Histone H3 Methylation Patterns Research Articles

Protein methylation characterization using NMR without isotopic labeling

Protein methylation is crucial in epigenetics, and targeting the involved methyltransferases shows great potential for therapeutic intervention with several inhibitors in clinical trials for oncology indications. Therefore, characterization of protein methylation is essential for understanding the methyltransferase function and discovering chemical inhibitors and antagonists. While NMR has been used to measure methylation rates, isotopic labeling of protein or methyl donors can be costly and cannot characterize demethylation of proteins extracted from natural sources. Our method employs a four-quantum filter 1H-13C experiment that selectively detects methyl groups, providing a simple way to characterize methylation and demethylation features of methyltransferases and demethylases, respectively, without requiring isotopic labeling. In our experiments, we successfully observed the methylation of H3 under lysate from various cells and tissues of mice with cancerous growth. The results revealed that H3 undergoes both mono- and dimethylation in all the tested lysates, but at varying rates and degrees. Significantly lower H3 methylation rates and levels were observed in both cervical tumor and breast tumor lysates compared with the corresponding cancerous cells and healthy cells lysates. These findings highlight the variability of histone H3 methylation patterns among healthy cells, cancerous cells, tumor tissues, and different tumor types, and suggest that this method has great potential in facilitating the development of effective interventions against these diseases. By characterizing the methylation features of suspected tumors or areas of concern, it provides valuable insights into the underlying mechanisms of cancer development and aids in identifying potential targets for therapeutic interventions.

H3K9 methylation patterns during somatic embryogenic competence expression in tamarillo (Solanum betaceum Cav.)

The capacity to regenerate is intrinsic to plants and is the basis of natural asexual propagation and artificial cloning. Despite there are different ways of plant regeneration, they all require a change in cell fate and pluripotency reacquisition, in particular somatic embryogenesis. The mechanisms underlying somatic cell reprogramming for embryogenic competence acquisition, expression and maintenance remain not fully understood. These complex processes have been often associated with epigenetic markers, mainly DNA methylation, while little is known about the possible role of histone modifications. In the present study, the dynamics of global levels and distribution patterns of histone H3 methylation at lysine 9 (H3K9), a major repressive histone modification, were analyzed in somatic embryogenesis-induced cell lines with different embryogenic capacities and during somatic embryo initiation, in the woody species Solanum betaceum. Quantification of global H3K9 methylation showed similar levels in the three types of proliferating calli (embryogenic, long-term and non-embryogenic), kept in high sucrose and auxin-containing medium. Microscopic analyzes revealed heterogeneous cell organization and different cell types, particularly evident in embryogenic callus. The H3K9 dimethylation (H3K9me2) immunofluorescence signal was lower in nuclei of cells showing embryogenic-like and proliferating features, while labeling was higher in vacuolated, non-embryogenic cells with higher proliferation rates. By auxin removal, somatic embryo development was promoted in the embryogenic cell line. During the initiation of this process, increasing levels of global H3K9 methylation were found, together with increasing H3K9me2 immunofluorescence signals, especially in cells of the developing embryo. These results suggest that H3K9 methylation is involved in somatic embryo development, a developmental pathway in which this epigenetic mark could play a role in the gene transcription variation that is associated with embryogenic competence expression in S. betaceum. Altogether, these data provide new insights into the role of this epigenetic mark in somatic embryogenesis in trees, where scarce information is available.

The pattern of histone H3 epigenetic posttranslational modifications is regulated by the VRK1 chromatin kinase

BackgroundDynamic chromatin remodeling is associated with changes in the epigenetic pattern of histone acetylations and methylations required for processes based on dynamic chromatin remodeling and implicated in different nuclear functions. These histone epigenetic modifications need to be coordinated, a role that may be mediated by chromatin kinases such as VRK1, which phosphorylates histones H3 and H2A.MethodsThe effect of VRK1 depletion and VRK1 inhibitor, VRK-IN-1, on the acetylation and methylation of histone H3 in K4, K9 and K27 was determined under different conditions, arrested or proliferating cells, in A549 lung adenocarcinoma and U2OS osteosarcoma cells.ResultsChromatin organization is determined by the phosphorylation pattern of histones mediated by different types of enzymes. We have studied how the VRK1 chromatin kinase can alter the epigenetic posttranslational modifications of histones by using siRNA, a specific inhibitor of this kinase (VRK-IN-1), and of histone acetyl and methyl transferases, as well as histone deacetylase and demethylase. Loss of VRK1 implicated a switch in the state of H3K9 posttranslational modifications. VRK1 depletion/inhibition causes a loss of H3K9 acetylation and facilitates its methylation. This effect is similar to that of the KAT inhibitor C646, and to KDM inhibitors as iadademstat (ORY-1001) or JMJD2 inhibitor. Alternatively, HDAC inhibitors (selisistat, panobinostat, vorinostat) and KMT inhibitors (tazemetostat, chaetocin) have the opposite effect of VRK1 depletion or inhibition, and cause increase of H3K9ac and a decrease of H3K9me3. VRK1 stably interacts with members of these four enzyme families. However, VRK1 can only play a role on these epigenetic modifications by indirect mechanisms in which these epigenetic enzymes are likely targets to be regulated and coordinated by VRK1.ConclusionsThe chromatin kinase VRK1 regulates the epigenetic patterns of histone H3 acetylation and methylation in lysines 4, 9 and 27. VRK1 is a master regulator of chromatin organization associated with its specific functions, such as transcription or DNA repair.

Chromatin Immunostaining of Plant Nuclei.

Recent evidence has demonstrated that specific epigenetic changes are also related to plant growth and development. Immunostaining enables the detection and characterization of chromatin modification, e.g., histone H4 acetylation (H4K5ac), histone H3 methylation (H3K4me2 and H3K9me2), and DNA methylation (5mC) with unique and specific patterns in plant tissues. Here we describe experimental procedures to determine the histone H3 methylation (H3K4me2 and H3K9me2) patterns in 3D-chromatin in whole roots tissue and 2D-chromatin in single nuclei in rice. To analyze both iron and salinity treatments, we show how to test for changes to the epigenetic chromatin landscape using heterochromatin (H3K9me2) and euchromatin (H3K4me) markers for chromatin immunostaining, especially in the proximal meristem region. To elucidate the epigenetic impact of environmental stress and external plant growth regulators, we demonstrate how to apply a combination of salinity, auxin, and abscisic acid treatments. The results of these experiments provide insights into the epigenetic landscape during rice root growth and development.

Abstract 2400: Identification of histone modifying enzymes associated with tamoxifen resistance in MCF7 breast cancer cells

Abstract Tamoxifen is widely used to treat estrogen receptor-alpha positive breast cancer. However, resistance to tamoxifen often prevent the success of endocrine therapy. Epigenetic change in tumors are associated with cancer development and progression. Although altered histone modification has been implicated in tumorigenesis, their role in endocrine resistance was little known. To identified tamoxifen-resistance associated histone methylation modifying enzyme, we examined the global histone H3 methylation patterns in tamoxifen-resistance MCF7 breast cancer cells. Our results show that, the levels of methylated histone H3 lysine 4 (H3K4) and histone H3 lysine 9 (H3K9) was upregulated in tamoxifen resistance MCF7 breast cancer cells. Next, we analysis expression levels of H3K4 and H3K9 methylation modifying enzyme in tamoxifen resistance breast cancer cells. qRT-PCR results show that H3K4 methyltransferases (KMT2A and KMT2C) is upregulated in tamoxifen resistance breast cancer cells. Moreover, we observed that si-RNA mediated knockdown of KMT2C was more susceptible to tamoxifen-induced growth inhibition. Finally, Analyses of publicly available patient data sets indicated that KMT2A and KMT2C levels are higher in patients after endocrine therapy failure (recurrence), compared to those of responders (non-recurrence). Our study indicates that elevated levels of KMT2A and KMT2C in the tamoxifen-resistance breast cancers may contributed tamoxifen resistance. Citation Format: Min-Ho Lee, Mi-Ock Lee. Identification of histone modifying enzymes associated with tamoxifen resistance in MCF7 breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2400. doi:10.1158/1538-7445.AM2017-2400

The ATP-dependent chromatin remodeler Chd1 is recruited by transcription elongation factors and maintains H3K4me3/H3K36me3 domains at actively transcribed and spliced genes.

Chd1 (Chromodomain Helicase DNA Binding Protein 1) is a conserved ATP-dependent chromatin remodeler that maintains the nucleosomal structure of chromatin, but the determinants of its specificity and its impact on gene expression are not well defined. To identify the determinants of Chd1 binding specificity in the yeast genome, we investigated Chd1 occupancy in mutants of several candidate factors. We found that several components of the PAF1 transcription elongation complex contribute to Chd1 recruitment to highly transcribed genes and identified Spt4 as a factor that appears to negatively modulate Chd1 binding to chromatin. We discovered that CHD1 loss alters H3K4me3 and H3K36me3 patterns throughout the yeast genome. Interestingly, the aberrant histone H3 methylation patterns were predominantly observed within 1 kb from the transcription start site, where both histone H3 methylation marks co-occur. A reciprocal change between the two marks was obvious in the absence of Chd1, suggesting a role for CHD1 in establishing or maintaining the boundaries of these largely mutually exclusive histone marks. Strikingly, intron-containing genes were most susceptible to CHD1 loss and exhibited a high degree of histone H3 methylation changes. Intron retention was significantly lower in the absence of CHD1, suggesting that CHD1 function as a chromatin remodeler could indirectly affect RNA splicing.

Pathology Tissue-quantitative Mass Spectrometry Analysis to Profile Histone Post-translational Modification Patterns in Patient Samples

Histone post-translational modifications (hPTMs) generate a complex combinatorial code that has been implicated with various pathologies, including cancer. Dissecting such a code in physiological and diseased states may be exploited for epigenetic biomarker discovery, but hPTM analysis in clinical samples has been hindered by technical limitations. Here, we developed a method (PAThology tissue analysis of Histones by Mass Spectrometry - PAT-H-MS) that allows to perform a comprehensive, unbiased and quantitative MS-analysis of hPTM patterns on formalin-fixed paraffin-embedded (FFPE) samples. In pairwise comparisons, histone extracted from formalin-fixed paraffin-embedded tissues showed patterns similar to fresh frozen samples for 24 differentially modified peptides from histone H3. In addition, when coupled with a histone-focused version of the super-SILAC approach, this method allows the accurate quantification of modification changes among breast cancer patient samples. As an initial application of the PAThology tissue analysis of Histones by Mass Spectrometry method, we analyzed breast cancer samples, revealing significant changes in histone H3 methylation patterns among Luminal A-like and Triple Negative disease subtypes. These results pave the way for retrospective epigenetic studies that combine the power of MS-based hPTM analysis with the extensive clinical information associated with formalin-fixed paraffin-embedded archives.

Changes in Methionine Metabolism and Histone H3 Trimethylation Are Linked to Mitochondrial Defects in Multiple Sclerosis.

For decades, it has been observed that vitamin B12 deficiency and multiple sclerosis (MS) share certain pathological changes, including conduction disturbances. In the present study, we have found that vitamin B12-dependent methionine metabolism is dysregulated in the MS brain. We found that concentrations of the methyl donor betaine are decreased in MS cortex and are correlated with reduced levels of the histone H3 methyl mark H3K4me3 in neurons. Cell culture and chromatin immunoprecipitation-seq data suggest that these changes may lead to defects in mitochondria and impact neuronal energetics. These data have uncovered a novel pathway linking methionine metabolism with mitochondrial respiration and have important implications for understanding mechanisms involved in neurodegeneration in MS.

In situ histone landscape of nephrogenesis

In the developing kidney, self-renewing progenitors respond to inductive signaling from the adjacent branching ureteric bud by undergoing mesenchyme-to-epithelium transition. Nascent nephrons subsequently undergo elongation, segmentation, and differentiation into a mature renal epithelium with diverse functions. Epigenetic mechanisms have been implicated in impacting cell fate decisions during nephrogenesis; however, the chromatin landscape of nephron progenitors and daughter differentiating cells are largely unknown. Here, we examined the spatiotemporal expression patterns of histone H3 methylation and histone methyltransferases in E15.5 mouse kidneys. Kidney sections were probed with antibodies against histone modifications, enzymes, and markers of progenitors and differentiation. The results revealed that: (1) nephron progenitor cells exhibit a broad histone methylation signature that comprises both “active” and “repressive” marks (H3K4me3/K9me3/K27me3/R2me2/R17me2); (2) nascent nephrons retain high H3K4me3 but show downregulation of H3K9/K27me3 and; (3) maturing epithelial tubules acquire high levels of H3K79me2/3. Consistent with respective histone marks, the H3K4 methyltransferase, Ash2l, is expressed in progenitors and nascent nephrons, whereas the H3K9/K27 methyltransferases, G9a/Ezh2, are more enriched in progenitors than nascent nephrons. We conclude that combinatorial histone signatures correlate with cell fate decisions during nephrogenesis.

The endoglycosidase heparanase enters the nucleus of T lymphocytes and modulates H3 methylation at actively transcribed genes via the interplay with key chromatin modifying enzymes

The methylation of histones is a fundamental epigenetic process regulating gene expression programs in mammalian cells. Dysregulated patterns of histone methylation are directly implicated in malignant transformation. Here, we report the unexpected finding that the invasive extracellular matrix degrading endoglycosidase heparanase enters the nucleus of activated human T lymphocytes and regulates the transcription of a cohort of inducible immune response genes by controlling histone H3 methylation patterns. It was found that nuclear heparanase preferentially associates with euchromatin. Genome-wide ChIP-on-chip analyses showed that heparanase is recruited to both the promoter and transcribed regions of a distinct cohort of transcriptionally active genes. Knockdown and overexpression of the heparanase gene also showed that chromatin-bound heparanase is a prerequisite for the transcription of a subset of inducible immune response genes in activated T cells. Furthermore, the actions of heparanase seem to influence gene transcription by associating with the demethylase LSD1, preventing recruitment of the methylase MLL and thereby modifying histone H3 methylation patterns. These data indicate that heparanase belongs to an emerging class of proteins that play an important role in regulating transcription in addition to their well-recognized extra-nuclear functions.

Epigenetic chromatin modifications inBrassicagenomes

Epigenetic modifications such as histone and DNA methylation are highly conserved among eukaryotes, although the nuclear patterns of these modifications vary between different species. Brassica species represent a very attractive model for analysis of epigenetic changes because of their differences in genome size, ploidy level, and the organization of heterochromatin blocks. Brassica rapa and B. oleracea are diploid species, and B. napus is an allotetraploid species that arose from the hybridization of these two diploids. We found that patterns of DNA and histone H3 methylation differ between Brassica species. The most prominent differences concern the two diploids. DNA methylation was present exclusively in the heterochromatin only in B. rapa. In B. oleracea and B. napus this modification was detected in both euchromatin and heterochromatin. A similar pattern was observed for dimethylation of lysine 9. Dimethylation of lysine 4 is a typical marker of euchromatin in Brassica species, like it is in other plant species. We conclude that the diploid species differ in patterns of analyzed epigenetic modifications and the allotetraploid B. napus has combined patterns from both diploids. Differences in patterns of DNA and histone H3 methylation between Brassica species can be attributed mainly to the genome structure and heterochromatin localization rather than ploidy level.

Lmo2 Induces T-Cell Leukemia with Epigenetic Deregulation of CD4.

The LIM domain Only-2 (Lmo2) oncogene is deregulated in the majority of human T-cell acute lymphoblastic leukemias (T-ALL). Enforced, constitutive expression of Lmo2 in transgenic mice using the T-cell-specific Cd2 promoter/enhancer induces T-ALL with median latency of 200 days and 100% penetrance. These transgenic mice have a pre-leukemic differentiation block at the double negative stage (CD4− CD8−) of T-cell differentiation. Despite this, T-ALLs in these mice arise from various stages of T-cell differentiation. To explore the relationship between T-cell development and Lmo2 oncogene function, we established cell lines from four independent T-cell tumors representing the double negative (DN), double positive (DP), and single positive (SP) stages. Two of the lines showed variegated expression of CD4 antigen. One line, 32080, was striking in that CD4 was expressed in 50% of cells whereas CD8 was uniformly present. The cell line expressed markers consistent with the intermediate single positive stage (ISP) to double positive (DP) stage transition. We sorted the 32080 line for highly CD4 positive and negative cells and observed them in culture. After one week, both sorted populations showed variegated CD4 expression. We determined CD4 expression was regulated at the transcriptional level and not due to post-transcriptional effects and that cell replication was necessary to re-establish CD4 variegation. Importantly, variegated CD4 expression was dependent on Lmo2 because its knockdown resulted in decreased CD4 expression. HDAC inhibition by trichostatin A had the reciprocal effect of inducing more CD4 expression suggesting that chromatin modification was playing a role. The CD4 locus was analyzed by chromatin immunoprecipitation and histone H3 methylation patterns consistent with silencing were found in the CD4 negative sorted cells but not in the CD4 positive population. The 32080 cell line is a striking model of ISP to DP T-cell plasticity and invokes a novel mechanism by which Lmo2 functions.

Dynamic and stable histone H3 methylation patterns at the Arabidopsis FLC and AP1 loci

Mechanisms that chemically modify nucleosomes leading to inheritable activation or repression of pertinent genes are defined as epigenetic. H3K4me3 and H3K27me3 are interpreted as ‘activating’ and ‘silencing’ marks, respectively. Here, we demonstrate that even for related genes neither modification, alone, could serve as an indicator of expression status: despite being members of the same gene family selectively activated by ATX1, FLC and AP1 nucleosomes may be similarly decorated but, also, surprisingly different. ‘Activating’ H3K4me3 and ‘silencing’ H3K27me3 modifications co-exist at 5′-end nucleosomes of transcriptionally active FLC-gene, while highly transcribed AP1 displays neither of the two marks. The results suggest that distinct mechanisms ‘read’ and operate at each locus. In a remarkable contrast, H3K4me3–H3K27me3 profiles at downstream FLC and AP1 gene sequences remain unchanged and transmitted as stable marks throughout development. We propose that H3K4me3 and H3K27me3 produce a distinct bi-modular ‘syllable’ in the histone ‘code’ conveying different meaning on specific genes. Evidence that certain chromatin modifications might be common for active or non-active genome regions but, also, that the same histone signs might have gene-specific ‘meaning’, as reported here, might be critically important for large-scale genome analyses. ATX1 and CLF encode enzyme activities involved in establishing the H3K4me3 and H3K27me3 marks, respectively. The potential involvement of ATX1 and CLF in generating the dual H3K4me3 and H3K27me3 marks on FLC and AP1 nucleosomes was investigated.

Developmental regulation of histone H3 methylation at lysine 4 in the porcine ovary

Follicular growth and oogenesis involve highly dynamic changes in morphogenesis, chromatin structure, and gene transcription. The tight coordination of these events leads to ovulation of a mature oocyte and formation of the luteal tissue necessary to regulate embryo implantation and development. This entire process is regulated by numerous endocrine and in situ mechanisms. The role of epigenetic mechanisms in folliculogenesis, such as the biochemical modification of the DNA packaging proteins, the histones, is not well understood. Our objective was to determine the cellular and follicular stage-specific patterns of histone H3 methylation at lysine 4 (K4) in porcine preovulatory follicles and during luteinization in pig ovaries. Ovary tissues were collected from slaughtered prepubertal and cyclic gilts at various stages of the estrous cycle, pregnancy, and from ovaries recovered from gonatropin-treated gilts at 0, 24, and 38 h post human chorionic gonadotropin (hCG) injection. Samples were fixed in 4% paraformaldehyde and processed for embedding in paraffin and sectioned using standard histological protocols. Immunofluorescent staining was performed on 3 microm thick sections. The immunostaining pattern of mono-, di-, and tri-methylated histone H3-K4 and lysine-specific demethylase 1 (LSD1, also known as KDM1 or AOF1) was assessed. Interestingly, H3-K4 mono-, di-, and tri-methylation in follicles of prepubertal gilts was specifically distributed and developmentally regulated. While granulosa cells of primary, secondary, and early antral follicles were negative for H3-K4 methylation those from large antral follicles showed a striking upregulation in the cells located in the proximity to the oocyte. Specifically, the cumulus oophorus displayed intense staining for H3-K4 methylation and signals were strongest in the granulosa cells in the inner two cell layers of the follicular wall. Although all oocytes from primary to large antral stage follicles were positive for H3-K4 mono-, di-, and tri-methylation, the patterns of distribution were altered through oocyte follicle development. H3-K4 methylation in granulosa cells was dramatically reduced as time to ovulation approached and was low to undetected at 38 h post hCG treatment. H3-K4 mono-, di-, and tri-methylation in large luteal cells increased as differentiation evolved but remained low in small luteal cells. Strikingly, LSD1 (KDM1) expression was found to be restricted to the corpus luteum. In summary, this study provides new information on histone H3-K4 methylation patterns in the oocyte and follicle during folliculogenesis, which suggests that these epigenetic markers serve an essential regulatory role during folliculogenesis.

The mouse C/EBPδ gene promoter is regulated by STAT3 and Sp1 transcriptional activators, chromatin remodeling and c‐Myc repression

CCAAT/enhancer binding proteindelta (C/EBPdelta) gene transcription is highly induced in G(0) growth arrested mammary epithelial cells and "loss of function" alterations in C/EBPdelta have been reported in human breast cancer. To gain a better understanding of the positive and negative factors that control C/EBPdelta gene expression we investigated the role of transcriptional activators, coactivators, repressors, histone modifications, chromatin remodeling and basal transcriptional machinery components in growing and growth arrested HC11 mouse mammary epithelial cells. Growth arrest treatments result in increased STAT3 activation (pSTAT3) and increased C/EBPdelta expression. Co-immunoprecipitation and chromatin immunoprecipitation (ChIP) assays demonstrated that pSTAT3 and Sp1 interact and bind to the transcriptionally active C/EBPdelta promoter. ChIP assays performed under exponentially growing (C/EBPdelta non-expressing) conditions demonstrated that the C/EBPdelta promoter is preloaded with transcriptional activators (Sp1 and CREB) and transcriptional machinery components (TBP and RNA Pol II). In contrast, under G(0) growth arrest (C/EBPdelta expressing) conditions ChIP analysis detected pSTAT3, Sp1, NCoA/SRC1, CBP/p300, pCREB, TBP, and serine 2 phosphorylated Pol II (pPol II) in association with the C/EBPdelta proximal promoter. C/EBPdelta promoter-associated histone post-translational modification analysis revealed histone H3 and H4 acetylation and methylation patterns consistent with a constitutively "open" chromatin conformation. Chromatin remodeling experiments demonstrated that BRG1, the ATPase component of the SWI/SNF chromatin remodeling complex, is required for C/EBPdelta transcription. Finally, C/EBPdelta expression is repressed in proliferating mammary epithelial cells by c-Myc via a mechanism that involves the binding of c-Myc:Max dimers to C/EBPdelta promoter-bound Miz-1. These results provide a molecular model of C/EBPdelta transcriptional regulation under G(0) growth arrest conditions.

MOM1 mediates DNA‐methylation‐independent silencing of repetitive sequences in Arabidopsis

The heterochromatic regions around centromeres of animal and plant chromosomes are composed of tandem repetitive sequences, interspersed with transposons and transposon derivatives. These sequences are largely transcriptionally silent and highly methylated, and are associated with specifically modified histones. Although embedded in heterochromatin, Arabidopsis 5S ribosomal RNA genes are among the most highly transcribed genes. However, some 5S genes are silenced, and we show here that this silencing can be suppressed by a reduction in CG methylation. Importantly, we show that mutation of MORPHEUS' MOLECULE 1 (MOM1) releases 5S repeat silencing independently of chromatin properties, as illustrated by the absence of detectable alteration of DNA and histone H3 methylation patterns. MOM1 also prevents transcription of 180-bp satellite repeats and 106B dispersed repeats but not of transposons. Our results provide evidence that transcription of densely methylated and highly repetitive heterochromatic sequences is controlled by two distinct epigenetic silencing pathways, one dependent on and the other independent of DNA methylation.

Mapping Global Histone Methylation Patterns in the Coding Regions of Human Genes

Histone methylation patterns in the human genome, especially in euchromatin regions, have not been systematically characterized. In this study, we examined the profile of histone H3 methylation (Me) patterns at different lysines (Ks) in the coding regions of human genes by genome-wide location analyses by using chromatin immunoprecipitation linked to cDNA arrays. Specifically, we compared H3-KMe marks known to be associated with active gene expression, namely, H3-K4Me, H3-K36Me, and H3-K79Me, as well as those associated with gene repression, namely, H3-K9Me, H3-K27Me, and H4-K20Me. We further compared these to histone lysine acetylation (H3-K9/14Ac). Our results demonstrated that: first, close correlations are present between active histone marks except between H3-K36Me2 and H3-K4Me2. Notably, histone H3-K79Me2 is closely associated with H3-K4Me2 and H3-K36Me2 in the coding regions. Second, close correlations are present between histone marks associated with gene silencing such as H3-K9Me3, H3-K27Me2, and H4-K20Me2. Third, a poor correlation is observed between euchromatin marks (H3-K9/K14Ac, H3-K4Me2, H3-K36Me2, and H3-K79Me2) and heterochromatin marks (H3-K9Me2, H3-K9Me3, H3-K27Me2, and H4-K20Me2). Fourth, H3-K9Me2 is neither associated with active nor repressive histone methylations. Finally, histone H3-K4Me2, H3-K4Me3, H3-K36Me2, and H3-K79Me2 are associated with hyperacetylation and active genes, whereas H3-K9Me2, H3-K9Me3, H3-K27Me2, and H4-K20Me2 are associated with hypoacetylation. These data provide novel new information regarding histone KMe distribution patterns in the coding regions of human genes.

Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin.

In mammals and plants, formation of heterochromatin is associated with hypermethylation of DNA at CpG sites and histone H3 methylation at lysine 9. Previous studies have revealed that maintenance of DNA methylation in Neurospora and Arabidopsis requires histone H3 methylation. A feedback loop from DNA methylation to histone methylation, however, is less understood. Its recent examination in Arabidopsis with a partial loss of function in DNA methyltransferase 1 (responsible for maintenance of CpG methylation) yielded conflicting results. Here we report that complete removal of CpG methylation in an Arabidopsis mutant null for DNA maintenance methyltransferase results in a clear loss of histone H3 methylation at lysine 9 in heterochromatin and also at heterochromatic loci that remain transcriptionally silent. Surprisingly, these dramatic alterations are not reflected in heterochromatin relaxation.

Dependence of heterochromatic histone H3 methylation patterns on the Arabidopsis gene DDM1.

The Arabidopsis gene DDM1 is required to maintain DNA methylation levels and is responsible for transposon and transgene silencing. However, rather than encoding a DNA methyltransferase, DDM1 has similarity to the SWI/SNF family of adenosine triphosphate-dependent chromatin remodeling genes, suggesting an indirect role in DNA methylation. Here we show that DDM1 is also required to maintain histone H3 methylation patterns. In wild-type heterochromatin, transposons and silent genes are associated with histone H3 methylated at lysine 9, whereas known genes are preferentially associated with methylated lysine 4. In ddm1 heterochromatin, DNA methylation is lost, and methylation of lysine 9 is largely replaced by methylation of lysine 4. Because DNA methylation has recently been shown to depend on histone H3 lysine 9 methylation, our results suggest that transposon methylation may be guided by histone H3 methylation in plant genomes. This would account for the epigenetic inheritance of hypomethylated DNA once histone H3 methylation patterns are altered.

Germline X Chromosomes Exhibit Contrasting Patterns of Histone H3 Methylation in Caenorhabditis elegans

In mammals, one of the two somatic X chromosomes in the female is inactivated, thereby equalizing X chromosome-derived transcription in the two sexes, a process known as dosage compensation. In the germline, however, the situation is quite different. Both X chromosomes are transcriptionally active during female oogenesis, whereas the X and Y chromosomes are transcriptionally silent during male spermatogenesis. Previous studies suggest that Caenorhabditis elegans germline X chromosomes might have different transcriptional activity in the two sexes in a manner similar to that in mammals. Using antibodies specific to H3 methylated at either lysine 4 or lysine 9, we show that the pattern of site-specific H3 methylation is different between X chromosomes and autosomes as well as between germline X chromosomes from the two sexes in C. elegans. We show that the pachytene germline X chromosomes in both sexes lack Me(K4)H3 when compared with autosomes, consistent with their being transcriptionally inactive. This transcriptional inactivity of germline X chromosomes is apparently transient in hermaphrodites because both X chromosomes stain brightly for Me(K4)H3 after germ nuclei exit pachytene. The male single X chromosome, on the other hand, remains devoid of Me(K4)H3 staining throughout the germline. Instead, the male germline X chromosome exhibits a high level of Me(K9)H3 that is not detected on any other chromosomes in either sex, consistent with stable silencing of this chromosome. Using mutants defective in the sex determination pathway, we show that X-chromosomal Me(K9)H3 staining is determined by the sexual phenotype, and not karyotype, of the animal. We detect a similar high level of Me(K9)H3 in male mouse XY bodies, suggesting an evolutionarily conserved mechanism for silencing the X chromosome specifically in the male germline.