Using ChIP-Based Approaches to Characterize FOXO Recruitment to its Target Promoters

  • Neeraj Kumar
  • Arnab MukhopadhyayEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1890)


Chromatin immunoprecipitation (ChIP) coupled to quantitative real-time PCR (ChIP-qPCR) or Next-Generation Sequencing (ChIP-seq) enables us to study the dynamics of chromatin recruitment of transcription factors (TFs). The popular model system Caenorhabditis elegans has provided us with fundamental understanding of the role of Insulin/IGF-1-like signaling (IIS) in metabolism and aging. The FOXO TF DAF-16 is the major output of the pathway that regulates most of the phenotypes associated with the IIS pathway. Here, we describe a ChIP protocol to study FOXO recruitment dynamics in whole C. elegans extracts. We discuss detailed practical procedures, including optimization, growth, harvesting, formaldehyde fixation, sonication of worms, TF immunoprecipitation for further downstream processing using qPCR as well as NGS for the analysis of FOXO-bound DNA.

Key words

Chromatin immunoprecipitation Quantitative real-time PCR Next-generation sequencing Transcription factor FOXO DAF-16 Gene promotor C. elegans 


  1. 1.
    Walhout AJ (2006) Unraveling transcription regulatory networks by protein-DNA and protein-protein interaction mapping. Genome Res 16(12):1445–1454CrossRefGoogle Scholar
  2. 2.
    Collas P, Dahl JA (2008) Chop it, ChIP it, check it: the current status of chromatin immunoprecipitation. Front Biosci 13(17):929–943CrossRefGoogle Scholar
  3. 3.
    Das PM, Ramachandran K, vanWert J et al (2004) Chromatin immunoprecipitation assay. BioTechniques 37(6):961–969CrossRefGoogle Scholar
  4. 4.
    Gade P, Kalvakolanu DV (2012) Chromatin immunoprecipitation assay as a tool for analyzing transcription factor activity. Methods Mol Biol 809:85–104CrossRefGoogle Scholar
  5. 5.
    Hoffman EA, Frey BL, Smith LM et al (2015) Formaldehyde crosslinking: a tool for the study of chromatin complexes. J Biol Chem 290(44):26404–26411CrossRefGoogle Scholar
  6. 6.
    Sambrook J, Russell DW (2006) Fragmentation of DNA by sonication. CSH Protoc 2006(4):pdb.prot4538PubMedGoogle Scholar
  7. 7.
    Duband-Goulet I (2016) Lamin ChIP from chromatin prepared by micrococcal nuclease digestion. Methods Mol Biol 1411:325–339CrossRefGoogle Scholar
  8. 8.
    Carey MF, Peterson CL, Smale ST (2009) Chromatin immunoprecipitation (ChIP). Cold Spring Harb Protoc 2009(9):pdb.prot5279PubMedGoogle Scholar
  9. 9.
    Sambrook J, Russell DW (2006) Purification of nucleic acids by extraction with phenol:chloroform. CSH Protoc 2006(1):pdb.prot4455PubMedGoogle Scholar
  10. 10.
    Collas P (2010) The current state of chromatin immunoprecipitation. Mol Biotechnol 45(1):87–100CrossRefGoogle Scholar
  11. 11.
    Kenyon C (2005) The plasticity of aging: insights from long-lived mutants. Cell 120(4):449–460CrossRefGoogle Scholar
  12. 12.
    Mukhopadhyay A, Oh SW, Tissenbaum HA (2006) Worming pathways to and from DAF-16/FOXO. Exp Gerontol 41(10):928–934CrossRefGoogle Scholar
  13. 13.
    Lin K, Hsin H, Libina N et al (2001) Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling. Nat Genet 28(2):139–145CrossRefGoogle Scholar
  14. 14.
    Grishok A, Sharp PA (2005) Negative regulation of nuclear divisions in Caenorhabditis elegans by retinoblastoma and RNA interference-related genes. Proc Natl Acad Sci U S A 102(48):17360–17365CrossRefGoogle Scholar
  15. 15.
    Whetstine JR, Ceron J, Ladd B et al (2005) Regulation of tissue-specific and extracellular matrix-related genes by a class I histone deacetylase. Mol Cell 18(4):483–490CrossRefGoogle Scholar
  16. 16.
    Lee MH, Hook B, Lamont LB et al (2006) LIP-1 phosphatase controls the extent of germline proliferation in Caenorhabditis elegans. EMBO J 25(1):88–96CrossRefGoogle Scholar
  17. 17.
    Ercan S, Giresi PG, Whittle CM et al (2007) X chromosome repression by localization of the C. elegans dosage compensation machinery to sites of transcription initiation. Nat Genet 39(3):403–408CrossRefGoogle Scholar
  18. 18.
    Riedel CG, Dowen RH, Lourenco GF et al (2013) DAF-16/FOXO employs the chromatin remodeller SWI/SNF to promote stress resistance and longevity. Nat Cell Biol 15(5):491CrossRefGoogle Scholar
  19. 19.
    Oh SW, Mukhopadhyay A, Dixit BL et al (2006) Identification of direct DAF-16 targets controlling longevity, metabolism and diapause by chromatin immunoprecipitation. Nat Genet 38(2):251–257CrossRefGoogle Scholar
  20. 20.
    Mukhopadhyay A, Deplancke B, Walhout AJ et al (2008) Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans. Nat Protoc 3(4):698–709CrossRefGoogle Scholar
  21. 21.
    Kumar N, Jain V, Singh A et al (2015) Genome-wide endogenous DAF-16/FOXO recruitment dynamics during lowered insulin signalling in C. elegans. Oncotarget 6(39):41418–41433CrossRefGoogle Scholar
  22. 22.
    Mikeska T, Dobrovic A (2009) Validation of a primer optimisation matrix to improve the performance of reverse transcription–quantitative real-time PCR assays. BMC Res Notes 2(1):112CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Reproductive BiologyAll India Institute of Medical Sciences (AIIMS)New DelhiIndia
  2. 2.Molecular Aging LaboratoryNational Institute of ImmunologyNew DelhiIndia

Personalised recommendations