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Hox Genes pp 291-306 | Cite as

cgChIP: A Cell Type- and Gene-Specific Method for Chromatin Analysis

  • Marios Agelopoulos
  • Daniel J. McKay
  • Richard S. MannEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1196)

Abstract

Hox and other homeobox-containing genes encode critical transcriptional regulators of animal development. Although these genes are well known for their roles in the body axis and appendage development, little is known regarding the mechanisms by which these factors influence chromatin landscapes. Chromatin structure can have a profound influence on gene expression during animal body formation. However, when applied to developing embryos, conventional chromatin analysis of genes and cis-regulatory modules (CRMs) typically lacks the required cell type-specific resolution due to the heterogeneous nature of animal bodies. Here we present a strategy to analyze both the composition and conformation of in vivo-tagged CRM sequences in a cell type-specific manner, using as a system Drosophila embryos. We term this method cgChIP (cell- and gene-specific Chromatin Immunoprecipitation) by which we access and analyze regulatory chromatin in specific cell types. cgChIP is an in vivo method designed to analyze genetic elements derived from limited cell populations. cgChIP can be used for both the analysis of chromatin structure (e.g., long-distance interactions between DNA elements) and the composition of histones and histone modifications and the occupancy of transcription factors and chromatin modifiers. This method was applied to the Hox target gene Distalless (Dll), which encodes for a homeodomain-containing transcription factor critical for the formation of appendages in Drosophila. However, cgChIP can be applied in diverse animal models to better dissect CRM-dependent gene regulation and body formation in developing animals.

Key words

Hox Chromatin Histone modifications DNA looping Cis-regulatory modules (CRMs) Enhancer-promoter communication Gene transcription LacI/lacO 

Notes

Acknowledgments

This work was supported by GM058575 and GM054510 awarded to R.S.M. D.J.M. was supported by 5T32DK07328 and M.A. was supported by a long-term EMBO fellowship. We thank D. Arvanitis and S. Tsiftsoglou for critical proofreading.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Marios Agelopoulos
    • 1
    • 2
  • Daniel J. McKay
    • 1
    • 3
  • Richard S. Mann
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular BiophysicsColumbia University Medical CenterNew YorkUSA
  2. 2.Institute of Molecular Biology, Genetics and BiotechnologyBiomedical Research Foundation, Academy of AthensAthensGreece
  3. 3.Department of BiologyUniversity of North Carolina at Chapel HillChapel HillUSA

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