ChIP is a powerful technique used to study the association of specific proteins, or their modified isoforms, with defined genomic regions. It is a fast growing research technique and is commonly used for mapping the DNA-protein interactions in cells which are crucial for correct gene regulation. For example, they may be used to determine whether proteins such as transcription factors and modified histones bind to a particular region of DNA of living cells or tissues.
Genome-wide mapping of protein-DNA interactions is essential for a complete understanding of gene regulation. A detailed map of epigenetic marks and transcription factor binding is necessary for deducing the regulatory networks that underpin gene expression in a variety of biological systems. The most widely used tool for examining these interactions is ChIP followed by massively parallel sequencing (ChIP-seq)
ChIP-seq begins with a traditional ChIP assay involving cell fixation (cross-linking), chromatin shearing, immunoprecipitation (IP), reverse-crosslinking and DNA purification. Living cells are fixed with a reversible crosslinking agent to retain protein-DNA interactions at their natural sites before being lysed in order to release the chromatin for shearing. Following crosslinking, the chromatin is sheared to a specific size range (100-500 bp) for optimal IP and ChIP-seq results. Either sonication or enzymatic shearing can be used to achieve fragment sizes between 100-500 bp, the chromatin is then immunoprecipitated using an antibody of interest and isolated.
ChIP will produce a library of target DNA sites that were in direct physical contact with regulatory mechanisms in vivo. Oligonucleotide adapters are then added to the fragments of DNA that were bound to the protein of interest to enable massively parallel sequencing. After size selection, all the resulting ChIP DNA fragments are sequenced simultaneously, scanning for genome-wide associations with high resolution. Mapping the sequenced fragments to whole genome sequence databases allows the DNA interaction pattern of any TF or epigenetic modification to be analysed quickly and effectively.
ChIP-seq has been made possible by technological advancements which enable us to map out entire genomes and pinpoint the exact binding sites of proteins by combining ChIP assay with next generation sequencing platforms. Pinpointing a specific gene sequence that a protein binds to using ChIP-seq helps epigenetic researchers advance their protein-DNA interaction studies and gleam valuable insights into disease development.
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