In order to determine how signaling pathways differentially regulate gene expression, it is necessary to identify the interactions between transcription factors (TFs) and their cognate cis-regulatory DNA elements. Here, we have outlined a chromatin immunoprecipitation (ChIP) protocol for use in whole Caenorhabditis elegans extracts. We discuss optimization of the procedure, including growth and harvesting of the worms, formaldehyde fixation, TF immunoprecipitation and analysis of bound sequences through real-time PCR. It takes ∼10–12 d to obtain the worm culture for ChIP; the ChIP procedure is spaced out over a period of 2.5 d with two overnight incubations.
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Walhout, A.J. Unraveling transcription regulatory networks by protein-DNA and protein–protein interaction mapping. Genome Res. 16, 1445–1454 (2006).
Collas, P. & Dahl, J.A. Chop it, ChIP it, check it: the current status of chromatin immunoprecipitation. Front. Biosci. 13, 929–943 (2008).
Das, P.M., Ramachandran, K., vanWert, J. & Singal, R. Chromatin immunoprecipitation assay. Biotechniques 37, 961–969 (2004).
Mukherjee, S. et al. Rapid analysis of the DNA-binding specificities of transcription factors with DNA microarrays. Nat. Genet. 36, 1331–1339 (2004).
Meng, X., Brodsky, M.H. & Wolfe, S.A. A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors. Nat. Biotechnol. 23, 988–994 (2005).
Deplancke, B., Dupuy, D., Vidal, M. & Walhout, A.J. A gateway-compatible yeast one-hybrid system. Genome Res. 14, 2093–2101 (2004).
Deplancke, B. et al. A gene-centered C. elegans protein-DNA interaction network. Cell 125, 1193–1205 (2006).
Johnson, D.S., Mortazavi, A., Myers, R.M. & Wold, B. Genome-wide mapping of in vivo protein-DNA interactions. Science 316, 1497–1502 (2007).
Dickmeis, T. & Müller, F. The identification and functional characterisation of conserved regulatory elements in developmental genes. Brief Funct. Genomic Proteomic 3, 332–350 (2005).
Ji, H. & Wong, W.H. Computational biology: toward deciphering gene regulatory information in mammalian genomes. Biometrics 62, 645–663 (2006).
Grishok, A. & Sharp, P.A. Negative regulation of nuclear divisions in Caenorhabditis elegans by retinoblastoma and RNA interference-related genes. Proc. Natl. Acad. Sci. USA 102, 17360–17365 (2005).
Whetstine, J.R. et al. Regulation of tissue-specific and extracellular matrix-related genes by a class I histone deacetylase. Mol. Cell 18, 483–490 (2005).
Lee, M.H., Hook, B., Lamont, L.B., Wickens, M. & Kimble, J. LIP-1 phosphatase controls the extent of germline proliferation in Caenorhabditis elegans. EMBO J. 25, 88–96 (2006).
Ercan, S. et al. X chromosome repression by localization of the C. elegans dosage compensation machinery to sites of transcription initiation. Nat. Genet. 39, 403–408 (2007).
Oh, S.W. et al. Identification of direct DAF-16 targets controlling longevity, metabolism and diapause by chromatin immunoprecipitation. Nat. Genet. 38, 251–257 (2006).
Rand, J.B. & Johnson, C.D. Genetic pharmacology: interactions between drugs and gene products in Caenorhabditis elegans. Methods Cell Biol. 48, 187–204 (1995).
Bustin, S.A., Benes, V., Nolan, T. & Pfaffl, M.W. Quantitative real-time RT-PCR—a perspective. J. Mol. Endocrinol. 34, 597–601 (2005).
Nolan, T., Hands, R.E. & Bustin, S.A. Quantification of mRNA using real-time RT-PCR. Nat. Protoc. 1, 1559–1582 (2006).
Ginzinger, D.G. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp. Hematol. 30, 503–512 (2002).
Breslauer, K.J., Frank, R., Blocker, H. & Marky, L.A. Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. USA 83, 3746–3750 (1986).
Murphy, C.T. et al. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature 424, 277–283 (2003).
Honda, Y. & Honda, S. The daf-2 gene network for longevity regulates oxidative stress resistance and Mn-superoxide dismutase gene expression in Caenorhabditis elegans. FASEB J. 13, 1385–1393 (1999).
McElwee, J.J., Schuster, E., Blanc, E., Thomas, J.H. & Gems, D. Shared transcriptional signature in Caenorhabditis elegans Dauer larvae and long-lived daf-2 mutants implicates detoxification system in longevity assurance. J. Biol. Chem. 279, 44533–44543 (2004).
Lin, K., Dorman, J.B., Rodan, A. & Kenyon, C. daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278, 1319–1322 (1997).
Lin, K., Hsin, H., Libina, N. & Kenyon, C. Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling. Nat. Genet. 28, 139–145 (2001).
Lee, R.Y., Hench, J. & Ruvkun, G. Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway. Curr. Biol. 11, 1950–1957 (2001).
Henderson, S.T. & Johnson, T.E. daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans. Curr. Biol. 11, 1975–1980 (2001).
H.A.T. is a William Randolph Hearst Young Investigator. This project was funded in part by a Burroughs Wellcome Career Award in the Biomedical Sciences to H.A.T., an endowment from the William Randolph Hearst Foundation, and grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK068429 to A.J.M.W.) and National Institute of Aging (AG25891 to H.A.T.).
About this article
Cite this article
Mukhopadhyay, A., Deplancke, B., Walhout, A. et al. Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans. Nat Protoc 3, 698–709 (2008). https://doi.org/10.1038/nprot.2008.38
The transcriptional coactivator CBP/p300 is an evolutionarily conserved node that promotes longevity in response to mitochondrial stress
Nature Aging (2021)
Journal of Molecular Neuroscience (2021)
Genome Research (2021)
Journal of Cellular Biochemistry (2020)
Whole-genome landscape of H3K4me3, H3K36me3 and H3K9ac and their association with gene expression during Paulownia witches’ broom disease infection and recovery processes
3 Biotech (2020)