Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, Yeast (S. cerevisiae)||ICC, IP, WB||M||Ascites||Monoclonal Antibody|
|Safety Information according to GHS|
|Material Size||100 µL|
|Anti-RNA Polymerase II, CTD, clone 8WG16 - 2137080||2137080|
|Anti-RNA Polymerase II, CTD, clone 8WG16 - 2366504||2366504|
|Anti-RNA Polymerase II, CTD, clone 8WG16 - 2424635||2424635|
|Anti-RNA Polymerase II, CTD, clone 8WG16 - DAM1797932||DAM1797932|
|Anti-RNA Polymerase II, CTD, -2519219||2519219|
|Anti-RNA Polymerase II, CTD, -2548160||2548160|
|Anti-RNA Polymerase II, CTD, -2620625||2620625|
|Anti-RNA Polymerase II, CTD, -2629272||2629272|
|Anti-RNA Polymerase II, CTD, -2653120||2653120|
|Anti-RNA Polymerase II, CTD, -2701525||2701525|
|Reference overview||Application||Species||Pub Med ID|
|Poised chromatin and bivalent domains facilitate the mitosis-to-meiosis transition in the male germline.|
Sin, HS; Kartashov, AV; Hasegawa, K; Barski, A; Namekawa, SH
BMC biology 13 53 2015
The male germline transcriptome changes dramatically during the mitosis-to-meiosis transition to activate late spermatogenesis genes and to transiently suppress genes commonly expressed in somatic lineages and spermatogenesis progenitor cells, termed somatic/progenitor genes.These changes reflect epigenetic regulation. Induction of late spermatogenesis genes during spermatogenesis is facilitated by poised chromatin established in the stem cell phases of spermatogonia, whereas silencing of somatic/progenitor genes during meiosis and postmeiosis is associated with formation of bivalent domains which also allows the recovery of the somatic/progenitor program after fertilization. Importantly, during spermatogenesis mechanisms of epigenetic regulation on sex chromosomes are different from autosomes: X-linked somatic/progenitor genes are suppressed by meiotic sex chromosome inactivation without deposition of H3K27me3.Our results suggest that bivalent H3K27me3 and H3K4me2/3 domains are not limited to developmental promoters (which maintain bivalent domains that are silent throughout the reproductive cycle), but also underlie reversible silencing of somatic/progenitor genes during the mitosis-to-meiosis transition in late spermatogenesis.
|Mechanism of Action of 2-Aminobenzamide HDAC Inhibitors in Reversing Gene Silencing in Friedreich's Ataxia.|
Soragni, E; Chou, CJ; Rusche, JR; Gottesfeld, JM
Frontiers in neurology 6 44 2015
The genetic defect in Friedreich's ataxia (FRDA) is the hyperexpansion of a GAA•TTC triplet in the first intron of the FXN gene, encoding the essential mitochondrial protein frataxin. Histone post-translational modifications near the expanded repeats are consistent with heterochromatin formation and consequent FXN gene silencing. Using a newly developed human neuronal cell model, derived from patient-induced pluripotent stem cells, we find that 2-aminobenzamide histone deacetylase (HDAC) inhibitors increase FXN mRNA levels and frataxin protein in FRDA neuronal cells. However, only compounds targeting the class I HDACs 1 and 3 are active in increasing FXN mRNA in these cells. Structural analogs of the active HDAC inhibitors that selectively target either HDAC1 or HDAC3 do not show similar increases in FXN mRNA levels. To understand the mechanism of action of these compounds, we probed the kinetic properties of the active and inactive inhibitors, and found that only compounds that target HDACs 1 and 3 exhibited a slow-on/slow-off mechanism of action for the HDAC enzymes. HDAC1- and HDAC3-selective compounds did not show this activity. Using siRNA methods in the FRDA neuronal cells, we show increases in FXN mRNA upon silencing of either HDACs 1 or 3, suggesting the possibility that inhibition of each of these class I HDACs is necessary for activation of FXN mRNA synthesis, as there appears to be redundancy in the silencing mechanism caused by the GAA•TTC repeats. Moreover, inhibitors must have a long residence time on their target enzymes for this activity. By interrogating microarray data from neuronal cells treated with inhibitors of different specificity, we selected two genes encoding histone macroH2A (H2AFY2) and Polycomb group ring finger 2 (PCGF2) that were specifically down-regulated by the inhibitors targeting HDACs1 and 3 versus the more selective inhibitors for further investigation. Both genes are involved in transcriptional repression and we speculate their involvement in FXN gene silencing. Our results shed light on the mechanism whereby HDAC inhibitors increase FXN mRNA levels in FRDA neuronal cells.
|The Menin-Bach2 axis is critical for regulating CD4 T-cell senescence and cytokine homeostasis.|
Kuwahara, M; Suzuki, J; Tofukuji, S; Yamada, T; Kanoh, M; Matsumoto, A; Maruyama, S; Kometani, K; Kurosaki, T; Ohara, O; Nakayama, T; Yamashita, M
Nature communications 5 3555 2014
Although CD4 T-cell senescence plays an important role in immunosenescence, the mechanism behind this process remains unclear. Here we show that T cell-specific Menin deficiency results in the premature senescence of CD4 T cells, which is accompanied by the senescence-associated secretory phenotype after antigenic stimulation and dysregulated cytokine production. Menin is required for the expansion and survival of antigen-stimulated CD4 T cells in vivo and acts by targeting Bach2, which is known to regulate immune homeostasis and cytokine production. Menin binds to the Bach2 locus and controls its expression through maintenance of histone acetylation. Menin binding at the Bach2 locus and the Bach2 expression are decreased in the senescent CD4 T cells. These findings reveal a critical role of the Menin-Bach2 pathway in regulating CD4 T-cell senescence and cytokine homeostasis, thus indicating the involvement of this pathway in the inhibition of immunosenescence.
|Thyroid hormone signaling in vivo requires a balance between coactivators and corepressors.|
Vella, KR; Ramadoss, P; Costa-E-Sousa, RH; Astapova, I; Ye, FD; Holtz, KA; Harris, JC; Hollenberg, AN
Molecular and cellular biology 34 1564-75 2014
Resistance to thyroid hormone (RTH), a human syndrome, is characterized by high thyroid hormone (TH) and thyroid-stimulating hormone (TSH) levels. Mice with mutations in the thyroid hormone receptor beta (TRβ) gene that cannot bind steroid receptor coactivator 1 (SRC-1) and Src-1(-/-) mice both have phenotypes similar to that of RTH. Conversely, mice expressing a mutant nuclear corepressor 1 (Ncor1) allele that cannot interact with TRβ, termed NCoRΔID, have low TH levels and normal TSH. We hypothesized that Src-1(-/-) mice have RTH due to unopposed corepressor action. To test this, we crossed NCoRΔID and Src-1(-/-) mice to create mice deficient for coregulator action in all cell types. Remarkably, NCoR(ΔID/ΔID) Src-1(-/-) mice have normal TH and TSH levels and are triiodothryonine (T(3)) sensitive at the level of the pituitary. Although absence of SRC-1 prevented T(3) activation of key hepatic gene targets, NCoR(ΔID/ΔID) Src-1(-/-) mice reacquired hepatic T(3) sensitivity. Using in vivo chromatin immunoprecipitation assays (ChIP) for the related coactivator SRC-2, we found enhanced SRC-2 recruitment to TR-binding regions of genes in NCoR(ΔID/ΔID) Src-1(-/-) mice, suggesting that SRC-2 is responsible for T(3) sensitivity in the absence of NCoR1 and SRC-1. Thus, T(3) targets require a critical balance between NCoR1 and SRC-1. Furthermore, replacement of NCoR1 with NCoRΔID corrects RTH in Src-1(-/-) mice through increased SRC-2 recruitment to T(3) target genes.
|The chromatin remodeling factor CHD5 is a transcriptional repressor of WEE1.|
Quan, J; Adelmant, G; Marto, JA; Look, AT; Yusufzai, T
PloS one 9 e108066 2014
Loss of the chromatin remodeling ATPase CHD5 has been linked to the progression of neuroblastoma tumors, yet the underlying mechanisms behind the tumor suppressor role of CHD5 are unknown. In this study, we purified the human CHD5 complex and found that CHD5 is a component of the full NuRD transcriptional repressor complex, which also contains methyl-CpG binding proteins and histone deacetylases. The CHD5/NuRD complex appears mutually exclusive with the related CHD4/NuRD complex as overexpression of CHD5 results in loss of the CHD4 protein in cells. Following a search for genes that are regulated by CHD5 in neuroblastoma cells, we found that CHD5 binds to and represses the G2/M checkpoint gene WEE1. Reintroduction of CHD5 into neuroblastoma cells represses WEE1 expression, demonstrating that CHD5 can function as a repressor in cells. A catalytically inactive mutant version of CHD5 is able to associate with a NuRD cofactor but fails to repress transcription. Our study shows that CHD5 is a NuRD-associated transcriptional repressor and identifies WEE1 as one of the CHD5-regulated genes that may link CHD5 to tumor suppression.
|Network of mutually repressive metastasis regulators can promote cell heterogeneity and metastatic transitions.|
Lee, J; Lee, J; Farquhar, KS; Yun, J; Frankenberger, CA; Bevilacqua, E; Yeung, K; Kim, EJ; Balázsi, G; Rosner, MR
Proceedings of the National Academy of Sciences of the United States of America 111 E364-73 2014
The sources and consequences of nongenetic variability in metastatic progression are largely unknown. To address these questions, we characterized a transcriptional regulatory network for the metastasis suppressor Raf kinase inhibitory protein (RKIP). We previously showed that the transcription factor BACH1 is negatively regulated by RKIP and promotes breast cancer metastasis. Here we demonstrate that BACH1 acts in a double-negative (overall positive) feedback loop to inhibit RKIP transcription in breast cancer cells. BACH1 also negatively regulates its own transcription. Analysis of the BACH1 network reveals the existence of an inverse relationship between BACH1 and RKIP involving both monostable and bistable transitions that can potentially give rise to nongenetic variability. Single-cell analysis confirmed monostable and bistable-like behavior. Treatment with histone deacetylase inhibitors or depletion of the polycomb repressor enhancer of zeste homolog 2 altered relative RKIP and BACH1 levels in a manner consistent with a prometastatic state. Together, our results suggest that the mutually repressive relationship between metastatic regulators such as RKIP and BACH1 can play a key role in determining metastatic progression in cancer.
|Deletion of a conserved cis-element in the Ifng locus highlights the role of acute histone acetylation in modulating inducible gene transcription.|
Balasubramani, A; Winstead, CJ; Turner, H; Janowski, KM; Harbour, SN; Shibata, Y; Crawford, GE; Hatton, RD; Weaver, CT
PLoS genetics 10 e1003969 2014
Differentiation-dependent regulation of the Ifng cytokine gene locus in T helper (Th) cells has emerged as an excellent model for functional study of distal elements that control lineage-specific gene expression. We previously identified a cis-regulatory element located 22 kb upstream of the Ifng gene (Conserved Non-coding Sequence -22, or CNS-22) that is a site for recruitment of the transcription factors T-bet, Runx3, NF-κB and STAT4, which act to regulate transcription of the Ifng gene in Th1 cells. Here, we report the generation of mice with a conditional deletion of CNS-22 that has enabled us to define the epigenetic and functional consequences of its absence. Deletion of CNS-22 led to a defect in induction of Ifng by the cytokines IL-12 and IL-18, with a more modest effect on induction via T-cell receptor activation. To better understand how CNS-22 and other Ifng CNSs regulated Ifng transcription in response to these distinct stimuli, we examined activation-dependent changes in epigenetic modifications across the extended Ifng locus in CNS-22-deficient T cells. We demonstrate that in response to both cytokine and TCR driven activation signals, CNS-22 and other Ifng CNSs recruit increased activity of histone acetyl transferases (HATs) that transiently enhance levels of histones H3 and H4 acetylation across the extended Ifng locus. We also demonstrate that activation-responsive increases in histone acetylation levels are directly linked to the ability of Ifng CNSs to acutely enhance Pol II recruitment to the Ifng promoter. Finally, we show that impairment in IL-12+IL-18 dependent induction of Ifng stems from the importance of CNS-22 in coordinating locus-wide levels of histone acetylation in response to these cytokines. These findings identify a role for acute histone acetylation in the enhancer function of distal conserved cis-elements that regulate of Ifng gene expression.
|Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos.|
Aoki, T; Wolle, D; Preger-Ben Noon, E; Dai, Q; Lai, EC; Schedl, P
Fly 8 43-51 2014
Chromatin immunoprecipitation (ChIP) is widely used for mapping DNA-protein interactions across eukaryotic genomes in cells, tissues or even whole organisms. Critical to this procedure is the efficient cross-linking of chromatin-associated proteins to DNA sequences that are in close proximity. Since the mid-nineties formaldehyde fixation has been the method of choice. However, some protein-DNA complexes cannot be successfully captured for ChIP using formaldehyde. One such formaldehyde refractory complex is the developmentally regulated insulator factor, Elba. Here we describe a new embryo fixation procedure using the bi-functional cross-linking reagents DSG (disuccinimidyl glutarate) and DSP (dithiobis[succinimidyl propionate). We show that unlike standard formaldehyde fixation protocols, it is possible to capture Elba association with insulator elements in 2-5 h embryos using this new cross-linking procedure. We show that this new cross-linking procedure can also be applied to localize nuclear proteins that are amenable to ChIP using standard formaldehyde cross-linking protocols, and that in the cases tested the enrichment was generally superior to that achieved using formaldehyde cross-linking.
|Optimal histone H3 to linker histone H1 chromatin ratio is vital for mesodermal competence in Xenopus.|
Lim, CY; Reversade, B; Knowles, BB; Solter, D
Development (Cambridge, England) 140 853-60 2013
Cellular differentiation during embryogenesis involves complex gene regulation to enable the activation and repression of genes. Here, we show that mesodermal competence is inhibited in Xenopus embryos depleted of histones H3 and H3.3, which fail to respond to Nodal/Activin signaling and exhibit concomitant loss of mesodermal gene expression. We find that transcriptional activation in gastrula embryos does not correlate with promoter deposition of H3.3. Instead, gastrulation defects in H3.3/H3-deficient embryos are partially rescued with concurrent depletion of the linker histone H1A. In addition, we show that linker histone H1-induced premature loss of mesodermal competence in animal cap explants can be abrogated with the overexpression of nucleosomal H3.3/H3. Our findings establish a chromatin-mediated regulatory mechanism in which a threshold level of H3 is required to prevent H1-induced gene repression, and thus facilitate mesodermal differentiation in response to inductive signaling.
|Epigenetic regulation of autophagy by the methyltransferase G9a.|
Artal-Martinez de Narvajas, A; Gomez, TS; Zhang, JS; Mann, AO; Taoda, Y; Gorman, JA; Herreros-Villanueva, M; Gress, TM; Ellenrieder, V; Bujanda, L; Kim, DH; Kozikowski, AP; Koenig, A; Billadeau, DD
Molecular and cellular biology 33 3983-93 2013
Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the cytoplasmic machinery that orchestrates autophagy induction during starvation, hypoxia, or receptor stimulation has been widely studied, the key epigenetic events that initiate and maintain the autophagy process remain unknown. Here we show that the methyltransferase G9a coordinates the transcriptional activation of key regulators of autophagosome formation by remodeling the chromatin landscape. Pharmacological inhibition or RNA interference (RNAi)-mediated suppression of G9a induces LC3B expression and lipidation that is dependent on RNA synthesis, protein translation, and the methyltransferase activity of G9a. Under normal conditions, G9a associates with the LC3B, WIPI1, and DOR gene promoters, epigenetically repressing them. However, G9a and G9a-repressive histone marks are removed during starvation and receptor-stimulated activation of naive T cells, two physiological inducers of macroautophagy. Moreover, we show that the c-Jun N-terminal kinase (JNK) pathway is involved in the regulation of autophagy gene expression during naive-T-cell activation. Together, these findings reveal that G9a directly represses genes known to participate in the autophagic process and that inhibition of G9a-mediated epigenetic repression represents an important regulatory mechanism during autophagy.