Introduction

Here we describe a modified 3C-qPCR protocol with four-fold increased efficiency of the 3C assay compared to the initially published protocols (Ref.1-4). Classical SybrGreen technique can then be used, instead of TaqMan probes, to quantify by quantitative PCR relevant 3C chimerical ligation products. It is optimized for nuclei preparation as starting material, coming from fresh tissues or cultured cells. In our hands, it was successfully used on human and mouse samples.

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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.

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Introduction

This is an alternative protocol for 3C that has been adopted by the author's lab. Much of it is identical to the previous version (PROT5). The major differences are in the amounts of DNA used at different steps. We reliably get the same results as previously, but also a greater yield of 3C material. The original yields are ample for real-time PCR analysis, but greater yields are required if the 3C material is going to be processed further. [...]

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Frank Neumann, Angela Taddei & Susan Gasse

Introduction

The visualisation of specific DNA sequences in living cells, achieved through the integration of lac operator arrays (lac^op) and expression of a GFP-lac repressor fusion, has provided new tools to examine how the nucleus is organised and how basic events like sister chromatid separation occur (Straight et al. 1996; Belmont 2001). In contrast to other methods, such as fluorescence in situ hybridisation, the lac^op/GFP-lac repressor (GFP-lacⁱ) technique is non-invasive, and therefore interferes minimally with nuclear structure and function. In addition, it facilitates analysis of the rapid dynamics of specific DNA loci (Gasser, 2002). Although this technique has been adapted to organisms from bacteria to man, the ease with which GFP fusions can be targeted to specific chromosomal sites depends on the organism's ability to carry out homologous recombination. This process is very efficient in budding yeast, allowing pairs of chromosomal loci to be analysed at the same time through the use of two bacterial repressors (lacⁱ and tetR) fused to different GFP variant. Given the relatively advanced state of the art in budding yeast, we present protocols optimised for this organism. These provide a starting point for adapting multi-locus tagging to other species. Moreover, the techniques described here for the quantitative analyses of locus dynamics are universally applicable.

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Introduction

An alternative protocol (PROT31) for 3C has been adopted by the author's lab. Much of it is identical to this version. The major differences are in the amounts of DNA used at different steps. We reliably get the same results as with this version, but also a greater yield of 3C material. Yields from this version are ample for real-time PCR analysis, but greater yields are required if the 3C material is going to be processed further.

The 3C (Chromosome Conformation Capture) technique generates a population average measurement of juxtaposition frequency between any two genomic loci, thus providing information on their relative proximity in the nucleus (Dekker et al., 2002). Cells are fixed with formaldehyde which forms DNA-protein and protein-protein cross-links between regions of the genome in proximity. Subsequent restriction enzyme digestion and intra-molecular ligation produces novel junctions between restriction fragments in proximity in the nucleus. Novel ligation products can be detected by PCR. We adapted the 3C assay (Dekker et al., 2002) to determine the conformation of mouse chromosome 7 and in particular the co-localization of actively transcribed genes in transcription factories (Osborne et al., 2004). The 3C assay can also be used to reveal proximity between active genes and distal genomic elements (Tolhuis et al., 2002). [...]

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