Chromatin Remodeling and Studying Protein-DNA Interactions with Chromatin Immunoprecipitation (ChIP)

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.

Use the ChromaFlash High-Sensitivity ChIP Kit for immunoprecipitation of chromatin (ChIP) from small amounts of mammalian cells or tissues. For downstream analysis, theEpiNext ChIP-Seq High-Sensitivity Kit (Illumina) provides streamlined ChIP sequencing NGS library preparation from low input cells.

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).