing product from the unmethylated substrate, suggest a distributive rather than processive mechanism for multiple methylations of a single arginine residue. In addition, we speculate that the increased catalytic efficiency of PRMT6 for methylated substrates combined with lower K(m) values for native protein methyl acceptors may obscure this distributive mechanism to produce an apparently processive mechanism.

The See-saw of Differentiation: Tipping the Chromatin Balance

Histone modifications, specifically, methylation of lysine residues in histone 3 (H3), determine chromatin structure and accessibility for transcription. A recent study by Bernstein and Lander identified a unique pattern of H3 methylation at the promoter region of developmental genes in pluripotent stem cells. The pattern consists of the simultaneous presence of a silencing mark [methylation of Lys(27) on H3 (H3K27me)] and an activation mark (H3K4me). This bivalent mark seems associated with a poised state of transcription and resolves upon differentiation largely into either H3K27me or H3K4me. Skin fibroblasts that can be reverted in culture to the pluripotent stage show re-establishment of the bivalent chromatin pattern. This switch in chromatin states may be key to our understanding of developmental and tissue specific regulation of gene expression and may be therapeutically useful in regenerative medicine.

MSL Complex Is Attracted to Genes Marked by H3K36 Trimethylation Using a Sequence-Independent Mechanism

In Drosophila, X chromosome dosage compensation requires the male-specific lethal (MSL) complex, which associates with actively transcribed genes on the single male X chromosome to upregulate transcription approximately 2-fold. We found that on the male X chromosome, or when MSL complex is ectopically localized to an autosome, histone H3K36 trimethylation (H3K36me3) is a strong predictor of MSL binding. We isolated mutants lacking Set2, the H3K36me3 methyltransferase, and found that Set2 is an essential gene in both sexes of Drosophila. In set2 mutant males, MSL complex maintains X specificity but exhibits reduced binding to target genes. Furthermore, recombinant MSL3 protein preferentially binds nucleosomes marked by H3K36me3 in vitro. Our results support a model in which MSL complex uses high-affinity sites to initially recognize the X chromosome and then associates with many of its targets through sequence-independent features of transcribed genes.

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.

Histone H3K4 demethylases are essential in development and differentiationThis paper is one of a selection of papers published in this Special Issue, entitled 28th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.

Lysine histone methylation is one of the most robust epigenetic marks and is essential for the regulation of multiple cellular processes. The methylation of Lys4 of histone H3 seems to be of particular significance. It is associated with active regions of the genome, and in Drosophila it is catalyzed by trithorax-group proteins that have become paradigms of developmental regulators at the level of chromatin. Like other histone methylation events, H3K4 methylation was considered irreversible until the identification of a large number of histone demethylases indicated that demethylation events play an important role in histone modification dynamics. However, the described demethylases had no strictly assigned biological functions and the identity of the histone demethylases that would contribute to the epigenetic changes specifying certain biological processes was unknown. Recently, several groups presented evidence that a family of 4 JmjC domain proteins results in the global changes of histone demethylation, and in elegant studies using model organisms, they demonstrated the importance of this family of histone demethylases in cell fate determination. All 4 proteins possess the demethylase activity specific to H3K4 and belong to the poorly described JARID1 protein family.

Effects of Chain Length and N‐Methylation on a Cation–π Interaction in a β‐Hairpin Peptide

The effects of N-methylation and chain length on a cation-pi interaction have been investigated within the context of a beta-hairpin peptide. Significant enhancement of the interaction and structural stabilization of the hairpin have been observed upon Lys methylation. Thermodynamic analysis indicates an increased entropic driving force for folding upon methylation of Lys residues. Comparison of lysine to analogues ornithine (Orn) and diaminobutyric acid (Dab) indicates that lysine provides the strongest cation-pi interaction and also provides the most stable beta-hairpin due to a combination of side chain-side chain interactions and beta-sheet propensities. These studies have significance for the recognition of methylated lysine in histone proteins.

The Fission Yeast Jmj2 Reverses Histone H3 Lysine 4 Trimethylation

Histone methylation regulates transcription, chromatin structure, and the epigenetic state of the cell. Recent studies identified the JmjC domain as a catalytic module for histone demethylation. Schizosaccharomyces pombe contains seven JmjC proteins, but it was unclear whether any of them functioned as histone demethylases. In this report, we show that the JmjC protein Jmj2, which is evolutionarily conserved from yeast to human, reversed trimethylated H3-Lys-4 to di- and mono-but not unmethylated products. Overexpression of Jmj2 but not a catalytically inactive mutant reduced H3-Lys-4 trimethylation levels in vivo and suppressed the toxicity caused by overexpression of the H3-Lys-4-me3-binding protein Yng1 in budding yeast. Genome-wide analysis showed that the loss of jmj2 was associated with an increase in the H3-Lys-4-me3 signal, which was enriched near the transcriptional start sites and the coding regions. At the mating-type locus, the loss of jmj2 or substitution of jmj2 with a catalytically inactive form is correlated with increased reporter gene transcription and H3-Lys-4-me3/2 levels, suggesting that Jmj2 and its demethylase activity may play a role in heterochromatin biology. Our findings identified a novel S. pombe histone demethylase with specificity toward di- and trimethylated histone H3-Lys-4 and a possible role in heterochromatin regulation.

Ubiquitylation of histone H2B controls RNA polymerase II transcription elongation independently of histone H3 methylation

Transcription by RNA polymerase II (polII) is accompanied by dramatic changes in chromatin structure. Numerous enzymatic activities contribute to these changes, including ATP-dependent nucleosome remodeling enzymes and histone modifying enzymes. Recent studies in budding yeast document a histone modification pathway associated with polII transcription, whereby ubiquitylation of histone H2B leads to methylation of histone H3 on specific lysine residues. Although this series of events appears to be highly conserved among eukaryotes, its mechanistic function in transcription is unknown. Here we document a significant functional divergence between ubiquitylation of H2B and methylation of Lys 4 on histone H3 in the fission yeast Schizosaccharomyces pombe. Loss of H2B ubiquitylation results in defects in cell growth, septation, and nuclear structure, phenotypes not observed in cells lacking H3 Lys 4 methylation. Consistent with these results, gene expression microarray analysis reveals a greater role for H2B ubiquitylation in gene regulation than for H3 Lys 4 methylation. Chromatin immunoprecipitation (ChIP) experiments demonstrate that loss of H2B ubiquitylation alters the distribution of polII and histones in gene coding regions. We propose that ubiquitylation of H2B impacts transcription elongation and nuclear architecture through its effects on chromatin dynamics.

Organismal Differences in Post-translational Modifications in Histones H3 and H4

Post-translational modifications (PTMs) of histones play an important role in many cellular processes, notably gene regulation. Using a combination of mass spectrometric and immunobiochemical approaches, we show that the PTM profile of histone H3 differs significantly among the various model organisms examined. Unicellular eukaryotes, such as Saccharomyces cerevisiae (yeast) and Tetrahymena thermophila (Tet), for example, contain more activation than silencing marks as compared with mammalian cells (mouse and human), which are generally enriched in PTMs more often associated with gene silencing. Close examination reveals that many of the better-known modified lysines (Lys) can be either methylated or acetylated and that the overall modification patterns become more complex from unicellular eukaryotes to mammals. Additionally, novel species-specific H3 PTMs from wild-type asynchronously grown cells are also detected by mass spectrometry. Our results suggest that some PTMs are more conserved than previously thought, including H3K9me1 and H4K20me2 in yeast and H3K27me1, -me2, and -me3 in Tet. On histone H4, methylation at Lys-20 showed a similar pattern as H3 methylation at Lys-9, with mammals containing more methylation than the unicellular organisms. Additionally, modification profiles of H4 acetylation were very similar among the organisms examined.

Two different Argonaute complexes are required for siRNA generation and heterochromatin assembly in fission yeast

The RNA-induced transcriptional silencing (RITS) complex, containing Ago1, Chp1, Tas3 and centromeric small interfering RNAs (siRNAs), is required for heterochromatic gene silencing at centromeres. Here, we identify a second fission yeast Argonaute complex (Argonaute siRNA chaperone, ARC), which contains, in addition to Ago1, two previously uncharacterized proteins, Arb1 and Arb2, both of which are required for histone H3 Lys9 (H3-K9) methylation, heterochromatin assembly and siRNA generation. Furthermore, whereas siRNAs in the RITS complex are mostly single-stranded, siRNAs associated with ARC are mostly double-stranded, indicating that Arb1 and Arb2 inhibit the release of the siRNA passenger strand from Ago1. Consistent with this observation, purified Arb1 inhibits the slicer activity of Ago1 in vitro, and purified catalytically inactive Ago1 contains only double-stranded siRNA. Finally, we show that slicer activity is required for the siRNA-dependent association of Ago1 with chromatin and for the spreading of histone H3-K9 methylation.

Id2 gene-targeted crosstalk between Wnt and retinoid signaling regulates proliferation in human keratinocytes

We investigated the effect of all-trans-retinoic acid (atRA) on proliferation in several human skin cell lines and found that antiproliferative potency of atRA correlated with the endogenous activity of canonical Wnt signaling. In HaCaT keratinocytes, we found that atRA significantly suppressed the expression of Id2, a member of the inhibitor of differentiation family of transcription factors that regulate cell growth and differentiation. However, no apparent change in the expression of other Wnt targets, like c-Myc or cyclin D1, was observed. Retinoid-induced Id2 gene suppression was associated with decreased levels of histone H3 and H4 acetylation and histone H3 Lys-4 methylation, and with recruitment of the LSD1 demethylase at the Wnt-response element (WRE) (TCF/LEF-binding site), in the Id2 gene promoter. None of such changes was detected at the WRE of c-Myc and cyclin D1 gene promoters. Inhibition of Id2 by short interfering RNA (siRNA) had a similar effect on the proliferation of HaCaT cells as exposure to atRA, whereas anti-beta-catenin siRNA significantly inhibited its antiproliferative effect. These data suggest that downregulation of Id2 gene expression through transcriptional convergence between Wnt and retinoid signaling pathways underlies the antiproliferative effect of retinoids in keratinocytes, and provide evidence of gene-targeted crosstalk between signaling pathways.