Chromosome Conformation Capture Carbon Copy (5C): A massively parallel solution for mapping interactions between genomic elements

Intense efforts are under way to map genes and regulatory elements throughout the human genome (ENCODE Project Consortium 2004). These studies are expected to identify many different types of elements, including those involved in gene regulation, DNA replication, and genome organizationin general. Analysis of only 1% of the human genome has already revealed that genes are surrounded by a surprisingly large number of putative regulatory elements (data available at http://genome.cse.ucsc.edu/encode/). In order to fully annotate the human genome and to understand its regulation, it is important to map all genes and functional elements and also to determine all relationships between them. For instance, all regulatory elements of each gene must be identified. This endeavor is complicated by the fact that the genomic positions of genes and elements do not provide direct information about functional relationships between them. A well-known example is provided by enhancers that can regulate multiple target genes that are located at large genomic distances or even on different chromosomes without affecting genes immediately next to them (Spilianakis et al. 2005; West and Fraser 2005). Recent evidence indicates that regulatory elements can act over large genomic distances by engaging in direct physical interactions with their target genes or with other elements (Dekker 2003; de Laat and Grosveld 2003; Chambeyron and Bickmore 2004; West and Fraser 2005). These observations indicate that the genome may be organized as a complex three-dimensional network that is determined by physical interactions between genes and elements. Therefore, we hypothesize that functional relationships between genes and regulatory elements can be determined by analyzing this network through mapping of chromatin interactions. Physical interactions between elements can be detected with the Chromosome Conformation Capture(3C) method (Dekker et al. 2002; Dekker 2003; Splinter et al. 2004; Miele et al. 2006). 3C uses formaldehyde cross-linking to covalently trap interacting chromatin segments throughout the genome. Interacting elements are then restriction-enzyme-digested and intramolecularly ligated (Fig. 1A). The frequency with which two restriction fragments become ligated is a measure of the frequency by which they interact in the nucleus (Dekker et al. 2002). Figure 1. Schematic representation of 5C. (A) A 3C library is generated by conventional 3C and then converted into a 5C library by annealing and ligating 5C oligonucleotides in a multiplex setting. 5C libraries are then analyzed on a microarray or by quantitative ... 3C was initially used to study the spatial organizationof yeast chromosome III (Dekker et al. 2002) and has since been applied to the analysis of several mammalian loci such as the β-globin locus (Tolhuis et al. 2002; Palstra et al. 2003; Vakoc et al. 2005), the T-helper type 2 cytokine locus (Spilianakis and Flavell 2004), the immunoglobulin κ locus (Liu and Garrard 2005), and the Igf2 imprinted locus (Murrell et al. 2004). These studies revealed direct interactions between enhancers and promoters of target genes, with the linking DNA looping outward. 3C was also used to detect trans interactions between yeast chromosomes (Dekker et al. 2002) and between functionally related elements located on different mouse chromosomes (Spilianakis et al. 2005; Ling et al. 2006; Xu et al. 2006). Together, these studies suggest that long-range cis and trans interactions play widespread roles in the regulation of the genome and that 3C is a convenient approach to map this network of interactions. 3C uses PCR to detect individual chromatin interactions, which is particularly suited for relatively small-scale studies focused on the analysis of interactions between a set of candidate elements. However, PCR detection is not conducive to ab initio and large- scale mappingof chromatin interactions. To overcome this problem, 3C libraries need to be analyzed using a high-throughput detection method such as microarrays or DNA sequencing. The extreme complexity of the 3C library and the low relative abundance of each specific ligation product make direct large-scale analysis difficult. Here we present a novel 3C-based methodology for large-scale parallel detection of chromatin interactions. We refer to this method as 3C- Carbon Copy, or “5C.” 5C uses highly multiplexed ligation-mediated amplification (LMA) to first “copy” and then amplify parts of the 3C library followed by detection on microarrays or by quantitative DNA sequencing. 5C was developed and validated by analyzing the human β-globin locusand a conserved gene desert region located on human chromosome 16. We find that 5C quantitatively detects several known DNA looping interactions. Interestingly, 5C analysis also identified a looping interaction between the β-globin Locus Control Region(LCR) and the γ–δ intergenic region. Previously, several lines of evidence have suggested that this region plays a role in regulating the developmentally controlled switching from γ-globin expression in fetal cells to β-globin expression in adult cells (Calzolari et al. 1999; Gribnau et al. 2000). 5C should be widely applicable to determine the cis and trans connectivity of regulatory elements throughout large genomic regions. In addition, 5C experiments can be designed so that complete interaction maps can be generated for any large genomic region of interest, which can reveal locations of novel gene regulatory elements and may also provide detailed insights into higher-order chromosome folding.