Advancing Epigenetics Towards Systems Biology

Sensitive detection of chromatin co-associations using enhanced chromosome conformation capture on chip (Prot 60)

Tom Sexton1, Sreenivasulu Kurukuti2, Jennifer A Mitchell3, David Umlauf4, Takashi Nagano5, Peter Fraser5

Introduction

Chromosome conformation capture (3C) is a powerful technique for analyzing spatial chromatin organization in vivo. Technical variants of the assay ('4C') allow the systematic detection of genome-wide co-associations with bait sequences of interest, enabling the nuclear environments of specific genes to be probed. We describe enhanced 4C (e4C, enhanced chromosome conformation capture on chip), a technique incorporating additional enrichment steps for bait-specific sequences, and thus improving sensitivity in the detection of weaker, distal chromatin co-associations. In brief, e4C entails the fixation, restriction digestion and ligation steps of conventional 3C, with an optional chromatin immunoprecipitation (ChIP) step to select for subsets of chromatin co-associations, followed by bait enrichment by biotinylated primer extension and pull-down, adapter ligation and PCR amplification. Chromatin co-associations with the bait sequence can then be assessed by hybridizing e4C products to microarrays or sequencing. The e4C procedure takes approximately 1 week to go from tissue to DNA ready for microarray hybridization.

PDF version or direct link to the protocol

Tom Sexton1, Sreenivasulu Kurukuti2, Jennifer A Mitchell3, David Umlauf4, Takashi Nagano5, Peter Fraser5

1 Laboratory of Chromatin and Cell Biology, Institute of Human Genetics, Montpellier, France.
2 Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India.
3 Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada.
4 Department of Functional Genomics, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.
5 Laboratory of Nuclear Dynamics and Function, Babraham Institute, Babraham Research Campus, Cambridge, UK.

Corresponding author: Tom Sexton
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Tom Sexton, Sreenivasulu Kurukuti, Jennifer A Mitchell, David Umlauf, Takashi Nagano, Peter Fraser