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  • ChIP (chromatin immunoprecipitation) analysis demonstrates co-ordinated binding of two transcription factors to the promoter of the p53 tumour-suppressor gene. 20446924

    p53 is a tumour-suppressor protein that plays a role in many cellular processes, including regulation of the cell cycle, DNA repair, transcriptional regulation of genes, chromosomal segregation, cell senescence and apoptosis. The protein's role as a transcription factor has shown that deregulated transcription, whether increased or decreased, has the potential to contribute to the formation of human cancers. It was previously reported that binding of two transcription factors, C/EBPbeta and RBP-Jkappa, to a regulatory site on the p53 promoter regulates its activity, in vitro, in a cell cycle-dependent manner. C/EBPbeta is a CCAAT enhancer-binding protein that is a member of the basic leucine zipper transcription factor (bZIP) family that plays an important role in mediating cell proliferation, differentiation and can also be involved in inflammatory responses, metabolism, cellular transformation, oncogene-induced senescence and tumorigenesis. RBP-Jkappa participates in the transcriptional regulation of target genes by interacting with the cytoplasmic domain of the Notch receptors. When RBP-Jkappa is released, transcriptional repression of its target genes occurs through the recruitment of co-repressor complexes and prevents transcription from occurring. Our reports, here and previously published, show that repression of p53 by RBP-Jkappa and activation of p53 by C/EBPbeta through differential binding of these two factors indicates a type of co-operative regulation in p53 expression. Here, we demonstrate through the use of chromatin immunoprecipitation (ChIP) assays that the co-ordinated binding of these two factors to the p53 promoter occurs in vivo and serves to regulate p53's activity during the cell cycle.
    Document Type:
    Reference
    Product Catalog Number:
    AB5790
    Product Catalog Name:
    Anti-RBP-Jk Antibody

  • Native chromatin immunoprecipitation (N-ChIP) and ChIP-Seq of Schistosoma mansoni: Critical experimental parameters. 19428675

    Histone modifications are important epigenetic marks that influence chromatin structure and consequently play a role in the control of eukaryotic transcription. Several histone modifying enzymes have been characterized in Schistosoma mansoni and it has been suggested that the regulation of gene transcription in schistosomes may require the action of these enzymes. However, the influence of chromatin structure on gene transcription in schistosomes has never been investigated. Chromatin immunoprecipitation (ChIP) is the technique of choice to study the relationship between histone modifications and gene expression. Although this technique has been widely used with cultured cells from model organisms and with many unicellular organisms, it remains challenging to apply this technique to non-conventional organisms that undergo complex life cycles. In this work, we describe a native ChIP procedure that is applicable to all the stages of the S. mansoni life cycle and does not require expensive equipment. Immunoprecipitated DNA was analysed on a whole-genome scale using massively parallel sequencing (ChIP-Sequencing or ChIP-Seq). We show that ChIP-Seq and conventional quantitative PCR deliver comparable results for a life-cycle regulated locus, smRHO, that encodes a guanine-protein coupled receptor. This is the first time that the ChIP-Seq procedure has been applied to a parasite. This technique opens new ways for analyzing epigenetic mechanisms in S. mansoni at a whole-genome scale and on the level of individual loci.
    Document Type:
    Reference
    Product Catalog Number:
    07-352
    Product Catalog Name:
    Anti-acetyl-Histone H3 (Lys9) Antibody

  • Large-scale chromatin immunoprecipitation with promoter sequence microarray analysis of the interaction of the NSs protein of Rift Valley fever virus with regulatory DNA ... 22896612

    Rift Valley fever virus (RVFV) is a highly pathogenic Phlebovirus that infects humans and ruminants. Initially confined to Africa, RVFV has spread outside Africa and presently represents a high risk to other geographic regions. It is responsible for high fatality rates in sheep and cattle. In humans, RVFV can induce hepatitis, encephalitis, retinitis, or fatal hemorrhagic fever. The nonstructural NSs protein that is the major virulence factor is found in the nuclei of infected cells where it associates with cellular transcription factors and cofactors. In previous work, we have shown that NSs interacts with the promoter region of the beta interferon gene abnormally maintaining the promoter in a repressed state. In this work, we performed a genome-wide analysis of the interactions between NSs and the host genome using a genome-wide chromatin immunoprecipitation combined with promoter sequence microarray, the ChIP-on-chip technique. Several cellular promoter regions were identified as significantly interacting with NSs, and the establishment of NSs interactions with these regions was often found linked to deregulation of expression of the corresponding genes. Among annotated NSs-interacting genes were present not only genes regulating innate immunity and inflammation but also genes regulating cellular pathways that have not yet been identified as targeted by RVFV. Several of these pathways, such as cell adhesion, axonal guidance, development, and coagulation were closely related to RVFV-induced disorders. In particular, we show in this work that NSs targeted and modified the expression of genes coding for coagulation factors, demonstrating for the first time that this hemorrhagic virus impairs the host coagulation cascade at the transcriptional level.
    Document Type:
    Reference
    Product Catalog Number:
    06-911

  • Chromatin remodeling gene AT-rich interactive domain-containing protein 1A suppresses gastric cancer cell proliferation by targeting PIK3CA and PDK1. 27323812

    The tumor suppressor gene AT-rich interactive domain-containing protein 1A (ARID1A) was frequently mutated in cancers. The modulation mechanism of ARID1A for PI3K/AKT signaling in gastric cancer (GC) remains elusive. Here, we found that depletion of endogenous ARID1A enhanced the in vitro proliferation, colony formation, cellular growth, nutrient uptake and in vivo xenograft tumor growth of GC cells. PI3K/AKT activation by ARID1A-silencing was profiled using a phospho-protein antibody array. The phosphorylation of PDK1, AKT, GSK3β and 70S6K, and the protein and mRNA expressions of PI3K and PDK1, were upregulated by ARID1A-silencing. Chromatin immunoprecipitation and luciferase reporter assay revealed that ARID1A-involved SWI/SNF complex inhibited PIK3CA and PDK1 transcription by direct binding to their promoters. Serial deletion mutation analyses revealed that the ARID1A central region containing the HIC1-binding domain, but not the ARID DNA-binding domain and the C-terminal domain, was essential for the inhibition of GC cell growth, PI3K/AKT pathway phosphorylation and its transcriptional modulation activity of PIK3CA and PDK1. The proliferation, cellular growth and glucose consumption of ARID1A-deficient GC cells were efficiently prohibited by allosteric inhibitors mk2206 and LY294002, which targeting AKT and PI3K, respectively. Both inhibitors also downregulated the phosphorylation of PI3K/AKT pathway in ARID1A-deficient GC cells. Such cells were sensitized to the treatment of LY294002, and AT7867, another inhibitor of AKT and p70S6K. The administration of LY294002 alone inhibited the in vivo growth of ARID1A- deficient GC cells in mouse xenograft model. Our study provides a novel insight into the modulatory function and mechanism of ARID1A in PI3K/AKT signaling in GC.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™

  • Chromatin interaction analysis using paired-end tag sequencing. 20069536

    Chromatin Interaction Analysis using Paired-End Tag sequencing (ChIA-PET) is a technique developed for large-scale, de novo analysis of higher-order chromatin structures. Cells are treated with formaldehyde to cross-link chromatin interactions, DNA segments bound by protein factors are enriched by chromatin immunoprecipitation, and interacting DNA fragments are then captured by proximity ligation. The Paired-End Tag (PET) strategy is applied to the construction of ChIA-PET libraries, which are sequenced by high-throughput next-generation sequencing technologies. Finally, raw PET sequences are subjected to bioinformatics analysis, resulting in a genome-wide map of binding sites and chromatin interactions mediated by the protein factor under study. This unit describes ChIA-PET for genome-wide analysis of chromatin interactions in mammalian cells, with the application of Roche/454 and Illumina sequencing technologies.
    Document Type:
    Reference
    Product Catalog Number:
    04-745
    Product Catalog Name:
    Anti-trimethyl-Histone H3 (Lys4) Antibody, clone MC315, rabbit monoclonal

  • Cocaine-induced chromatin remodeling increases brain-derived neurotrophic factor transcription in the rat medial prefrontal cortex, which alters the reinforcing efficacy ... 20810894

    Cocaine self-administration alters patterns of gene expression in the brain that may underlie cocaine-induced neuronal plasticity. In the present study, male Sprague Dawley rats were allowed to self-administer cocaine (0.25 mg/infusion) 2 h/d for 14 d, followed by 7 d of forced abstinence. Compared with yoked saline control rats, cocaine self-administration resulted in increased brain-derived neurotrophic factor (BDNF) protein levels in the rat medial prefrontal cortex (mPFC). To examine the functional relevance of this finding, cocaine self-administration maintained under a progressive ratio schedule of reinforcement was assessed after short hairpin RNA-induced suppression of BDNF expression in the mPFC. Decreased BDNF expression in the mPFC increased the cocaine self-administration breakpoint. Next, the effect of cocaine self-administration on specific BDNF exons was assessed; results revealed selectively increased BDNF exon IV-containing transcripts in the mPFC. Moreover, there were significant cocaine-induced increases in acetylated histone H3 (AcH3) and phospho-cAMP response element binding protein (pCREB) association with BDNF promoter IV. In contrast, there was decreased methyl-CpG-binding protein 2 (MeCP2) association with BDNF promoter IV in the mPFC of rats that previously self-administered cocaine. Together, these results indicate that cocaine-induced increases in BDNF promoter IV transcript in the mPFC are driven by increased binding of AcH3 and pCREB as well as decreased MeCP2 binding at this BDNF promoter. Collectively, these results indicate that cocaine self-administration remodels chromatin in the mPFC, resulting in increased expression of BDNF, which appears to represent a compensatory neuroadaptation that reduces the reinforcing efficacy of cocaine.
    Document Type:
    Reference
    Product Catalog Number:
    AB1779
    Product Catalog Name:
    Anti-Brain Derived Neurotrophic Factor Antibody

  • Chromatin modulation at the FLO11 promoter of Saccharomyces cerevisiae by HDAC and Swi/Snf complexes. 22542969

    Cell adhesion and biofilm formation are critical processes in the pathogenicity of fungi and are mediated through a family of adhesin proteins conserved throughout yeasts and fungi. In Saccharomyces cerevisiae, Flo11 is the main adhesin involved in cell adhesion and biofilm formation, making the study of its function and regulation in this nonpathogenic budding yeast highly relevant. The S. cerevisiae FLO11 gene is driven by a TATA-box-containing promoter that is regulated through one of the longest regulatory upstream regions (3 kb) in yeast. We reported recently that two chromatin cofactor complexes, the Rpd3L deacetylase and the Swi/Snf chromatin-remodeling complexes, contribute significantly to the regulation of FLO11. Here, we analyze directly how these complexes impact on FLO11 promoter chromatin structure and dissect further the interplay between histone deacetylases, chromatin remodeling, and the transcriptional repressor Sfl1. We show that the regulation of chromatin structure represents an important layer of control in the highly complex regulation of the FLO11 promoter.
    Document Type:
    Reference
    Product Catalog Number:
    06-866
    Product Catalog Name:
    Anti-acetyl-Histone H4 Antibody

  • Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions. 19804873

    We established a novel method, insertional chromatin immunoprecipitation (iChIP), for isolation of specific genomic regions. In iChIP, specific genomic domains are immunoprecipitated with antibody against a tag, which is fused to the DNA-binding domain of an exogenous DNA-binding protein, whose recognition sequence is inserted into the genomic domains of interest. The iChIP method will be a useful tool for dissecting chromatin structure of genomic region of interest.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Stwl modifies chromatin compaction and is required to maintain DNA integrity in the presence of perturbed DNA replication. 19056684

    Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent accumulation of this type of DNA damage that coincides with hampered DNA synthesis. Here, we report the identification of stonewall (stwl) as a novel hydroxyurea-hypersensitive mutant. We demonstrate that Stwl is required to prevent accumulation of DNA damage induced by hydroxyurea; yet, Stwl is not involved in S/M checkpoint regulation. We show that Stwl is a heterochromatin-associated protein with transcription-repressing capacities. In stwl mutants, levels of trimethylated H3K27 and H3K9 (two hallmarks of silent chromatin) are decreased. Our data provide evidence for a Stwl-dependent epigenetic mechanism that is involved in the maintenance of the normal balance between euchromatin and heterochromatin and that is required to prevent accumulation of DNA damage in the presence of DNA replication stress.
    Document Type:
    Reference
    Product Catalog Number:
    17-622
    Product Catalog Name:
    ChIPAb+ Trimethyl-Histone H3 (Lys27) - ChIP Validated Antibody and Primer Set

  • A fast carrier chromatin immunoprecipitation method applicable to microdissected tissue samples. 18502516

    Transcriptional regulation studies of CNS neurons are complicated by both cellular diversity and plasticity. Microdissection of specific functionally related populations of neurons can greatly reduce these issues, but typically excludes the use of many technologies due to tissue requirements, such as Chromatin Immunoprecipitation (ChIP), a powerful tool for studying in vivo protein-DNA interactions. We have developed a fast carrier ChIP (Fast CChIP) method for analyzing specific in vivo transcription factor-DNA interactions in as little as 0.2 mm(3) brain tissue. Using an antibody against phosphorylated cyclic-AMP response element binding (CREB) protein, we confirmed phospho-CREB (pCREB) binding at the c-fos gene promoter. Then we further demonstrated the applicability of Fast CChIP in determining hypertension-induced pCREB binding at the c-fos gene promoter in the rat nucleus tractus solitarius (NTS), confirming CREB's role in mediating hypertension-induced c-fos expression. This method will be broadly applicable to individual brain nucleus and biopsy/surgical samples.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple