ChIP-Seq is chromatin immunoprecipitation paired with DNA sequencing. These interactions are processed using quantitative polymerase chain reaction (qPCR), ChIP-chip, or next-generation sequencing (NGS).
This method provides information about accessible binding sites for particular proteins, often transcription factors, on a genome. DNA in eukaryotes is not floating freely in the nucleus. It is wrapped around an octamer of histone proteins and compacted into higher order structures.
The complex of DNA and histones is known as chromatin. The degree of compaction or relaxation of chromatin is a major factor in determining which genes are expressed. The field of study concerned with these extragenic modifications is known as epigenetics.
There are many kinds of signals that unite to alter the compaction of chromatin. Methylation of cytosine residues in DNA is associated with tightly compacted or closed chromatin. In this case, the chromatin is inaccessible to transcription factors.
Another modification that contributes to chromatin accessibility is signaled through the exposed tails of the histone proteins around which the DNA strands are wrapped. Many types of proteins are closely associated with DNA, not just the ones that comprise chromatin. Proteins involved in transcription, replication, and gene regulation are all associated with DNA depending on the region analyzed.
With this approach, it is possible to learn if a promoter is bound by specific transcription factors and if the gene is likely to be expressed.
Alterations in DNA accessibility are associated with neurological disorders, autoimmune diseases, and cancers. Many cancers display altered patterns in gene expression that are not caused by alterations in the DNA sequence. ChIP-Seq has been a key tool in identifying potential prognostic signatures in prostate cancer and profiling breast cancer tumors at the epigenetic level.
ChIP-Seq is an improvement over ChIP-chip, the technology preceding it. ChIP-chip used the output from immunoprecipitation to screen a tiling microarray. That array approach lacked sensitivity, and DNA regions detected were limited to probes on the array. One study reported that 10-30% of functional transcription factor binding sites were lost when ChIP-chip was used.
In contrast, ChIP-Seq uses an NGS readout. It lacks the limitations in target detection range and sensitivity prevalent with ChIP-chip. With single-base resolution across the whole genome, ChIP-Seq provides comprehensive coverage but it is limited by the quality of the antibody used to pull down the target protein.
ChIP-Seq has a variety of advantages, including the ability to:
ChIP-Seq allows for population analysis of protein-DNA interactions on a genomic scale. It has also allowed for the investigation of the evolution of transcription factor binding. Both of these use cases were impossible with previously available methods. As a mature technology with compatibility across various input DNA samples, ChIP-Seq is the preferred method for genome-wide profiling across multiple samples.
The Illumina platform breaks ChIP-Seq down into three simple steps: Library Preparation, Sequencing, and Analysis.
ChIP-Seq can be completed on any Next Generation Sequencing (NGS) platform. However, the vast majority of papers published so far have been done on the Illumina platform. At Psomagen, we also use the Illumina platform for many of our sequencing needs.
Illumina has a clear workflow, that can essentially be explained as:
While ChIP-Seq is a relatively mature technology, its cost and availability were concerns as recently as a few years ago. As cost has decreased and the availability of ChIP-Seq has increased, ChIP-Seq has become the primary method used to analyze protein interactions with DNA. ChIP-Seq is expected to have a major role in epigenetic sequencing and continue to reveal key cancer genes.
Psomagen thanks Dr. Stacy Matthews Branch for her contributions to the research and writing of the original version of this article. Dr. Branch is a biomedical consultant, medical writer, and veterinary medical doctor. She owns Djehuty Biomed Consulting and has published research articles and book chapters in the areas of molecular, developmental, reproductive, forensic, and clinical toxicology. Dr. Matthews Branch received her DVM from Tuskegee University and her Ph.D. from North Carolina State University.