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Epigenetics
 
  • Overview
  • Histones
  • Enzymes
  • Control DNA
    The Epigenetics Code
    The Epigenetic Code
    Epigenetics studies heritable changes in gene expression that are not actually encoded in the DNA of the genome. These effects are mediated by the covalent attachment of chemical groups to DNA and its associated proteins, histones and chromatin. Types of modification include methylation, acetylation, phosphorylation, ubiquitination and ADP-ribosylation. Post-translational modifications have been linked to gene regulation (1), cellular stress events (1), aging and DNA repair (2). To facilitate epigenetic research and discovery, New England Biolabs offers a line of research reagents, including McrBC, methyltransferases, control DNAs and histones.

    References:
    1. Huang J., et al. (2006) Nature, 444, 629–632.
    2. Pahlich, S., Zakaryan, R.P. and Gehring, H. (2006) Biochim. Biophys. Acta., 1764, 1890–1903.
    Purity of NEB Histones
    Purity of NEB Histones

    Mass Spec. of Histone H2A
    Mass spectrometry of octamer; Histone H2A: 13990 Da; Histone H2B: 13789 Da; Histone H3: 15273 Da; Histone H4: 11236 Da
    Histones:
    In eukaryotes, nuclear DNA is found assembled into chromatin by histones. Two molecules of each histone H2A, H2B, H3 and H4 combine to form an octamer complex and package approximately 147 base pair segments of nuclear DNA into nucleosome core particles (NCP). Histone H1 further condenses the DNA by binding the linker segments between the nucleosome core particles (3,4).

    The core human histones (Histone H2A, H2B, H3 and H4) as well as the human linker histone (Histone H1) have been individually cloned in E. coli and each recombinant human histone has been purified from E. coli cell extracts. Mass spectrometry (MS) analysis indicates that these recombinant histones are free of post-translational modifications. These histones are an ideal substrate for the purification and characterization of enzymes that modify histone proteins. Additionally, we have demonstrated that the core histones can form an octamer (5,6). The octamer can be assembled with DNA to form unmodified nucleosome core particles (NCP). These NCPs may be modified by enzymes that are inactive on individual histones or DNA. They also may be used as carrier chromatin in CChIP (carrier chromatin immunoprecipitation) assays (7).

    References:
    1. Kornberg, R.D. (1977) Annu. Rev. Biochem., 46, 931–954.
    2. van Holde, K.E. (1989) Chromatin, 1–497.
    3. Luger et al. (1999) Methods in Mol. Biol., 119, 1–16.
    4. Comb, D. unpublished observation.
    5. O'Neill et al. (2006) Nature Genetics, 38, 835–841.
    DNA Methyltransferase
    NEB offers histone, protein and DNA Methyltransferases.
    Enzymes:
    In eukaryotic chromatin, histones may be enzymatically modified at many sites. The predominate modifications are methylation, acetylation, phosphorylation, ubiquitination, sumoylation and ADP ribosylation. These occur on the flexible N-and C-terminal tails of the histones or within their globular folds in the nucleosome core (8). The lysine or arginine residues in the histone undergo enzymatic methylation via attachment of one, two or three methyl groups. The timing of the appearance of these modifications will depend on the signaling condition of the cell and are often dynamic. Many of the covalent modifications on the histone tail are enzymatically reversible (9). Histone modifications participate in transcription, repair, replication and chromatin condensation. Acting individually or combinatorially, histone modifications in conjunction with DNA modification, are thought to encipher an epigenetic code of gene regulation. Identifying enzymes that can modify histones has been a research focus of interest (10).

    A variety of our existing restriction enzymes can be used to study epigenetic modifications of DNA such as, DpnI and DpnII that recognize the same sequence but different methylation patterns. McrBC also only cleaves DNA that is methylated on one or both strands.

    References:
    1. Peterson, C. L. and Laniel, M. A. (2004) Curr. Biol.,14, R546–51.
    2. Shi, Y. and Whetstine J.R. (2007) Mol. Cell, 25, 1–14.
    3. Kouzarides, T. (2007) Cell, 128, 802.
     
    Control DNA:
    Altered epigenetic patterns are a hallmark of cancer. Many genes are silenced in cancerous cells due to newly acquired de novo methylation of CpG islands (11). Currently, there are several methods that detect methylated CpGs. Methylation-specific PCR (MSP) is a technology used for the sensitive detection of gene methylation in the genome (12). MSP uses an initial bisulfite reaction to modify the DNA by converting unmethylated cytosines to uracils while 5-methylcytosines remain unaltered. This reaction is followed by PCR amplification with specific primers designed to distinguish methylated from unmethylated DNA. Since this is an extremely sensitive PCR-based assay, the use of control DNA is necessary to determine the quality of the bisulfite conversion and to identify artifacts such as primer-dimer pairing and mispriming that can cloud the interpretation of results.

    To create a methylation-positive DNA control, all cytosine residues (C5) within the double-stranded dinucleotide recognition sequence 5΄…CG…3΄ in HeLa, NIH-3T3 and Jurkat genomic DNA have been enzymatically methylated with CpG Methylase (M.SssI). These modified DNAs are extensively tested for complete methylation by an additional methyl group transfer assay and MSP. A partially demethylated DNA control has also been created by incubating Jurkat cells with a potent methyltransferase inhibitor, 5-aza-2-deoxycytidine (5-Azadc). Bisulfite conversion (13) and sequencing of a section of intergenic (IGS) repetitive DNA (rDNA) that is normally methylated revealed significant CpG demethylation. These DNA controls are powerful tools in the investigation of genomic DNA methylation and epigenomic research.

    References:
    1. Bird, A. P. (1986) Nature, 321, 209-213.
    2. Herman, ,J. G. et al. (1996) Proc. Natl. Acad. Sci. USA, 18, 9821–9826.
    3. Clark, S. J. and Frommer M. (1997). In G. R.Taylor (Ed.), Laboratory Methods (pp. 151–162). Florida: CRC press.




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