The State-of-the-Art of Chromatin Immunoprecipitation
The biological significance of interactions of nuclear proteins with DNA in the context of gene expression,cell differentiation, or disease has immensely been enhanced by the advent of chromatin immunoprecipita-tion (ChIP). ChIP is a technique whereby a protein of interest is selectively immunoprecipitated from achromatin preparation to determine the DNA sequences associated with it. ChIP has been widely used tomap the localization of post-translationally modified histones, histone variants, transcription factors, orchromatin-modifying enzymes on the genome or on a given locus. In spite of its power, ChIP has for a longtime remained a cumbersome procedure requiring large number of cells. These limitations have sparkedthe development of modifications to shorten the procedure, simplify the sample handling, and make theChIP amenable to small number of cells. In addition, the combination of ChIP with DNA microarray,paired-end ditag, and high-throughput sequencing technologies has in recent years enabled the profilingof histone modifications and transcription factor occupancy on a genome-wide scale. This review high-lights the variations on the theme of the ChIP assay, the various detection methods applied downstream ofChIP, and examples of their application.
Key words: Chromatin immunoprecipitation, ChIP, acetylation, methylation, transcription factor,DNA binding, epigenetics.
1. Introduction:Modifications ofDNA and HistoneProteins The interaction between proteins and DNA is essential for many
cellular functions such as DNA replication and repair, maintenanceof genomic stability, chromosome segregation at mitosis, andregulation of gene expression. Transcription is controlled by thedynamic association of transcription factors and chromatin modi-fiers with target DNA sequences. These associations take place notonly within regulatory regions of genes (promoters and enhan-cers), but also within coding sequences. They are modulated by
Philippe Collas (ed.), Chromatin Immunoprecipitation Assays, Methods in Molecular Biology 567,DOI 10.1007/978-1-60327-414-2_1, Humana Press, a part of Springer Science+Business Media, LLC 2009
modifications of DNA such as methylation of CpG dinucleotides(1), by post-translational modifications of histones (2), and byincorporation of histone variants (37). These alterations are com-monly referred to as epigenetic modifications: they modify thecomposition of DNA and chromatin without altering genomesequence, and they are passed onto daughter cells (they areheritable).
DNA methylation is generally seen as a hallmark of long-termgene silencing (8, 9). Methyl groups on the cytosine in CpGdinucleotides create target sites for methyl-binding proteins,which induce transcriptional repression by recruiting transcrip-tional repressors such as histone deacetylases or histone methyl-transferases (9). DNA methylation largely contributes to generepression and as such it is essential for development (1012),X chromosome inactivation (13), and genomic imprinting (14,15). The relationship between DNA methylation and gene expres-sion is intricate, and recent evidence based on genome-wide CpGmethylation profiling has highlighted CpG content and density ofpromoters as one component of this complexity (16, 17).
In addition to DNA methylation, post-translational modifica-tions of histone proteins regulate gene expression. The core ele-ment of chromatin is the nucleosome, which consists of DNAwrapped around two subunits of histone H2A, H2B, H3, andH4. Nucleosomes are spaced by the linker histone H1. Theamino-terminal tails of histones are post-translationally modifiedto confer physical properties that affect their interactions withDNA. Histone modifications not only influence chromatin packa-ging, but are also read by adaptor molecules, chromatin-modifyingenzymes, transcription factors, and transcriptional repressors, andthereby contribute to the regulation of transcription (2, 1820).
Histone modifications have been best characterized so far forH3 and H4. They include combinatorial lysine acetylation, lysinemethylation, arginine methylation, serine phosphorylation, lysineubiquitination, lysine SUMOylation, proline isomerization, andglutamate ADP-ribosylation (2) (Fig. 1.1). In particular, di- andtrimethylation of H3 lysine 9 (H3K9me2, H3K9me3) and tri-methylation of H3K27 (H3K27me3) elicit the formation ofrepressive heterochromatin through the recruitment of hetero-chromatin protein 1 (21) and polycomb group (PcG) proteins,respectively (2224). However, whereas H3K9me3 marks consti-tutive heterochromatin (25), H3K27me3 characterizes facultativeheterochromatin, or chromatin domains containing transcription-ally repressed genes that can potentially be activated, for exampleupon differentiation (26, 27). In contrast, acetylation of histonetails loosens their interaction with DNA and creates a chromatinconformation accessible to targeting of transcriptional activators(28, 29). Thus, acetylation on H3K9 (H3K9ac) and H4K16(H4K16ac), together with di- or trimethylation of H3K4
(H3K4me2, H3K4me3), is found in euchromatin, often in asso-ciation with transcriptionally active genes (27, 3033). The com-bination of DNA methylation and histone modifications has beenproposed to constitute a code read by effector proteins to turnon, turn off, or modulate transcription (20, 34). Increasing evi-dence also indicates that specific histone modification and DNAmethylation patterns mark promoters for potential activation inundifferentiated cells (17, 26, 27, 35).
2. Analysis of DNA-Bound Proteinsby ChromatinImmunoprecipi-tation
Chromatin immunoprecipitation (ChIP) has become the techni-que of choice to investigate proteinDNA interactions inside thecell (36, 37). ChIP has been used for mapping the localization ofpost-translationally modified histones and histone variants in thegenome and for mapping DNA target sites for transcription factorsand other chromosome-associated proteins.
The principle of the ChIP assay is outlined in Fig. 1.2. DNAand proteins are commonly reversibly cross-linked with formalde-hyde (which is heat-reversible) to covalently attach proteins totarget DNA sequences. Formaldehyde cross-links proteins andDNA molecules within 2 A of each other, and thus is suitablefor looking at proteins which directly bind DNA. The short cross-linking arm of formaldehyde, however, is not suitable for examin-ing proteins that indirectly associate with DNA, such as thosefound in larger complexes. As a remedy to this limitation, a varietyof long-range bifunctional cross-linkers have been used in combi-nation with formaldehyde to detect proteins on target sequences,which could not be detected with formaldehyde alone (38). Incontrast to cross-link ChIP, native ChIP (NChIP) omits cross-linking (37, 39). NChIP is well suited for the analysis of histonesbecause of their high affinity for DNA. In both cross-link ChIPand NChIP, chromatin is subsequently fragmented, either byenzymatic digestion with micrococcal nuclease (MNase, whichdigests DNA at the level of the linker, leaving nucleosomes intact)or by sonication of whole cells or nuclei, into fragments of
Fig. 1.1. Known post-translational modifications of histones.
The State-of-the-Art of Chromatin Immunoprecipitation 3
2001,000 base pair (bp), with an average of 500 bp. The lysate iscleared by sedimentation and proteinDNA complexes are immu-noprecipitated from the supernatant (chromatin) using antibodiesto the protein of interest. Immunoprecipitated complexes arewashed under stringent conditions to remove non-specificallybound chromatin, the cross-link is reversed, proteins are digested,and the precipitated ChIP-enriched DNA is purified. DNAsequences associated with the precipitated protein can be identi-fied by end-point polymerase chain reaction (PCR), quantitative(q)PCR, labeling and hybridization to genome-wide or tilingDNA microarrays (ChIP-on-chip) (4042), molecular cloningand sequencing (43, 44), or direct high-throughput sequencing(ChIP-seq) (45) (Fig. 1.2).
Development of techniques leading to the ChIP assay as weknow it since the mid-1990s has occurred over many years[reviewed in (46)]. The use of formaldehyde to cross-link proteinswith proteins or proteins with DNA, however, was first reported in
Fig. 1.2. Outline of the chromatin immunoprecipitation (ChIP) assay and various methodsof analysis.
the 1960s and its application to study histoneDNA interactionswithin the nucleosome goes back to the mid-late 1970s. Thedevelopment of anti-histone antibodies 20 years ago, to investi-gate the association of histones with DNA in relation to transcrip-tion, led the path to the ChIP assay (47). Pioneering studiesshowed that during heat shock, histone H4 remained associatedwith the HSP70 gene (47). Subsequent improvements in theprocedure enabled the demonstration that the interaction of his-tone H1 with DNA was altered during changes in transcriptionalactivity in Tetrahymena (48). The availability of antibodies to post-translationally modified histones, in combination with ChIP, hasbeen instrumental in the understanding of transcription regulationin the early 1990s. For instance, antibodies to acetylated histoneshave been used to show that, using the b-globin locus as a targetgenomic sequence, core histone acetylation is associated withchromatin that is active or poised for transcription (4952). TheChIP assays have since been extended to non-histone proteins,including less-abundant protein complexes, and to a wide range oforganisms such as protozoa, yeast, sea urchin, flies, fish, and avianand mammalian cells (46).
For well over a decade, ChIP has remained a cumbersomeprotocol, requiring 34 days and large number of cells in themulti-million range per immunoprecipitation. These limitationshave restricted the application of ChIP to large cell samples. Clas-sical ChIP assays also involve extensive sample handling (37, 53),which is a source of loss of material, creates opportunities fortechnical errors, and enhances inconsistency between replicates.As a remedy to these limitations, modifications have been made tomake ChIP protocols shorter, simpler, and allow analysis of smallcell samples (39, 5457).
This introductory review addresses modifications of conven-tional ChIP assays, which have recently been introduced to sim-plify and accelerate the procedure and enable the analysis ofDNA-bound proteins in small cell samples. Analytical tools thatcan be combined with ChIP to address the landscape of proteinDNA interactions are also presented.
3. ChIP Assaysfor Small CellNumbers
A major drawback of ChIP has for a long time been the require-ment for large cell numbers. This has been necessary to compen-sate for the loss of cells upon recovery after cross-linking, for theoverall inefficiency of ChIP, and for the relative insensitivity ofdetection of ChIP-enriched DNA. The need for elevated cellnumbers has hampered the application of ChIP to rare cell
The State-of-the-Art of Chromatin Immunoprecipitation 5
samples, such as cells from small tissue biopsies, rare stem cellpopulations, or cells from embryos. Several recent publicationshave addressed this issue and reported alterations of conventionalChIP protocols to make the technique applicable to smaller num-ber of cells.
3.1. CChIP The rationale behind the carrier ChIP, or CChIP, is that theimmunoprecipitation of a small amount of chromatin preparedfrom few mammalian cells (1001,000) is facilitated by the addi-tion of carrier chromatin from Drosophila or any other speciessufficiently evolutionarily distant from the species investigated(39). CChIP involves the mixing of cultured Drosophila cellswith a small number of mammalian cells. Native chromatin frag-ments are prepared from purified nuclei by partial MNase diges-tion and immunoprecipitated using antibodies to modifiedhistones. To compensate for the small amount of target DNAprecipitated, the ChIP DNA is detected by radioactive PCR andphosphorimaging. Specificity of amplification is monitored foreach ChIP by determination of the size of the DNA fragmentproduced (39).
CChIP has proven to be suitable for the analysis of 100-cellsamples. A limitation, however, is that analysis of multiple histonemodifications requires multiple aliquots of 100 cells which may ormay not be identical. Furthermore, in its published form, CChIP isbased on the NChIP procedure (37) and as such is not suited forprecipitation of transcription factors. Nonetheless, there is noreason to believe that CChIP is not compatible with cross-linking,and thereby becomes more versatile. Despite these limitations,however, the benefit of CChIP for analyzing small cell samples isalready clear.
Using CChIP, ONeill et al. (39) have reported an analysis ofactive and repressive histone modifications on a handful of targetloci in mouse inner cell mass and trophectoderm cells the two celltypes of the blastocyst. Application of CChIP to embryonic tran-scription factors in embryos and embryonic stem (ES) cells tounravel common and distinct target genes should enhance ourunderstanding of the molecular basis of pluripotency.
3.2. Q2ChIP As an alternative to CChIP, a quick and quantitative (Q2)ChIPprotocol suitable for up to 1,000 histone ChIPs or up to 100transcription factor ChIPs from as few as 100,000 cells has beendeveloped in our laboratory (56). Q2ChIP involves a chromatinpreparation from a larger number of cells than CChIP, butincludes chromatin dilution and aliquoting steps which allow forstorage of many identical chromatin aliquots from a single pre-paration. Because Q2ChIP involves a cross-linking step, chromatinsamples are also suitable for immunoprecipitation of transcription
factors or other non-histone DNA-bound proteins. ProteinDNAcross-linking in suspension in the presence of a histone deacetylaseinhibitor, elimination of essentially all non-specific backgroundchromatin through a tube-shift after washes of the ChIP material,and combination of cross-linking reversal, protein digestion, andDNA elution into a single 2-h step considerably shorten the pro-cedure and enhance the ChIP efficiency (56). Suitability ofQ2ChIP to small amounts of chromatin has been attributed tothe reduction of the number of steps in the procedure and increasein the ratio of antibody-to-target epitope, resulting in an enhancedsignal-to-noise ratio. Q2ChIP has been validated against the con-ventional ChIP assay from which it was derived (53). It has beenused to illustrate changes in histone H3K4, K9, and K27 acetyla-tion and methylation associated with differentiation of embryonalcarcinoma cells on developmentally regulated promoters (56).
3.3. mChIP With the aim of further reducing the number of cells used, wesubsequently devised a micro (m)ChIP protocol suitable for up tonine parallel ChIPs of modified histones and/or RNA polymeraseII (RNAPII) from...