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Expertise | Chromatin function
The major components of chromatin are DNA and histone proteins. Two copies of each histone core protein (H2A, H2B, H3 & H4) are assembled into an octamer that has 145- 147 base pairs of DNA wrapped around it to form a nucleosome core. The nucleosome controls the accessibility of DNA to the transcription machinery and chromatin remodelling factors (Luger et al. 1997).
Epigenetic research has been defined as the “sum of the alterations to the chromatin template that collectively establish and propagate different patterns of gene expression (transcription) and silencing from the same genome” (Allis et al. 2007).
Histone modifications are site-specific and reversible epigenetic marks (e.g. acetylation, methylation, phosphorylation, ubiquitination, sumoylation) (Peterson and Laniel 2004, Allis et al. 2007). They commonly occur in the histone tail modifications and over 50 histone epigenetic marks have by now been mapped (Jenuwein 2006).
In order to regulate specific DNA-dependent processes, chromatin can be remodelled in three different ways: by removing or mobilizing the histones by means of ATPdependent nucleosome remodelling machines (Smith and Peterson, 2005); by altering chromatin structure via the post-translational modification of histones (Strahl and Allis, 2000); or by the replacement of specific histones (Henikoff and Ahmad 2005, Cavalli 2006).
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Learn more about The main histone modifications and their role in gene expression
Chromatin Immunoprecipitation (ChIP)
Diagenode offers a wide range of kits for chromatin immunoprecipitation a method used to determine the location of DNA binding sites on the genome for a particular protein of interest. The ChIP assay offers great potential to improve knowledge about the regulation of gene expression. This technique is now used in a variety of life science disciplines including cellular differentiation, tumor suppressor gene silencing, and the effect of histone modifications on gene expression.
The chromatin-bound proteins are formaldehyde fixed to the DNA (xChIP) or ChIP is performed on native chromatin (nChIP). Afterwards, the chromatin is sheared to small fragment sizes (200bp – 1kb) and immunoprecipitated using a specific ChIP-grade antibody. Crosslinking is reversed followed by proteinase K treatment or DNA purification using magnetic beads (IPure kit). The purified DNA is analyzed to identify the genomic regions where the specific protein was located (next generation sequencing, qPCR, hybridization on microarray).
The structure of cellular chromatin is capable of undergoing rapid changes to respond to the metabolic requirements of the cell. These changes have a direct impact on gene expression and therefore, the chromatin context must be considered when biochemical reactions that involve DNA are studied. DNA-binding factors have evolved with chromatin. It is therefore more desirable to study the molecular mechanisms of DNA-directed processes with chromatin than with naked DNA templates (Lusser and Kadonaga, 2004). To this end, it is necessary to reconstitute DNA and histones into chromatin.