Active Histone Methylation Research Articles

The STAT4/MLL1 Epigenetic Axis Regulates the Antimicrobial Functions of Murine Macrophages

Macrophages are critical immune cells for the clearance of microbial pathogens and cellular debris from peripheral tissues. Macrophage inflammatory responses are governed by gene expression patterns, and these patterns are often subject to epigenetic control. Chromatin modifications, such as histone methylation, regulate gene accessibility in macrophages, and macrophage polarization is governed in part by the expression and function of chromatin-modifying enzymes. The histone methyltransferase mixed-lineage leukemia 1 (MLL1) preferentially modifies lysine residue 4 on the unstructured protein tail of histone H3. MLL1 expression and function have been shown to be governed by signal transduction pathways that are activated by inflammatory stimuli, such as NF-κB. Therefore, we sought to investigate the role of MLL1 in mediating macrophage inflammatory responses. Bone marrow-derived macrophages from mice with a targeted MLL1 gene knockout (Lys2-Cre+/- MLL1fx/fx) exhibited decreased proinflammatory gene expression with concurrent decreases in activating histone methylation. However, MLL1-deficient macrophages also exhibited increased phagocytic and bacterial killing activity in vitro. RNA profiling of MLL1-knockout macrophages identified numerous genes involved with inflammatory responses whose expression was altered in response to TLR ligands or proinflammatory cytokines, including STAT4. STAT4-dependent cytokines, such as type I IFNs were able to drive MLL1 expression in macrophages, and MLL1-knockout macrophages exhibited decreased activating histone methylation in the STAT4 promoter. These results implicate an important role for MLL1-dependent epigenetic regulation of macrophage antimicrobial functions.

Multi-dimensional histone methylations for coordinated regulation of gene expression under hypoxia.

Hypoxia increases both active and repressive histone methylation levels via decreased activity of histone demethylases. However, how such increases coordinately regulate induction or repression of hypoxia-responsive genes is largely unknown. Here, we profiled active and repressive histone tri-methylations (H3K4me3, H3K9me3, and H3K27me3) and analyzed gene expression profiles in human adipocyte-derived stem cells under hypoxia. We identified differentially expressed genes (DEGs) and differentially methylated genes (DMGs) by hypoxia and clustered the DEGs and DMGs into four major groups. We found that each group of DEGs was predominantly associated with alterations in only one type among the three histone tri-methylations. Moreover, the four groups of DEGs were associated with different TFs and localization patterns of their predominant types of H3K4me3, H3K9me3 and H3K27me3. Our results suggest that the association of altered gene expression with prominent single-type histone tri-methylations characterized by different localization patterns and with different sets of TFs contributes to regulation of particular sets of genes, which can serve as a model for coordinated epigenetic regulation of gene expression under hypoxia.

Abstract 4052: Enhancing estrogen receptor beta mediated tumor suppression by lysine specific demethylase 1A inhibitor for the treatment of ovarian cancer

Abstract Ovarian cancer (OCa) is the deadliest of all gynecologic cancers in the United States. Although patients with OCa respond initially to standard combinations of surgical and cytotoxic therapy, nearly 90% will develop recurrence and inevitably succumb to chemotherapy-resistant disease. Estrogen mediates its effects through the estrogen receptor α (ESR1) and estrogen receptor β (ESR2). Recent studies suggest that OCa cells and OCa stem cells express ESR2, which functions as a tumor suppressor and identified synthetic compound LY500307 as a potent ESR2 specific agonist. However, ESR2 expression decreases during tumor progression and under the selection pressure of chemotherapy; this decrease occurs via epigenetic mechanisms. The lysine-specific histone demethylase 1A (KDM1A/LSD1) is overexpressed in OCa and plays a vital role in metastasis and cancer stemness. Our group recently developed a novel KDM1A-specific inhibitor (NCD-38) based on an innovative concept of direct delivery of phenylcyclopropylamine to the KDM1A active site. We reason that agents that restore the expression and functions of ESR2 by increasing active histone marks may provide a novel therapeutic opportunity to reactivate ESR2 for suppression of OCa. The objective of this study is to test whether KDM1A inhibitor could enhance the expression and tumor suppressive functions of ESR2 and to test the combination of KDM1A inhibitor along with ESR2 agonist using in vitro and in vivo preclinical models. Our results demonstrated that combination of ESR2 agonist and KDM1A inhibitor reduces the cell viability of OCa cells synergistically. Further caspase-3/7 activity and Annexin V assays showed synergistic induction of apoptosis upon combination therapy. Matrigel invasion assays revealed reduction in cell invasion of OCa cell lines following ESR2 agonist and KDM1A inhibitor treatment. RT-qPCR analysis demonstrated that KDM1A inhibitor treatment significantly increases the expression of ESR2 and its target genes leading to increased tumor suppression. Further, co-immunoprecipitation analysis revealed that KDM1A interacts with ESR2 and Chromatin immunoprecipitation assays demonstrated that KDM1A inhibition increases the recruitment of active histone methylation mark H3K4-me2 at ESR2 0N promoter. RNA-sequencing analysis demonstrated that ESR2 agonist treatment modulates the several pathways related to cell cycle, apoptosis and DNA damage check point regulation. Finally, ESR2 agonist and KDM1A inhibitor treatment significantly reduced the in vivo tumor growth in orthotopic models. Since ESR2 agonists and KDM1A inhibitors are currently in clinical trials for other clinical indications and are well tolerated, identification of KDM1A inhibitor and ESR2 agonist combination therapy as a novel therapeutic can be readily transferred to clinical use for enhancing survival in OCa patients. Citation Format: Gangadhara Reddy R. Sareddy, Jinyou Liu, Lauren Garcia, Suryavathi Viswanadhapalli, Mei Zhou, Takayoshi Suzuki, Edward Kost, Ratna K. Vadlamudi. Enhancing estrogen receptor beta mediated tumor suppression by lysine specific demethylase 1A inhibitor for the treatment of ovarian cancer [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 4052. doi:10.1158/1538-7445.AM2017-4052

KDM5A controls bone morphogenic protein 2-induced osteogenic differentiation of bone mesenchymal stem cells during osteoporosis.

Bone morphogenetic protein 2 (BMP2) has been used to induce bone regeneration by promoting osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSCs). However, its effect is attenuated in osteoporotic conditions by unknown mechanisms. In this study, we investigated the molecular mechanisms of reduced osteogenic effect of BMP2 in osteoporotic conditions. By interrogating the microarray data from osteoporosis patients, we revealed an upregulation of the epigenetic modifying protein lysine (K)-specific demethylase 5A (KDM5A) and decreased Runt-related transcription factor 2 (RUNX2) expression. Further studies were focused on the role of KDM5A in osteoporosis. We first established ovariectomized (OVX) mouse model and found that the BMP2-induced osteogenic differentiation of osteoporotic MSCs was impaired. The elevated level of KDM5A was confirmed in osteoporotic MSCs. Overexpression of KDM5A in normal MSCs inhibited BMP2-induced osteogenesis. Moreover, osteogenic differentiation of osteoporotic MSCs was restored by specific KDM5A short hairpin RNA or inhibitor. Furthermore, by chromatin immunoprecipitation assay we demonstrated that KDM5A functions as endogenous modulator of osteogenic differentiation by decreasing H3K4me3 levels on promoters of Runx2, depend on its histone methylation activity. More importantly, we found an inhibitory role of KDM5A in regulating bone formation in osteoporotic mice, and pretreatment with KDM5A inhibitor partly rescued the bone loss during osteoporosis. Our results show, for the first time, that KDM5A-mediated H3K4me3 modification participated in the etiology of osteoporosis and may provide new strategies to improve the clinical efficacy of BMP2 in osteoporotic conditions.

Smyd3 open & closed lock mechanism for substrate recruitment: The hinge motion of C-terminal domain inferred from μ-second molecular dynamics simulations

BackgroundThe human lysine methyltransferase Smyd3, a member of the SET and MYND domain containing protein family, harbors methylation activity on both histone and non-histone targets in a tightly regulated manner. The mechanism of how Smyd3 dynamically regulates substrate recognition is still not fully unveiled. MethodsHere, we employed molecular dynamics simulations on full length human Smyd3, performed to a total of 1.2μ-second, in the presence (holo) and absence (apo) of the S-Adenosyl methionine (AdoMet) cofactor. The dynamical features of Smyd3 in apo and holo states have been examined and compared via examining geometrical and electrostatic properties. ResultsThe results show a distinct dynamics of the C-terminal domain (CTD) in the two states. In the apo state, the CTD undergoes a large hinge like motion and samples more opened configurations, thus acting like a loosened clamp and resulting in expanded substrate binding crevice. In the holo state, the CTD exhibits a restricted motion while the overall structure remains compact, mimicking a closed clamp. This leads to a localized increase in the negative potential at the substrate binding cleft. Further, solvent accessibility of critical residues at the target lysine access channel, important for methylation activity, is increased. ConclusionsWe postulate that AdoMet cofactor acts like a key and locks Smyd3 in a closed conformation. In effect, the cofactor binding restricts the elasticity of the CTD, presenting a compact substrate binding cleft with high negative potential, which may have implications on substrate recruitment via long range electrostatics. General significanceThe deletion of the CTD from Smyd3 has been shown to abolish the basal histone methylation activity. Our study highlights the importance of the CTD elasticity in shaping the substrate binding site for recognition and supports the previously proposed role of the CTD in stabilizing the active site for methylation activity.

Bacterial Short Chain Fatty Acids Push All The Buttons Needed To Reactivate Latent Viruses.

The genomes of herpesviruses and HIV become silent during latency through multiple chromatin silencing mechanisms including: histone deacetylation, repressive histone methylation, and DNA methylation. Reactivation of the latent virus requires removal of the chromatin silencing marks and their replacement by activating modifications such as histone acetylation and activating histone methylation. In a complementary mechanism, RNA Polymerase II (RNAP II) elongation is regulated by the positive transcription elongation factor b (P-TEFb)-dependent phosphorylation of Ser2 residues on its C-terminal domain. In resting T-cells latently infected by HIV, expression of P-TEFb is restricted. We found that a group of short chain fatty acids (SCFAs) produced by oral bacteria not only promote histone acetylation but also change the histone methylation dynamics by decreasing repressive histone methylation while increasing activating histone methylation. SCFAs also block DNA methylation and activate P-TEFb to enable elongation of stalled RNA polymerase II. Thus these molecules do not simply act as histone deacetylase (HDAC) inhibitors as previously claimed. Instead, they impact multiple complementary epigenetic regulatory mechanisms to promote highly efficient reactivation of latent viruses.

Evidence for intermediate mesoderm and kidney progenitor cell specification by Pax2 and PTIP dependent mechanisms

Activation of the Pax2 gene marks the intermediate mesoderm shortly after gastrulation, as the mesoderm becomes compartmentalized into paraxial, intermediate, and lateral plate. Using an EGFP knock-in allele of Pax2 to identify and sort cells of the intermediate mesodermal lineage, we compared gene expression patterns in EGFP positive cells that were heterozygous or homozygous null for Pax2. Thus, we identified critical regulators of intermediate mesoderm and kidney development whose expression depended on Pax2 function. In cell culture models, Pax2 is thought to recruit epigenetic modifying complex to imprint activating histone methylation marks through interactions with the adaptor protein PTIP. In kidney organ culture, conditional PTIP deletion showed that many Pax2 target genes, which were activated early in renal progenitor cells, remained on once activated, whereas Pax2 target genes expressed later in kidney development were unable to be fully activated without PTIP. In Pax2 mutants, we also identified a set of genes whose expression was up-regulated in EGFP positive cells and whose expression was consistent with a cell fate transformation to paraxial mesoderm and its derivatives. These data provide evidence that Pax2 specifies the intermediate mesoderm and renal epithelial cells through epigenetic mechanisms and in part by repressing paraxial mesodermal fate.

The Role of Histone Demethylases in the Transcription Regulation of HOX Genes in PML-RARa+ AML Patients

Homeobox genes (HOX) encode transcription factors that are frequently deregulated in leukemias. Our previous findings described that HOX gene expression differs among genetically characterized subtypes of pediatric AML with PML-RARa+ patients having the lowest overall HOX gene expression. We observed that HOX gene expression positively correlated with expression of histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX and negatively with DNA methyltransferase DNMT3b. Interestingly, it has been shown that JMJD3 is a direct target of PML-RARa protein (Martens, JH et al, 2010, Cancer Cell). These findings led us to postulate the hypothesis that reduced levels of HOX genes in PML-RARa+ AML can be caused by the suppressed expression of histone demethylases, such as JMJD3 and UTX, resulting in increased H3K27 methylation and transcription inhibition.We chose PML-RARa+ NB4 cell line to study the role of PML-RARa fusion gene in the regulation of HOX gene expression. To inhibit the effect of PML-RARa we used all-trans retinoic acid (ATRA; 1 uM, 10 uM) which was described to release the block caused by this fusion protein. Expression of particular HOX genes (e.g., HOXA1, HOXA3, HOXA5, HOXA7) together with that of JMJD3 and UTX assessed by qPCR was significantly elevated after ATRA treatment, while gene expression of DNMT3b was decreased.To test whether the reduction in HOX gene expression is directly related to the levels of JMJD3 and UTX, we cultured NB4 cells with a specific inhibitor of these histone demethylases, GSK-J4 (1 uM, 10 uM), in combination with ATRA. This co-treatment led to inhibition of JMJD3 and UTX proteins, followed by significant reduction of HOX genes expression (e.g., HOXA1, HOXA3, HOXA5, HOXA7). This result supports our hypothesis that HOX genes expression is directly related to JMJD3/UTX activity.To determine the effect of ATRA and GSK-J4 on histone marks we have isolated histones by acid extraction and detected the levels of histones by western blot in NB4 ATRA or GSK-J4/ATRA treated cells. We observed that the level of repressive histone methylation mark (trimethylated H3K27; H3K27me3) was decreased after ATRA treatment (activation of JMJD3/UTX) and increased after GSK-J4/ATRA co-treatment (inhibition of JMJD3/UTX). The opposite effect was observed in active histone methylation marks where di- and tri-methylated H3K4 (H3K4me2, H3K4me3) increased after ATRA treatment and decreased after GSK-J4/ATRA co-treatment. H3K9 dimethylated (another repressive histone methylation mark) levels did not change.Next, to investigate the histone code directly in particular HOX genes regions we performed chromatin immunoprecipitation (ChIP) assays. We studied the presence of H3K27me3 and H3K4me2 in 5´UTR genomic region of particular HOX genes (HOXA1, HOXA2, HOXA3, HOXA5, HOXA7) in cells treated with ATRA alone or in the combination with GSK-J4. Preliminary results showed reduction in repressive marks (H3K27me3) upon ATRA treatment, whereas addition of GSK-J4 prevented this decrease. Accordingly, we observed that ATRA/GSK-J4 co-treatment reduced active histone mark H3K4me2.To evaluate the role of DNA methylation in observed expression changes after ATRA treatment we performed bisulfite sequencing of particular promoter sites of HOX genes (e.g., HOXA7, HOXA5). Although we detected decreased DNMT3b gene expression after ATRA treatment there was no change in DNA methylation of CpGs in studied regions.Our results demonstrate that changes in chromatin activity correspond with changes in HOX gene expression. Moreover, ChIP data show direct binding of the modified histones and HOX 5´UTR sites. Our data implicate histone demethylases in regulation of HOX gene expression in PML-RARa+ leukemic blasts. DNA methylation in these particular HOX genes is not involved in the regulation. Elucidating the mechanism of regulation of HOX genes expression can help to understand their role in the leukemogenic process.Supported by GACR P304/12/2214 and GAUK 568213. DisclosuresNo relevant conflicts of interest to declare.

DNA polymerase α interacts with PrSet7 and mediates H4K20 monomethylation in Drosophila.

In human cells, appropriate monomethylation of histone H4 lysine 20 by PrSet7 (also known as SET8 and SETD7) is important for the correct transcription of specific genes and timely progression through the cell cycle. Over-methylation appears to be prevented through the interaction of PrSet7 with proliferating cell nuclear antigen (PCNA), which targets PrSet7 for destruction through the pathway mediated by CRL4(C) (dt2) (the cullin ring finger ligase-4 complex containing Cdt2). However, the factors involved in positive regulation of PrSet7 histone methylation remain undefined. Here, we present biochemical and genetic evidence for a previously undocumented interaction between Drosophila PrSet7 (dPrSet7) and DNA polymerase α in Drosophila. Depletion of the polymerase reduces H4K20 monomethylation suggesting that it is required for dPrSet7 histone methylation activity. We also show that the interaction between PCNA and PrSet7 is conserved in Drosophila, but is only detectable in chromatin fractions. Consistent with this, S2 cells show a significant loss of chromatin-bound dPrSet7 protein as S phase progresses. Based on these data we suggest that interaction with the DNA polymerase represents an important route for stimulation of PrSet7 histone methylase activity that is mediated by allowing loading of dPrSet7 onto chromatin or its subsequent activation.

Heated spermatozoa: effects on embryonic development and epigenetics

Sperm chromatin is highly condensed and relatively resistant to chemical and physical treatments. The purpose of this study was to explore the highest temperature that sperm can tolerate and still produce live offspring. Mouse sperm were heated in a water bath at 50, 65, 80 or 95°C for 30 min before they were microinjected into mouse oocytes. Fertilization, embryo development and 1-cell embryo karyotypes were evaluated. Epigenetic reprogramming including DNA methylation and histone H3K4-trimethylation were evaluated by immunofluorescent staining. The ability of mouse sperm to activate the egg after ICSI was heat sensitive; only 20% of eggs were activated by sperm that had been heated to 50°C and none was activated by sperm heated to 80°C. However, if eggs were activated artificially, mouse sperm subjected to 80°C for 30 min were able to produce live offspring, while 95°C treatment disabled sperm decondensation after ICSI. Once the heat-treated sperm nucleus had developed into a pronucleus, sperm chromatin was able to undergo normal active DNA demethylation and histone methylation. Aberrant chromosome rates increased from 16.3 to 100% when the temperature was raised from 50 to 95°C. Heat treatment destroys integrity of sperm chromatin in a temperature-dependent manner. Eighty degree Celsius was the highest temperature that mouse sperm could withstand and still produce live offspring.

Human Th1 and Th17 Cells Exhibit Epigenetic Stability at Signature Cytokine and Transcription Factor Loci

The linear model of Th cell lineage commitment is being revised due to reports that mature Th cells can trans-differentiate into alternate lineages. This ability of Th cells to reprogram is thought to be regulated by epigenetic mechanisms that control expression of transcription factors characteristic of opposing lineages. It is unclear, however, to what extent this new model of Th cell plasticity holds true in human Th cell subsets that develop under physiological conditions in vivo. We isolated in vivo-differentiated human Th1 and Th17 cells, as well as intermediate Th1/17 cells, and identified distinct epigenetic signatures at cytokine (IFNG and IL17A) and transcription factor (TBX21, RORC, and RORA) loci. We also examined the phenotypic and epigenetic stability of human Th17 cells exposed to Th1-polarizing conditions and found that although they could upregulate TBX21 and IFN-γ, this occurred without loss of IL-17 or RORC expression, and resulted in cells with a Th1/17 phenotype. Similarly, Th1 cells could upregulate IL-17 upon enforced expression of RORC2, but did not lose expression of IFN-γ or TBX21. Despite alterations in expression of these signature genes, epigenetic modifications were remarkably stable aside from the acquisition of active histone methylation marks at cytokine gene promoters. The limited capacity of human Th17 and Th1 cells to undergo complete lineage conversion suggests that the bipotent Th1/17 cells may arise from Th1 and/or Th17 cells. These data also question the broad applicability of the new model of Th cell lineage plasticity to in vivo-polarized human Th cell subsets.

Methylation and acetylation of histones during spermatogenesis

Spermatogenesis is a highly complex and unique differentiation process. This process involves development of spermatogonia into spermatocytes, formation of haploid spermatids, and maturation of spermatozoa. It features stage- and testis-specific gene expression, mitotic and meiotic divisions, and the histone-protamine transition. The epigenetic modification plays an important role in meiotic recombination, formation of the synaptonemal complex, sister chromatid cohesion, spermiogenesis during postmeiotic stages, gene expression repression, and heterochromatin formation. The mark of the repressive and/or activating histone methylation and acetylation has a defined composition. It not only ensures proper chromosome pairing and successful bivalent segregation but also mediates highly orchestrated expression of meiosis-specific genes. The incorrect histone methylation and/or acetylation during spermatogenesis will directly affect the establishment and maintenance of epigenetic patterns, resulting in abnormal spermatogenic cells and even male infertility. This article is an effort to review the dynamic changes of methylation and acetylation of histones during spermatogenesis, as well as the regulatory mechanism of the enzymes involved in these processes, which provides some basic information for further study of the epigenetic events during spermatogenesis and the prevention of male infertility.

Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa

In higher eukaryotes, histone methylation is involved in maintaining cellular identity during somatic development. As most nucleosomes are replaced by protamines during spermatogenesis, it is unclear whether histone modifications function in paternal transmission of epigenetic information. Here we show that two modifications important for Trithorax- and Polycomb-mediated gene regulation have methylation-specific distributions at regulatory regions in human spermatozoa. Histone H3 Lys4 dimethylation (H3K4me2) marks genes that are relevant in spermatogenesis and cellular homeostasis. In contrast, histone H3 Lys27 trimethylation (H3K27me3) marks developmental regulators in sperm, as in somatic cells. However, nucleosomes are only moderately retained at regulatory regions in human sperm. Nonetheless, genes with extensive H3K27me3 coverage around transcriptional start sites in particular tend not to be expressed during male and female gametogenesis or in preimplantation embryos. Promoters of orthologous genes are similarly modified in mouse spermatozoa. These data are compatible with a role for Polycomb in repressing somatic determinants across generations, potentially in a variegating manner.

Abstract 4613: Targeting ER-KDM1 axis to sensitize therapy resistant breast cancer cells to hormonal therapy

Abstract Estrogen-induced breast carcinogenesis is characterized by global changes in histone modifications. KDM1 (also known as LSD1) a histone demethylase enzyme, plays a key role in establishing specific histone methylation marks at ERα target gene promoters. Emerging evidence suggests histone methylation imposes ligand dependency; and deregulation of this epigenetic pathway potentially contributes to hormonal independence and adaptive resistance in breast cancer. Our study examined the therapeutic efficacy of pargyline, a KDM1 inhibiting drug, and evaluated the therapeutic benefit to curb growth of therapy resistant breast cancer cells. We used several model cell lines shown to exhibit resistance to hormonal therapy including MCF7-HER2, MCF7-Tam, MCF7-Ca-LTLT, MCF7-PELP1 and parental MCF7 cells as controls. Reporter gene assays showed KDM1 enhanced ERα- mediated transcription in therapy resistant cells. KDM1 functionally interacts with ERα coregulator PELP1 and is co-recruited with PELP1 to ERα target genes. Pargyline treatment substantially inhibited ERα transactivation functions. ChIP analysis revealed distinct activating histone methylation modifications at growth regulatory ERα target genes in aggressively growing and therapy resistant breast cancer cells. Breast cancer models treated with pargyline facilitated reversal of the observed specific modifications and thereby inhibited the growth of breast cancer cells in vitro and in vivo nude mice models. Pargyline also showed significant effect in blocking local estrogen synthesis via alterations in histone methylation modifications at the aromatase promoter. Combinational therapy using three agents that: (a) block ERα's nuclear actions (tamoxifen or letrozole), (b) ERα's extranuclear actions (dasatinib) and (c) ERα's epigenetic modifications (pargyline) showed most promising therapeutic effect compared to single agent therapy on the growth of therapy resistant cells. Our results suggest that histone methylation modifications play a role in therapy resistance and validate the therapeutic potential of pargyline in combinational therapies. Collectively, our results suggests targeting KDM1 axis with pargyline in combination with current endocrine therapies will have better therapeutic effect and may inhibit or delay development of hormonal resistance. This study is funded by Komen grant KG090447. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4613.

Gene Regulation and Epigenetic Remodeling in Murine Embryonic Stem Cells by c-Myc

BackgroundThe Myc oncoprotein, a transcriptional regulator involved in the etiology of many different tumor types, has been demonstrated to play an important role in the functions of embryonic stem (ES) cells. Nonetheless, it is still unclear as to whether Myc has unique target and functions in ES cells.Methodology/Principal FindingsTo elucidate the role of c-Myc in murine ES cells, we mapped its genomic binding sites by chromatin-immunoprecipitation combined with DNA microarrays (ChIP-chip). In addition to previously identified targets we identified genes involved in pluripotency, early development, and chromatin modification/structure that are bound and regulated by c-Myc in murine ES cells. Myc also binds and regulates loci previously identified as Polycomb (PcG) targets, including genes that contain bivalent chromatin domains. To determine whether c-Myc influences the epigenetic state of Myc-bound genes, we assessed the patterns of trimethylation of histone H3-K4 and H3-K27 in mES cells containing normal, increased, and reduced levels of c-Myc. Our analysis reveals widespread and surprisingly diverse changes in repressive and activating histone methylation marks both proximal and distal to Myc binding sites. Furthermore, analysis of bulk chromatin from phenotypically normal c-myc null E7 embryos demonstrates a 70–80% decrease in H3-K4me3, with little change in H3-K27me3, compared to wild-type embryos indicating that Myc is required to maintain normal levels of histone methylation.Conclusions/SignificanceWe show that Myc induces widespread and diverse changes in histone methylation in ES cells. We postulate that these changes are indirect effects of Myc mediated by its regulation of target genes involved in chromatin remodeling. We further show that a subset of PcG-bound genes with bivalent histone methylation patterns are bound and regulated in response to altered c-Myc levels. Our data indicate that in mES cells c-Myc binds, regulates, and influences the histone modification patterns of genes involved in chromatin remodeling, pluripotency, and differentiation.

Analyzing DNA Replication Timing in the Human Genome

The time of replication in the human genome is of interest due to its interaction with chromatin structure, gene density, and origin activity. We developed a method to profile this timing using DNA tiling microarrays. Areas of the genome were categorized to replicate in early S‐phase, mid S‐phase, late S‐phase, or to exhibit inter‐allelic variation in replication timing. Further, a continuous profile of replication timing was reconstructed for areas that did not exhibit inter‐allelic variation. The robustsness of the computational algorithm was tested on simulated positive control data and random negative control data. Early replicating regions showed strong positive correlation with activating histone methylation and acetylation marks and high gene density. Late replicating areas displayed strong positive correlation with a repressive histone marker, and low gene density. Interestingly, areas exhibiting inter‐allelic variation in replication timing showed positive correlations with both repressive and activating histone marks, consistent with the hypothesis that they represent areas where the two alleles are in different epigenetic environments. This work was supported by funding from the NIH.

Enhancer requirement for histone methylation linked with gene activation

Enhancers cause a high level of transcription and activation of chromatin structure at target genes. Hyperacetylation of histones H3 and H4, a mark of active chromatin, is established broadly across target loci by enhancers that function over long distances. In the present study, we studied the role of an enhancer in methylation of various lysine residues on H3 by comparing a model gene locus having an active enhancer with one in which the enhancer has been inactivated within the context of minichromosomes. The intact enhancer affected histone methylation at K4, K9 and K36 in distinct ways depending on the methylation level and the location in the locus. All three lysine residues were highly tri-methylated in the coding region of the gene linked to the active enhancer but not the inactive enhancer. However di-methylation of K9 and K36 was not affected by the enhancer. The enhancer region itself was marked by mono-methylation at K4 and K9, distinguishing it from the methyl marks in the gene coding region. These results indicate that an enhancer has roles in establishing active histone methylation patterns linked with gene transcription rather than removing methylation linked with gene inactivation.

EVI1 Is a Transcriptional Repressor Able To Recruit SUV39H1 Histone Methylation Activity.

The Evi1 (Ecotropic viral integration site 1) gene is located on chromosome 3q26 and encodes a highly conserved nuclear protein that belongs to the Kruppel family of transcription factors, containing two Cys2-His2 repeat zinc finger motifs domains. EVI1 is not expressed in normal hematopoietic tissues, but can be found overexpressed in acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) cells as a consequence of chromosome 3 rearrangements. In vitro and in vivo studies have shown that a number of biological properties can be attributed to the EVI1 protein that might contribute to leukemogenesis: deregulation of cell proliferation, inhibition of TGF-ß signaling pathway, and suppression of stress induced apoptosis. We and others reported that EVI1 acts as a transcriptional repressor through its interaction with a subset of co-repressor proteins. More recently, we showed that EVI1 directly interacts with RUNX1 and GATA1 transcription factors. In both cases this interaction alters the ability of the transcription factors to bind their recognition sites on the DNA, leading to the deregulation of their target genes and to the impairment of the normal hematopoietic development. Interestingly, EVI1-mediated transcriptional silencing in association with HDAC proteins can be only partially rescued by HDAC inhibitors, suggesting that additional chromosomal modification might occur for gene silencing. Methylation by histone methyltransferase proteins (HMTs) is one type of these modifications, and when it occurs on certain residues can lead both to repression or activation of gene expression. The SUV39H1 (Suppressor of Variegation 3–9 Homolog 1) gene encodes a histone 3 specific methyltransferase. SUV39H1 selectively methylates lysine 9 of the amino terminus of the histone 3. Tri-methylation of histone H3 is important for recruiting heterochromatin protein 1 (HP1), thereby regulating gene expression, chromatin packaging and heterochromatin formation. Here we investigate the hypothesis that histone methylation may contribute to the transcriptional repressive potential of EVI1. We found that EVI1 physically interacts with the histone methyltransferase SUV39H1. The interaction between the two proteins requires the N-terminus of EVI1 where the proximal zinc finger domain is located; in particular, out of the seven motifs that compose this domain, the distal three motifs seem to be responsible for the binding between the two proteins. By pull-down assay with in vitro translated EVI1 this interaction appears to be direct. Futhermore, using in vitro histone methylation assay we show that EVI1 forms a complex with SUV39H1 that is able to methylate a recombinant H3, suggesting that the EVI1-SUV39H1 interaction does not affect the histone methyltransferase activity of SUV39H1. We suggest here that EVI1 interacts with SUV39H1 and that this binding could have a role in the epigenetic remodellig of the chromatin. According to this model, it is possible that EVI1 recruits histone methylation activity to selected genomic loci to silence tumor suppressor genes and repress differentiation factors. Because lysine methylation by SUV39H1 cannot take place on an already acetylated lysine, deacetylation activity associated with EVI1, through its interaction with HDACs, becomes a necessary preceeding step to SUV39H1 mediated histone methylation.

Phosphorylation-mediated inactivation of coactivator-associated arginine methyltransferase 1

Multiple protein arginine methyltransferases are involved in transcriptional activation of nuclear receptors. Coactivator-associated arginine methyltransferase 1 (CARM1)-mediated histone methylation has been shown to activate nuclear receptor-dependent transcription; however, little is known about the regulation of its enzymatic activity. Here, we report that the methyltransferase activity of CARM1 is negatively regulated through phosphorylation at a conserved serine residue. When the serine residue is mutated to glutamic acid, which mimics the phosphorylated serine residue, the mutant CARM1 exhibits diminished ability to bind the methyl donor adenosylmethionine and diminished histone methylation activity. Moreover, such mutation leads to the inhibition of CARM1 transactivation of estrogen receptor-dependent transcription. Our results provide an example for the regulation of protein arginine methyltransferase activity by phosphorylation. As CARM1 is a potent transcriptional coactivator of estrogen receptor, our results suggest that phosphorylation of CARM1 serves as a unique mechanism for inactivating CARM1-regulated estrogen-dependent gene expression.

Histone methylation controls telomerase-independent telomere lengthening in cells undergoing dedifferentiation

Cellular dedifferentiation underlies topical issues in biology such as regeneration and nuclear cloning and has common features in plants and animals. In plants, this process characterizes the transition of differentiated leaf cells to protoplasts (plant cells devoid of cell walls) and is accompanied by global chromatin reorganization associated with reprogramming of gene expression. A screen for mutants defective in proliferation and callus formation identified kyp-2—a mutant in the KRYPTONITE ( KYP)/SUVH4 gene encoding a histone H3 lysine 9 (H3K9) methyltransferase. Analysis of telomere length revealed stochastic telomerase-independent lengthening of telomeres in wild type but not in kyp-2 protoplasts. In kyp-2 mutant, telomeric repeats were no longer associated with dimethylated H3K9. The Arabidopsis telomerase reverse transcriptase ( tert) mutant displayed accelerated proliferation despite its short telomeres, though it also showed accelerated cell death. Microarray analysis uncovered several components of the ubiquitin proteolytic system, which are downregulated in kyp-2 compared to wild-type protoplasts. Thus, our results suggest that histone methylation activity is required for the establishment/maintenance of the dedifferentiated state and/or reentry into the cell cycle, at least partly, through activation of genes whose products are involved in the ubiquitin proteolytic pathway. In addition, our results illuminate the complexity of cellular dedifferentiation, particularly the occurrence of DNA recombination that can lead to genome instability.