• My NEB
  • Print
  • PDF
  • Chromatin Analysis

    Return to Epigenetics

    Apart from studies involving DNA methylation, there are numerous methods for the analysis of chromatin, the biological template for epigenetic inheritance. In the nucleus of the cell, genomic DNA wraps around two copies of each of the four core histone proteins, H2A, H2B H3, H4 to form a nucleosome which is the functional unit of chromatin. During cellular development or gene expression, chromatin modifying enzymes are recruited to specifically modify an amino acid on the histone tail or on DNA resulting in transcriptional regulation of the associated genes. For example, H3K4me3 marks are associated with gene activation while H3K9me3 and H3K27me3 are involved in gene repression (1). Some of these marks also serve as binding sites for other effector proteins involved in chromatin modeling popularly known as the reader proteins (2). Therefore the dynamic modifications of chromatin can be studied by using modification specific antibodies in immunocytochemistry and immunoprecipitation techniques, such as chromatin immunoprecipitation (ChIP). Once modification specific chromatin and/or DNA is isolated, ChIP on Chip assays (microarray) and ChIP-Seq (next generation sequencing) methods could be used for genome wide studies. Furthermore, positioning of nucleosomes on DNA can be determined by nucleosome foot-printing using GpC methyltransferases (3). Open regions of the genome can be assayed by their sensitivity to nucleases, such as DNAseI or micrococcoal nuclease. Similarly, high resolution mapping of protein factors binding is obtained by combining ChIP with Exonuclease I (ChIP-exo) (4). Apart from direct chromatin studies, in vitro assembled recombinant nucleosomes are also used for biological studies of chromatin enzymes as some histone modification enzymes require nucleosome substrates. (5)

    All of the above applications are used in exploring epigenetic mechanisms and understanding epigenome profiles, with the goals of identifying diagnostic or therapeutic biomarkers and developing new therapeutics.

    References

    1. Lachner, M. et al. (2003) J Cell Sci. 116(Pt 11): 2117-24. PMID: 12730288
    2. Banerjee, T. and Chakravarti D. (2011) Mol. Cell. Biol. 31(24):4858-73. PMID: 22006017
    3. Fatemi, M. et al. (2005) Nucleic Acids Research, Vol. 33, No. 20 e176 PMID: 16314307
    4. Rhee HS and Pugh BF, (2011) Cell 147(6):1408-19. PMID: 22153082
    5. Fang, J. et al (2002) Current Biology, 12, 1086–1099 PMID: 12121615