Mapping networks of physical interactions between genomic elements using 5C technology

Genomic elements separated by large genomic distances can physically interact to mediate long-range gene regulation and other chromosomal processes. Interactions between genomic elements can be detected using the chromosome conformation capture (3C) technology. We recently developed a high-throughput adaptation of 3C, 3C-carbon copy (5C), that is used to measure networks of millions of chromatin interactions in parallel. As in 3C, cells are treated with formaldehyde to cross-link chromatin interactions. The chromatin is solubilized, digested with a restriction enzyme and ligated at low DNA concentration to promote intra-molecular ligation of cross-linked DNA fragments. Ligation products are subsequently purified to generate a 3C library. The 5C technology then employs highly multiplexed ligation-mediated amplification (LMA) to detect and amplify 3C ligation junctions. The resulting 5C library of ligated primers is analyzed using either microarray detection or ultra-high-throughput DNA sequencing. The 5C protocol described here can be completed in 13 d.     

Quantitative analysis of genomic element interactions by molecular colony technique

Distant genomic elements were found to interact within the folded eukaryotic genome. However, the used experimental approach (chromosome conformation capture, 3C) enables neither determination of the percentage of cells in which the interactions occur nor demonstration of simultaneous interaction of >2 genomic elements. Each of the above can be done using in-gel replication of interacting DNA segments, the technique reported here. Chromatin fragments released from formaldehyde–cross-linked cells by sodium dodecyl sulfate extraction and sonication are distributed in a polyacrylamide gel layer followed by amplification of selected test regions directly in the gel by multiplex polymerase chain reaction. The fragments that have been cross-linked and separate fragments give rise to multi- and monocomponent molecular colonies, respectively, which can be distinguished and counted. Using in-gel replication of interacting DNA segments, we demonstrate that in the material from mouse erythroid cells, the majority of fragments containing the promoters of active β-globin genes and their remote enhancers do not form complexes stable enough to survive sodium dodecyl sulfate extraction and sonication. This indicates that either these elements do not interact directly in the majority of cells at a given time moment, or the formed DNA–protein complex cannot be stabilized by formaldehyde cross-linking.     

tRNA gene identity affects nuclear positioning

The three-dimensional organization of genomes is dynamic and plays a critical role in the regulation of cellular development and phenotypes. Here we use proximity-based ligation methods (i.e. chromosome conformation capture [3C] and circularized chromosome confrmation capture [4C]) to explore the spatial organization of tRNA genes and their locus-specific interactions with the ribosomal DNA. Directed replacement of one lysine and two leucine tRNA loci shows that tRNA spatial organization depends on both tRNA coding sequence identity and the surrounding chromosomal loci. These observations support a model whereby the three-dimensional, spatial organization of tRNA loci within the nucleus utilizes tRNA gene-specific signals to affect local interactions, though broader organization of chromosomal regions are determined by factors outside the tRNA genes themselves.     

Cell-type-specific long-range looping interactions identify distant regulatory elements of the CFTR gene

Identification of regulatory elements and their target genes is complicated by the fact that regulatory elements can act over large genomic distances. Identification of long-range acting elements is particularly important in the case of disease genes as mutations in these elements can result in human disease. It is becoming increasingly clear that long-range control of gene expression is facilitated by chromatin looping interactions. These interactions can be detected by chromosome conformation capture (3C). Here, we employed 3C as a discovery tool for identification of long-range regulatory elements that control the cystic fibrosis transmembrane conductance regulator gene, CFTR. We identified four elements in a 460-kb region around the locus that loop specifically to the CFTR promoter exclusively in CFTR expressing cells. The elements are located 20 and 80 kb upstream; and 109 and 203 kb downstream of the CFTR promoter. These elements contain DNase I hypersensitive sites and histone modification patterns characteristic of enhancers. The elements also interact with each other and the latter two activate the CFTR promoter synergistically in reporter assays. Our results reveal novel long-range acting elements that control expression of CFTR and suggest that 3C-based approaches can be used for discovery of novel regulatory elements.     

染色体构象俘获技术及其研究进展

真核生物中远距离的调控元件往往通过相互作用形成复杂的染色体相互作用网络,对基因的表达进行三维调节,染色体构象俘获是研究染色体相互作用的有力工具。简要综述了染色体构象俘获技术的基本原理及其研究进展,并对相关技术存在的问题进行了分析,对发展趋势进行了展望。     

Higher-order chromatin structure: bridging physics and biology

Advances in microscopy and genomic techniques have provided new insight into spatial chromatin organization inside of the nucleus. In particular, chromosome conformation capture data has highlighted the relevance of polymer physics for high-order chromatin organization. In this context, we review basic polymer states, discuss how an appropriate polymer model can be determined from experimental data, and examine the success and limitations of various polymer models of higher-order interphase chromatin organization. By taking into account topological constraints acting on the chromatin fiber, recently developed polymer models of interphase chromatin can reproduce the observed scaling of distances between genomic loci, chromosomal territories, and probabilities of contacts between loci measured by chromosome conformation capture methods. Polymer models provide a framework for the interpretation of experimental data as ensembles of conformations rather than collections of loops, and will be crucial for untangling functional implications of chromosomal organization.     

Insulators, not Polycomb response elements, are required for long-range interactions between Polycomb targets in Drosophila melanogaster

The genomic binding sites of Polycomb group (PcG) complexes have been found to cluster, forming Polycomb "bodies" or foci in mammalian or fly nuclei. These associations are thought to be driven by interactions between PcG complexes and result in enhanced repression. Here, we show that a Polycomb response element (PRE) with strong PcG binding and repressive activity cannot mediate trans interactions. In the case of the two best-studied interacting PcG targets in Drosophila, the Mcp and the Fab-7 regulatory elements, we find that these associations are not dependent on or caused by the Polycomb response elements they contain. Using functional assays and physical colocalization by in vivo fluorescence imaging or chromosome conformation capture (3C) methods, we show that the interactions between remote copies of Mcp or Fab-7 elements are dependent on the insulator activities present in these elements and not on their PREs. We conclude that insulator binding proteins rather than PcG complexes are likely to be the major determinants of the long-range higher-order organization of PcG targets in the nucleus.     

HiCORE: Hi-C Analysis for Identification of Core Chromatin Looping Regions with Higher Resolution

Genome-wide chromosome conformation capture (3C)-based high-throughput sequencing (Hi-C) has enabled identification of genome-wide chromatin loops. Because the Hi-C map with restriction fragment resolution is intrinsically associated with sparsity and stochastic noise, Hi-C data are usually binned at particular intervals; however, the binning method has limited reliability, especially at high resolution. Here, we describe a new method called HiCORE, which provides simple pipelines and algorithms to overcome the limitations of single-layered binning and predict core chromatin regions with three-dimensional physical interactions. In this approach, multiple layers of binning with slightly shifted genome coverage are generated, and interacting bins at each layer are integrated to infer narrower regions of chromatin interactions. HiCORE predicts chromatin looping regions with higher resolution, both in human and Arabidopsis genomes, and contributes to the identification of the precise positions of potential genomic elements in an unbiased manner.     

r3Cseq: an R/Bioconductor package for the discovery of long-range genomic interactions from chromosome conformation capture and next-generation sequencing data

The coupling of chromosome conformation capture (3C) with next-generation sequencing technologies enables the high-throughput detection of long-range genomic interactions, via the generation of ligation products between DNA sequences, which are closely juxtaposed in vivo. These interactions involve promoter regions, enhancers and other regulatory and structural elements of chromosomes and can reveal key details of the regulation of gene expression. 3C-seq is a variant of the method for the detection of interactions between one chosen genomic element (viewpoint) and the rest of the genome. We present r3Cseq, an R/Bioconductor package designed to perform 3C-seq data analysis in a number of different experimental designs. The package reads a common aligned read input format, provides data normalization, allows the visualization of candidate interaction regions and detects statistically significant chromatin interactions, thus greatly facilitating hypothesis generation and the interpretation of experimental results. We further demonstrate its use on a series of real-world applications.     

Physical Origin of the Contact Frequency in Chromosome Conformation Capture Data

Physical proximity between each pair of genomic loci in a nucleus is measured as a form of contact frequency in chromosome conformation capture-based methods. Complexity of chromosome structure in interphase can be characterized by measuring a statistical property of physical distance between genomic loci according to genomic separation along single chromatids. To find a relationship between the physical distance and the contact frequency, we propose a polymer model derived from the Langevin equation. The model is derived by considering a structure of a chromosome as a trajectory of a particle, where each consecutive segment in the chromosome corresponds to a transient position in the trajectory over time. Using chromosome conformation capture data, we demonstrate the functional relationship between the two quantities. The physical distances derived from the mean contact frequencies by the model show a good correlation with those from experimental data. From the model, we present that the mean contact frequency curve can be divided into three components that arise from different physical origins and show that the contact frequency is proportional to the contact surface area, not to the volume of segments suggested by the fractal globule model. The model explains both a decaying pattern of the contact frequency and the biphasic relationship between the physical distance and the genomic length.     

Physical Origin of the Contact Frequency in Chromosome Conformation Capture Data

Physical proximity between each pair of genomic loci in a nucleus is measured as a form of contact frequency in chromosome conformation capture-based methods. Complexity of chromosome structure in interphase can be characterized by measuring a statistical property of physical distance between genomic loci according to genomic separation along single chromatids. To find a relationship between the physical distance and the contact frequency, we propose a polymer model derived from the Langevin equation. The model is derived by considering a structure of a chromosome as a trajectory of a particle, where each consecutive segment in the chromosome corresponds to a transient position in the trajectory over time. Using chromosome conformation capture data, we demonstrate the functional relationship between the two quantities. The physical distances derived from the mean contact frequencies by the model show a good correlation with those from experimental data. From the model, we present that the mean contact frequency curve can be divided into three components that arise from different physical origins and show that the contact frequency is proportional to the contact surface area, not to the volume of segments suggested by the fractal globule model. The model explains both a decaying pattern of the contact frequency and the biphasic relationship between the physical distance and the genomic length.     

Chromosome conformation capture technologies and their impact in understanding genome function

It has been more than a decade since the first chromosome conformation capture (3C) assay was described. The assay was originally devised to measure the frequency with which two genomic loci interact within the three-dimensional (3D) nuclear space. Over time, this method has evolved both qualitatively and quantitatively, from detection of pairwise interaction of two unique loci to generating maps for the global chromatin interactome. Combined with the analysis of the epigenetic chromatin context, these advances led to the unmasking of general genome folding principles. The evolution of 3C-based methods has been supported first by the revolution in ChIP and then by sequencing-based approaches, methods that were primarily tools to study the unidimensional genome. The gradual improvement of 3C-based methods illustrates how the field adapted to the need to gradually address more subtle questions, beginning with enquiries of reductionist nature to reach more holistic perspectives, as the technology advanced, in a process that is greatly improving our knowledge on genome behavior and regulation. Here, we describe the evolution of 3C and other 3C-based methods for the analysis of chromatin interactions, along with a brief summary of their contribution in uncovering the significance of the three-dimensional world within the nucleus. We also discuss their inherent limitations and caveats in order to provide a critical view of the power and the limits of this technology.