ChIP-on-chip

ChIP-on-chip is an exciting new application that has significantly improved the amount of information generated from chromatin immunoprecipitation experiments. Researchers can now generate genome-wide maps of histone modifications and chromatin associated proteins. This will serve to provide us with a comprehensive understanding of where regulatory proteins interact with the genome in vivo and will greatly increase our understanding of mechanisms such as transcription, DNA replication, recombination and DNA repair.

1. Background

Chromatin immunoprecipitation (ChIP) is a powerful experimental approach for identifying the proteins associated with specific regions of the genome. ChIP can be used to locate both non-histone proteins and histones carrying specific covalent modifications. Using appropriate antibodies chromatin is precipitated and the associated DNA is purified. Analysis for enrichment of given genomic regions can be performed in a number of ways.

Conventional slot blotting: The immunoprecipitated DNA is applied to a membrane and hybridised using the DNA regions of interest (Dedon et al., 1991).
Southern Blot analysis: This permits the identification of binding sites across large genomic regions without relying on multiple hybridisations or PCR reactions by using the precipitated DNA as a probe (Orlando and Paro, 1993).
PCR based strategies: Perhaps the most popular, whether quantitative (Hecht et al., 1996), real-time quantitative (Litt et al., 2001) or semi-quantitative (Breiling et al., 2001).
Microarray analysis: Generating high resolution genome-wide maps of patterns of histone modification or non-histone proteins using the relatively new technique of Chip-on-chip.

2. What is ChIP-on-chip?

Traditional methods of investigation (e.g. approaches 1-3 above) have failed to create high-resolution, genome-wide maps of the interaction between a DNA-binding protein and DNA, often relying on handpicked genomic loci and multiple PCR reactions. The development of glass slide DNA microarrays has allowed whole genome analysis of the purified DNA in ChIP-on-chip analysis.

In brief DNA is purified from the precipitated chromatin and a sample representative of the starting chromatin and fluorescently labelled with different dyes using ligation mediated PCR (see figure 1). The dye-labelled DNA is applied to the same microarray. The array(s) are scanned and the enrichment of the regulatory protein relative to the input is recorded at each genomic locus.

Figure 1: ChIP-on-chip schematic

3. The importance of antibodies in ChIP-on-chip

Given that ChIP-on-chip is an extension of chromatin immunoprecipitation, the importance of the antibody cannot be underestimated. A well characterised antibody is crucial in ChIP because not only must it recognise its antigen in free solution but also under fixed conditions. Successful application of an antibody in immunoprecipitation (or in immunhistochemistry) gives a good indicator as to its potential success using ChIP. At Abcam we operate a ChIP testing programme run by a postdoctoral scientist in our laboratory using our recommended ChIP protocol. When an antibody is demonstrated to immunoprecipite cross-linked chromatin it is assigned a “ChIP-grade” status. Abcam also qualifies antibodies applied using ChIP in publications and those given favourable Abreviews for use in ChIP.

4. Abcam antibodies for ChIP-on-chip

Recently Abcam’s antibodies have been used to analyse patterns of histone modifications (Pokolok et al., 2005) and nucleosomal occupancy across the entire yeast genome (Bernstein et al., 2004; Lee et al., 2004).

Richard Young’s group at the Whitehead Institute, Cambridge, MA profiled nucleosomal modifications across the yeast genome and produced high-resolution genome-wide maps of histone acetylation and methylation. They demonstrate that both modifications are associated with transcriptional activity. However, histone acetylation occurs predominantly at the beginning of genes whereas histone methylation can occur throughout the transcribed region. Most notable there were striking differences in the distribution of mono-, di- and tri-methylation and the lysine that is methylated. The Abcam antibodies used in this study were:

Bradley Bernstein’s (Bauer Center for Genomics Research, Cambridge, MA) and Jason Lieb’s group (Carolina Center for the Genome Sciences, University of North Carolina at Chapel Hill, North Carolina) have shown depletion in nucleosomal occupancy at the promoter regions of active genes in the yeast genome by ChIP-on-chip. Both groups performed chromatin immunoprecipitation using Abcam’s antibody against unmodified histone H3 (ab1791) before applying the DNA to custom designed microarrays incorporating ORFs and intergenic regions. Both groups found an indirect correlation between the degree of nucleosomal occupancy and the level of transcription.

5. DNA microarrays today

The ChIP-on-chip technique was first applied successfully almost four years ago (Lieb et al., 2001; Ren et al., 2000; Iyer et al., 2001) to identify binding sites for individual transcription factors in yeast and on a genome wide level a couple of years later (Lee et al., 2002). Experiments in mammalian genomes have proven more difficult due to their large and repetitive nature. Indeed many of the ChIP-on-chip studies in mammalian cells have analyzed only selected predicted promoter regions and have not interrogated the entire genome.

Mammalian whole genome arrays have recently become commercially available at Nimblegen. Agilent offer custom arrays, mammalian promoter arrays in addition to the whole yeast genome array used by Richard Young’s group in the aforementioned study. Agilent also offer the ENCODE array (Encyclopedia of DNA Elements) which covers regions of the genome specifically selected by a committee of the National Human Genome Research Institute (NHGRI). The aim of the ENCODE project is to identify all functional elements in the human genome sequence. The ENCODE array is comprised of 30 Mb of DNA, or approximately 1 percent of the human genome. Affymetrix also offer custom arrays and the ENCODE array.

6. Summary

ChIP-on-chip will ultimately provide researchers with a genome-wide map of the histone modifications and non-histone proteins in mammalian systems. It will also serve to further develop our understanding of the affects of cancer on the genome wide chromatin landscape.

At Abcam we are excited to see our antibodies being applied in ChIP-on-chip. In order to let you know whether our antibodies have been applied using ChIP-on-chip in publications or in collaborative experiments we qualify these products with “ChIP-on-chip” in the list of tested applications. However, we have every confidence that our “ChIP grade” antibodies can be applied in ChIP-on-chip analyses.

7. References

Bernstein BE, Liu CL, Humphrey EL, Perlstein EO, Schreiber SL. (2004) Global nucleosome occupancy in yeast. Genome Biol. 5(9), R62.

Breiling A, Turner BM, Bianchi ME, Orlando V. (2001) General transcription factors bind promoters repressed by Polycomb group proteins. Nature 412, 651-5.

Dedon PC, Soults JA, Allis CD, Gorovsky MA. (1991) Formaldehyde cross-linking and immunoprecipitation demonstrate developmental changes in H1 association with transcriptionally active genes. Mol. Cell Biol. 11(3), 1729-1733.

Hecht A, Strahl-Bolsinger S, Grunstein M. (1996) Spreading of transcriptional repressor SIR3 from telomeric heterochromatin. Nature 383(6595), 92-6.

Iyer VR, Horak CE, Scafe CS, Botstein D, Snyder M, Brown PO. (2001) Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF. Nature. 409(6819), 533-8.

Lee CK, Shibata Y, Rao B, Strahl BD, Lieb JD. (2004) Evidence for nucleosome depletion at active regulatory regions genome-wide. Nat. Genet. 36(8), 900-5.

Lee TI, Rinaldi NJ, Robert F, Odom DT, Bar-Joseph Z, Gerber GK, Hannett NM, Harbison CT, Thompson CM, Simon I, Zeitlinger J, Jennings EG, Murray HL, Gordon DB, Ren B, Wyrick JJ, Tagne JB, Volkert TL, Fraenkel E, Gifford DK, Young RA. (2002) Transcriptional regulatory networks in Saccharomyces cerevisiae. Science. 298(5594):799-804.

Lieb JD, Liu X, Botstein D, Brown PO. (2001) Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association. Nat. Genet. 29(1), 100.

Litt MD, Simpson M, Gaszner M, Allis CD, Felsenfeld G. (2001) Correlation between histone lysine methylation and developmental changes at the chicken beta-globin locus. Science 28 293(5539), 2453-5.

Orlando V, Paro R. (1993) Mapping Polycombrepressed domains in the BX-C using in vivo formaldehyde crosslinked chromatin Cell 75,1187–1198

Pokholok DK, Harbison CT, Levine S, Cole M, Hannett NM, Lee TI, Bell GW, Walker K, Rolfe PA, Herbolsheimer E, Zeitlinger J, Lewitter F, Gifford DK, Young RA. (2005) Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122(4), 517-27.

Ren B, Robert F, Wyrick JJ, Aparicio O, Jennings EG, Simon I, Zeitlinger J, Schreiber J, Hannett N, Kanin E, Volkert TL, Wilson CJ, Bell SP, Young RA. (2000) Genome-wide location and function of DNA binding proteins. Science. 290(5500), 2306-9.

8. ChIP-on-chip publications using Abcam antibodies