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Histone H4


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  • Efficient organic monoliths prepared by γ-radiation induced polymerization in the evaluation of histone deacetylase inhibitors by capillary(nano)-high performance liquid ... 21561626

    New monolithic HPLC columns were prepared by γ-radiation-triggered polymerization of hexyl methacrylate and ethylene glycol dimethacrylate monomers in the presence of porogenic solvents. Polymerization was carried out directly within capillary (250-200μm I.D.) and nano (100-75μm I.D.) fused-silica tubes yielding highly efficient columns for cap(nano)-LC applications. The columns were applied in the complete separation of core (H2A, H2B, H3, and H4) and linker (H1) histones under gradient elution with UV and/or electrospray ionization (ESI) ion trap mass spectrometry (MS) detections. Large selectivity towards H1, H2A-1, H2A-2, H2B, H3-1, H3-2 and H4 histones and complete separation were obtained within 8min time windows, using fast gradients and very high linear flow velocities, up to 11mm/s for high throughput applications. The method developed was the basis of a simple and efficient protocol for the evaluation of post-translational modifications (PTMs) of histones from NCI-H460 human non-small-cell lung cancer (NSCLC) and HCT-116 human colorectal carcinoma cells. The study was extended to monitoring the level of histone acetylation after inhibition of Histone DeACetylase (HDAC) enzymes with suberoylanilide hydroxamic acid (SAHA), the first HDAC inhibitor approved by the FDA for cancer therapy. Attractive features of our cap(nano)-LC/MS approach are the short analysis time, the minute amount of sample required to complete the whole procedure and the stability of the polymethacrylate-based columns. A lab-made software package ClustMass was ad hoc developed and used to elaborate deconvoluted mass spectral data (aligning, averaging, clustering) and calculate the potency of HDAC inhibitors, expressed through a Relative half maximal Inhibitory Concentration parameter, namely R_IC(50) and an averaged acetylation degree.Copyright © 2011 Elsevier B.V. All rights reserved.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Genome-wide H4 K16 acetylation by SAS-I is deposited independently of transcription and histone exchange. 21908408

    The MYST HAT Sas2 is part of the SAS-I complex that acetylates histone H4 lysine 16 (H4 K16Ac) and blocks the propagation of heterochromatin at the telomeres of Saccharomyces cerevisiae. In this study, we investigated Sas2-mediated H4 K16Ac on a genome-wide scale. Interestingly, H4 K16Ac loss in sas2Δ cells outside of the telomeric regions showed a distinctive pattern in that there was a pronounced decrease of H4 K16Ac within the majority of open reading frames (ORFs), but little change in intergenic regions. Furthermore, regions of low histone H3 exchange and low H3 K56 acetylation showed the most pronounced loss of H4 K16Ac in sas2Δ, indicating that Sas2 deposited this modification on chromatin independently of histone exchange. In agreement with the effect of Sas2 within ORFs, sas2Δ caused resistance to 6-azauracil, indicating a positive effect on transcription elongation in the absence of H4 K16Ac. In summary, our data suggest that Sas2-dependent H4 K16Ac is deposited into chromatin independently of transcription and histone exchange, and that it has an inhibitory effect on the ability of PolII to travel through the body of the gene.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-329
    Nombre del producto:
    Anti-acetyl-Histone H4 (Lys16) Antibody

  • MOF and H4 K16 acetylation play important roles in DNA damage repair by modulating recruitment of DNA damage repair protein Mdc1. 20837706

    MOF (MYST1) is the major enzyme to catalyze acetylation of histone H4 lysine 16 (K16) and is highly conserved through evolution. Using a conditional knockout mouse model and the derived mouse embryonic fibroblast cell lines, we showed that loss of Mof led to a global reduction of H4 K16 acetylation, severe G(2)/M cell cycle arrest, massive chromosome aberration, and defects in ionizing radiation-induced DNA damage repair. We further showed that although early DNA damage sensing and signaling by ATM were normal in Mof-null cells, the recruitment of repair mediator protein Mdc1 and its downstream signaling proteins 53bp1 and Brca1 to DNA damage foci was completely abolished. Mechanistic studies suggested that Mof-mediated H4 K16 acetylation and an intact acidic pocket on H2A.X were essential for the recruitment of Mdc1. Removal of Mof and its associated proteins phenocopied a charge-neutralizing mutant of H2A.X. Given the well-characterized H4-H2A trans interactions in regulating higher-order chromatin structure, our study revealed a novel chromatin-based mechanism that regulates the DNA damage repair process.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Histone exchange activity and its correlation with histone acetylation status in porcine oocytes. 21239526

    In mammalian oocytes, histone H3 and histone H4 (H4) in the chromatin are highly acetylated at the germinal vesicle (GV) stage, and become globally deacetylated after GV breakdown (GVBD). Although nuclear core histones can be exchanged by cytoplasmic free histones in somatic cells, it remains unknown whether this is also the case in mammalian oocytes. In this study, we examined the histone exchange activity in maturing porcine oocytes before and after GVBD, and investigated the correlations between this activity and both the acetylation profile of the H4 N-terminal tail and the global histone acetylation level in the chromatin. We injected Flag-tagged H4 (H4-Flag) mRNA into GV oocytes, and found that the Flag signal was localized to the chromatin. We next injected mRNAs of mutated H4-Flag, which lack all acetylation sites and the whole N-terminal tail, and found that the H4 N-terminal tail and its modification were not necessary for histone incorporation into chromatin. Despite the lack of acetylation sites, the mutated H4-Flag mRNA injection did not decrease the acetylation level on the chromatin, indicating that the histone exchange occurs partially in the GV chromatin. In contrast to GV oocytes, the Flag signal was not detected on the chromatin after the injection of H4-Flag protein into the second meiotic metaphase oocytes. These results suggest that histone exchange activity changes during meiotic maturation in porcine oocytes, and that the acetylation profile of the H4 N-terminal tail has no effect on histone incorporation into chromatin and does not affect the global level of histone acetylation in it.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AP181R
    Nombre del producto:
    Goat Anti-Mouse IgG Antibody, Rhodamine conjugate, Species Adsorbed

  • Expression, purification, and structural characterization of human histone H4. 11922758

    Recombinant human histone H4 (hH4) was produced in milligrams quantities in Escherichia coli, without altering the codons of the original cDNA sequence. The hH4 cDNA was subcloned into the pQE30 expression vector, in frame with a sequence encoding an N-terminal stretch of six histidine residues. Purification to electrophoretic homogeneity was obtained by nickel-chelating chromatography, followed by gel filtration. The final yield of the entire expression and purification process was about 1 mg of pure histone H4 per liter of bacterial culture. SDS-PAGE analysis showed for the recombinant H4 a molecular weight corresponding to the expected one (12,535 Da). Circular dichroism spectroscopy was used to estimate the secondary structural composition of recombinant histone, when it is isolated from the physiological core particle. It was observed that under these conditions histone H4 exhibits an altered secondary conformation. In order to induce the recombinant histone to assume a conformation more similar to the one measured when it is organized inside the nucleosome, we resuspended it in buffers at increasing ionic strengths and in the presence of different concentrations of trifluoroethanol. We tried also to mimic the physiological situation of histone H4 by adding an equimolar amount of a commercial DNA to the protein solution. Finally, an estimation of protein thermal stability was evaluated by spectropolarimetry.
    Tipo de documento:
    Referencia
    Referencia del producto:
    14-697
    Nombre del producto:
    Histone H4 Protein, human recombinant, 1 mg

  • Purification and characterization of C-terminal truncated forms of histone H2A in monocytic THP-1 cells. 16979371

    Histones are key components of chromatin. We investigated histone H2A-immunoreactive proteins in acute monocytic leukemia THP-1 cells using three polyclonal antibodies raised against peptides corresponding to distinct regions of H2A. Two unknown immunoreactive proteins (9- and 12-kDa proteins), H2A (14kDa) and ubiquitinated H2A (23kDa) were found in the cell lysates prepared by immediate direct addition of SDS-PAGE sample buffer to the cells as well as in the nuclear and chromatin fractions. However, they were not found in the cytoplasmic fraction. The unknown proteins were successfully purified by immunoaffinity chromatography from the cell nucleus extract and identified as 9-kDa H2A(1-87) and 12-kDa H2A(1-114), suggesting that both were produced by limited proteolysis of intact H2A(1-129). The truncated forms of H2A probably persisted as chromatin constituents, since the stability of H2A(1-87) in the chromatin fraction was sensitive to treatment with micrococcal nuclease, and H2A(1-114) was solubilized with lower ionic strength from the chromatin fraction obtained by micrococcal nuclease treatment. Truncated H2A proteins in THP-1 cells were transiently increased in amount by short-term treatment with phorbol 12-myristate 13-acetate or all-trans-retinoic acid, both of which induce macrophage-like differentiation. Furthermore, these increases were suppressed by preceding treatment with carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132) but not with carbobenzoxy-l-isoleucyl-gamma-t-butyl-l-glutamyl-l-alanyl-l-leucinal (PSI), both of which are generally known as proteasome inhibitors. Our results suggest that histone H2A is cleaved at least at two sites by protease(s) that remain obscure, and might affect chromatins in the early stage of THP-1 cell differentiation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Genome-wide mapping of histone H4 serine-1 phosphorylation during sporulation in Saccharomyces cerevisiae. 20375100

    We previously showed that histone H4 serine-1 phosphorylation (H4S1ph) is evolutionarily conserved during gametogenesis, and contributes to post-meiotic nuclear compaction and to full completion of sporulation in the yeast Saccharomyces cerevisiae. Previous studies showed that H4S1ph and another modification of the same histone, H4 acetylation (H4ac), do not occur together and have opposing roles during DNA double-strand break (DSB) repair. In this study, we investigated the relationship between these marks during yeast sporulation. H4S1ph and H4ac co-exist globally during later stages of sporulation, in contrast to DSB repair. Genome-wide mapping during sporulation reveals accumulation of both marks over promoters of genes. Prevention of H4S1ph deposition delays the decline in transcription that normally occurs during spore maturation. Taken together, our results indicate that H4S1ph deposition reinforces reduced transcription that coincides with full spore compaction, without disrupting the local acetylation signature. These studies indicate distinctive features of a histone H4 modification marking system during sporulation compared with DSB repair.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • The G2/M regulator histone demethylase PHF8 is targeted for degradation by the anaphase-promoting complex containing CDC20. 23979597

    Monomethylated histone H4 lysine 20 (H4K20me1) is tightly regulated during the cell cycle. The H4K20me1 demethylase PHF8 transcriptionally regulates many cell cycle genes and is therefore predicted to play key roles in the cell cycle. Here, we show that PHF8 protein levels are the highest during G2 phase and mitosis, and we found PHF8 protein stability to be regulated by the ubiquitin-proteasome system. Purification of the PHF8 complex led to the identification of many subunits of the anaphase-promoting complex (APC) associated with PHF8. We showed that PHF8 interacts with the CDC20-containing APC (APC(cdc20)) primarily during mitosis. In addition, we defined a novel, KEN- and D-box-independent, LXPKXLF motif on PHF8 that is required for binding to CDC20. Through various in vivo and in vitro assays, we demonstrate that mutations of the LXPKXLF motif abrogate polyubiquitylation of PHF8 by the APC. APC substrates are typically cell cycle regulators, and consistent with this, the loss of PHF8 leads to prolonged G2 phase and defective mitosis. Furthermore, we provide evidence that PHF8 plays an important role in transcriptional activation of key G2/M genes during G2 phase. Taken together, these findings suggest that PHF8 is regulated by APC(cdc20) and plays an important role in the G2/M transition.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • The fission yeast inhibitor of growth (ING) protein Png1p functions in response to DNA damage. 20299455

    In budding yeast and human cells, ING (inhibitor of growth) tumor suppressor proteins play important roles in response to DNA damage by modulating chromatin structure through collaborating with histone acetyltransferase or histone deacetylase complexes. However, the biological functions of ING family proteins in fission yeast are poorly defined. Here, we report that Png1p, a fission yeast ING homolog protein, is required for cell growth under normal and DNA-damaged conditions. Png1p was further confirmed to regulate histone H4 acetylation through collaboration with the MYST family histone acetyltransferase 1 (Mst1). Additionally, both fission yeast PNG1 and MST1 can functionally complement their budding yeast correspondence homologs YNG2 and ESA1, respectively. These results suggest that ING proteins in fission yeast might also conserve function, similar to ING proteins in budding yeast and human cells. We also showed that decreased acetylation in Deltapng1 cells resulted in genome-wide down-regulation of 756 open reading frames, including the central DNA repair gene RAD22. Overexpression of RAD22 partially rescued the png1 mutant phenotype under both normal and DNA-damaged conditions. Furthermore, decreased expression of RAD22 in Deltapng1 cells was confirmed to be caused by decreased H4 acetylation at its promoter. Altogether, these results indicate that Png1p is required for histone H4 acetylation and functions upstream of RAD22 in the DNA damage response pathway.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Histone H4 lysine 20 mono- and tri-methylation define distinct biological processes in SV40 minichromosomes. 20404477

    The methylation profile of histone h4 on lysine 20 in sV40 chromatin during an infection was investigated using ChIp analyses with antibodies to monomethyl (h4K20me1), dimethyl (h4K20me2), and trimethyl (h4K20me3) histone h4. h4K20me1 was found in late-transcribing, uncoating, encapsidating and replicating minichromosomes as well as in the sV40 chromatin present in virions. Its prevalence was greatest in virions and least in minichromosomes present between 4 and 24 hours post-infection. In contrast, h4K20me2 did not appear to be present and h4K20me3 appeared to be present only in minichromosomes obtained 30 minutes post-infection. The presence of h4K20me1 late in infection in replicating minichromosomes and its relative enrichment in virions suggested that it played a role in the encapsidation process. In contrast, the presence of h4K20me3 at the earliest stages of the infection and its subsequent relatively rapid loss along with sV40 chromatin suggested that it was functioning during the uncoating process
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-463
    Nombre del producto:
    Anti-trimethyl-Histone H4 (Lys20) Antibody