What Is ChIP-Seq?
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.
How Does ChIP-Seq Work?
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.
- ChIP assays begin by crosslinking DNA to its associated proteins.
- The DNA:protein complex is fragmented to enable immunoprecipitation.
- An antibody against the target protein is used to pull down only fragments that contain DNA crosslinked to the target protein.
- The captured DNA:protein complexes are de-crosslinked and the proteins are washed away leaving DNA.
- The captured DNA is used to generate libraries for next generation sequencing.
- Sequences are mapped onto the source genome to identify regions closely associated with the target protein.
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.
Advancements in ChIP Technologies
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.
What Can You Do with ChIP-Seq?
ChIP-Seq has a variety of advantages, including the ability to:
- Identify global corepressor binding sites, which play an important role in regulating transcription. A team of scientists at Singapore’s A*STAR used ChIP-Seq to identify a global network of transcriptional repressors that impact Androgen receptor-mediated gene regulation. These exciting results offer clues to mechanisms involved in prostate cancer progression.
- Reveal gene networks when combined with RNA sequencing as in this first-time characterization of placental changes that could be the source of human labor. Researchers identified epigenetic changes between early, mid-trimester, and full term placental samples that correlate with gene expression changes seen in term labor.
- Targeting histone modifications of the entire genome is a massive undertaking given that there are 130 post-translational modifications that occur on histones. That said, the ENCODE and Roadmap Epigenomics Projects are repositories of epigenomic data spanning many types of human cells available for other researchers due to the scalability of ChIP-Seq.
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.
Using Illumina Platforms for ChIP-Seq
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 be found in greater detail here, but essentially it can be explained as:
- TruSeq ChIP Library Preparation – Illumina’s simple, cost-effective kit is used to generate ChIP-Seq libraries.
- Sequencing – The NovaSeq6000 and NovaSeq X Plus offer the largest read capacities to date. Psomagen has validated both platforms and either can be used for ChIP-Seq library pools, with the appropriate flow cell and system selected to maximize data output and the lowest cost to our customers.
- Analyze and Store Data – BaseSpace apps can be used to analyze and store data once it’s sequenced. Apps like the ChIP-Seq app, Genomatix Pathway System (GePS), BaseSpace Sequence Hub, and BaseSpace Correlation Engine can be used in the analysis process.
ChIP-Seq and the Future of Epigenetics
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.