Key Specifications Table
|Safety Information according to GHS|
|Storage and Shipping Information|
|Material Size||1 kit|
|Material Package||Kit capacity: 22 assays|
Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit SDS
|Reference overview||Application||Species||Pub Med ID|
|The inhibitor of histone deacetylases sodium butyrate enhances the cytotoxicity of mitomycin C.|
Anastas Gospodinov,Stanislava Popova,Ivelina Vassileva,Boyka Anachkova
Molecular cancer therapeutics 11 2012
The use of histone deacetylase inhibitors has been proposed as a promising approach to increase the cell killing effect of DNA damage-inducing drugs in chemotherapy. However, the molecular mechanism of their action remains understudied. In the present article, we have assessed the effect of the histone deacetylase inhibitor sodium butyrate on the DNA damage response induced by the crosslinking agent mitomycin C. Sodium butyrate increased mitomycin C cytotoxicity, but did not impair the repair pathways required to remove mitomycin C-induced lesions as neither the rate of nucleotide excision repair nor the homologous recombination repair rate were diminished. Sodium butyrate treatment abrogated the S-phase cell-cycle checkpoint in mitomycin C-treated cells and induced the G(2)-M checkpoint. However, sodium butyrate treatment alone resulted in accumulation of reactive oxygen species, double-strand breaks in DNA, and apoptosis. These results imply that the accumulation of reactive oxygen species-mediated increase in DNA lesion burden may be the major mechanism by which sodium butyrate enhances the cytotoxicity of mitomycin C. Mol Cancer Ther; 11(10); 2116-26. ©2012 AACR.
|SUV420H2-mediated H4K20 trimethylation enforces RNA polymerase II promoter-proximal pausing by blocking hMOF-dependent H4K16 acetylation.|
Kapoor-Vazirani, P; Kagey, JD; Vertino, PM
Molecular and cellular biology 31 1594-609 2011
Many human genes exhibit evidence of initiated RNA polymerase II (Pol II) at their promoters, despite a lack of significant full-length transcript. Such genes exhibit promoter-proximal "pausing," wherein initiated Pol II accumulates just downstream of the transcription start site due to a rate-limiting step mediating the transition to elongation. The mechanisms that regulate the escape of Pol II from pausing and the relationship to chromatin structure remain incompletely understood. Recently, we showed that CpG island hypermethylation and epigenetic silencing of TMS1/ASC in human breast cancers are accompanied by a local shift from histone H4 lysine 16 acetylation (H4K16Ac) to H4 lysine 20 trimethylation (H4K20me3). Here, we show that hMOF-mediated H4K16Ac and SUV420H2-mediated H4K20me3 play opposing roles in the regulation of Pol II pausing. We found that H4K16Ac promoted the release of Pol II from pausing through the recruitment of BRD4 and pTEFb. Aberrant methylation of CpG island DNA blocked Pol II recruitment to gene promoters. Whereas the inhibition of DNA methylation allowed for the reassociation and initiation of Pol II at the TMS1 promoter, Pol II remained paused in the presence of H4K20me3. Combined inhibition of H4K20me3 and DNA methylation resulted in the rerecruitment of hMOF and subsequent H4K16Ac, release of Pol II into active elongation, and synergistic reactivation of TMS1 expression. Marking by H4K20me3 was not restricted to TMS1 but also occurred at other genes independently of DNA methylation, where it similarly imposed a block to Pol II promoter escape through a mechanism that involved the local inhibition of H4K16Ac. These data indicate that H4K20me3 invokes gene repression by antagonizing hMOF-mediated H4K16Ac and suggest that overcoming Pol II pausing might be a rate-limiting step in achieving tumor suppressor gene reactivation in cancer therapy.Full Text Article
|A possible inflammatory role of twist1 in human white adipocytes.|
Pettersson AT, Laurencikiene J, Mejhert N, NÃ¤slund E, BouloumiÃ© A, Dahlman I, Arner P, RydÃ©n M
Diabetes 59 564-71. Epub 2009 Dec 10. 2010
OBJECTIVE: Twist1 is a transcription factor that is highly expressed in murine brown and white adipose tissue (WAT) and negatively regulates fatty acid oxidation in mice. The role of twist1 in WAT is not known and was therefore examined. RESEARCH DESIGN AND METHODS: The expression of twist1 was determined by quantitative real-time PCR in different tissues and in different cell types within adipose tissue. The effect of twist1 small interfering RNA on fatty acid oxidation, lipolysis, adipokine secretion, and mRNA expression was determined in human adipocytes. The interaction between twist1 and specific promoters in human adipocytes was investigated by chromatin immunoprecipitation (ChIP) and reporter assays. RESULTS: Twist1 was highly expressed in human WAT compared with a set of other tissues and found predominantly in adipocytes. Twist1 levels increased during in vitro differentiation of human preadipocytes. Gene silencing of twist1 in human white adipocytes had no effect on lipolysis or glucose transport. Unexpectedly, and in contrast with results in mice, twist1 RNA interference reduced fatty acid oxidation. Furthermore, the expression and secretion of the inflammatory factors tumor necrosis factor-alpha, interleukin-6, and monocyte chemoattractant protein-1 were downregulated by twist1 silencing. ChIP and reporter assays confirmed twist1 interaction with the promoters of these genes. CONCLUSIONS: Twist1 may play a role in inflammation of human WAT because it can regulate the expression and secretion of inflammatory adipokines via direct transcriptional effects in white adipocytes. Furthermore, twist1 may, in contrast to findings in mice, be a positive regulator of fatty acid oxidation in human white adipocytes.Full Text Article
|Epigenetic analysis reveals a euchromatic configuration in the FMR1 unmethylated full mutations.|
Tabolacci, E; Moscato, U; Zalfa, F; Bagni, C; Chiurazzi, P; Neri, G
European journal of human genetics : EJHG 16 1487-98 2008
Fragile X syndrome (FXS) is caused by the expansion of a CGG repeat in the 5'UTR of the FMR1 gene and the subsequent methylation of all CpG sites in the promoter region. We recently identified, in unrelated FXS families, two rare males with an unmethylated full mutation, that is, with an expanded CGG repeat (greater than 200 triplets) lacking the typical CpG methylation in the FMR1 promoter. These individuals are not mentally retarded and do not appear to be mosaic for premutation or methylated full mutation alleles. We established lymphoblastoid and fibroblast cell lines that showed essentially normal levels of the FMR1-mRNA but reduced translational efficiency of the corresponding mRNA. Epigenetic analysis of the FMR1 gene demonstrated the lack of DNA methylation and a methylation pattern of lysines 4 and 27 on histone H3 similar to that of normal controls, in accordance with normal transcription levels and consistent with a euchromatic configuration. On the other hand, histone H3/H4 acetylation and lysine 9 methylation on histone H3 were similar to those of typical FXS cell lines, suggesting that these epigenetic changes are not sufficient for FMR1 gene inactivation. These findings demonstrate remarkable consistency and suggest a common genetic mechanism causing this rare FMR1 epigenotype. The discovery of such a mechanism may be important in view of therapeutic attempts to convert methylated into unmethylated full mutations, restoring the expression of the FMR1 gene.
|Chromatin Immunoprecipitation (ChIP)||Human||18628788|
|Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification.|
Pinnoji, RC; Bedadala, GR; George, B; Holland, TC; Hill, JM; Hsia, SC
Virology journal 4 56 2007
During primary infection of its human host, Herpes Simplex Virus Type-1 (HSV-1) establishes latency in neurons where the viral genome is maintained in a circular form associated with nucleosomes in a chromatin configration. During latency, most viral genes are silenced, although the molecular mechanisms responsible for this are unclear. We hypothesized that neuronal factors repress HSV-1 gene expression during latency. A search of the HSV-1 DNA sequence for potential regulatory elements identified a Repressor Element-1/Neuronal Restrictive Silencer Element (RE-1/NRSE) located between HSV-1 genes ICP22 and ICP4. We predicted that the Repressor Element Silencing Transcription Factor/Neuronal Restrictive Silencer Factor (REST/NRSF) regulates expression of ICP22 and ICP4.Transient cotransfection indicated that REST/NRSF inhibited the activity of both promoters. In contrast, cotransfection of a mutant form of REST/NRSF encoding only the DNA-binding domain of the protein resulted in less inhibition. Stably transformed cell lines containing episomal reporter plasmids with a chromatin structure showed that REST/NRSF specifically inhibited the ICP4 promoter, but not the ICP22 promoter. REST/NRSF inhibition of the ICP4 promoter was reversed by histone deacetylase (HDAC) inhibitor Trichostatin A (TSA). Additionally, chromatin immuno-precipitation (ChIP) assays indicated that the corepressor CoREST was recruited to the proximity of ICP4 promoter and that acetylation of histone H4 was reduced in the presence of REST/NRSF.Since the ICP4 protein is a key transactivator of HSV-1 lytic cycle genes, these results suggest that REST/NRSF may have an important role in the establishment and/or maintenance of HSV-1 gene silencing during latency by targeting ICP4 expression.Full Text Article
|Cyclin D1 activation in B-cell malignancy: association with changes in histone acetylation, DNA methylation, and RNA polymerase II binding to both promoter and distal sequences.|
Hui Liu, Jin Wang, Elliot M Epner
Blood 104 2505-13 2004
Cyclin D1 expression is deregulated by chromosome translocation in mantle cell lymphoma and a subset of multiple myeloma. The molecular mechanisms involved in long-distance gene deregulation remain obscure, although changes in acetylated histones and methylated CpG dinucleotides may be important. The patterns of DNA methylation and histone acetylation were determined at the cyclin D1 locus on chromosome 11q13 in B-cell malignancies. The cyclin D1 promoter was hypomethylated and hyperacetylated in expressing cell lines and patient samples, and methylated and hypoacetylated in nonexpressing cell lines. Domains of hyperacetylated histones and hypomethylated DNA extended over 120 kb upstream of the cyclin D1 gene. Interestingly, hypomethylated DNA and hyperacetylated histones were also located at the cyclin D1 promoter but not the upstream major translocation cluster region in cyclin D1-nonexpressing, nontumorigenic B and T cells. RNA polymerase II binding was demonstrated both at the cyclin D1 promoter and 3' immunoglobulin heavy-chain regulatory regions only in malignant B-cell lines with deregulated cyclin D1 expression. Our results suggest a model where RNA polymerase II bound at IgH regulatory sequences can activate the cyclin D1 promoter by either long-range polymerase transfer or tracking.
|Counterregulation of chromatin deacetylation and histone deacetylase occupancy at the integrated promoter of human immunodeficiency virus type 1 (HIV-1) by the HIV-1 repressor YY1 and HIV-1 activator Tat|
He, G. and Margolis, D. M.
Mol Cell Biol, 22:2965-73 (2002) 2002
|Chromatin Immunoprecipitation (ChIP)||11940654|
|How should I resuspend my pellet prior to PCR?||You should resuspend your pellet in water and not TE as the EDTA found in the TE may interfere with PCR.|
|Is there ever a time when I do not need to cross-link Histones?||In native ChIP, Histone H3 and Histone H4 do not need to be crosslinked as they are very tightly associated. Histone H2A and Histone H2B are not as tightly associated, but will still work in native ChIP.|
|What were your conditions for PCR?||Please see the manual for The EZ ChIP Kit (Catalog #17-371) for more information.|
|If I wanted to quantitate my immunoprecipitated DNA, how would I do so?||DNA purified from ChIP experiments can be quantitated by PCR, providing the amplifying oligos meet specific criteria. Oligos should be 24 mers, with a GC content of 50% (+/- 4) and a Tm of 60.0C (+/- 2.0). You must be certain that the PCR reactions are within the linear range of amplification. Generally it takes time to achieve this. Too much input DNA will affect your results, so set up several tubes for each experiment to optimize the input DNA. Generally, this is about 1/25th to 1/100th for yeast, approximately 1/10 for mammalian cells, but depends on the amount of antibody and input chromatin.
Also, do not use more than 20 cycles, making sure that dNTP's always remain in excess. Also, include each reaction a control primer (to compare your experimental band against-make sure the sizes are sufficiently different to allow proper separation-75 base pairs is usually OK) set to a region of the genome that should not change throughout your experimental conditions. Also PCR from purified input DNA (no ChIP) and include no antibody control PCR's as well. PCR products should be no more than 500 base pairs and should span the area of interest (where you think you will see changes in acetylation or methylation of histones). All PCR products should be run on 7-8% acrylamide gels and stained with SYBR Green 1 (Molecular Probes) at a dilution of 1:10,000 (in 1X Tris-borate-EDTA buffer, pH 7.5) for 30 minutes-no destaining is required.
Quantitation is carried out subsequent to scanning of the gel on a Molecular Dynamics Storm 840 or 860 in Blue fluorescence mode with PMT voltage at 900 with ImageQuant software. This has distinct advantages over ethidium bromide staining. SYBR Green is much more sensitive, and illumination of ethidium stained gels can vary across the gel based on the quality of UV bulbs in your in your light box. For further info, see Strahl-Bolsinger et al. (1997) Genes Dev. 11: 83-93. A radioactive quantitation m
|I am not getting amplification with input DNA. What did I do wrong?||Your input DNA sample should be taken just prior to adding the antibody. It is considered the starting material. If you are not seeing amplification with your input DNA, either you have not successfully reversed the cross links or the PCR is not working for reasons other than the kit.|
|How would you recommend eluting Antibody-protein-DNA complexes from agarose (or sepharose) in order to perform a Re-ChIP experiment?||The complex is removed with the elution buffer that you find in the ChIP assay kit. For a re-CHIP, it might make sense to add protease inhibitors to the IP wash buffers and the elution buffer and the second set of dilution buffers. Make sure everything stays cold so that the proteins aren't degraded during the collection of the first complex or during the second IP.|
|Do you have any tips for sonication?||Keep cells on ice throughout the procedure - even during sonication. Be sure that you don't sonicate for to long (more than 30 seconds could cause sample overheating and denaturation).|
|Why is more DNA is precipitated in my no-antibody control than for my test sample?||To eliminate banding in your negative controls you can do several things:
A) Pre-clear the 2ml diluted cell pellet suspension with 80 microliters of Salmon Sperm DNA/Protein A Agarose-50% Slurry for 30 minutes at 4ºC with agitation. You could try to preclear the lysate longer or with more clearings.
B) Titrate your input DNA, to see when the bands in the NFA disappear.
C) Use an alternative lysis procedure: Resuspend cell pellet in 200 microliters of 5mM Pipes pH 8.0, 85mM KCl, 0.5% NP40 containing protease inhibitors. Place on ice for 10 minutes. Pellet by centrifugation (5 minutes at 5000 rpm). Resuspend pellet in 200 microliters of 1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1 containing protease inhibitors. Incubate on ice for 10 minutes.
D) Block the Salmon Sperm DNA Agarose prior to use in 1-5% BSA and Chip dilution buffer (mix at room temperature for 30 minutes). After incubation, spin the agarose and remove the 1% BSA/ChIP assay buffer supernatant. Wash once in ChIP assay buffer and continue.
|What is 'Input DNA', and why no 'Output DNA'?||Input DNA is DNA obtained from chromatin that has been cross-link reversed similar to your samples. It is a control for PCR effectiveness. Output DNA is the DNA from each of your ChIP experiments.|
|What types of controls do I need to run in the IP and the PCR portions of the ChIP?||ChIP control: use Anti-acetyl H3 primary antibody and PCR for the GAPDH gene promoter. This will ensure that each step of the procedure is working. PCR amplification: Control for PCR amplification using primers designed against a sequence that would not be enriched by your chromatin IP.
Liner Range PCR controls:
Ensure that PCR amplification is in the linear range by setting up each reaction at different dilutions of DNA for various amplification cycle numbers, and select the final PCR conditions accordingly. The assays are typically done in duplicate or triplicate. The average fragment size after sonication is ~500 bp (Kondo, et al. Molecular and Cellular Biology, January 2003, p. 206-215, Vol. 23, No. 1)
1) ChIP analysis of a transcribed region of the gene of interest which is >40 kb away from the promoter you are looking at. This may reveal that the activation level (e.g., acetylation level) may be very low or more importantly, not affected by your treatment.
2) Control for specificity of an induced local Histone hyperacetylation, you could analyze the acetylation level of another promoter (Sachs, et al. Proc. Natl. Acad. Sci. USA 97:2000, 13138-13143).
No primary antibody control:
This is the control in which you run the ChIP assay but don't add the primary immunoprecipitating antibody. It will ensure that you are not seeing sequences that bind non-specifically to the beads and that the recognition of your protein by the antibody you are using is required for enrichment of the target sequence
Negative antibody control:
A normal serum, normal IgG, or an antibody to a distant protein (all from the same species) is a good negative antibody control. The best control if using a polyclonal antibody is pre-immune antiserum of the animal that has been immunized.