Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Specificity||Histone H3 acetylated at position 9.|
|Presentation||Purified rabbit polyclonal in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide with 30% glycerol.|
|Safety Information according to GHS|
|Material Size||200 µg|
References | 29 Available | See All References
|Reference overview||Application||Pub Med ID|
|Association of the interferon-β gene with pericentromeric heterochromatin is dynamically regulated during virus infection through a YY1-dependent mechanism. |
T Josse,H Mokrani-Benhelli,R Benferhat,E Shestakova,Z Mansuroglu,H Kakanakou,A Billecocq,M Bouloy,Eliette Bonnefoy
Nucleic acids research 40 2012
Nuclear architecture as well as gene nuclear positioning can modulate gene expression. In this work, we have analyzed the nuclear position of the interferon-β (IFN-β) locus, responsible for the establishment of the innate antiviral response, with respect to pericentromeric heterochromatin (PCH) in correlation with virus-induced IFN-β gene expression. Experiments were carried out in two different cell types either non-infected (NI) or during the time course of three different viral infections. In NI cells, we showed a monoallelic IFN-β promoter association with PCH that strongly decreased after viral infection. Dissociation of the IFN-β locus away from these repressive regions preceded strong promoter transcriptional activation and was reversible within 12 h after infection. No dissociation was observed after infection with a virus that abnormally maintained the IFN-β gene in a repressed state. Dissociation induced after virus infection specifically targeted the IFN-β locus without affecting the general structure and nuclear distribution of PCH clusters. Using cell lines stably transfected with wild-type or mutated IFN-β promoters, we identified the proximal region of the IFN-β promoter containing YY1 DNA-binding sites as the region regulating IFN-β promoter association with PCH before as well as during virus infection.
|An essential role for a mammalian SWI/SNF chromatin-remodeling complex during male meiosis. |
Yuna Kim,Andrew M Fedoriw,Terry Magnuson
Development (Cambridge, England) 139 2012
Germ cell development and gametogenesis require genome-wide transitions in epigenetic modifications and chromatin structure. These changes include covalent modifications to the DNA and histones as well as remodeling activities. Here, we explore the role of the mammalian SWI/SNF chromatin-remodeling complex during spermatogenesis using a conditional allele of the ATPase subunit, brahma-related gene 1 (Brg1, or Smarca4). Not only do BRG1 levels peak during the early stages of meiosis, genetic ablation of Brg1 in murine embryonic gonocytes results in arrest during prophase of meiosis I. Coincident with the timing of meiotic arrest, mutant spermatocytes accumulate unrepaired DNA and fail to complete synapsis. Furthermore, mutant spermatocytes show global alterations to histone modifications and chromatin structure indicative of a more heterochromatic genome. Together, these data demonstrate a requirement for BRG1 activity in spermatogenesis, and suggest a role for the mammalian SWI/SNF complex in programmed recombination and repair events that take place during meiosis.
|Epigenetic reprogramming as a key contributor to melanocyte malignant transformation. |
Molognoni F, Cruz AT, Meliso FM, Morais AS, Souza CF, Xander P, Bischof JM, Costa FF, Soares MB, Liang G, Jones PA, Jasiulionis MG.
Epigenetics : official journal of the DNA Methylation Society 6 450-64 2011
Melanoma progression requires deregulation of gene expression by currently uncharacterized epigenetic mechanisms. A mouse model based on changes in cell microenvironment was developed by our group to study melanocyte malignant transformation. Melanoma cell lines (4C11- and 4C11+) were obtained as result of 5 sequential anchorage blockades of non-tumorigenic melan-a melanocytes. Melan-a cells submitted to 4 de-adhesion cycles were also established (4C), are non-tumorigenic and represent an intermediary phase of tumor progression. The aim of this work was to identify factors contributing to epigenetic modifications in early and later phases of malignant transformation induced by anchorage impediment. Epigenetic alterations occur early in tumorigenesis; 4C cell line shows changes in global and gene-specific DNA methylation and histone marks. Many histone modifications differ between melan-a, 4C, 4C11- (non-metastatic melanoma cell line) and 4C11+ (metastatic melanoma cell line) which could be associated with changes in gene and microRNA expression. These epigenetic alterations seem to play a key role in malignant transformation since melanocytes treated with 5-Aza-2'-deoxycytidine before each anchorage blockade do not transform. Some epigenetic changes seem to be also responsible for the maintenance of malignant phenotype, since melanoma cell lines (4C11- and 4C11+) treated in vitro with 5-Aza-2'-deoxycytidine or Trichostatin A showed reduction of tumor growth in vivo. Changes in gene expression reflecting cell adaptation to new environment were also observed. We propose a model in which sustained microenvironmental stress in melanocytes results in epigenetic reprogramming. Thus, after adaptation, cells may acquire epigenetic marks that could contribute to the establishment of a malignant phenotype.
|Mouse Rankl expression is regulated in T cells by c-Fos through a cluster of distal regulatory enhancers designated the T cell control region. |
Bishop KA, Coy HM, Nerenz RD, Meyer MB, Pike JW
The Journal of biological chemistry 2011
Rankl is a TNF-like factor that induces the formation of osteoclasts responsible for bone resorption. While T cell activation upregulates this gene, the molecular mechanism of its transcriptional control remains unknown. We used ChIP-chip analysis in mouse primary T cells and a T cell hybridoma to define the regulatory enhancers responsible for this upregulation and to characterize their properties. Elevated H3/H4 acetylation and increased RNA polymerase II (RNA pol II) density were evident at mRL-D5, a known enhancer located 76 kb upstream of the TSS, as well as at a cluster of regulatory sites located even further upstream between -123 to -156 kb, termed the T cell control region (TCCR). Based upon the ability of calcium signaling and MAPK inhibitors to block Rankl expression, we conducted further ChIP-chip analysis of the transcriptional mediators c-Fos, NF-κB and NFAT. T cell activation induced c-Fos binding at the mRL-D5 enhancer and within the TCCR. The interaction of NF-κB was observed at the TSS and at mRL-D5. Both mRL-D5 and segments of the TCCR exhibited robust transcriptional activity in reporter assays, and site-specific mutagenesis of c-Fos and Nfat elements abrogated reporter activity suggesting a role for both factors in the control of enhancer-mediated Rankl transcription. Finally, 3C analysis confirmed that mRL-D5 and segments of the TCCR were located in proximity to and thus potentially able to influence directly Rankl gene promoter activity. We conclude that both mRL-D5 and the TCCR represent control segments that play an integral role in Rankl expression in T cells.
|Post-transcription initiation function of the ubiquitous SAGA complex in tissue-specific gene activation. |
Weake, Vikki M, et al.
Genes Dev., 25: 1499-509 (2011) 2011
The Spt-Ada-Gcn5-acetyltransferase (SAGA) complex was discovered from Saccharomyces cerevisiae and has been well characterized as an important transcriptional coactivator that interacts both with sequence-specific transcription factors and the TATA-binding protein TBP. SAGA contains a histone acetyltransferase and a ubiquitin protease. In metazoans, SAGA is essential for development, yet little is known about the function of SAGA in differentiating tissue. We analyzed the composition, interacting proteins, and genomic distribution of SAGA in muscle and neuronal tissue of late stage Drosophila melanogaster embryos. The subunit composition of SAGA was the same in each tissue; however, SAGA was associated with considerably more transcription factors in muscle compared with neurons. Consistent with this finding, SAGA was found to occupy more genes specifically in muscle than in neurons. Strikingly, SAGA occupancy was not limited to enhancers and promoters but primarily colocalized with RNA polymerase II within transcribed sequences. SAGA binding peaks at the site of RNA polymerase pausing at the 5' end of transcribed sequences. In addition, many tissue-specific SAGA-bound genes required its ubiquitin protease activity for full expression. These data indicate that in metazoans SAGA plays a prominent post-transcription initiation role in tissue-specific gene expression.
|SIRT6 in mouse spermatogenesis is modulated by diet-induced obesity. |
Palmer NO, Fullston T, Mitchell M, Setchell BP, Lane M.
Reproduction, fertility, and development 23 929-39 2011
Male obesity is associated with reduced sperm function and increased incidence of sperm DNA damage; however, the underlying molecular mechanisms have not yet been identified. Mammalian SIRT6 protein is involved in caloric-dependant DNA damage repair in other tissue types, yet a possible role for SIRT6 in male obesity and subfertility has not been investigated previously. To assess SIRT6 levels and activity in the testes, male mice (n=12 per diet) were fed either a control diet (CD; 6% fat) or a high-fat diet (HFD; 21% fat) for 16 weeks before the collection of testes and spermatozoa. SIRT6 protein was localised to the nucleus of transitional spermatids and the acrosome of mature spermatozoa, with levels significantly decreased in HFD-fed male mice (P<0.05). This decrease in SIRT6 protein was associated with transitional spermatids having increased levels of acetylated H3K9 in the nucleus (P<0.01) and increased DNA damage (P<0.001). We propose a role for SIRT6 in spermiogenesis and potentially protamination processes, which are known to be compromised by male obesity.
|MDM2 recruitment of lysine methyltransferases regulates p53 transcriptional output. |
Chen L, Li Z, Zwolinska AK, Smith MA, Cross B, Koomen J, Yuan ZM, Jenuwein T, Marine JC, Wright KL, Chen J
EMBO J 2010
MDM2 is a key regulator of the p53 tumor suppressor acting primarily as an E3 ubiquitin ligase to promote its degradation. MDM2 also inhibits p53 transcriptional activity by recruiting histone deacetylase and corepressors to p53. Here, we show that immunopurified MDM2 complexes have significant histone H3-K9 methyltransferase activity. The histone methyltransferases SUV39H1 and EHMT1 bind specifically to MDM2 but not to its homolog MDMX. MDM2 mediates formation of p53-SUV39H1/EHMT1 complex capable of methylating H3-K9 in vitro and on p53 target promoters in vivo. Furthermore, MDM2 promotes EHMT1-mediated p53 methylation at K373. Knockdown of SUV39H1 and EHMT1 increases p53 activity during stress response without affecting p53 levels, whereas their overexpression inhibits p53 in an MDM2-dependent manner. The p53 activator ARF inhibits SUV39H1 and EHMT1 binding to MDM2 and reduces MDM2-associated methyltransferase activity. These results suggest that MDM2-dependent recruitment of methyltransferases is a novel mechanism of p53 regulation through methylation of both p53 itself and histone H3 at target promoters.
|Chromatin states of core pluripotency-associated genes in pluripotent, multipotent and differentiated cells. |
S Barrand, P Collas
Biochemical and biophysical research communications 391 762-7 2010
Oct4, Nanog and Sox2 constitute a core of transcription factors controlling pluripotency. Differentiation and reprogramming studies have unraveled a few epigenetic modifications associated in relation to the expression state of OCT4, NANOG and SOX2. There is, however, no comprehensive map of chromatin states on these genes in human primary cells at different stages of differentiation. We report here a profile of DNA methylation and of 10 histone modifications on regulatory regions of OCT4, NANOG and SOX2 in embryonal carcinoma cells, mesenchymal stem cells and fibroblasts. Bisulfite sequencing reveals correlation between promoter CpG methylation and repression of OCT4, but not NANOG or SOX2, suggesting distinct repression mechanisms. Whereas none of these genes, even when inactive, harbor repressive trimethylated H3K9, CpG hypomethylated NANOG and SOX2, but not CpG methylated OCT4, are enriched in repressive H3K27me3. H3K79me1 and H3K79me3 tend to parallel each other and are linked to repression. Moreover, we highlight an inverse relationship between H3K27me3 occupancy on promoters and H3K36me3 occupancy on coding regions of OCT4, NANOG and SOX2, suggesting a cross-talk between K27 and K36 methylation. Establishment of distinct repression mechanisms for pluripotency-associated genes may constitute a safeguard system to prevent promiscuous reactivation during development or differentiation.
|Polycomb target genes are silenced in multiple myeloma. |
Kalushkova A, Fryknäs M, Lemaire M, Fristedt C, Agarwal P, Eriksson M, Deleu S, Atadja P, Osterborg A, Nilsson K, Vanderkerken K, Oberg F, Jernberg-Wiklund H
PLoS One 5 e11483. 2010
Multiple myeloma (MM) is a genetically heterogeneous disease, which to date remains fatal. Finding a common mechanism for initiation and progression of MM continues to be challenging. By means of integrative genomics, we identified an underexpressed gene signature in MM patient cells compared to normal counterpart plasma cells. This profile was enriched for previously defined H3K27-tri-methylated genes, targets of the Polycomb group (PcG) proteins in human embryonic fibroblasts. Additionally, the silenced gene signature was more pronounced in ISS stage III MM compared to stage I and II. Using chromatin immunoprecipitation (ChIP) assay on purified CD138+ cells from four MM patients and on two MM cell lines, we found enrichment of H3K27me3 at genes selected from the profile. As the data implied that the Polycomb-targeted gene profile would be highly relevant for pharmacological treatment of MM, we used two compounds to chemically revert the H3K27-tri-methylation mediated gene silencing. The S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin (DZNep) and the histone deacetylase inhibitor LBH589 (Panobinostat), reactivated the expression of genes repressed by H3K27me3, depleted cells from the PRC2 component EZH2 and induced apoptosis in human MM cell lines. In the immunocompetent 5T33MM in vivo model for MM, treatment with LBH589 resulted in gene upregulation, reduced tumor load and increased overall survival. Taken together, our results reveal a common gene signature in MM, mediated by gene silencing via the Polycomb repressor complex. The importance of the underexpressed gene profile in MM tumor initiation and progression should be subjected to further studies.Full Text Article
|Chromatin environment of histone variant H3.3 revealed by quantitative imaging and genome-scale chromatin and DNA immunoprecipitation. |
Delbarre E, Jacobsen BM, Reiner AH, Sørensen AL, Küntziger T, Collas P
Mol Biol Cell 21 1872-84. Epub 2010 Apr 7. 2010
In contrast to canonical histones, histone variant H3.3 is incorporated into chromatin in a replication-independent manner. Posttranslational modifications of H3.3 have been identified; however, the epigenetic environment of incorporated H3.3 is unclear. We have investigated the genomic distribution of epitope-tagged H3.3 in relation to histone modifications, DNA methylation, and transcription in mesenchymal stem cells. Quantitative imaging at the nucleus level shows that H3.3, relative to replicative H3.2 or canonical H2B, is enriched in chromatin domains marked by histone modifications of active or potentially active genes. Chromatin immunoprecipitation of epitope-tagged H3.3 and array hybridization identified 1649 H3.3-enriched promoters, a fraction of which is coenriched in H3K4me3 alone or together with H3K27me3, whereas H3K9me3 is excluded, corroborating nucleus-level imaging data. H3.3-enriched promoters are predominantly CpG-rich and preferentially DNA methylated, relative to the proportion of methylated RefSeq promoters in the genome. Most but not all H3.3-enriched promoters are transcriptionally active, and coenrichment of H3.3 with repressive H3K27me3 correlates with an enhanced proportion of expressed genes carrying this mark. H3.3-target genes are enriched in mesodermal differentiation and signaling functions. Our data suggest that in mesenchymal stem cells, H3.3 targets lineage-priming genes with a potential for activation facilitated by H3K4me3 in facultative association with H3K27me3.Full Text Article
|Characterization of an antagonistic switch between histone H3 lysine 27 methylation and acetylation in the transcriptional regulation of Polycomb group target genes. |
Pasini D, Malatesta M, Jung HR, Walfridsson J, Willer A, Olsson L, Skotte J, Wutz A, Porse B, Jensen ON, Helin K
Nucleic Acids Res 2010
Polycomb group (PcG) proteins are transcriptional repressors, which regulate proliferation and cell fate decisions during development, and their deregulated expression is a frequent event in human tumours. The Polycomb repressive complex 2 (PRC2) catalyzes trimethylation (me3) of histone H3 lysine 27 (K27), and it is believed that this activity mediates transcriptional repression. Despite the recent progress in understanding PcG function, the molecular mechanisms by which the PcG proteins repress transcription, as well as the mechanisms that lead to the activation of PcG target genes are poorly understood. To gain insight into these mechanisms, we have determined the global changes in histone modifications in embryonic stem (ES) cells lacking the PcG protein Suz12 that is essential for PRC2 activity. We show that loss of PRC2 activity results in a global increase in H3K27 acetylation. The methylation to acetylation switch correlates with the transcriptional activation of PcG target genes, both during ES cell differentiation and in MLL-AF9-transduced hematopoietic stem cells. Moreover, we provide evidence that the acetylation of H3K27 is catalyzed by the acetyltransferases p300 and CBP. Based on these data, we propose that the PcG proteins in part repress transcription by preventing the binding of acetyltransferases to PcG target genes.
|Histone modifiers, YY1 and p300, regulate the expression of cartilage-specific gene, chondromodulin-I, in mesenchymal stem cells. |
Aoyama T, Okamoto T, Fukiage K, Otsuka S, Furu M, Ito K, Jin Y, Ueda M, Nagayama S, Nakayama T, Nakamura T, Toguchida J
J Biol Chem 285 29842-50. Epub 2010 Jul 27. 2010
Elucidating the regulatory mechanism for tissue-specific gene expression is key to understanding the differentiation process. The chondromodulin-I gene (ChM-I) is a cartilage-specific gene, the expression of which is regulated by the transcription factor, Sp3. The binding of Sp3 to the core-promoter region is regulated by the methylation status of the Sp3-binding motif as we reported previously. In this study, we have investigated the molecular mechanisms of the down-regulation of ChM-I expression in mesenchymal stem cells (MSCs) and normal mesenchymal tissues other than cartilage. The core-promoter region of cells in bone and peripheral nerve tissues was hypermethylated, whereas the methylation status in cells of other tissues including MSCs did not differ from that in cells of cartilage, suggesting the presence of inhibitory mechanisms other than DNA methylation. We found that a transcriptional repressor, YY1, negatively regulated the expression of ChM-I by recruiting histone deacetylase and thus inducing the deacetylation of associated histones. As for a positive regulator, we found that a transcriptional co-activator, p300, bound to the core-promoter region with Sp3, inducing the acetylation of histone. Inhibition of YY1 in combination with forced expression of p300 and Sp3 restored the expression of ChM-I in cells with a hypomethylated promoter region, but not in cells with hypermethylation. These results suggested that the expression of tissue-specific genes is regulated in two steps; reversible down-regulation by transcriptional repressor complex and tight down-regulation via DNA methylation.
|Epigenetics of multiple myeloma after treatment with cytostatics and gamma radiation. |
Jana Krejcí, Andrea Harnicarová, Denisa Streitová, Roman Hájek, Ludek Pour, Stanislav Kozubek, Eva Bártová, Jana Krejcí, Andrea Harnicarová, Denisa Streitová, Roman Hájek, Ludek Pour, Stanislav Kozubek, Eva Bártová
Leukemia research 33 1490-8 2009
Genetic and epigenetic changes in multiple myeloma (MM) correlate with the stage of the disease. Therefore, we investigated how cytostatics and gamma radiation influence MM-associated histone modifications. ChIP-PCR and ChIP-on-chip technologies were used to quantify H3K9 acetylation and H3K9 dimethylation at select loci in MM patients, lymphoblastoid ARH-77, and myeloma MOLP-8 cells. Genome-wide analysis revealed that the cytostatic, melphalan, increased H3K9 acetylation at multiple gene promoters in ARH-77 cells. Melphalan and gamma radiation also influenced histone modification of prognostically important c-myc and CCND1 genes in ARH-77 and MOLP-8 cells. Moreover, H3K9 acetylation at c-myc and CCND1 promoters was increased in individual MM patients after melphalan treatment. Western blotting revealed that these effects were accompanied by changes in c-MYC and cyclin D1 protein levels. Taken together, we showed that cytostatics significantly alter histone modification of tumor-related genes which is indispensable for understanding cancer therapies.
|N-Myc regulates expression of pluripotency genes in neuroblastoma including lif, klf2, klf4, and lin28b. |
Rebecca Cotterman,Paul S Knoepfler
PloS one 4 2009
myc genes are best known for causing tumors when overexpressed, but recent studies suggest endogenous myc regulates pluripotency and self-renewal of stem cells. For example, N-myc is associated with a number of tumors including neuroblastoma, but also plays a central role in the function of normal neural stem and precursor cells (NSC). Both c- and N-myc also enhance the production of induced pluripotent stem cells (iPSC) and are linked to neural tumor stem cells. The mechanisms by which myc regulates normal and neoplastic stem-related functions remain largely open questions. Here from a global, unbiased search for N-Myc bound genes using ChIP-chip assays in neuroblastoma, we found lif as a putative N-Myc bound gene with a number of strong N-Myc binding peaks in the promoter region enriched for E-boxes. Amongst putative N-Myc target genes in expression microarray studies in neuroblastoma we also found lif and three additional important embryonic stem cell (ESC)-related factors that are linked to production of iPSC: klf2, klf4, and lin28b. To examine the regulation of these genes by N-Myc, we measured their expression using neuroblastoma cells that contain a Tet-regulatable N-myc transgene (TET21N) as well as NSC with a nestin-cre driven N-myc knockout. N-myc levels closely correlated with the expression of all of these genes in neuroblastoma and all but lif in NSC. Direct ChIP assays also indicate that N-Myc directly binds the lif promoter. N-Myc regulates trimethylation of lysine 4 of histone H3 in the promoter of lif and possibly in the promoters of several other stem-related genes. Together these findings indicate that N-Myc regulates overlapping stem-related gene expression programs in neuroblastoma and NSC, supporting a novel model by which amplification of the N-myc gene may drive formation of neuroblastoma. They also suggest mechanisms by which Myc proteins more generally contribute to maintenance of pluripotency and self-renewal of ESC as well as to iPSC formation.Full Text Article
|Histone H3 modifications associated with differentiation and long-term culture of mesenchymal adipose stem cells. |
Agate Noer, Leif C Lindeman, Philippe Collas, Agate Noer, Leif C Lindeman, Philippe Collas, Agate Noer, Leif C Lindeman, Philippe Collas
Stem cells and development 18 725-36 2009
Long-term culture of mesenchymal stem cells leads to a loss of differentiation capacity, the molecular mechanism of which remains not understood. We show here that expansion of adipose stem cells (ASCs) to late passage (replicative senescence) is associated with promoter-specific and global changes in epigenetic histone modifications. In undifferentiated ASCs, inactive adipogenic and myogenic promoters are enriched in a repressive combination of trimethylated H3K4 (H3K4m3) and H3K27m3 in the absence of H3K9m3, a heterochromatin mark. Sequential chromatin immunoprecipitation assays indicate that H3K4m3 and H3K27m3 co-occupy a fraction of nucleosomes on some but not all lineage-specific promoters examined. However in cultured primary keratinocytes, adipogenic and myogenic promoters are enriched in trimethylated H3K4, K27, and K9, illustrating two distinct epigenetic states of inactive promoters related to potential for activation. H3K4m3 and H3K27m3 stably mark promoters during long-term ASC culture indicating that loss of differentiation capacity is not due to alterations in these histone modifications on these loci. Adipogenic differentiation in early passage results in H3K27 demethylation and H3K9 acetylation specifically on adipogenic promoters. On induction of differentiation in late passage, however, transcriptional upregulation is impaired, H3K27 trimethylation is maintained and H3K9 acetylation is inhibited on promoters. In addition, the polycomb proteins Ezh2 and Bmi1 are targeted to promoters. This correlates with global cellular Ezh2 increase and H3K9 deacetylation. Promoter targeting by Ezh2 and Bmi1 in late passage ASCs suggests the establishment of a polycomb-mediated epigenetic program aiming at repressing transcription.
|Topoisomerase IIbeta negatively modulates retinoic acid receptor alpha function: a novel mechanism of retinoic acid resistance. |
Suzan McNamara, Hongling Wang, Nessrine Hanna, Wilson H Miller
Molecular and cellular biology 28 2066-77 2008
Interactions between retinoic acid (RA) receptor alpha (RARalpha) and coregulators play a key role in coordinating gene transcription and myeloid differentiation. In patients with acute promyelocytic leukemia (APL), the RARalpha gene is fused with the promyelocytic leukemia (PML) gene via the t(15;17) translocation, resulting in the expression of a PML/RARalpha fusion protein. Here, we report that topoisomerase II beta (TopoIIbeta) associates with and negatively modulates RARalpha transcriptional activity and that increased levels of and association with TopoIIbeta cause resistance to RA in APL cell lines. Knockdown of TopoIIbeta was able to overcome resistance by permitting RA-induced differentiation and increased RA gene expression. Overexpression of TopoIIbeta in clones from an RA-sensitive cell line conferred resistance by a reduction in RA-induced expression of target genes and differentiation. Chromatin immunoprecipitation assays indicated that TopoIIbeta is bound to an RA response element and that inhibition of TopoIIbeta causes hyperacetylation of histone 3 at lysine 9 and activation of transcription. Our results identify a novel mechanism of resistance in APL and provide further insight to the role of TopoIIbeta in gene regulation and differentiation.Full Text Article
|Positive histone marks are associated with active transcription from a methylated ICSBP/IRF8 gene. |
Marina Tshuikina, Kenneth Nilsson, Fredrik Oberg
Gene 410 259-67 2008
Epigenetic modifications are critical for regulating many different aspects of normal cell biology and tumourigenesis. Gene expression may be epigenetically silenced by DNA-methylation and histone modifications, resulting in remodelling of chromatin into a repressed state. We performed DNA-methylation analysis of the ICSBP/IRF8 gene, a member of the IRF family of transcriptional regulators expressed in monocytic and lymphocytic cells, in human monoblastic U-937 cells. We found complete methylation of all 39 CpG positions located in a 308 bp sequence encompassing the proximal promoter and transcriptional start site of the ICSBP/IRF8 gene. However, strikingly, the ICSBP/IRF8 gene is still expressed. Chromatin Immuno-precipitation (ChIP) showed that RNA-Polymerase II was present at the major transcriptional start site. Investigating the histone modifications across the ICSBP/IRF8 gene we found the positive histone marks H3K9ac and H3K4me3 to be enriched at the promoter, whereas the level of H3K9me3 was low. This suggests that an active chromatin structure, indicated by histone H3 modifications and enrichment for RNA Pol II, can over-ride the silencing effect of DNA-methylation at the promoter, thereby permitting transcription of the ICSBP/IRF8 gene.
|Loss of RB compromises specific heterochromatin modifications and modulates HP1alpha dynamics. |
Hasan Siddiqui, Sejal R Fox, Ranjaka W Gunawardena, Erik S Knudsen
Journal of cellular physiology 211 131-7 2007
Heterochromatin domains are important for gene silencing, centromere organization, and genomic stability. These genomic domains are marked with specific histone modifications, heterochromatin protein 1 (HP1) binding and DNA methylation. The retinoblastoma tumor suppressor, RB mediates transcriptional repression and functionally interacts with a number of factors that are involved in heterochromatin biology including HP1, Suv39h1, DNMT1, and components of the SWI/SNF chromatin remodeling complex. To analyze the specific influence of RB loss on chromatin modification, mouse adult fibroblasts (MAFs) derived from Rb(loxP/loxP) mice were utilized to acutely knockout RB. In this setting, target genes of RB are deregulated. Additionally, changes in histone modifications were observed. Specifically, histone H4 lysine 20 trimethylation was absent from heterochromatin domains following loss of RB and there were changes in the relative levels of histone modifications between RB-proficient and deficient cells. While RB loss significantly altered the modifications associated with heterochromatin domains, these domains were readily identified and efficiently mediated the recruitment of HP1alpha. Kinetic analyses of HP1alpha within the heterochromatin domains present in RB-deficient cells indicated that loss of RB retarded HP1alpha dynamics, indicating that HP1alpha is paradoxically more tightly associated with heterochromatin in the absence of RB function. Combined, these analyses demonstrate that loss of RB has global effects on chromatin modifications and dynamics.
|Distinct patterns of histone methylation and acetylation in human interphase nuclei. |
M Skalníková, E Bártová, V Ulman, P Matula, D Svoboda, A Harnicarová, M Kozubek, S Kozubek
Physiological research / Academia Scientiarum Bohemoslovaca 56 797-806 2007
To study 3D nuclear distributions of epigenetic histone modifications such as H3(K9) acetylation, H3(K4) dimethylation, H3(K9) dimethylation, and H3(K27) trimethylation, and of histone methyltransferase Suv39H1, we used advanced image analysis methods, combined with Nipkow disk confocal microscopy. Total fluorescence intensity and distributions of fluorescently labelled proteins were analyzed in formaldehyde-fixed interphase nuclei. Our data showed reduced fluorescent signals of H3(K9) acetylation and H3(K4) dimethylation (di-me) at the nuclear periphery, while di-meH3(K9) was also abundant in chromatin regions closely associated with the nuclear envelope. Little overlapping (intermingling) was observed for di-meH3(K4) and H3(K27) trimethylation (tri-me), and for di-meH3(K9) and Suv39H1. The histone modifications studied were absent in the nucleolar compartment with the exception of H3(K9) dimethylation that was closely associated with perinucleolar regions which are formed by centromeres of acrocentric chromosomes. Using immunocytochemistry, no di-meH3(K4) but only dense di-meH3(K9) was found for the human acrocentric chromosomes 14 and 22. The active X chromosome was observed to be partially acetylated, while the inactive X was more condensed, located in a very peripheral part of the interphase nuclei, and lacked H3(K9) acetylation. Our results confirmed specific interphase patterns of histone modifications within the interphase nuclei as well as within their chromosome territories.
|Epigenetic reprogramming of OCT4 and NANOG regulatory regions by embryonal carcinoma cell extract. |
Christel T Freberg, John Arne Dahl, Sanna Timoskainen, Philippe Collas
Molecular biology of the cell 18 1543-53 2007
Analyses of molecular events associated with reprogramming somatic nuclei to pluripotency are scarce. We previously reported the reprogramming of epithelial cells by extract of undifferentiated embryonal carcinoma (EC) cells. We now demonstrate reprogramming of DNA methylation and histone modifications on regulatory regions of the developmentally regulated OCT4 and NANOG genes by exposure of 293T cells to EC cell extract. OCT4 and NANOG are transcriptionally up-regulated and undergo mosaic cytosine-phosphate-guanosine demethylation. OCT4 demethylation occurs as early as week 1, is enhanced by week 2, and is most prominent in the proximal promoter and distal enhancer. Targeted OCT4 and NANOG demethylation does not occur in 293T extract-treated cells. Retinoic acid-mediated differentiation of reprogrammed cells elicits OCT4 promoter remethylation and transcriptional repression. Chromatin immunoprecipitation analyses of lysines K4, K9, and K27 of histone H3 on OCT4 and NANOG indicate that primary chromatin remodeling determinants are acetylation of H3K9 and demethylation of dimethylated H3K9. H3K4 remains di- and trimethylated. Demethylation of trimethylated H3K9 and H3K27 also occurs; however, trimethylation seems more stable than dimethylation. We conclude that a central epigenetic reprogramming event is relaxation of chromatin at loci associated with pluripotency to create a conformation compatible with transcriptional activation.Full Text Article
|Transcriptional regulation of the human NRIP1/RIP140 gene by estrogen is modulated by dioxin signalling. |
Patrick Augereau, Eric Badia, Maryse Fuentes, Fanja Rabenoelina, Marine Corniou, Danièle Derocq, Patrick Balaguer, Vincent Cavailles
Molecular pharmacology 69 1338-46 2006
Receptor interacting protein 140 (RIP140) is a negative transcriptional regulator of nuclear hormone receptors that is required for the maintenance of energy homeostasis and ovulation. In this study, we investigated the mechanisms by which RIP140 expression is controlled by estrogens in breast cancer cells. We first analyzed by real time reverse transcription-polymerase chain reaction the regulation of RIP140 mRNA accumulation by estrogen receptor (ER) ligands in MCF-7 cells. We showed that the induction by estradiol (E2) was rapid and did not affect the apparent stability of the mRNA, suggesting a direct transcriptional regulation. To further study the underlying regulatory mechanisms, we then characterized the human RIP140 gene. We identified several noncoding exons with alternative splicing and localized the promoter region more than 100 kilobases upstream from the coding exon. Although we mapped a perfect consensus estrogen response element able to bind ERalpha in gel shift and in chromatin immunoprecipitation experiments, the effect of E2 on RIP140 gene transcription was very modest. This might result at least in part from the presence of an overlapping aryl hydrocarbon receptor (AhR) binding site, which interfered with the E2 response on both the transiently transfected reporter construct and the accumulation of the endogenous RIP140 mRNA. Altogether, our data indicate that the RIP140 gene exhibits a complex structure with several noncoding exons and supports transcriptional cross-talk and feedback involving the ERalpha and AhR nuclear receptors.
|NFATc2 and T-bet contribute to T-helper-cell-subset-specific regulation of IL-21 expression. |
Devangi S Mehta, Andrea L Wurster, Amy S Weinmann, Michael J Grusby
Proceedings of the National Academy of Sciences of the United States of America 102 2016-21 2005
T helper (Th) 2 cells selectively express IL-21 in addition to the classic Th2 cytokines IL-4, IL-5, and IL-13. In contrast to these clustered Th2 cell cytokine genes, the IL-21 gene resides on a different chromosome and is not coordinately regulated by the same locus control region that directs the expression of other Th2 cytokines. We demonstrate that the proximal promoter of IL-21 controls its Th-cell-subset-specific expression through the action of NFATc2 and T-bet. Whereas NFATc2 directly binds to and activates transcription of the IL-21 promoter in Th2 cells, T-bet represses IL-21 transcription by inhibiting the binding of NFATc2 to the promoter in Th1 cells. These data suggest that there are multiple mechanisms by which Th-cell-subset-specific cytokine genes are regulated.Full Text Article
|Family members p53 and p73 act together in chromatin modification and direct repression of alpha-fetoprotein transcription. |
Rutao Cui, Thi T Nguyen, Joseph H Taube, Sabrina A Stratton, Miriam H Feuerman, Michelle Craig Barton, Rutao Cui, Thi T Nguyen, Joseph H Taube, Sabrina A Stratton, Miriam H Feuerman, Michelle Craig Barton
The Journal of biological chemistry 280 39152-60 2005
Aberrant expression of the alpha-fetoprotein (AFP) gene is a diagnostic tumor marker of hepatocellular carcinoma. We find that AFP gene expression is repressed by the TP53 family member p73 during normal hepatic development and when p73alpha or p73beta is introduced into cultured hepatoma cells that express AFP. Transient co-transfection of p53 family members showed that p53 and transactivating (TA)-p73, but not TA-p63, repress endogenous AFP transcription additively or independently. p53-independent functions of p73 are further supported by delayed, p73-associated compensation of AFP repression during development of the p53-null mouse. Chromatin immunoprecipitation assays of normal and p53-null mouse liver tissue showed that TA-p73 binds at a previously identified p53 repressor site (-860/-830) within the distal promoter of AFP at a level equivalent to p53 in wild type liver, with increased binding of TA-p73 to chromatin in the absence of p53. Sequential chromatin immunoprecipitation analyses revealed that TA-p73 and p53 bind simultaneously to their shared regulatory site in wild type liver. Like the founding family member p53, TA-p73 represses AFP expression by chromatin structure alteration, targeting reduction of acetylated histone H3 lysine 9 and increased dimethylated histone H3 lysine 9 levels. However, chromatin-bound TA-p73 is associated with elevated di- and tri-methylated histone H3 lysine 4 levels in p53-null liver and hepatoma cells, concomitant with a reduced ability to repress transcription compared with p53.
|Tumor promoter arsenite stimulates histone H3 phosphoacetylation of proto-oncogenes c-fos and c-jun chromatin in human diploid fibroblasts. |
Li, Ji, et al.
J. Biol. Chem., 278: 13183-91 (2003) 2003
Although epidemiological studies have long established that inorganic arsenic is a potent human carcinogen, the underlying mechanisms are still poorly understood. Recent studies suggest that inorganic arsenic may act as a tumor promoter by perturbing key signaling transduction pathways. We have shown previously that arsenite can potently activate the mitogen-activated protein kinase cascades and induce the expression of proliferation-associated genes, including proto-oncogenes c-jun and c-fos. In order to elucidate further the molecular mechanisms underlying its tumor-promoting properties, we investigated the signaling events involved in arsenite-mediated induction of c-fos and c-jun. We found that induction of both c-fos and c-jun by arsenite can be substantially inhibited by the MEK- selective inhibitor U0126, suggesting that the ERK pathway is critically involved in their up-regulation. Interestingly, arsenite dramatically induced the phosphorylation and acetylation of histone H3 preceding the induction of mRNAs encoding c-fos and c-jun. Finally, chromatin immunoprecipitation assays revealed that arsenite treatment markedly induced the phosphorylation/acetylation of histone H3 associated with the c-fos and c-jun genes through an ERK-dependent pathway. Our results strongly suggest that arsenic-triggered alterations in chromatin structure perturb specific gene transcription, including that of proto-oncogenes c-jun and c-fos, and may thereby contribute to the carcinogenic process.
|Ultraviolet B-induced phosphorylation of histone H3 at serine 28 is mediated by MSK1. |
Zhong, S, et al.
J. Biol. Chem., 276: 33213-9 (2001) 2001
N-terminal tail phosphorylation of histone H3 plays an important role in gene expression, chromatin remodeling, and chromosome condensation. Phosphorylation of histone H3 at serine 10 was shown to be mediated by RSK2, mitogen- and stress-activated protein kinase-1 (MSK1), and mitogen-activated protein kinases depending on the specific stimulation or stress. Our previous study showed that mitogen-activated protein kinases MAP kinases are involved in ultraviolet B-induced phosphorylation of histone H3 at serine 28 (Zhong, S., Zhong, Z., Jansen, J., Goto, H., Inagaki, M., and Dong, Z., J. Biol. Chem. 276, 12932-12937). However, downstream effectors of MAP kinases remain to be identified. Here, we report that H89, a selective inhibitor of the nucleosomal response, totally inhibits ultraviolet B-induced phosphorylation of histone H3 at serine 28. H89 blocks MSK1 activity but does not inhibit ultraviolet B-induced activation of MAP kinases p70/85(S6K), p90(RSK), Akt, and protein kinase A. Furthermore, MSK1 markedly phosphorylated serine 28 of histone H3 and chromatin in vitro. Transfection experiments showed that an N-terminal mutant MSK1 or a C-terminal mutant MSK1 markedly blocked MSK1 activity. Compared with wild-type MSK1, cells transfected with N-terminal or C-terminal mutant MSK1 strongly blocked ultraviolet B-induced phosphorylation of histone H3 at serine 28 in vivo. These data illustrate that MSK1 mediates ultraviolet B-induced phosphorylation of histone H3 at serine 28.
|Regulation of global acetylation in mitosis through loss of histone acetyltransferases and deacetylases from chromatin. |
Kruhlak, M J, et al.
J. Biol. Chem., 276: 38307-19 (2001) 2001
Histone acetylation, a reversible modification of the core histones, is widely accepted to be involved in remodeling chromatin organization for genetic reprogramming. Histone acetylation is a dynamic process that is regulated by two classes of enzymes, the histone acetyltransferases (HATs) and histone deacetylases (HDACs). Although promoter-specific acetylation and deacetylation has received most of the recent attention, it is superimposed upon a broader acting and dynamic acetylation that profoundly affects many nuclear processes. In this study, we monitored this broader histone acetylation as cells enter and exit mitosis. In contrast to the hypothesis that HATs and HDACs remain bound to mitotic chromosomes to provide an epigenetic imprint for postmitotic reactivation of the genome, we observed that HATs and HDACs are spatially reorganized and displaced from condensing chromosomes as cells progress through mitosis. During mitosis, HATs and HDACs are unable to acetylate or deacetylate chromatin in situ despite remaining fully catalytically active when isolated from mitotic cells and assayed in vitro. Our results demonstrate that HATs and HDACs do not stably bind to the genome to function as an epigenetic mechanism of selective postmitotic gene activation. Our results, however, do support a role for spatial organization of these enzymes within the cell nucleus and their relationship to euchromatin and heterochromatin postmitotically in the reactivation of the genome.
|Technology development and implementation in the public health institutions: a strategic and pedagogical task for the learning organization. |
H S Wentzer
Studies in health technology and informatics 77 264-70 2000
The article presents two overall problem issues for IT in the health sector. A strategic problem: Which organization structure and which work routines should be enhanced by technology? A pedagogical problem: How does the personnel learn to think, act and express work through IT-tools? The suggested solution is to put technology development and implementation together within a theoretical frame for the learning organization where reciprocal demands and terms exist for both the IT-tools and the personnel. The personnel makes demands to which goals should be reached through technology and technology demands that the personnel is acquainted with hardware and software. As a communication system, technology offers new possibilities of realizing other forms of cooperation. The suggested solution is a synthesis between the strategy for organization development through technology and the implementation of IT as a learning process for the personnel. The synthesis between the mutual demands of the organization and its personnel provides for IT-tools which are in concordance with the principal values of the health practice rather than merely economic and management values. The analysis builds on a qualitative, in-depth investigation of IT-implementation in the secondary sector. The investigation indicated that the use of IT is a learning process which requires three types of knowledge and that continued IT-development in the organization requires a new fourth knowledge.
|Gcn5p is involved in the acetylation of histone H3 in nucleosomes |
Ruiz-García, A B, et al
FEBS Lett, 403:186-90 (1997) 1997
|Transcription-linked acetylation by Gcn5p of histones H3 and H4 at specific lysines. |
Kuo, M H, et al.
Nature, 383: 269-72 (1996) 1996
The yeast transcriptional adaptor, Gcn5p, is a catalytic subunit of a nuclear (type A) histone acetyltransferase linking histone acetylation to gene activation. Here we report that Gcn5p acetylates histones H3 and H4 non-randomly at specific lysines in the amino-terminal domains. Lysine 14 of H3 and lysines 8 and 16 of H4 are highly preferred acetylation sites for Gcn5p. We also demonstrate that lysine 9 is the preferred position of acetylation in newly synthesized yeast H3 in vivo. This finding, along with the fact that lysines 5 and 12 in H4 are predominant acetylation sites during chromatin assembly of many organisms, indicates that Gcn5p acetylates a distinct set of lysines that do not overlap with those sites characteristically used by type B histone acetyltransferases for histone deposition and chromatin assembly.
|Anti-acetyl-Histone H3 (Lys9) ChIP-Seq Analysis|