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Background Frequent mutations in the nuclear receptor binding SET domain protein 1 (NSD1) gene have been observed in head and neck squamous cell carcinomas (HNSCC). NSD1 encodes a histone 3 lysine-36 methyltransferase. NSD1 mutations are correlated with improved clinical outcomes and increased sensitivity to platinum-based chemotherapy agents in human papillomavirus-negative (HPV-) tumors, despite weak T-cell infiltration. However, the role of NSD1 and related family members NSD2 and NSD3 in human papillomavirus-positive (HPV+) HNSCC is unclear. Methods Using data from over 500 HNSCC patients from The Cancer Genome Atlas (TCGA), we compared the relative level of mRNA expression of NSD1, NSD2, and NSD3 in HPV+ and HPV- HNSCC. Correlation analyses were performed between T-cell infiltration and the relative level of expression of NSD1, NSD2, and NSD3 mRNA in HPV+ and HPV- HNSCC. In addition, overall survival outcomes were compared for both the HPV+ and HPV- subsets of patients based on stratification by NSD1, NSD2, and NSD3 expression levels. Results Expression levels of NSD1, NSD2 or NSD3 were not correlated with altered lymphocyte infiltration in HPV+ HNSCC. More importantly, low expression of NSD1, NSD2, or NSD3 correlated with significantly reduced overall patient survival in HPV+, but not HPV- HNSCC. Conclusion These results starkly illustrate the contrast in molecular features between HPV+ and HPV- HNSCC tumors and suggest that NSD1, NSD2, and NSD3 expression levels should be further investigated as novel clinical metrics for improved prognostication and patient stratification in HPV+ HNSCC.

Background SETD8 is the sole methyltransferase capable of mono-methylating histone H4, lysine 20. SETD8 and H4K20me1 play a role in a number of essential biologic processes, including cell cycle progression, establishment of higher order chromatin structure, and transcriptional regulation. SETD8 is highly expressed in erythroid cells and erythroid deletion of Setd8 is embryonic lethal by embryonic day 11.5 (E11.5) due to profound anemia, suggesting that it has an erythroid-specific function. The function of SETD8 in the hemopoietic system is poorly understood. The goal of our study was to gain insights into the function of SETD8 during erythroid differentiation. Results We performed ATAC-seq (assay for transposase-accessible chromatin) on sorted populations of E10.5 Setd8 mutant and control erythroblasts. Accessibility profiles were integrated with expression changes and a mark of heterochromatin (H3K27me3) performed in wild-type E10.5 erythroblasts to further understand the role of SETD8 in erythropoiesis. Data integration identified regions of greater chromatin accessibility in Setd8 mutant cells that co-located with H3K27me3 in wild-type E10.5 erythroblasts suggesting that these regions, and their associated genes, are repressed during normal erythropoiesis. The majority of these more accessible regions were located in promoters and they frequently co-located with the NFY complex. Pathway analysis of genes identified through data integration revealed stemness-related pathways. Among those genes were multiple transcriptional regulators active in multipotent progenitors, but repressed during erythroid differentiation including Hhex, Hlx, and Gata2. Consistent with a role for SETD8 in erythroid specification, SETD8 expression is up-regulated upon erythroid commitment, and Setd8 disruption impairs erythroid colony forming ability. Conclusion Taken together, our results suggest that SETD8 is an important regulator of the chromatin landscape during erythroid differentiation, particularly at promoters. Our results also identify a novel role for Setd8 in the establishment of appropriate patterns of lineage-restricted gene expression during erythroid differentiation.

BackgroundEuchromatic histone-lysine-N-methyltransferases 1 and 2 (EHMT1/2, aka GLP/G9A) catalyze dimethylation of histone H3 lysine 9 (H3K9me2) and have roles in epigenetic silencing of gene expression. EHMT1/2 also have direct roles in DNA repair and are implicated in chemoresistance in several cancers. Resistance to chemotherapy and PARP inhibitors (PARPi) is a major cause of mortality in high-grade serous ovarian carcinoma (HGSOC), but the contribution of the epigenetic landscape is unknown.ResultsTo identify epigenetic mechanisms of PARPi resistance in HGSOC, we utilized unbiased exploratory techniques, including RNA-Seq and mass spectrometry profiling of histone modifications. Compared to sensitive cells, PARPi-resistant HGSOC cells display a global increase of H3K9me2 accompanied by overexpression of EHMT1/2. EHMT1/2 overexpression was also observed in a PARPi-resistant in vivo patient-derived xenograft (PDX) model. Genetic or pharmacologic disruption of EHMT1/2 sensitizes HGSOC cells to PARPi. Cell death assays demonstrate that EHMT1/2 disruption does not increase PARPi-induced apoptosis. Functional DNA repair assays show that disruption of EHMT1/2 ablates homologous recombination (HR) and non-homologous end joining (NHEJ), while immunofluorescent staining of phosphorylated histone H2AX shows large increases in DNA damage. Propidium iodide staining and flow cytometry analysis of cell cycle show that PARPi treatment increases the proportion of PARPi-resistant cells in S and G2 phases, while cells treated with an EHMT1/2 inhibitor remain in G1. Co-treatment with PARPi and EHMT1/2 inhibitor produces an intermediate phenotype. Immunoblot of cell cycle regulators shows that combined EHMT1/2 and PARP inhibition reduces expression of specific cyclins and phosphorylation of mitotic markers. These data suggest DNA damage and altered cell cycle regulation as mechanisms of sensitization. RNA-Seq of PARPi-resistant cells treated with EHMT1/2 inhibitor showed significant gene expression changes enriched in pro-survival pathways that remain unexplored in the context of PARPi resistance, including PI3K, AKT, and mTOR.ConclusionsThis study demonstrates that disrupting EHMT1/2 sensitizes HGSOC cells to PARPi, and suggests a potential mechanism through DNA damage and cell cycle dysregulation. RNA-Seq identifies several unexplored pathways that may alter PARPi resistance. Further study of EHMT1/2 and regulated genes will facilitate development of novel therapeutic strategies to successfully treat HGSOC.

Background Wolf-Hirschhorn syndrome candidate gene-1 (WHSC1), a histone methyltransferase, has been found to be upregulated and its expression to be correlated with expression of enhancer of zeste homolog 2 (EZH2) in several cancers. In this study, we evaluated the role of WHSC1 and its therapeutic significance in ovarian clear cell carcinoma (OCCC). Methods First, we analyzed WHSC1 expression by quantitative PCR and immunohistochemistry using 23 clinical OCCC specimens. Second, the involvement of WHSC1 in OCCC cell proliferation was evaluated by MTT assays after siRNA-mediated WHSC1 knockdown. We also performed flow cytometry (FACS) to address the effect of WHSC1 on cell cycle. To examine the functional relationship between EZH2 and WHSC1, we knocked down EZH2 using siRNAs and checked the expression levels of WHSC1 and its histone mark H3K36m2 in OCCC cell lines. Finally, we checked WHSC1 expression after treatment with the selective inhibitor, GSK126. Results Both quantitative PCR and immunohistochemical analysis revealed that WHSC1 was significantly overexpressed in OCCC tissues compared with that in normal ovarian tissues. MTT assay revealed that knockdown of WHSC1 suppressed cell proliferation, and H3K36me2 levels were found to be decreased in immunoblotting. FACS revealed that WHSC1 knockdown affected the cell cycle. We also confirmed that WHSC1 expression was suppressed by EZH2 knockdown or inhibition, indicating that EZH2 is upstream of WHSC1 in OCCC cells. Conclusions WHSC1 overexpression induced cell growth and its expression is, at least in part, regulated by EZH2. Further functional analysis will reveal whether WHSC1 is a promising therapeutic target for OCCC. Electronic supplementary material The online version of this article (10.1186/s12885-019-5638-9) contains supplementary material, which is available to authorized users.

Background Rewriting of the epigenome has risen as a promising alternative to gene editing for precision medicine. In nature, epigenetic silencing can result in complete attenuation of target gene expression over multiple mitotic divisions. However, persistent repression has been difficult to achieve in a predictable manner using targeted systems. Results Here, we report that persistent epigenetic memory required both a DNA methyltransferase (DNMT3A-dCas9) and a histone methyltransferase (Ezh2-dCas9 or KRAB-dCas9). We demonstrate that the histone methyltransferase requirement can be locus specific. Co-targeting Ezh2-dCas9, but not KRAB-dCas9, with DNMT3A-dCas9 and DNMT3L induced long-term HER2 repression over at least 50 days (approximately 57 cell divisions) and triggered an epigenetic switch to a heterochromatic environment. An increase in H3K27 trimethylation and DNA methylation was stably maintained and accompanied by a sustained loss of H3K27 acetylation. Interestingly, substitution of Ezh2-dCas9 with KRAB-dCas9 enabled long-term repression at some target genes (e.g., SNURF) but not at HER2, at which H3K9me3 and DNA methylation were transiently acquired and subsequently lost. Off-target DNA hypermethylation occurred at many individual CpG sites but rarely at multiple CpGs in a single promoter, consistent with no detectable effect on transcription at the off-target loci tested. Conversely, robust hypermethylation was observed at HER2. We further demonstrated that Ezh2-dCas9 required full-length DNMT3L for maximal activity and that co-targeting DNMT3L was sufficient for persistent repression by Ezh2-dCas9 or KRAB-dCas9. Conclusions These data demonstrate that targeting different combinations of histone and DNA methyltransferases is required to achieve maximal repression at different loci. Fine-tuning of targeting tools is a necessity to engineer epigenetic memory at any given locus in any given cell type. Electronic supplementary material The online version of this article (10.1186/s13072-019-0275-8) contains supplementary material, which is available to authorized users.

Background Glioblastoma multiforme (GBM) is the common primary brain tumor classified the most malignant glioma. Long non-coding RNAs (LncRNAs) are important epigenetic regulators with critical roles in cancer initiation and progression. LncRNA HOTAIRM1 transcribes from the antisense strand of HOXA gene cluster which locus in chromosome 7p15.2. Recent studies have shown that HOTAIRM1 is involved in acute myeloid leukemia and colorectal cancer. Here we sought to investigate the role of HOTAIRM1 in GBM and explore its mechanisms of action. Methods The expressions of HOTAIRM1 and HOXA1 in GBM tissues and cells were determined by qRT-PCR, and the association between HOTAIRM1, HOXA1 transcription and tumor grade were analyzed. The biological function of HOTAIRM1 in GBM was evaluated both in vitro and in vivo. Chromatin immunoprecipitation (ChIP) assay and quantitative Sequenom MassARRAY methylation analysis were performed to explore whether HOTAIRM1 could regulate histone and DNA modification status of the HOXA1 gene transcription start sites (TSS) and activate its transcription. ChIP and RNA-ChIP were further performed to determine the molecular mechanism of HOTAIRM1 in epigenetic regulation of the HOXA1 gene. Results HOTAIRM1 was abnormally up-regulated in GBM tissues and cells, and this up-regulation was correlated with grade malignancy in glioma patients. HOTAIRM1 silencing caused tumor suppressive effects via inhibiting cell proliferation, migration and invasion, and inducing cell apoptosis. In vivo experiments showed knockdown of HOTAIRM1 lessened the tumor growth. Additionally, HOTAIRM1 action as regulating the expression of the HOXA1 gene. HOXA1, as an oncogene, it’s expression levels were markedly elevated in GBM tissues and cell lines. Mechanistically, HOTAIRM1 mediated demethylation of histone H3K9 and H3K27 and reduced DNA methylation levels by sequester epigenetic modifiers G9a and EZH2, which are H3K9me2 and H3K27me3 specific histone methyltransferases, and DNA methyltransferases (DnmTs) away from the TSS of HOXA1 gene. Conclusions We investigated the potential role of HOTAIRM1 to promote GBM cell proliferation, migration, invasion and inhibit cell apoptosis by epigenetic regulation of HOXA1 gene that can be targeted simultaneously to effectively treat GBM, thus putting forward a promising strategy for GBM treatment. Meanwhile, this finding provides an example of transcriptional control over the chromatin state of gene and may help explain the role of lncRNAs within the HOXA gene cluster. Electronic supplementary material The online version of this article (10.1186/s13046-018-0941-x) contains supplementary material, which is available to authorized users.

Background While the genomes of eukaryotes and Archaea both encode the histone-fold domain, only eukaryotes encode the core histone paralogs H2A, H2B, H3, and H4. With DNA, these core histones assemble into the nucleosomal octamer underlying eukaryotic chromatin. Importantly, core histones for H2A and H3 are maintained as neofunctionalized paralogs adapted for general bulk chromatin (canonical H2 and H3) or specialized chromatin (H2A.Z enriched at gene promoters and cenH3s enriched at centromeres). In this context, the identification of core histone-like “doublets” in the cytoplasmic replication factories of the Marseilleviridae (MV) is a novel finding with possible relevance to understanding the origin of eukaryotic chromatin. Here, we analyze and compare the core histone doublet genes from all known MV genomes as well as other MV genes relevant to the origin of the eukaryotic replisome. Results Using different phylogenetic approaches, we show that MV histone domains encode obligate H2B-H2A and H4-H3 dimers of possible proto-eukaryotic origin. MV core histone moieties form sister clades to each of the four eukaryotic clades of canonical and variant core histones. This suggests that MV core histone moieties diverged prior to eukaryotic neofunctionalizations associated with paired linear chromosomes and variant histone octamer assembly. We also show that MV genomes encode a proto-eukaryotic DNA topoisomerase II enzyme that forms a sister clade to eukaryotes. This is a relevant finding given that DNA topo II influences histone deposition and chromatin compaction and is the second most abundant nuclear protein after histones. Conclusions The combined domain architecture and phylogenomic analyses presented here suggest that a primitive origin for MV histone genes is a more parsimonious explanation than horizontal gene transfers + gene fusions + sufficient divergence to eliminate relatedness to eukaryotic neofunctionalizations within the H2A and H3 clades without loss of relatedness to each of the four core histone clades. We thus suggest MV histone doublet genes and their DNA topo II gene possibly were acquired from an organism with a chromatinized replisome that diverged prior to the origin of eukaryotic core histone variants for H2/H2A.Z and H3/cenH3. These results also imply that core histones were utilized ancestrally in viral DNA compaction and/or protection from host endonucleases. Electronic supplementary material The online version of this article (10.1186/s13072-017-0162-0) contains supplementary material, which is available to authorized users.

Background Gametogenesis in mammals entails profound re-patterning of the epigenome. In the female germline, DNA methylation is acquired late in oogenesis from an essentially unmethylated baseline and is established largely as a consequence of transcription events. Molecular and functional studies have shown that imprinted genes become methylated at different times during oocyte growth; however, little is known about the kinetics of methylation gain genome wide and the reasons for asynchrony in methylation at imprinted loci. Results Given the predominant role of transcription, we sought to investigate whether transcription timing is rate limiting for de novo methylation and determines the asynchrony of methylation events. Therefore, we generated genome-wide methylation and transcriptome maps of size-selected, growing oocytes to capture the onset and progression of methylation. We find that most sequence elements, including most classes of transposable elements, acquire methylation at similar rates overall. However, methylation of CpG islands (CGIs) is delayed compared with the genome average and there are reproducible differences amongst CGIs in onset of methylation. Although more highly transcribed genes acquire methylation earlier, the major transitions in the oocyte transcriptome occur well before the de novo methylation phase, indicating that transcription is generally not rate limiting in conferring permissiveness to DNA methylation. Instead, CGI methylation timing negatively correlates with enrichment for histone 3 lysine 4 (H3K4) methylation and dependence on the H3K4 demethylases KDM1A and KDM1B, implicating chromatin remodelling as a major determinant of methylation timing. We also identified differential enrichment of transcription factor binding motifs in CGIs acquiring methylation early or late in oocyte growth. By combining these parameters into multiple regression models, we were able to account for about a fifth of the variation in methylation timing of CGIs. Finally, we show that establishment of non-CpG methylation, which is prevalent in fully grown oocytes, and methylation over non-transcribed regions, are later events in oogenesis. Conclusions These results do not support a major role for transcriptional transitions in the time of onset of DNA methylation in the oocyte, but suggest a model in which sequences least dependent on chromatin remodelling are the earliest to become permissive for methylation. Electronic supplementary material The online version of this article (doi:10.1186/s13072-017-0133-5) contains supplementary material, which is available to authorized users.

SETD8/SET8/Pr-SET7/KMT5A is the only known lysine methyltransferase (KMT) that monomethylates lysine 20 of histone H4 (H4K20) in vivo. Lysine residues of non-histone proteins including proliferating cell nuclear antigen (PCNA) and p53 are also monomethylated. As a consequence, the methyltransferase activity of the enzyme is implicated in many essential cellular processes including DNA replication, DNA damage response, transcription modulation, and cell cycle regulation. This review aims to provide an overview of the roles of SETD8 in physiological and pathological pathways and to discuss the progress made to date in inhibiting the activity of SETD8 by small molecules, with an emphasis on their discovery, selectivity over other methyltransferases and cellular activity.

Histone H1.0 belongs to the class of linker histones (H1), although it is substantially distinct from other histone H1 family members. The differences can be observed in the chromosomal location and organization of the histone H1.0 encoding gene, as well as in the length and composition of its amino acid chain. Whereas somatic (H1.1-H1.5) histone H1 variants are synthesized in the cell cycle S-phase, histone H1.0 is synthesized throughout the cell cycle. By replacing somatic H1 variants during cell maturation, histone H1.0 is gradually deposited in low dividing cells and achieves the highest level of expression in the terminally differentiated cells. Compared to other differentiation-specific H1 histone (H5) characteristic for unique tissue and organisms, the distribution of histone H1.0 remains non-specific. Classic investigations emphasize that histone H1.0 is engaged in the organization of nuclear chromatin accounting for formation and maintenance of its nucleosomal and higher-order structure, and thus influences gene expression. However, the recent data confirmed histone H1.0 peculiar localization in the nucleolus and unexpectedly revealed its potential for regulation of nucleolar, RNA-dependent, activity via interaction with other proteins. According to such findings, histone H1.0 participates in the formation of gene-coded information through its control at both transcriptional and translational levels. In order to reappraise the biological significance of histone H1.0, both aspects of its activity are presented in this review.

Background In human hosts, Entamoeba histolytica cysts can develop into trophozoites, suggesting that the life cycle of this parasite are regulated by changes in gene expression. To date, some evidence has suggested that epigenetic mechanisms such as DNA methylation and histone modification are involved in the regulation of gene expression in Entamoeba. Some post–translational modifications (PTMs) at the N-terminus of E. histolytica’s histones have been reported experimentally, including tri-methylation in the lysine 4 of histone H3 (H3K4me3) and dimethylation in the lysine 27 of histone H3 (H3K27me2), dimethylation of arginine 3 (H4R3me2) and the indirect acetylation of histone H4 in the N-terminal region. However, it is not known which residues of histone H4 are subject to acetylation and/or methylation or where in the nucleus these epigenetic marks are located. Methods Histones from trophozoites of E. histolytica were obtained and analyzed by LC-MS/MS. WB assays were performed using antibodies against epigenetic marks (acetylated lysines and methylated arginines). Immunofluorescence assays (IFA) were carried out to determine the distribution of PTMs and the localization of DNA methylation as a heterochromatin marker. Nuclear bodies such as the nucleolus were identified by using antibodies against fibrillarin and nucleolin and speckles by using anti-PRP6 antibody. Results Some new PTMs in histone H4 of E. histolytica, such as the acetylation of lysines 5, 8, 12 and 16 and the monomethylation of arginine 3, were identified by WB. IFA demonstrated that some marks are associated with transcriptional activity (such as acetylation and/or methylation) and that these marks are distributed throughout the E. histolytica nucleus. Staining with antibodies against anti-pan-acetylated lysine H4 histone and 5-methyl cytosine showed that the activation and transcriptional repression marks converge. Additionally, two nuclear bodies, the nucleolus and speckles, were identified in this parasite. Conclusions This study provides the first evidence that the nucleus of E. histolytica is not compartmentalized and contains two nuclear bodies, the nucleolus and speckles, the latter of which was not identified previously. The challenge is now to understand how these epigenetic marks and nuclear bodies work together to regulate gene expression in E. histolytica. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1298-7) contains supplementary material, which is available to authorized users.

Background In eukaryotes, histone arginine methylation associates with both active and repressed chromatin states depending on the residues involved and the status of methylation. Even when the amino-terminus of Entamoeba histolytica histones diverge from metazoan sequences, these regions contain arginine residues that are potential targets for methylation. However, histone arginine methylation as well as the activity of arginine methyltransferases (PRMTs) has not been studied in this parasite. The aim of this work was to examine the dimethylation of arginine 3 of H4 histone (H4R3me2) and to identify the parasite PRMT that could be responsible for this modification (EhPRMT1). Methods To examine the presence of H4R3me2 in E histolytica, we performed Western blot and immunofluorescence assays on trophozoites using an antibody against this epigenetic mark. To recognize the PRMT1 enzyme of this parasite that possibly perform that modification, we first performed a phylogenetic analysis of E. histolytica and human PRMTs. RT-PCR assays were carried out to analyze the expression of the putative PRMT1 genes. One of these genes was cloned and expressed in Escherichia coli. The recombinant protein was tested by its recognition by an antibody against human PRMT1 and in its ability to form homodimers and to methylate commercial histones. Results The arginine 3 of human H4, which is subjected to post translational methylation, was aligned with the arginine 8 of E. histolytica H4, suggesting that this residue could be methylated. The recognition of an 18 kDa nuclear protein of E. histolytica by an antibody against H4R3me2 confirmed this assumption. We found that this parasite expresses three phylogenetic and structural proteins related to PRMT1. Antibodies against the human PRMT1 detected E. histolytica proteins in cytoplasm and nuclei and recognized a recombinant PRMT1 of this parasite. The recombinant protein was able to form homodimers and homotetramers and displayed methyltransferase activity on arginine 3 of chicken H4. Conclusion All these results suggest that E. histolytica contains as a minimum one structural and functional protein ortholog to PRMT1, enzyme that potentially dimethylates H4R8. This modification may play an important role in the gene expression regulation of this microorganism.

Histone modifications are increasingly being used as biomarkers of cancer prognosis and survival. However, we identified a cleavage product of histone H3 in human peripheral blood mononuclear cells, which interferes with measures of certain H3 modifications. Therefore, the potential for enzymatic cleavage of histones should be considered when measuring histone modifications in human samples. Furthermore, the enzymatic cleavage of human H3 is itself a fascinating area of research and two important questions remain to be answered: 1) Does cleavage of human H3 occur in vivo, as it does in other organisms? and 2) Does it serve a biologically important function?

Background Ovarian cancer (OCa) peritoneal metastasis is the leading cause of cancer–related deaths in women with limited therapeutic options available for treating it and poor prognosis, as the underlying mechanism is not fully understood. Method The clinicopathological correlation of G9a expression was assessed in tumor specimens of ovarian cancer patients. Knockdown or overexpression of G9a in ovarian cancer cell lines was analysed with regard to its effect on adhesion, migration, invasion and anoikis-resistance. In vivo biological functions of G9a were tested by i.p. xenograft ovarian cancer models. Microarray and quantitative RT-PCR were used to analyze G9a-regulated downstream target genes. Results We found that the expression of histone methyltransferase G9a was highly correlated with late stage, high grade, and serous-type OCa. Higher G9a expression predicted a shorter survival in ovarian cancer patients. Furthermore, G9a expression was higher in metastatic lesions compared with their corresponding ovarian primary tumors. Knockdown of G9a expression suppressed prometastatic cellular activities including adhesion, migration, invasion and anoikis-resistance of ovarian cancer cell lines, while G9a over-expression promoted these cellular properties. G9a depletion significantly attenuated the development of ascites and tumor nodules in a peritoneal dissemination model. Importantly, microarray and quantitative RT-PCR analysis revealed that G9a regulates a cohort of tumor suppressor genes including CDH1, DUSP5, SPRY4, and PPP1R15A in ovarian cancer. Expression of these genes was also inversely correlated with G9a expression in OCa specimens. Conclusion We propose that G9a contributes to multiple steps of ovarian cancer metastasis and represents a novel target to combat this deadly disease. Electronic supplementary material The online version of this article (doi:10.1186/1476-4598-13-189) contains supplementary material, which is available to authorized users.

Background Stored, soluble histones in eggs are essential for early development, in particular during the maternally controlled early cell cycles in the absence of transcription. Histone post-translational modifications (PTMs) direct and regulate chromatin-templated transactions, so understanding the nature and function of pre-deposition maternal histones is essential to deciphering mechanisms of regulation of development, chromatin assembly, and transcription. Little is known about histone H2A pre-deposition modifications nor known about the transitions that occur upon the onset of zygotic control of the cell cycle and transcription at the mid-blastula transition (MBT). Results We isolated histones from staged Xenopus laevis oocytes, eggs, embryos, and assembled pronuclei to identify changes in histone H2A modifications prior to deposition and in chromatin. Soluble and chromatin-bound histones from eggs and embryos demonstrated distinct patterns of maternal and zygotic H2A PTMs, with significant pre-deposition quantities of S1ph and R3me1, and R3me2s. We observed the first functional distinction between H2A and H4 S1 phosphorylation, as we showed that H2A and H2A.X-F (also known as H2A.X.3) serine 1 (S1) is phosphorylated concomitant with germinal vesicle breakdown (GVBD) while H4 serine 1 phosphorylation occurs post-MBT. In egg extract H2A/H4 S1 phosphorylation is independent of the cell cycle, chromatin assembly, and DNA replication. H2AS1ph is highly enriched on blastula chromatin during repression of zygotic gene expression while H4S1ph is correlated with the beginning of maternal gene expression and the lengthening of the cell cycle, consistent with distinct biological roles for H2A and H4 S1 phosphorylation. We isolated soluble H2A and H2A.X-F from the egg and chromatin-bound in pronuclei and analyzed them by mass spectrometry analysis to quantitatively determine abundances of S1ph and R3 methylation. We show that H2A and H4 S1ph, R3me1 and R3me2s are enriched on nucleosomes containing both active and repressive histone PTMs in human A549 cells and Xenopus embryos. Conclusions Significantly, we demonstrated that H2A phosphorylation and H4 arginine methylation form a new class of bona fide pre-deposition modifications in the vertebrate embryo. We show that S1ph and R3me containing chromatin domains are not correlated with H3 regulatory PTMs, suggesting a unique role for phosphorylation and arginine methylation.

Materials and methods To analyze the catalytic activity of PHGDH on possible alternative substrates, we conducted in vitro biochemical assays on recombinant human PHGDH. The reaction rate in the reductive direction with various substrates was monitored based on NADH consumption, measured by absorbance at 340 nm. The identity and chirality of products were analyzed by liquid chromatography-mass spectrometry (LC-MS) and by gas chromatography-mass spectrometry (GC-MS) after (R)-2-butanol derivatization. To analyze the intracellular function of PHGDH, we generated stable PHGDH knockdown cell lines, using two shRNA sequences. Metabolite levels in the knockdown cells were analyzed by LC-MS [4], and DNA and histone methylation was analyzed by immunoblotting.

Results Genome-wide analysis of histone modifications documented that adult hCSCs are characterized by a bivalent chromatin configuration similar to that of embryonic stem cells with repressive and activating marks at lysine residues of histones H3 and H4. The high levels of di-(H3K4me2) and tri-methylation (H3K4me3) at lysine 4 of histone H3 prompted us to test the function of histone methyltransferase Smyd3, which is downregulated by the non-functional mutation in the c-kit receptor, in hCSCs. Inhibition of Smyd3 by siRNA strategy decreased the fraction (60%) of cycling hCSCs and increased apoptosis (2.5-fold). Attenuation of hCSC growth was coupled with a decline in the expression of telomerase, hTERT, and the myocyte specific transcription factors hNkx2.5 and hGata6. By immunolabeling, Smyd3 co-localized with hTERT, hNkx2.5 and hGATA6 in hCSC nuclei. Conversely, transfection of hCSCs with a plasmid carrying Smyd3 was coupled with upregulation of hTERT and hNkx2.5. Chromatin immunoprecipitation assay show that Smyd3 binds to hTERT, hGata6 and hNkx2.5 promoter regions that contains the putative binding sites for Smyd3. Moreover, the mutations at the Smyd3 DNA binding sites, decreased the promoter activity of hTERT and hGata6. Smyd3 siRNA treatment decreased the reporter activity of luciferase reporter plasmids containing hTERT.

A serious effort has begun to develop therapies that may be capable of eradicating established HIV infection in man. Because of the biological complexity of HIV infection that persists despite potent antiretroviral therapy, it is widely believed that if such therapies can be developed they will involve complex, multimodality approaches. An inhibitor of histone deacetylase has been demonstrated to disrupt latency in man, and new histone deacetylase inhibitors have been identified. Other potential targets, such as histone methyltransferase, protein kinase C, and BRD4, have been recently studied. Model systems, both in primary cells and in animal models, are beginning to be validated. In the clinic, immune-based therapies to aid in the clearance of persistent infection are also being tested. It is too early to know what combination eradication therapies for HIV infection will look like in the future, but candidate therapies and model systems to perform preclinical validation are beginning to take shape.

Epithelial-mesenchymal transition (EMT) programs require the expression of a variety of so-called master regulators of EMT, including members of the Snail, Zeb, and Twist transcription factor families. Teleologically, the requirement for such a diverse group of 'master regulators' seems evolutionarily cumbersome, and emerging evidence indicates that these transcription factors do in fact mediate unique and specialized functions, suggesting the existence of higher-order 'masters' that truly direct and coordinate EMT programs. Accordingly, Tiwari and colleagues recently delineated an elegant pathway wherein transforming growth factor-beta stimulates Sox4 expression, which induces that of the histone methyltransferase, Ezh2, thereby reprogramming the epigenome to elicit EMT programs and metastasis of breast cancers. This viewpoint highlights Sox4 as a 'new' master of EMT programs and metastatic breast cancer.

Background Chromatin, residing in the nuclei of eukaryotic cells, comprises DNA and histones to make up chromosomes. Chromatin condenses to compact the chromosomes and loosens to facilitate gene transcription and DNA replication/repair. Chemical modifications to the histones mediate changes in chromatin structure. Histone-modifying enzymes are potential drug targets. How herbs affect phenotypes through histone modifications is interesting. Methods Two public traditional Chinese medicine (TCM) databases were accessed to retrieve the chemical constituents and TCM natures of 3,294 TCM medicinals. NCBI taxonomy database was accessed to build the phylogenetic tree of the TCM medicinals. Statistical test was used to test if TCM natures of the medicinals cluster in the phylogenetic tree. A public chemical-protein interaction database was accessed to identify TCM medicinals whose constituent chemicals interact with human histone-modifying enzymes. For each histone modification, a correlation coefficient was calculated between the medicinals’ TCM natures and modification modulabilities. Information of the ingredient medicinals of 200 classical TCM formulas was accessed from a public database. Results It was found that 1,170 or 36% of the 3,294 TCM medicinals interact with human histone-modifying enzymes. Among the histone-modifying medicinals, 56% of them promote chromatin condensation. The cold-hot natures of TCM medicinals were found to be phylogenetically correlated. Furthermore, cold (hot) TCM medicinals were found to be associated with heterochromatinization (euchromatinization) through mainly H3K9 methylation and H3K4 demethylation. The associations were weak yet statistically significant. On the other hand, analysis of TCM formulas, the major form of TCM prescriptions in clinical practice, found that 99% of 200 government approved TCM formulas are histone-modifying. Furthermore, in formula formation, heterochromatic medicinals were found to team up with other heterochromatic medicinals to enhance the heterochromatinization of the formula. The synergy was mainly through concurrent DNMT and HDAC inhibition, co-inhibition of histone acetylation and H3S10 phosphorylation, or co-inhibition of H3K4 demethylation and H3K36 demethylation. Conclusions TCM prescriptions’ modulation of the human epigenome helps elucidation of phyto-pharmacology and discovery of epigenetic drugs. Furthermore, as TCM medicinals’ properties are closely tied to patient TCM syndromes, results of this materia-medica-wide, bioinformatic analysis of TCM medicinals may have implications for molecular differentiation of TCM syndromes.

Background Histone post-translational modifications are critical determinants of chromatin structure and function, impacting multiple biological processes including DNA transcription, replication, and repair. The post-translational acetylation of histone H4 at lysine 16 (H4K16ac) was initially identified in association with dosage compensation of the Drosophila male X chromosome. However, in mammalian cells, H4K16ac is not associated with dosage compensation and the genomic distribution of H4K16ac is not precisely known. Therefore, we have mapped the genome-wide H4K16ac distribution in human cells. Results We performed H4K16ac chromatin immunoprecipitation from human embryonic kidney 293 (HEK293) cells followed by hybridization to whole-genome tiling arrays and identified 25,893 DNA regions (false discovery rate ±1.5-fold) were identified as potential H4K16ac-regulated genes. Seventeen transcription factor-binding sites were significantly associated with H4K16ac occupancy (p

The 300 kb of the Drosophila bithorax complex (BX-C) is the original model for studying Hox gene repression by Polycomb group proteins. Decades of genetic experiments have led to the hypothesis that Polycomb organizes BX-C chromatin differently in each of the abdominal parasegments, and the structure of the chromatin is critical for maintaining cell identity. Studying the molecular organization of chromatin in individual parasegments has been technically difficult. We solved this problem by developing a sorted nuclei ChIP-seq pipeline. In this system, transgenic embryos produce tagged nuclei in single parasegments. Using FACS, we sorted these tagged nuclei and performed small-scale ChIP-seq. Initial results show that a histone mark catalyzed by Polycomb group proteins, trimethylated histone H3 at lysine 27 (H3K27me3), covers less of the BX-C as we move from PS5 to PS7. The boundaries of the H3K27 methylation correlate precisely with previously identified CTCF binding sites, a protein known to act as a chromatin barrier. Correspondingly, a mark that correlates with gene activation, H3K4me3, appears over the transcription start sites of BX-C genes that are not associated with Polycomb histone methyltransferase activity. Going forward, we will assay the localization of Polycomb group proteins and many other chromatin regulatory proteins to gain a complete picture of the chromatin environment that regulates Hox gene expression in the developing embryo. Still, the current results offer a compelling first look at BX-C chromatin in vivo, and provide molecular support to a long-standing genetic hypothesis.

The genetic information in eukaryotic cells is organized in a specific structure called chromatin. The basic unit of chromatin is the nucleosome, which consist of four histone proteins and ~147 bp of DNA [1]. The N-terminal tails of these proteins contain sited for post-translational modifications directly linked to gene expression. The modifications include acetylation, methylation, phosphorylation, ubiquitinylation, sumoylation and ribosylation. Specific enzymes mediate each modification. LSD1 (Lysine Specific Demethylase I) is one of the histone demethylases, which removes one methyl group from mono- or di-methylated lysine residue. It has recently been demonstrated that androgen receptor (AR)-LSD1 complex demethylates a repressive histone mark (H3K9) promoting genes activation [2]. Experimental data show, also, that LSD1 is strongly expressed in prostate cancers [3]. For these reasons, specific modulation of LSD1 might be a promising therapeutic strategy in tissues where AR has a key physiological role. LSD1 is a flavin-dependent amine oxidase, which shares sequence identity with other flavin dependent amine oxidases like monoamine oxidase (MAO), and polyamine oxidase (PAO). After we analyzed structural differences and similarities among these enzymes several docking studies were evaluated using the available crystal structures of LSD1 and the related oxidases to discover novel LSD1 inhibitors. For the evaluation studies we selected different ligand data sets containing known inhibitors of MAO and PAO. The docking setup that showed the highest accuracy and enrichment factors was selected for virtual screening of LSD1 inhibitors. Preliminary biological data were obtained and will be discussed in the context of the target structure.

During the process of transcription elongation, the chromatin structure of transcribed sequences can be perturbed, exposing cryptic promoter-like sequences within the body of transcribed genes to function as initiation sites. Re-establishing a stable repressive structure of open reading frames requires histone chaperones, methyltransferases, deacetylases and chromatin remodeling complexes. The Set2/Rpd3S pathway is used by elongating RNA polymerase II to signal for histone deacetylation in its wake. Set2 associates with the elongating form of RNA polymerase II and co-transcriptionally methylates histone H3K36. H3K36 is recognized by the Rpd3S deacetylase complex to deacetylate histones in transcribed sequences. In recent work, we have found that a major source of cotranscriptional histone acetylation is the incorporation of soluble, Rtt109 H3K56-acetylated histones by the Asf1 histone chaperone. Set2 methylation of H3K36 promotes retention of the original histones and suppresses the incorporation of soluble histones by Asf1. By identifying factors that interact with H3K36 methylated nucleosomes, we have found that chromatin remodeling is also required to stabilize the chromatin structures of open reading frames following transcription elongation. Our studies identified the ATP-dependent chromatin remodelers Isw1 and Chd1 as two factors involved in this pathway as deletion of ISW1 and CHD1 enhanced the cryptic transcript phenotype caused by set2. Moreover, loss of ISW1 and CHD1 also enhanced the incorporation of new histones from the soluble pool into chromatin. Thus, retention of original histones, deacetylation of any new ones and their organization by chromatin remodeling are all required to re-establish stable chromatin resistant to cryptic transcription initiation.

Genes transcribed by RNA polymerase II show a stereotypical pattern of histone H3 methylations. H3K4 trimethylation peaks near the transcription start site, with dimethylation peaking further downstream. H3K36 methylation levels increase with distance from the promoter. It is not yet completely understood how this pattern is generated, but the mechanism involves direct interactions between the transcribing polymerase and the methyltransferases Set1/COMPASS (for H3K4) and Set2 (for H3K36). We are working towards a better understanding of how this coupling works. We are also very interested in the function of these co-transcriptional histone modifications. Previous work has shown that methylated H3K36 recruits the histone deacetylase complex Rpd3S and that H3K4me2 recruits the Set3C histone deacetylase complex. Therefore, a major function of co-transcriptional histone methylation is to reset transcribed chromatin to a state less conducive to nucleosome turnover. This pathway represses cryptic promoters inside the transcribed gene, but also slows transcription elongation through this region. We and others have found that these histone methylation-deacetylation pathways can be targeted via overlapping non-coding transcription to regulate nearby promoters. In contrast to H3K4me2 and H3K36me2/3, H3K4me3 correlates with high turnover nucleosomes near transcription start sites. Available data suggests that H3K4me3 is recognized by three complexes containing ING/Yng proteins via their PHD finger domains. The yeast proteins Yng1 and Yng2 (as well as their metazoan counterparts) are subunits of histone acetyltransferase complexes, suggesting that H3K4me3 can target histone acetylation. Since acetylation is correlated with nucleosome remodeling, this putative pathway could be important for gene activation. Surprisingly, the third Yng protein (Pho23, as well as its metazoan counterparts) is a subunit of the Rpd3L histone deacetylase complex, hinting at a more complex series or cycle of acetylation and deacetylation. We are continuing to probe the functions of these pathways and some of our latest work will be presented.

Early prostate cancer (PCa) is generally treatable and associated with good prognosis. After a variable time, PCa evolves into a highly metastatic and treatment-refractory disease: castration-resistant PCa (CRPC). Currently, few prognostic factors are available to predict the emergence of CRPC, and no curative option is available. Epigenetic gene regulation has been shown to trigger PCa metastasis and androgen-independence. Most epigenetic studies have focused on DNA and histone methyltransferases. While DNA methylation leads to gene silencing, histone methylation can trigger gene activation or inactivation, depending on the target amino acid residues and the extent of methylation (me1, me2, or me3). Interestingly, some histone modifiers are essential for PCa tumor-initiating cell (TIC) self-renewal. TICs are considered the seeds responsible for metastatic spreading and androgen-independence. Histone Lysine Demethylases (KDMs) are a novel class of epigenetic enzymes which can remove both repressive and activating histone marks. KDMs are currently grouped into 7 major classes, each one targeting a specific methylation site. Since their discovery, KDM expression has been found to be deregulated in several neoplasms. In PCa, KDMs may act as either tumor suppressors or oncogenes, depending on their gene regulatory function. For example, KDM1A and KDM4C are essential for PCa androgen-dependent proliferation, while PHF8 is involved in PCa migration and invasion. Interestingly, the possibility of pharmacologically targeting KDMs has been demonstrated. In the present paper, we summarize the emerging role of KDMs in regulating the metastatic potential and androgen-dependence of PCa. In addition, we speculate on the possible interaction between KDMs and other epigenetic effectors relevant for PCa TICs. Finally, we explore the role of KDMs as novel prognostic factors and therapeutic targets. We believe that studies on histone demethylation may add a novel perspective in our efforts to prevent and cure advanced PCa.

Background The SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domain is an evolutionarily conserved sequence of approximately 130-150 amino acids, and constitutes the catalytic site of lysine methyltransferases (KMTs). KMTs perform many crucial biological functions via histone methylation of chromatin. Histone methylation marks are interpreted differently depending on the histone type (i.e. H3 or H4), the lysine position (e.g. H3K4, H3K9, H3K27, H3K36 or H4K20) and the number of added methyl groups (i.e. me1, me2 or me3). For example, H3K4me3 and H3K36me3 are associated with transcriptional activation, but H3K9me2 and H3K27me3 are associated with gene silencing. The substrate specificity and activity of KMTs are determined by sequences within the SET domain and other regions of the protein. Results Here we identified 49 SET-domain proteins from the recently sequenced Brassica rapa genome. We performed sequence similarity and protein domain organization analysis of these proteins, along with the SET-domain proteins from the dicot Arabidopsis thaliana, the monocots Oryza sativa and Brachypodium distachyon, and the green alga Ostreococcus tauri. We showed that plant SET-domain proteins can be grouped into 6 distinct classes, namely KMT1, KMT2, KMT3, KMT6, KMT7 and S-ET. Apart from the S-ET class, which has an interrupted SET domain and may be involved in methylation of nonhistone proteins, the other classes have characteristics of histone methyltransferases exhibiting different substrate specificities: KMT1 for H3K9, KMT2 for H3K4, KMT3 for H3K36, KMT6 for H3K27 and KMT7 also for H3K4. We also propose a coherent and rational nomenclature for plant SET-domain proteins. Comparisons of sequence similarity and synteny of B. rapa and A. thaliana SET-domain proteins revealed recent gene duplication events for some KMTs. Conclusion This study provides the first characterization of the SET-domain KMT proteins of B. rapa. Phylogenetic analysis data allowed the development of a coherent and rational nomenclature of this important family of proteins in plants, as in animals. The results obtained in this study will provide a base for nomenclature of KMTs in other plant species and facilitate the functional characterization of these important epigenetic regulatory genes in Brassica crops.

BackgroundHistone post-translational modifications are critical for gene expression and cell viability. A broad spectrum of histone lysine residues have been identified in yeast that are targeted by a variety of modifying enzymes. However, the regulation and interaction of these enzymes remains relatively uncharacterized. Previously we demonstrated that deletion of either the histone acetyltransferase (HAT)GCN5or the histone deacetylase (HDAC)HDA1exacerbated the temperature sensitive (ts) mutant phenotype of the Anaphase Promoting Complex (APC)apc5CAallele. Here, theapc5CAmutant background is used to study a previously uncharacterized functional antagonistic genetic interaction between Gcn5 and Hda1 that is not detected inAPC5cells.ResultsUsing Northerns, Westerns, reverse transcriptase PCR (rtPCR), chromatin immunoprecipitation (ChIP), and mutant phenotype suppression analysis, we observed that Hda1 and Gcn5 appear to compete for recruitment to promoters. We observed that the presence of Hda1 can partially occlude the binding of Gcn5 to the same promoter. Occlusion of Gcn5 recruitment to these promoters involved Hda1 and Tup1. Using sequential ChIP we show that Hda1 and Tup1 likely form complexes at these promoters, and that complex formation can be increased by deletingGCN5.ConclusionsOur data suggests large Gcn5 and Hda1 containing complexes may compete for space on promoters that utilize the Ssn6/Tup1 repressor complex. We predict that inapc5CAcells the accumulation of an APC target may compensate for the loss of bothGCN5andHDA1.