Chromatin immunoprecipitation (ChIP-qPCR)

Inside the nucleus, DNA is wrapped around histone proteins and bound by transcription factors that switch genes on and off. Chromatin immunoprecipitation (ChIP) asks which of these proteins, or which chemical marks on the histones, are present on a particular stretch of DNA in the living cell. Cells are first treated with formaldehyde, which crosslinks proteins to the DNA they touch (Solomon et al., 1988; Orlando et al., 1997). The chromatin is broken into short pieces, an antibody pulls out the pieces carrying the protein of interest, the crosslinks are reversed, and the recovered DNA is measured, here by quantitative PCR (qPCR); detailed protocols are published (Nelson et al., 2006).

The example is the Fos gene, an immediate early gene switched on within minutes of strong neuronal activity (Morgan and Curran, 1991). Its promoter contains a calcium and cAMP response element (CRE) bound by the transcription factor CREB; depolarisation and calcium entry activate Fos transcription through phosphorylation of CREB (Sheng et al., 1990), at serine 133 (Gonzalez and Montminy, 1989; Sheng et al., 1991). Genome-wide ChIP in mouse cortical neurons found CREB already bound at many enhancers and promoters before depolarisation (Kim et al., 2010), whereas acetylation of histone H3 at lysine 27 (H3K27ac), a mark of active regulatory elements (Creyghton et al., 2010), increased at the Fos promoter after 2 h in 55 mM KCl (Malik et al., 2014). In rat hippocampus, histone H4 acetylation at the c-fos promoter tracked the level of c-fos mRNA after electroconvulsive seizures (Tsankova et al., 2004).

The result of a ChIP is usually expressed as percent input: the fraction of all copies of a region in the starting chromatin that the antibody recovered, compared with an IgG control and a region that should not be bound (Haring et al., 2007). Every step, from the crosslinking time and the fragment size to the antibody and the washes, changes how much specific and nonspecific DNA is recovered. ChIP can also be read out genome-wide by sequencing (ChIP-seq), and newer methods such as CUT&RUN avoid crosslinking and sonication altogether.

Unlike in a real laboratory, the true occupancy of the promoter is known in the simulation, and the final station shows which decisions changed the result.

Learning objectives

• Design a ChIP experiment with the independent culture or the animal as the unit, matched pairs where possible, random allocation and coded tubes
• Explain what formaldehyde crosslinking does, why its time matters and why glycine stops it
• Shear chromatin to approximately 200–500 bp, read the size on a gel and a fragment trace, and explain the costs of under- and over-shearing
• Choose a ChIP-grade antibody, an IgG control, an input and a negative-control region, and explain what each controls for
• Calculate percent input and fold enrichment from Ct values
• Compare conditions on a log scale with the right test across biological replicates, and avoid pseudoreplication

Ethics and safety

Neuronal cultures are prepared from embryonic or newborn mice killed humanely by trained staff under the institution's approved procedures; one dissection supplies many dishes, which reduces the number of animals used (Reduction, one of the 3Rs). Electroconvulsive seizures in rats are a regulated procedure that needs approval by an Institutional Animal Ethics Committee (in India, the CCSEA under the Prevention of Cruelty to Animals Act 1960; elsewhere, frameworks such as EU Directive 2010/63/EU, the US Animal Welfare Act and PHS Policy, and the UK Animals (Scientific Procedures) Act 1986), with sham animals handled identically; group sizes are justified in advance and the work is reported following the ARRIVE guidelines, developed by the NC3Rs in the United Kingdom and endorsed by journals internationally (Percie du Sert et al., 2020). At the bench, formaldehyde is toxic and a carcinogen and is handled only in a fume hood; phenol and chloroform, if used, also need a fume hood; sonicators require ear protection; gloves, a lab coat and eye protection are worn throughout.

Key terms

• Chromatin: DNA together with the proteins packed on it in the nucleus, chiefly histones, plus the transcription factors and other proteins bound to it.
• Histone and nucleosome: Histones are small proteins around which DNA is wrapped; about 147 bp of DNA around eight histones form one nucleosome. Their tails carry chemical marks such as acetylation.
• Transcription factor: A protein that binds a specific DNA sequence, such as CREB on the CRE, and helps switch a nearby gene on or off.
• Promoter: The stretch of DNA at the start of a gene where the machinery that copies the gene into RNA assembles.
• Crosslinking: Formaldehyde forms covalent bonds between proteins and DNA that are very close, fixing their contacts; heat at 65 °C reverses them.
• Sonication: Breaking chromatin into short fragments with ultrasound; the fragment size sets the resolution of the experiment.
• Input: A small portion of the sheared chromatin kept before the immunoprecipitation and processed alongside it; it shows how much of each region the starting chromatin held.
• Immunoprecipitation (IP): Pulling one protein, and whatever is attached to it, out of a mixture with an antibody bound to beads.
• IgG control: An antibody of the same species that recognises nothing in the cell, used at the same amount, to measure nonspecific recovery.
• ChIP-grade antibody: An antibody shown to recognise its target in crosslinked chromatin and to be specific for it.
• Amplicon: The stretch of DNA that a pair of qPCR primers copies; for ChIP it is usually short, approximately 80–150 bp, so that it fits inside most fragments.
• Ct (threshold cycle): The qPCR cycle at which a sample's fluorescence crosses a set threshold; one cycle less means about twice as many starting copies.
• Percent input: 100 × the fraction of all copies of a region in the chromatin that the IP recovered: 100 × 2^(adjusted input Ct − IP Ct).
• Fold enrichment: The IP signal divided by the IgG signal (or by the signal at a negative-control region).
• Gene desert: A long stretch of DNA with no genes, used as a region where the factor or mark should be absent.