Quantitative analysis of chromosome conformation capture assays (3C-qPCR)

Abstract

Chromosome conformation capture (3C) technology is a pioneering methodology that allows in vivo genomic organization to be explored at a scale encompassing a few tens to a few hundred kilobase-pairs. Understanding the folding of the genome at this scale is particularly important in mammals where dispersed regulatory elements, like enhancers or insulators, are involved in gene regulation. 3C technology involves formaldehyde fixation of cells, followed by a polymerase chain reaction (PCR)-based analysis of the frequency with which pairs of selected DNA fragments are crosslinked in the population of cells. Accurate measurements of crosslinking frequencies require the best quantification techniques. We recently adapted the real-time TaqMan PCR technology to the analysis of 3C assays, resulting in a method that more accurately determines crosslinking frequencies than current semiquantitative 3C strategies that rely on measuring the intensity of ethidium bromide-stained PCR products separated by gel electrophoresis. Here, we provide a detailed protocol for this method, which we have named 3C-qPCR. Once preliminary controls and optimizations have been performed, the whole procedure (3C assays and quantitative analyses) can be completed in 7-9 days. INTRODUCTIONInsight into genomic organization is key to understanding gene regulation in mammals. However, owing to technical limitations, we still have little idea about how the mammalian genome is structured in vivo at the scale at which long-range physical interactions between genes and dispersed regulatory elements most often take place (1-10 3 kbp). The recent development of the ''Tagging and recovery of associated proteins'' 1 and 3C (see ref.2) assays allowed the very first glimpse into this crucial level of organization of the genome 3-5 . However, the RNA-TRAP technique, which is based on the targeting of peroxidase activity to nascent transcripts, is restricted to physical interactions occurring with actively transcribed genes, while 3C assays potentially allow identification of physical interactions between any chromatin segments. 3C technology is particularly suited to identify chromatin loops formed in genomic regions of up to several hundreds of kilobases in size. 5C technology 6,7 offers a robust high-throughput alternative for this analysis, based on large-scale sequencing or microarray analysis. 5C is however more laborious to set up. To identify DNA segments that interact over distances larger than several hundreds of kilobases, we recommend using 4C technology [8][9][10][11] , which allows for an unbiased genome-wide screen for DNA elements that interact with a genomic site of choice.The principle of 3C technology 2 (Fig. 1) is based on formaldehyde crosslinking of interacting chromatin segments, followed by restriction digestion and intramolecular ligation of crosslinked fragments. Ligation products are subsequently analyzed by PCR using primers specific for the restriction fragments of interest. The mere detection of a ligation product between two segmen...

TET2 repression by androgen hormone regulates global hydroxymethylation status and prostate cancer progression

“…3C assay. 3C assay was basically performed as previously described 51 . Briefly, crosslinked samples were incubated with HindIII restriction enzyme at 37°C overnight and then incubated with T4 DNA ligase (Promega) at 16°C for 4 h. Samples were diluted and immunoprecipitated with AR antibody at 4°C overnight.…”

An evolutionarily conserved three-dimensional structure in the vertebrate Irx clusters facilitates enhancer sharing and coregulation

“…For the zebrafi sh 3C experiments, which required precise quantifi cations, we followed the quantitative PCR protocol indicated in ref. 47. In all cases, product values were related to a control composed of bacterial artifi cial chromosomes (BACs) that encompass all our regions of interest (RP23-127L21, RP23-347L10, RP23-93E17, RP23-52B16 and RP23-131B17 for mouse genome; OAAA043I21 and OAAA098B15 for Xenopus ; and DKEY-103G18 and CH211-25M11 for zebrafi sh).…”

Unifying model for molecular determinants of the preselection Vβ repertoire

“…Interaction between the nearest neighbor fragments in the ERCC3 gene was set as 1. Cross-linking frequencies were calculated as described in Hagège et al (58). A list of primers, probe sequences, and BAC clones are provided in Table S3.…”

“…3C assays were performed on 10 7 RAG1-deficient C57BL/6 DN thymocytes or CD19 + pro-B cells using HindIII as described in Hagège et al (58). Primers and probes designed for HindIII fragments corresponding to each vantage point in the recombination center (Dβ1, Dβ2, and Eβ) were used in Taqman assays with primers specific for each Vβ gene-containing fragment.…”