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  • Protocol: methodology for chromatin immunoprecipitation (ChIP) in Chlamydomonas reinhardtii. 22050920

    We report on a detailed chromatin immunoprecipitation (ChIP) protocol for the unicellular green alga Chlamydomonas reinhardtii. The protocol is suitable for the analysis of nucleosome occupancy, histone modifications and transcription factor binding sites at the level of mononucleosomes for targeted and genome-wide studies. We describe the optimization of conditions for crosslinking, chromatin fragmentation and antibody titer determination and provide recommendations and an example for the normalization of ChIP results as determined by real-time PCR.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • The human histone gene expression regulator HBP/SLBP is required for histone and DNA synthesis, cell cycle progression and cell proliferation in mitotic cells. 15546920

    Histone proteins are essential for chromatin formation, and histone gene expression is coupled to DNA synthesis. In metazoans, the histone RNA binding protein HBP/SLBP is involved in post-transcriptional control of histone gene expression. In vitro assays have demonstrated that human HBP/SLBP is involved in histone mRNA 3' end formation and translation. We have inhibited human HBP/SLBP expression by RNA interference to determine its function during the mitotic cell cycle. Inhibition of HBP/SLBP expression resulted in the inhibition of histone gene expression and DNA synthesis, the inhibition of cell cycle progression in S phase and the inhibition of cell proliferation. These findings indicate that human HBP/SLBP is essential for the coordinate synthesis of DNA and histone proteins and is required for progression through the cell division cycle.
    Tipo de documento:
    Referencia
    Referencia del producto:
    06-138
    Nombre del producto:
    Anti-Cyclin A Antibody

  • BTG1 regulates glucocorticoid receptor autoinduction in acute lymphoblastic leukemia. 20354172

    Resistance to glucocorticoids (GCs) is a major clinical problem in the treatment of acute lymphoblastic leukemia (ALL), but the underlying mechanisms are not well understood. Although mutations in the glucocorticoid receptor (GR) gene can give rise to therapy resistance in vitro, acquired somatic mutations in the GR are rarely encountered in patients. Here we report that the protein encoded by the BTG1 gene, which is frequently deleted in (pediatric) ALL, is a key determinant of GC responsiveness. Using RNA interference, we show that loss of BTG1 expression causes GC resistance both by decimating GR expression and by controlling GR-mediated transcription. Conversely, reexpression of BTG1 restores GC sensitivity by potentiating GC-induced GR expression, a phenomenon known as GR autoinduction. In addition, the arginine methyltransferase PRMT1, a BTG1-binding partner and transcriptional coactivator, is recruited to the GR gene promoter in a BTG1-dependent manner. These results implicate the BTG1/PRMT1 complex in GR-mediated gene expression and reveal that deregulation of a nuclear receptor coactivator complex can give rise to GC resistance. Further characterization of this complex as part of the GR regulatory circuitry could offer novel opportunities for improving the efficacy of GC-based therapies in ALL and other hematologic malignancies.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-404
    Nombre del producto:
    Anti-PRMT1 Antibody

  • SWI/SNF-Brg1 regulates self-renewal and occupies core pluripotency-related genes in embryonic stem cells. 19056910

    The SWI/SNF-Brg1 chromatin remodeling protein plays critical roles in cell-cycle control and differentiation through regulation of gene expression. Loss of Brg1 in mice results in early embryonic lethality, and recent studies have implicated a role for Brg1 in somatic stem cell self-renewal and differentiation. However, little is known about Brg1 function in preimplantation embryos and embryonic stem (ES) cells. Here we report that Brg1 is required for ES cell self-renewal and pluripotency. RNA interference-mediated knockdown of Brg1 in blastocysts caused aberrant expression of Oct4 and Nanog. In ES cells, knockdown of Brg1 resulted in phenotypic changes indicative of differentiation, downregulation of self-renewal and pluripotency genes (e.g., Oct4, Sox2, Sall4, Rest), and upregulation of differentiation genes. Using genome-wide promoter analysis (chromatin immunoprecipitation) we found that Brg1 occupied the promoters of key pluripotency-related genes, including Oct4, Sox2, Nanog, Sall4, Rest, and Polycomb group (PcG) proteins. Moreover, Brg1 co-occupied a subset of Oct4, Sox2, Nanog, and PcG protein target genes. These results demonstrate an important role for Brg1 in regulating self-renewal and pluripotency in ES cells.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • The role of HIRA and maternal histones in sperm nucleus decondensation in the gibel carp and color crucian carp. 21337451

    The histone H3.3 chaperone HIRA is essential for chromatin assembly during male pronucleus formation in Drosophila. However, the role of HIRA during fertilization in vertebrates remains unclear. The gibel carp (Carassius auratus gibelio) is a unique gynogenetic crucian carp (gyno-carp). Heterologous sperm nuclei cannot decondense when incorporated in the egg, thus the eggs produce a clonal lineage of all females by typical gynogenesis. In contrast, after entering the egg, homologous sperm can undergo decondensation and sexual reproduction is activated, which may produce both female and male offspring. Therefore, this fish is a useful model for studying the mechanisms of fertilization. Herein, we first compared HIRA expression during embryogenesis between gyno-carp and the gonochoristic color crucian carp (Carassius auratus; gono-carp). In gono-carp, a dramatic reduction of HIRA protein occurs shortly after fertilization, whereas HIRA protein is consistently expressed during embryogenesis of gyno-carp. Next, we used immunodepletion and an in vitro sperm decondensation system, and found that complete removal of HIRA inhibited sperm decondensation in both of the fish. Immunofluorescence localization showed that in the condensed sperm nuclei of gono-carp incubated in gyno-carp egg extracts, HIRA was detected, but neither the histone H2A variant H2af1o nor acetylated histone H4 was observed. These results suggest that HIRA may be a critical factor required for sperm nucleus decondensation, while the defect in deposition of some maternal histones in the sperm nucleus could be one reason why heterologous sperm cannot decondense in the gibel carp egg.Copyright © 2011 Wiley-Liss, Inc.
    Tipo de documento:
    Referencia
    Referencia del producto:
    06-598
    Nombre del producto:
    Anti-acetyl-Histone H4 Antibody

  • Comparative analysis of mitosis-specific antibodies for bulk purification of mitotic populations by fluorescence-activated cell sorting. 24502799

    Mitosis entails complex chromatin changes that have garnered increasing interest from biologists who study genome structure and regulation-fields that are being advanced by high-throughput sequencing (Seq) technologies. The application of these technologies to study the mitotic genome requires large numbers of highly pure mitotic cells, with minimal contamination from interphase cells, to ensure accurate measurement of phenomena specific to mitosis. Here, we optimized a fluorescence-activated cell sorting (FACS)-based method for isolating formaldehyde-fixed mitotic cells-at virtually 100% mitotic purity and in quantities sufficient for high-throughput genomic studies. We compared several commercially available antibodies that react with mitosis-specific epitopes over a range of concentrations and cell numbers, finding antibody MPM2 to be the most robust and cost-effective.
    Tipo de documento:
    Referencia
    Referencia del producto:
    05-368
    Nombre del producto:
    Anti-phospho-Ser/Thr-Pro MPM-2 Antibody

  • Asymmetrically modified nucleosomes. 23021224

    Mononucleosomes, the basic building blocks of chromatin, contain two copies of each core histone. The associated posttranslational modifications regulate essential chromatin-dependent processes, yet whether each histone copy is identically modified in vivo is unclear. We demonstrate that nucleosomes in embryonic stem cells, fibroblasts, and cancer cells exist in both symmetrically and asymmetrically modified populations for histone H3 lysine 27 di/trimethylation (H3K27me2/3) and H4K20me1. Further, we obtained direct physical evidence for bivalent nucleosomes carrying H3K4me3 or H3K36me3 along with H3K27me3, albeit on opposite H3 tails. Bivalency at target genes was resolved upon differentiation of ES cells. Polycomb repressive complex 2-mediated methylation of H3K27 was inhibited when nucleosomes contain symmetrically, but not asymmetrically, placed H3K4me3 or H3K36me3. These findings uncover a potential mechanism for the incorporation of bivalent features into nucleosomes and demonstrate how asymmetry might set the stage to diversify functional nucleosome states.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-449
    Nombre del producto:
    Anti-trimethyl-Histone H3 (Lys27) 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.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-352
    Nombre del producto:
    Anti-acetyl-Histone H3 (Lys9) Antibody

  • Jmjd3 activates Mash1 gene in RA-induced neuronal differentiation of P19 cells. 20506217

    Covalent modifications of histone tails have fundamental roles in chromatin structure and function. Tri-methyl modification on lysine 27 of histone H3 (H3K27me3) usually correlates with gene repression that plays important roles in cell lineage commitment and development. Mash1 is a basic helix-loop-helix regulatory protein that plays a critical role in neurogenesis, where it expresses as an early marker. In this study, we have shown a decreased H3K27me3 accompanying with an increased demethylase of H3K27me3 (Jmjd3) at the promoter of Mash1 can elicit a dramatically efficient expression of Mash1 in RA-treated P19 cells. Over-expression of Jmjd3 in P19 cells also significantly enhances the RA-induced expression and promoter activity of Mash1. By contrast, the mRNA expression and promoter activity of Mash1 are significantly reduced, when Jmjd3 siRNA or dominant negative mutant of Jmjd3 is introduced into the P19 cells. Chromatin immunoprecipitation assays show that Jmjd3 is efficiently recruited to a proximal upstream region of Mash1 promoter that is overlapped with the specific binding site of Hes1 in RA-induced cells. Moreover, the association between Jmjd3 and Hes1 is shown in a co-Immunoprecipitation assay. It is thus likely that Jmjd3 is recruited to the Mash1 promoter via Hes1. Our results suggest that the demethylase activity of Jmjd3 and its mediator Hes1 for Mash1 promoter binding are both required for Jmjd3 enhanced efficient expression of Mash1 gene in the early stage of RA-induced neuronal differentiation of P19 cells.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB15582

  • 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.
    Tipo de documento:
    Referencia
    Referencia del producto:
    04-745
    Nombre del producto:
    Anti-trimethyl-Histone H3 (Lys4) Antibody, clone MC315, rabbit monoclonal