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  • H3K9 methyltransferase G9a negatively regulates UHRF1 transcription during leukemia cell differentiation. 25765655

    Histone H3K9 methyltransferase (HMTase) G9a-mediated transcriptional repression is a major epigenetic silencing mechanism. UHRF1 (ubiquitin-like with PHD and ring finger domains 1) binds to hemimethylated DNA and plays an essential role in the maintenance of DNA methylation. Here, we provide evidence that UHRF1 is transcriptionally downregulated by H3K9 HMTase G9a. We found that increased expression of G9a along with transcription factor YY1 specifically represses UHRF1 transcription during TPA-mediated leukemia cell differentiation. Using ChIP analysis, we found that UHRF1 was among the transcriptionally silenced genes during leukemia cell differentiation. Using a DNA methylation profiling array, we discovered that the UHRF1 promoter was hypomethylated in samples from leukemia patients, further supporting its overexpression and oncogenic activity. Finally, we showed that G9a regulates UHRF1-mediated H3K23 ubiquitination and proper DNA replication maintenance. Therefore, we propose that H3K9 HMTase G9a is a specific epigenetic regulator of UHRF1.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • DNA methyltransferase 1 knockdown activates a replication stress checkpoint. 17015478

    DNA methyltransferase 1 (DNMT1) is an important component of the epigenetic machinery and is responsible for copying DNA methylation patterns during cell division. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming. Knockdown of DNMT1 leads to inhibition of DNA replication, but the mechanism has been unclear. Here we show that depletion of DNMT1 with either antisense or small interfering RNA (siRNA) specific to DNMT1 activates a cascade of genotoxic stress checkpoint proteins, resulting in phosphorylation of checkpoint kinases 1 and 2 (Chk1 and -2), gammaH2AX focus formation, and cell division control protein 25a (CDC25a) degradation, in an ataxia telangiectasia mutated-Rad3-related (ATR)-dependent manner. siRNA knockdown of ATR blocks the response to DNMT1 depletion; DNA synthesis continues in the absence of DNMT1, resulting in global hypomethylation. Similarly, the response to DNMT1 knockdown is significantly attenuated in human mutant ATR fibroblast cells from a Seckel syndrome patient. This response is sensitive to DNMT1 depletion, independent of the catalytic domain of DNMT1, as indicated by abolition of the response with ectopic expression of either DNMT1 or DNMT1 with the catalytic domain deleted. There is no response to short-term treatment with 5-aza-deoxycytidine (5-aza-CdR), which causes demethylation by trapping DNMT1 in 5-aza-CdR-containing DNA but does not cause disappearance of DNMT1 from the nucleus. Our data are consistent with the hypothesis that removal of DNMT1 from replication forks is the trigger for this response.
    Document Type:
    Reference
    Product Catalog Number:
    05-636
    Product Catalog Name:
    Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301

  • A methyltransferase required for proper timing of the vernalization response in Arabidopsis. 25605879

    Prolonged exposure to winter cold enables flowering in many plant species through a process called vernalization. In Arabidopsis, vernalization results from the epigenetic silencing of the floral repressor flowering locus C (FLC) via a Polycomb Repressive Complex 2 (PRC2)-mediated increase in the density of the epigenetic silencing mark H3K27me3 at FLC chromatin. During cold exposure, a gene encoding a unique, cold-specific PRC2 component, vernalization insensitive 3 (VIN3), which is necessary for PRC2-mediated silencing of FLC, is induced. Here we show that set domain group 7 (SDG7) is required for proper timing of VIN3 induction and of the vernalization process. Loss of SDG7 results in a vernalization-hypersensitive phenotype, as well as more rapid cold-mediated up-regulation of VIN3. In the absence of cold, loss of SDG7 results in elevated levels of long noncoding RNAs, which are thought to participate in epigenetic repression of FLC. Furthermore, loss of SDG7 results in increased H3K27me3 deposition on FLC chromatin in the absence of cold exposure and enhanced H3K27me3 spreading during cold treatment. Thus, SDG7 is a negative regulator of vernalization, and loss of SDG7 creates a partially vernalized state without cold exposure.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • DNA methyltransferase 1 knock down induces gene expression by a mechanism independent of DNA methylation and histone deacetylation. 15087453

    DNA methyltransferase 1 (DNMT1) catalyzes the post-replication methylation of DNA and is responsible for maintaining the DNA methylation pattern during cell division. A long list of data supports a role for DNMT1 in cellular transformation and inhibitors of DNMT1 were shown to have antitumorigenic effects. It was long believed that DNMT1 promoted tumorigenesis by maintaining the hypermethylated and silenced state of tumor suppressor genes. We have previously shown that DNMT1 knock down by either antisense oligonucleotides directed at DNMT1 or expressed antisense activates a number of genes involved in stress response and cell cycle arrest by a DNA methylation-independent mechanism. In this report we demonstrate that antisense knock down of DNMT1 in human lung carcinoma A549 and embryonal kidney HEK293 cells induces gene expression by a mechanism that does not involve either of the known epigenomic mechanisms, DNA methylation, histone acetylation, or histone methylation. The mechanism of activation of the cell cycle inhibitor p21 and apoptosis inducer BIK by DNMT1 inhibition is independent of the mechanism of activation of the same genes by histone deacetylase inhibition. We determine whether DNMT1 knock down activates one of the nodal transcription activation pathways in the cell and demonstrate that DNMT1 activates Sp1 response elements. This activation of Sp1 response does not involve an increase in either Sp1 or Sp3 protein levels in the cell or the occupancy of the Sp1 elements with these proteins. The methylation-independent regulation of Sp1 elements by DNMT1 unravels a novel function for DNMT1 in gene regulation. DNA methylation was believed to be a mechanism for suppression of CG-rich Sp1-bearing promoters. Our data suggest a fundamentally different and surprising role for DNMT1 regulation of CG-rich genes by a mechanism independent of DNA methylation and histone acetylation. The implications of our data on the biological roles of DNMT1 and the therapeutic potential of DNMT1 inhibitors as anticancer agents are discussed.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • DNA methyltransferase 1 may be a therapy target for attenuating diabetic nephropathy and podocyte injury. 28318634

    The contribution of DNA methylation to diabetic nephropathy, especially the effect on podocyte integrity, is not clarified. Here we found that albuminuria in a db/db mouse model was markedly attenuated after treatment with a DNA methylation inhibitor. This was accompanied by alleviation of glomerular hypertrophy, mesangial matrix expansion, and podocyte injury. The expression of DNA methyltransferase 1 (Dnmt1), nuclear factor Sp1, and nuclear factor kappa B (NFκB)-p65 markedly increased in podocytes in vivo and in vitro under the diabetic state. The increased expression of Dnmt1 was attenuated after treatment with 5-azacytidine or 5-aza-2'-deoxycytidine or Dnmt1 knockdown, accompanied by restored decreased podocyte slit diaphragm proteins resulting from hypermethylation and improved podocyte motility. Further studies found that increased Sp1 and NFκB-p65 interacted in the nucleus of podocytes incubated with high glucose, and Sp1 bound to the Dnmt1 promoter region. The involvement of the Sp1/NFκB-p65 complex in Dnmt1 regulation was confirmed by the observation that Sp1 knockdown using mithramycin A or siRNA decreased Dnmt1 protein levels. The luciferase reporter assay further indicated that Dnmt1 was a direct target of Sp1. Thus, inhibition of DNA methylation may be a new therapeutic avenue for treating diabetic nephropathy. Hence, the Sp1/NFκB p65-Dnmt1 pathway may be exploited as a therapeutic target for protecting against podocyte injury in diabetic nephropathy.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™

  • DNA methyltransferase 1-associated protein (DMAP1) is a co-repressor that stimulates DNA methylation globally and locally at sites of double strand break repair. 20864525

    Correction of double strand DNA breaks proceeds in an error-free pathway of homologous recombination (HR), which can result in gene silencing of half of the DNA molecules caused by action by DNA methyltransferase 1 (DNMT1) (Cuozzo, C., Porcellini, A., Angrisano, T., Morano, A., Lee, B., Di Pardo, A., Messina, S., Iuliano, R., Fusco, A., Santillo, M. R., Muller, M. T., Chiariotti, L., Gottesman, M. E., and Avvedimento, E. V. (2007) PLoS Genet. 3, e110). To explore the mechanism that leads to HR-induced silencing, a genetic screen was carried out based on the silencing of a GFP reporter to identify potential partners. DMAP1, a DNMT1 interacting protein, was identified as a mediator of this process. DMAP1 is a potent activator of DNMT1 methylation in vitro, suggesting that DMAP1 is a co-repressor that supports the maintenance and de novo action of DNMT1. To examine critical roles for DMAP1 in vivo, lentiviral shRNA was used to conditionally reduce cellular DMAP1 levels. The shRNA transduced cells grew poorly and eventually ceased their growth. Analysis of the tumor suppressor gene p16 methylation status revealed a clear reduction in methylated CpGs in the shRNA cells, suggesting that reactivation of a tumor suppressor gene pathway caused the slow growth phenotype. Analysis of HR, using a fluorescence-based reporter, revealed that knocking down DMAP1 also caused hypomethylation of the DNA repair products following gene conversion. DMAP1 was selectively enriched in recombinant GFP chromatin based on chromatin immunoprecipitation analysis. The picture that emerges is that DMAP1 activates DNMT1 preferentially at sites of HR repair. Because DMAP1 depleted cells display enhanced HR, we conclude that it has additional roles in genomic stability.
    Document Type:
    Reference
    Product Catalog Number:
    05-714
    Product Catalog Name:
    Anti-Lamin A/C Antibody, clone 14

  • The methyltransferase Setdb2 mediates virus-induced susceptibility to bacterial superinfection. 25419628

    Immune responses are tightly regulated to ensure efficient pathogen clearance while avoiding tissue damage. Here we report that Setdb2 was the only protein lysine methyltransferase induced during infection with influenza virus. Setdb2 expression depended on signaling via type I interferons, and Setdb2 repressed expression of the gene encoding the neutrophil attractant CXCL1 and other genes that are targets of the transcription factor NF-κB. This coincided with occupancy by Setdb2 at the Cxcl1 promoter, which in the absence of Setdb2 displayed diminished trimethylation of histone H3 Lys9 (H3K9me3). Mice with a hypomorphic gene-trap construct of Setdb2 exhibited increased infiltration of neutrophils during sterile lung inflammation and were less sensitive to bacterial superinfection after infection with influenza virus. This suggested that a Setdb2-mediated regulatory crosstalk between the type I interferons and NF-κB pathways represents an important mechanism for virus-induced susceptibility to bacterial superinfection.
    Document Type:
    Reference
    Product Catalog Number:
    07-352
    Product Catalog Name:
    Anti-acetyl-Histone H3 (Lys9) Antibody

  • Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells. 22284370

    DNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear.Here we show that G9a is not essential for maintenance of DNA methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to nucleosomes containing methylated DNA even in the absence of G9a, ensuring faithful propagation of methylated states in cooperation with DNMT1 through somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B and DNA methylation-independent manner. However, G9a knockdown synergizes with pharmacologic inhibition of DNMTs resulting in increased hypomethylation and inhibition of cell proliferation.Taken together, these data suggest that G9a is not involved in maintenance of DNA methylation in somatic cells but might play a role in re-initiation of de novo methylation after treatment with hypomethylating drugs, thus serving as a potential target for combinatorial treatments strategies involving DNMTs inhibitors.
    Document Type:
    Reference
    Product Catalog Number:
    05-724
    Product Catalog Name:
    Anti-Myc Tag Antibody, clone 4A6

  • DNA methyltransferase DNMT3a contributes to neuropathic pain by repressing Kcna2 in primary afferent neurons. 28270689

    Nerve injury induces changes in gene transcription in dorsal root ganglion (DRG) neurons, which may contribute to nerve injury-induced neuropathic pain. DNA methylation represses gene expression. Here, we report that peripheral nerve injury increases expression of the DNA methyltransferase DNMT3a in the injured DRG neurons via the activation of the transcription factor octamer transcription factor 1. Blocking this increase prevents nerve injury-induced methylation of the voltage-dependent potassium (Kv) channel subunit Kcna2 promoter region and rescues Kcna2 expression in the injured DRG and attenuates neuropathic pain. Conversely, in the absence of nerve injury, mimicking this increase reduces the Kcna2 promoter activity, diminishes Kcna2 expression, decreases Kv current, increases excitability in DRG neurons and leads to spinal cord central sensitization and neuropathic pain symptoms. These findings suggest that DNMT3a may contribute to neuropathic pain by repressing Kcna2 expression in the DRG.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™

  • DNA methyltransferase 3b preferentially associates with condensed chromatin. 20923784

    In mammals, DNA methylation is catalyzed by DNA methyltransferases (DNMTs) encoded by Dnmt1, Dnmt3a and Dnmt3b. Since, the mechanisms of regulation of Dnmts are still largely unknown, the physical interaction between Dnmt3b and chromatin was investigated in vivo and in vitro. In embryonic stem cell nuclei, Dnmt3b preferentially associated with histone H1-containing heterochromatin without any significant enrichment of silent-specific histone methylation. Recombinant Dnmt3b preferentially associated with nucleosomal DNA rather than naked DNA. Incorporation of histone H1 into nucleosomal arrays promoted the association of Dnmt3b with chromatin, whereas histone acetylation reduced Dnmt3b binding in vitro. In addition, Dnmt3b associated with histone deacetylase SirT1 in the nuclease resistant chromatin. These findings suggest that Dnmt3b is preferentially recruited into hypoacetylated and condensed chromatin. We propose that Dnmt3b is a 'reader' of higher-order chromatin structure leading to gene silencing through DNA methylation.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple