Chromatin Remodeling and Studying Protein-DNA Interactions with Chromatin Immunoprecipitation (ChIP)
Investigate heterochromatin and euchromatin and use ChIP to study protein-DNA interactions
DNA is tightly associated with histone proteins in the nucleosome. The nucleosome is composed of an octamer of histone
proteins tightly wrapped around with ~147 bp DNA and a linker histone H1 protein. The octamer is composed of two histone
H2A, two histone H2B, two histone H3, and two histone H4 proteins. These DNA-histone complexes or nucleosomes constitute
the basic unit of chromatin, and they are linked together forming the chromatin fiber. How closely bound together the
nucleosomes are will influence transcription and gene expression.
Chromatin exists in two main forms. Heterochromatin is highly compacted chromatin, and is inaccessible for the
transcriptional machinery, leading to silenced chromatin. It is mostly found in the telomeres and centromeres of
chromosomes, as well as in permanently silenced regions of the genome, such as imprinted X-chromosome in females. On the
other hand, euchromatin is looser, more accessible chromatin that is mostly present in areas with high
transcriptional activity and gene expression.
For quantitative chromatin analysis and determination of euchromatin or
heterochromatin states use the EpiQuik Chromatin
Accessibility Assay Kit. This kit is ideal for gene-specific analysis of chromatin accessibility including
nucleosome/transcription factor positioning from various biological samples via real time PCR.
Heterochromatin is tightly compact whereas euchromatin is looser and more accessible. An abundance of histone
acetylation leads to looser chromatin.
DNA methylation and histone modifications are often responsible for keeping chromatin in these states
with concomitant regulation of expression. Besides the DNA-associated histones, transcription factors and other
DNA-binding proteins also interact with DNA, altering chromatin structure and regulating gene expression.
Chromatin Immunoprecipitation (ChIP)
DNA-protein interactions can be studied by chromatin immunoprecipitation (ChIP).
This technique involves the immunoprecipitation or pull down of DNA with antibodies specific for the DNA-associated
proteins. Histone modifications, as well as transcription factors, enzymes, and other important proteins in chromatin
remodeling complexes can be studied in this manner.
Generally in a ChIP experiment, sample is first crosslinked to fix the DNA and protein interactions, and chromatin is
then isolated and sheared into smaller fragments. The DNA and associated proteins are pulled down with specific
antibodies to the proteins of interest (histones, transcription factors, others). Then the crosslinks are reversed,
proteins are digested and the associated DNA is eluted. The obtained DNA can be analyzed by several downstream
applications, including qPCR (ChIP-PCR) or NGS (ChIP-Seq).