Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, M, R, Ch||DB, ICC, Mplex, PIA, WB, ChIP-seq||Rb||Purified||Polyclonal Antibody|
|Presentation||Purified rabbit polyclonal IgG in buffer containing 0.07 M Tris-glycine, pH 7.4, 0.105 M NaCl, 0.035% sodium azide and 30% glycerol.|
|Safety Information according to GHS|
|Material Size||100 µg|
Anti-trimethyl-Histone H3 (Lys9) Antibody SDS
|Anti-trimethyl-Histone H3 (Lys9)||2475742|
|Anti-trimethyl-Histone H3 (Lys9) - 2120113||2120113|
|Anti-trimethyl-Histone H3 (Lys9) - 2383148||2383148|
|Anti-trimethyl-Histone H3 (Lys9) - 2441949||2441949|
|Anti-trimethyl-Histone H3 (Lys9) - 0609040589||0609040589|
|Anti-trimethyl-Histone H3 (Lys9) - 1971290||1971290|
|Anti-trimethyl-Histone H3 (Lys9) - 2017310||2017310|
|Anti-trimethyl-Histone H3 (Lys9) - 2043528||2043528|
|Anti-trimethyl-Histone H3 (Lys9) - 2054862||2054862|
|Anti-trimethyl-Histone H3 (Lys9) - 2190636||2190636|
|Reference overview||Application||Species||Pub Med ID|
|DGCR14 induces Il17a gene expression through the RORγ/BAZ1B/RSKS2 complex.|
Molecular and cellular biology 35 344-55 2015
The Dgcr14/Es2 gene is located in a chromosomal region the loss of which has been associated with DiGeorge syndrome, a cause of immunodeficiency, heart defects, and skeletal abnormalities. However, the role of DGCR14 protein remains to be elucidated. Here, I found that DGCR14 protein acts as a coactivator of RORγt in TH17 cells. Biochemical purification of the RORγ coregulator complex allowed me to identify the associated DGCR14 protein by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Overexpression of Dgcr14 mRNA enhanced RORγt-mediated transcriptional activity and facilitated TH17 cell differentiation. Furthermore, knockdown of Dgcr14 reduced Il17a mRNA expression. I also found that DGCR14 associated with ribosomal S6 kinase 2 (RSK2, also called RpS6ka3) and BAZ1B, both of which were recruited to the Il17a promoter during TH17 cell differentiation. Knockdown of Baz1b or RpS6ka3 also reduced Il17a mRNA expression, and Baz1b knockdown increased transcriptional suppressive histone marks (histone H3K9me3) on the Il17a promoter. My findings showed the roles of DGCR14, RSK2, and BAZ1B in the transcriptional regulation of Il17a mRNA during TH17 cell differentiation.
|Epigenetic silencing of Oct4 by a complex containing SUV39H1 and Oct4 pseudogene lncRNA.|
Scarola, M; Comisso, E; Pascolo, R; Chiaradia, R; Maria Marion, R; Schneider, C; Blasco, MA; Schoeftner, S; Benetti, R
Nature communications 6 7631 2015
Pseudogene-derived, long non-coding RNAs (lncRNAs) act as epigenetic regulators of gene expression. Here we present a panel of new mouse Oct4 pseudogenes and demonstrate that the X-linked Oct4 pseudogene Oct4P4 critically impacts mouse embryonic stem cells (mESCs) self-renewal. Sense Oct4P4 transcription produces a spliced, nuclear-restricted lncRNA that is efficiently upregulated during mESC differentiation. Oct4P4 lncRNA forms a complex with the SUV39H1 HMTase to direct the imposition of H3K9me3 and HP1α to the promoter of the ancestral Oct4 gene, located on chromosome 17, leading to gene silencing and reduced mESC self-renewal. Targeting Oct4P4 expression in primary mouse embryonic fibroblasts causes the re-acquisition of self-renewing features of mESC. We demonstrate that Oct4P4 lncRNA plays an important role in inducing and maintaining silencing of the ancestral Oct4 gene in differentiating mESCs. Our data introduces a sense pseudogene-lncRNA-based mechanism of epigenetic gene regulation that controls the cross-talk between pseudogenes and their ancestral genes.
|Dgcr8 and Dicer are essential for sex chromosome integrity during meiosis in males.|
Modzelewski, AJ; Hilz, S; Crate, EA; Schweidenback, CT; Fogarty, EA; Grenier, JK; Freire, R; Cohen, PE; Grimson, A
Journal of cell science 128 2314-27 2015
Small RNAs play crucial roles in regulating gene expression during mammalian meiosis. To investigate the function of microRNAs (miRNAs) and small interfering RNAs (siRNAs) during meiosis in males, we generated germ-cell-specific conditional deletions of Dgcr8 and Dicer in mice. Analysis of spermatocytes from both conditional knockout lines revealed that there were frequent chromosomal fusions during meiosis, always involving one or both sex chromosomes. RNA sequencing indicates upregulation of Atm in spermatocytes from miRNA-deficient mice, and immunofluorescence imaging demonstrates an increased abundance of activated ATM kinase and mislocalization of phosphorylated MDC1, an ATM phosphorylation substrate. The Atm 3'UTR contains many potential microRNA target sites, and, notably, target sites for several miRNAs depleted in both conditional knockout mice were highly effective at promoting repression. RNF8, a telomere-associated protein whose localization is controlled by the MDC1-ATM kinase cascade, normally associates with the sex chromosomes during pachytene, but in both conditional knockouts redistributed to the autosomes. Taken together, these results suggest that Atm dysregulation in microRNA-deficient germ lines contributes to the redistribution of proteins involved in chromosomal stability from the sex chromosomes to the autosomes, resulting in sex chromosome fusions during meiotic prophase I.
|An Lnc RNA (GAS5)/SnoRNA-derived piRNA induces activation of TRAIL gene by site-specifically recruiting MLL/COMPASS-like complexes.|
He, X; Chen, X; Zhang, X; Duan, X; Pan, T; Hu, Q; Zhang, Y; Zhong, F; Liu, J; Zhang, H; Luo, J; Wu, K; Peng, G; Luo, H; Zhang, L; Li, X; Zhang, H
Nucleic Acids Res 43 3712-25 2015
PIWI-interacting RNA (piRNA) silences the transposons in germlines or induces epigenetic modifications in the invertebrates. However, its function in the mammalian somatic cells remains unknown. Here we demonstrate that a piRNA derived from Growth Arrest Specific 5, a tumor-suppressive long non-coding RNA, potently upregulates the transcription of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a proapoptotic protein, by inducing H3K4 methylation/H3K27 demethylation. Interestingly, the PIWIL1/4 proteins, which bind with this piRNA, directly interact with WDR5, resulting in a site-specific recruitment of the hCOMPASS-like complexes containing at least MLL3 and UTX (KDM6A). We have indicated a novel pathway for piRNAs to specially activate gene expression. Given that MLL3 or UTX are frequently mutated in various tumors, the piRNA/MLL3/UTX complex mediates the induction of TRAIL, and consequently leads to the inhibition of tumor growth.
|Glycolytic metabolism influences global chromatin structure.|
Liu, XS; Little, JB; Yuan, ZM
Oncotarget 6 4214-25 2015
Metabolic rewiring, specifically elevated glycolytic metabolism is a hallmark of cancer. Global chromatin structure regulates gene expression, DNA repair, and also affects cancer progression. But the interrelationship between tumor metabolism and chromatin architecture remain unclear. Here we show that increased glycolysis in cancer cells promotes an open chromatin configuration. Using complementary methods including Micrococcal nuclease (MNase) digestion assay, electron microscope and immunofluorescence staining, we demonstrate that glycolysis inhibition by pharmacological and genetic approaches was associated with induction of compacted chromatin structure. This condensed chromatin status appeared to result chiefly from histone hypoacetylation as restoration of histone acetylation with an HDAC inhibitor reversed the compacted chromatin state. Interestingly, glycolysis inhibition-induced chromatin condensation impeded DNA repair efficiency leading to increased sensitivity of cancer cells to DNA damage drugs, which may represent a novel molecular mechanism that can be exploited for cancer therapy.
|Nonantibiotic Effects of Fluoroquinolones in Mammalian Cells.|
Badal, S; Her, YF; Maher, LJ
The Journal of biological chemistry 290 22287-97 2015
Fluoroquinolones (FQ) are powerful broad-spectrum antibiotics whose side effects include renal damage and, strangely, tendinopathies. The pathological mechanisms underlying these toxicities are poorly understood. Here, we show that the FQ drugs norfloxacin, ciprofloxacin, and enrofloxacin are powerful iron chelators comparable with deferoxamine, a clinically useful iron-chelating agent. We show that iron chelation by FQ leads to epigenetic effects through inhibition of α-ketoglutarate-dependent dioxygenases that require iron as a co-factor. Three dioxygenases were examined in HEK293 cells treated with FQ. At sub-millimolar concentrations, these antibiotics inhibited jumonji domain histone demethylases, TET DNA demethylases, and collagen prolyl 4-hydroxylases, leading to accumulation of methylated histones and DNA and inhibition of proline hydroxylation in collagen, respectively. These effects may explain FQ-induced nephrotoxicity and tendinopathy. By the same reasoning, dioxygenase inhibition by FQ was predicted to stabilize transcription factor HIF-1α by inhibition of the oxygen-dependent hypoxia-inducible transcription factor prolyl hydroxylation. In dramatic contrast to this prediction, HIF-1α protein was eliminated by FQ treatment. We explored possible mechanisms for this unexpected effect and show that FQ inhibit HIF-1α mRNA translation. Thus, FQ antibiotics induce global epigenetic changes, inhibit collagen maturation, and block HIF-1α accumulation. We suggest that these mechanisms explain the classic renal toxicities and peculiar tendinopathies associated with FQ antibiotics.
|Conserved Epigenetic Mechanisms Could Play a Key Role in Regulation of Photosynthesis and Development-Related Genes during Needle Development of Pinus radiata.|
Valledor, L; Pascual, J; Meijón, M; Escandón, M; Cañal, MJ
PloS one 10 e0126405 2015
Needle maturation is a complex process that involves cell growth, differentiation and tissue remodelling towards the acquisition of full physiological competence. Leaf induction mechanisms are well known; however, those underlying the acquisition of physiological competence are still poorly understood, especially in conifers. We studied the specific epigenetic regulation of genes defining organ function (PrRBCS and PrRBCA) and competence and stress response (PrCSDP2 and PrSHMT4) during three stages of needle development and one de-differentiated control. Gene-specific changes in DNA methylation and histone were analysed by bisulfite sequencing and chromatin immunoprecipitation (ChIP). The expression of PrRBCA and PrRBCS increased during needle maturation and was associated with the progressive loss of H3K9me3, H3K27me3 and the increase in AcH4. The maturation-related silencing of PrSHMT4 was correlated with increased H3K9me3 levels, and the repression of PrCSDP2, to the interplay between AcH4, H3K27me3, H3K9me3 and specific DNA methylation. The employ of HAT and HDAC inhibitors led to a further determination of the role of histone acetylation in the regulation of our target genes. The integration of these results with high-throughput analyses in Arabidopsis thaliana and Populus trichocarpa suggests that the specific epigenetic mechanisms that regulate photosynthetic genes are conserved between the analysed species.
|Increased SHP-1 expression results in radioresistance, inhibition of cellular senescence, and cell cycle redistribution in nasopharyngeal carcinoma cells.|
Sun, Z; Pan, X; Zou, Z; Ding, Q; Wu, G; Peng, G
Radiation oncology (London, England) 10 152 2015
Radioresistance is the main limit to the efficacy of radiotherapy in nasopharyngeal carcinoma (NPC). SHP-1 is involved in cancer progression, but its role in radioresistance and senescence of NPC is not well understood. This study aimed to assess the role of SHP-1 in the radioresistance and senescence of NPC cells.SHP-1 was knocked-down and overexpressed in CNE-1 and CNE-2 cells using lentiviruses. Cells were irradiated to observe their radiosensitivity by colony forming assay. BrdU incorporation assay and flow cytometry were used to monitor cell cycle. A β-galactosidase assay was used to assess senescence. Western blot was used to assess SHP-1, p21, p53, pRb, Rb, H3K9Me3, HP1γ, CDK4, cyclin D1, cyclin E, and p16 protein expressions.Compared with CNE-1-scramble shRNA cells, SHP-1 downregulation resulted in increased senescence (+107%, P less than 0.001), increased radiosensitivity, higher proportion of cells in G0/G1 (+33%, P less than 0.001), decreased expressions of CDK4 (-44%, P less than 0.001), cyclin D1 (-41%, P = 0.001), cyclin E (-97%, P less than 0.001), Rb (-79%, P less than 0.001), and pRb (-76%, P = 0.001), and increased expression of p16 (+120%, P = 0.02). Furthermore, SHP-1 overexpression resulted in radioresistance, inhibition of cellular senescence, and cell cycle arrest in the S phase. Levels of p53 and p21 were unchanged in both cell lines (all P greater than 0.05).SHP-1 has a critical role in radioresistance, cell cycle progression, and senescence of NPC cells. Down-regulating SHP-1 may be a promising therapeutic approach for treating patients with NPC.
|Chromatin decompaction by the nucleosomal binding protein HMGN5 impairs nuclear sturdiness.|
Furusawa, T; Rochman, M; Taher, L; Dimitriadis, EK; Nagashima, K; Anderson, S; Bustin, M
Nature communications 6 6138 2015
In most metazoan nuclei, heterochromatin is located at the nuclear periphery in contact with the nuclear lamina, which provides mechanical stability to the nucleus. We show that in cultured cells, chromatin decompaction by the nucleosome binding protein HMGN5 decreases the sturdiness, elasticity and rigidity of the nucleus. Mice overexpressing HMGN5, either globally or only in the heart, are normal at birth but develop hypertrophic heart with large cardiomyoctyes, deformed nuclei and disrupted lamina and die of cardiac malfunction. Chromatin decompaction is seen in cardiomyocytes of newborn mice but misshaped nuclei with disrupted lamina are seen only in adult cardiomyocytes, suggesting that loss of heterochromatin diminishes the ability of the nucleus to withstand the mechanical forces of the contracting heart. Thus, heterochromatin enhances the ability of the nuclear lamina to maintain the sturdiness and shape of the eukaryotic nucleus; a structural role for chromatin that is distinct from its genetic functions.
|Novel Scaffolds of Cell-Active Histone Demethylase Inhibitors Identified from High-Throughput Screening.|
Wang, W; Marholz, LJ; Wang, X
Journal of biomolecular screening 20 821-7 2015
Jumonji C domain-containing histone demethylases (JHDMs) are epigenetic proteins capable of demethylating methylated lysine residues on histones proteins and for which high-quality chemical probes and eventual therapeutic leads are highly desirable. To expand the extent of known scaffolds targeting JHDMs, we initiated an unbiased high-throughput screening approach using a fluorescence polarization (FP)-based competitive binding assay we recently reported for JHDM1A (aka KDM2A). In total, 14,400 compounds in the HitFinder collection v.11 were screened, which represent all the distinct skeletons of the Maybridge Library. An eventual three compounds with two new scaffolds were discovered and further validated, which not only show in vitro binding for two different JHDMs, JHDM1A and JMJD2A (aka KDM4A), but also induce hypermethylation of their substrate in cells. These represent novel scaffolds as JHDM inhibitors and provide a basis for future optimization of affinity and selectivity.