Abstract
Immunoprecipitation of cross-linked chromatin in combination with microarrays (ChIP-chip) or ultra high-throughput sequencing (ChIP-seq) is widely used to map genome-wide in vivo transcription factor binding. Both methods employ initial steps of in vivo cross-linking, chromatin isolation, DNA fragmentation, and immunoprecipitation. For ChIP-chip, the immunoprecipitated DNA samples are then amplified, labeled, and hybridized to DNA microarrays. For ChIP-seq, the immunoprecipitated DNA is prepared for a sequencing library, and then the library DNA fragments are sequenced using ultra high-throughput sequencing platform. The protocols described here have been developed for ChIP-chip and ChIP-seq analysis of sequence-specific transcription factor binding in Drosophila embryos. A series of controls establish that these protocols have high sensitivity and reproducibility and provide a quantitative measure of relative transcription factor occupancy. The quantitative nature of the assay is important because regulatory transcription factors bind to highly overlapping sets of thousands of genomic regions and the unique regulatory specificity of each factor is determined by relative moderate differences in occupancy between factors at commonly bound regions.
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References
Biggin MD, Tjian R (2001) Transcriptional regulation in Drosophila: the post-genome challenge. Funct Integr Genomics. 1, 223–234.
Gilmour, D. S., Rougvie, A. E., and Lis, J. T. (1991) Protein-DNA cross-linking as a means to determine the distribution of proteins on DNA in vivo. Meth. Cell Biol. 35, 369–381.
Carr, A. and Biggin, M.D. (1999) An in vivo UV crosslinking assay that detects DNA binding by sequence-specific transcription factors. Methods Mol Biol. 119, 497–508.
Orlando, V. (2000) Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation, Trends Biochem. Sci. 25, 99–104.
Ren, B., Robert, F., Wyrick, J.J., Aparicio, O., Jennings, E.G., Simon, I., Zeitlinger, J., Schreiber, J., Hannett, N., Kanin, E., Volkert, T.L., Wilson, C.J., Bell, S.P., and Young, R.A. (2000) Genome-wide location and function of DNA binding proteins. Science 290, 2306–9.
Iyer, V.R., Horak, C.E., Scafe, C.S., Botstein, D., Snyder, M., et al. (2001) Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF. Nature 409, 533-538.
Metzker, M.L. (2010) Sequencing technologies — the next generation. Nature Reviews Genetics 11, 31–46.
Johnson, D.S., Mortazavi, A., Myers, R.M.,and Wold, B. (2007) Genome-wide mapping of in vivo protein–DNA interactions. Science 316, 1497–1502.
Barski, A., Cuddapah, S., Cui, K., Roh, T.Y., D.E. Schones et al., (2007) High-resolution profiling of histone methylations in the human genome, Cell 129, 823–837.
Robertson, G., Hirst, M., Bainbridge, M., Bilenky, M., Zhao Y. et al. (2007) Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing, Nat. Methods 4, 651–657.
Toth, J., and Biggin, M.D. (2000) The specificity of protein-DNA crosslinking by formaldehyde: in vitro and in drosophila embryos. Nucleic Acids Res. 15, e4.
Auerbach, R.K., Euskirchen, G., Rozowsky, J., Lamarre-Vincent, N., Moqtaderi, Z., et al. (2009) Mapping accessible chromatin regions using Sono-Seq. Proc Natl Acad Sci USA 106, 14926–14931.
Li, X.-Y., Thomas,S., Sabo, P.J., Eisen, M.B., Stamatoyannopoulos, J.A., and Biggin, M.D. (2011) The role of chromatin accessibility in directing the widespread, overlapping patterns of Drosophila transcription factor binding. Genome Biology. 12, R34.
Kaplan, T., Li, X.-Y., Sabo, P.J., Thomas, S., Stamatoyannopoulos, J.A., Biggin, M.D. , and Eisen, M.B. (2011) Predicting the Landscape of Transcription Factor Binding During Early Drosophila Development. PLoS Genet. 7, e1001290.
Li, X.-Y., Macarthur, S., Bourgon, R., Nix, D., Pollard, D.A., et al. (2008) Transcription Factors Bind Thousands of Active and Inactive Regions in the Drosophila Blastoderm. Plos Biol. 6, e27.
Macarthur, S., Li, X.-Y., Li, J., Brown, J.B., Chu, H.C., et al. (2009) Developmental roles of 21 Drosophila transcription factors are determined by quantitative differences in binding to an overlapping set of thousands of genomic regions. Genome Biol . 10, R80.
Bradley, R.K., Li, X.-Y., Trapnell, C., Davidson, S., Pachter, L., et al. (2010) Binding site turnover produces pervasive quantitative changes in transcription factor binding between closely related Drosophila species. Plos Biol. 8, e1000343.
Quail, M.A., Kozarewa, I., Smith, F., Scally, A., Stephens, P.J. et al., (2008) A large genome center’s improvements to the Illumina sequencing system, Nat. Methods 5, 1005–1010.
O’Geen, H., Nicolet, C.M., Blahnik, K., Green, R., Farnham, P.J. (2006) Comparison of sample preparation methods for ChIP-chip assays. Biotechniques 41, 577–80.
Li, H., Ruan, J. & Durbin, R. (2008) Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 18, 1851–1858.
Langmead B, Trapnell C, Pop M, Salzberg SL. (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25.
Ji, H., Jian, H., Ma., W., Johnson, D.S., Myers, R.M., and Wong, W,H. (2008) An integrated software system for analyzing CHIP-chip and ChIP-seq data. Nat. Biotechnol. 26, 1293–1300.
Rozowsky, J., Euskirchen, G., Auerbach, R.K., Zhang, Z.D., Gibson, T., Bjornson, R., Carriero, N., Snyder, M., Gerstein, M.B. (2009) PeakSeq enables systematic scoring of ChIP-seq experiments relative to controls. Nat Biotechnol. 27, 66–75.
Jothi, R., Cuddapah, S., Barski, A., Cui, K., Zhao, K. (2008) Genome-wide identification of in vivo protein-DNA binding sites from ChIP-Seq data. Nucleic Acids Res. 36, 5221–31.
Zhang ,Y., Liu, T., Meyer, C.A., Eeckhoute, J., Johnson, D.S., Bernstein, B.E., Nussbaum, C., Myers, R.M., Brown, M., Li, W., et al. (2008) Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137.
Tuteja, G., White, P., Schug, J., Kaestner, K.H.. (2009) Extracting transcription factor targets from ChIP-Seq data. Nucleic Acids Res. 37, e113.
Blahnik, K.R., Dou, L., O’Geen, H., McPhillips, T., Xu, X., Cao, A.R., Iyengar, S., Nicolet, C.M., Ludäscher, B., Korf, I., and Farnham, P.J. (2010) Sole-Search: an integrated analysis program for peak detection and functional annotation using ChIP-seq data. Nucleic Acids Res. 38, e13.
Carr. A., Biggin, M.D. (1999) A comparison of in vivo and in vitro DNA-binding specificities suggests a new model for homeoprotein DNA binding in Drosophila embryos. EMBO J. 18,1598–608.
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Li, XY., Biggin, M.D. (2012). Genome-Wide In Vivo Cross-linking of Sequence-Specific Transcription Factors. In: Vancura, A. (eds) Transcriptional Regulation. Methods in Molecular Biology, vol 809. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-376-9_1
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DOI: https://doi.org/10.1007/978-1-61779-376-9_1
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