High-resolution circular chromosome conformation capture assay

The pioneering chromosome conformation capture (3C) method provides the opportunity to study chromosomal folding in the nucleus. It is based on formaldehyde cross-linking of living cells followed by enzyme digestion, intramolecular ligation and quantitative (Q)-PCR analysis. However, 3C requires prior knowledge of the bait and interacting sequence (termed interactor) rendering it less useful for genome-wide studies. As several recent reports document, this limitation has been overcome by exploiting a circular intermediate in a variant of the 3C method, termed 4C (for circular 3C). The strategic positioning of primers within the bait enables the identification of unknown interacting sequences, which form part of the circular DNA. Here, we describe a protocol for our 4C method, which produces a high-resolution interaction map potentially suitable for the analysis of cis-regulatory elements and for comparison with chromatin marks obtained by chromatin immunoprecipitation (ChIP) on chip at the sites of interaction. Following optimization of enzyme digestions and amplification conditions, the protocol can be completed in 2-3 weeks.     

三维基因组染色质构象捕获及其衍生技术

线性染色质经过多重折叠凝缩到真核生物的细胞核中,染色质的三维构象直接决定了真核生物的基因表达,因此染色质可以在局部或远程空间上发生互作调控基因转录。折叠成环状构象的染色质可以借助染色质构象捕获 (Chromosome conformation capture,3C) 技术来研究,基于3C技术扩展的4C/5C/Hi-C从单个位点延伸到全基因组捕捉三维构象,在此基础上,染色质构象核心技术可以与免疫共沉淀、核酸分子杂交、单细胞、基因组测序等技术偶联而产生新的衍生技术和应用,这极大地推动了染色质构象技术在基因时空特异性表达调控上的研究。文中将以3C和Hi-C等三维基因组核心技术为基础,重点介绍染色质构象捕获及其衍生技术的原理和前沿应用。     

A rapid simple approach to quantify chromosome conformation capture

Chromosome conformation capture (3C) is a powerful tool to study DNA looping. The procedure generates chimeric DNA templates after ligation of restriction enzyme fragments juxtaposed in vivo by looping. These unique ligation products (ULPs) are typically quantified by gel-based methods, which are practically inefficient. Taqman probes may be used, but are expensive. Cycle threshold (Ct) determined using SYBR Green, an inexpensive alternative, is hampered by non-specific products and/or background fluorescence, both due to high template/ULP ratio. SYBR Green melting curve analysis (MCA) is a well-known qualitative tool for assessing PCR specificity. Here we present for the first time MCA as a quantitative tool (qMCA) to compare template concentrations across different samples and apply it to 3C to assess looping among remote elements identified by STAT1 and IRF1 ChIP-chip at the interferon-γ responsive CIITA and SOCS1 loci. This rapid, inexpensive approach provided highly reproducible identification and quantification of ULPs over a significant linear range. Therefore, qMCA is a robust method to assess chromatin looping in vivo, and overcomes several drawbacks associated with other approaches. Our data suggest that basal and induced looping is a involving remote enhancers is a common mechanism at IFNγ-regulated targets.     

Global identification of yeast chromosome interactions using Genome conformation capture

The association of chromosomes with each other and other nuclear components plays a critical role in nuclear organization and Genome function. Here, using a novel and generally applicable methodology (Genome conformation capture [GCC]), we reveal the network of chromosome interactions for the yeast Saccharomyces cerevisiae. Inter- and intra-chromosomal interactions are non-random and the number of interactions per open reading frame depends upon the dispensability of the gene product. Chromosomal interfaces are organized and provide evidence of folding within chromosomes. Interestingly, the genomic connections also involve the 2 μm plasmid and the mitochondrial genome. Mitochondrial interaction partners include genes of α-proteobacterial origin and the ribosomal DNA. Organization of the 2 μm plasmid aligns two inverted repeats (IR1 and IR2) and displays the stability locus on a prominent loop thus making it available for plasmid clustering. Our results form the first global map of chromosomal interactions in a eukaryotic nucleus and demonstrate the highly connected nature of the yeast genome. These results have significant implications for understanding eukaryotic genome organization.     

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

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.     

Exploiting native forces to capture chromosome conformation in mammalian cell nuclei

Mammalian interphase chromosomes fold into a multitude of loops to fit the confines of cell nuclei, and looping is tightly linked to regulated function. Chromosome conformation capture (3C) technology has significantly advanced our understanding of this structure‐to‐function relationship. However, all 3C‐based methods rely on chemical cross‐linking to stabilize spatial interactions. This step remains a “black box” as regards the biases it may introduce, and some discrepancies between microscopy and 3C studies have now been reported. To address these concerns, we developed “i3C”, a novel approach for capturing spatial interactions without a need for cross‐linking. We apply i3C to intact nuclei of living cells and exploit native forces that stabilize chromatin folding. Using different cell types and loci, computational modeling, and a methylation‐based orthogonal validation method, “TALE‐iD”, we show that native interactions resemble cross‐linked ones, but display improved signal‐to‐noise ratios and are more focal on regulatory elements and CTCF sites, while strictly abiding to topologically associating domain restrictions.     

Sensitive detection of chromatin coassociations using enhanced chromosome conformation capture on chip

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 coassociations 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 coassociations. 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 coassociations, followed by bait enrichment by biotinylated primer extension and pull-down, adapter ligation and PCR amplification. Chromatin coassociations 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.     

Techniques for and challenges in reconstructing 3D genome structures from 2D chromosome conformation capture data

Chromosome conformation capture technologies that provide frequency information for contacts between genomic regions have been crucial for increasing our understanding of genome folding and regulation. However, such data do not provide direct evidence of the spatial 3D organization of chromatin. In this opinion article, we discuss the development and application of computational methods to reconstruct chromatin 3D structures from experimental 2D contact data, highlighting how such modeling provides biological insights and can suggest mechanisms anchored to experimental data. By applying different reconstruction methods to the same contact data, we illustrate some state-of-the-art of these techniques and discuss our gene resolution approach based on Brownian dynamics and Monte Carlo sampling.     

Relationship between spatial organization and biological function,analyzed using gene ontology and chromosome conformation capture of human and fission yeast genomes

Cells regulate functionally related genes cis- and trans-contacts in order to perform specific biological roles. To understand the cryptic spatial genomic contexts underlying these biological functions, we analyzed the gene association data from the gene ontology (GO) database and the genomic spatial organization data obtained by analysis of chromosome conformation capture (3C)-based data from the Sequence Read Archive, where GO and 3C-based data were used to measure functional similarity and spatial proximity, respectively, between genomic loci. In the human genome and the fission yeast genome, we observed that correlation between the two measures was statistically significant on a genome-wide scale. Specifically, it is also confirmed that the genomic spatial architecture is affected by functional similarity of genes by showing better correlation of functional similarities with spatial distances estimated by contact frequencies than those estimated by genomic distances for cis-contacts. Furthermore, we analyzed distances between the genomic segments sharing the same GO term using the two-sample t test, found that the genomic segments identified by various GO terms are spatially located closer than the average distance over statistically-valid contacts, and provided a list of the GO terms. The results suggested that genomic loci with similar biological functions are situated in close proximity to each other in the nuclear space by aggregating functionally related genes in a short spatial range.     

Enzyme inhibition by allosteric capture of an inactive conformation

All members of the human herpesvirus protease (HHV Pr) family are active as weakly associating dimers but inactive as monomers. A small-molecule allosteric inhibitor of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) traps the enzyme in an inactive monomeric state where the C-terminal helices are unfolded and the hydrophobic dimer interface is exposed. NMR titration studies demonstrate that the inhibitor binds to KSHV Pr monomers with low micromolar affinity. A 2.0-Å-resolution X-ray crystal structure of a C-terminal truncated KSHV Pr-inhibitor complex locates the binding pocket at the dimer interface and displays significant conformational perturbations at the active site, 15 Å from the allosteric site. NMR and CD data suggest that the small molecule inhibits human cytomegalovirus protease via a similar mechanism. As all HHV Prs are functionally and structurally homologous, the inhibitor represents a class of compounds that may be developed into broad-spectrum therapeutics that allosterically regulate enzymatic activity by disrupting protein-protein interactions.     

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.     

The three vibrio cholerae chromosome II-encoded ParE toxins degrade chromosome I following loss of chromosome II

Three homologues of the plasmid RK2 ParDE toxin-antitoxin system are present in the Vibrio cholerae genome within the superintegron on chromosome II. Here we found that these three loci-two of which have identical open reading frames and regulatory sequences-encode functional toxin-antitoxin systems. The ParE toxins inhibit bacterial division and reduce viability, presumably due to their capacity to damage DNA. The in vivo effects of ParE1/3 mimic those of ParE2, which we have previously demonstrated to be a DNA gyrase inhibitor in vitro, suggesting that ParE1/3 is likewise a gyrase inhibitor, despite its relatively low degree of sequence identity. ParE-mediated DNA damage activates the V. cholerae SOS response, which in turn likely accounts for ParE's inhibition of cell division. Each toxin's effects can be prevented by the expression of its cognate ParD antitoxin, which acts in a toxin-specific fashion both to block toxicity and to repress the expression of its parDE operon. Derepression of ParE activity in ΔparAB2 mutant V. cholerae cells that have lost chromosome II contributes to the prominent DNA degradation that accompanies the death of these cells. Overall, our findings suggest that the ParE toxins lead to the postsegregational killing of cells missing chromosome II in a manner that closely mimics postsegregational killing mediated by plasmid-encoded homologs. Thus, the parDE loci aid in the maintenance of the integrity of the V. cholerae superintegron and in ensuring the inheritance of chromosome II.     

Comparative chromosome morphology in three Callitrichid genera: Cebuella, Callithrix, and Leontopithecus

A G-band karyotypic analysis was carried out in individual species groups of three Callitrichid primate genera: Cebuella, Callithrix, and Leontopithecus. Within Callithrix, the karyotypes of the morphologically distinct and geographically isolated morphotypes C. jacchus jacchus and C. jacchus penicillata were identical. Within the lion tamarin genus, Leontopithecus, the karyotypes of the three morphotypes (L. rosalia rosalia, L. rosalia chrysomelas and L. rosalia chrysopygus) were also indistinguishable from one another. These results are consistent with the taxonomic designation of subspecies rank to the different morphotypes. A comparison of type specimens among the three Callitrichid genera showed that their phyletic radiation has been paralleled by a limited number of chromosome rearrangements and a relatively high amount of karyotypic invariance. A fusion/fission event has been postulated to account for the difference in diploid number between Cebuella (2n = 44) and the other species (2n = 46). The karyotype of Callithrix jacchus was found to be more directly derived from Cebuella than was that of Leontopithecus. These findings differ from the previous proposition that Leontopithecus might have diverged from a common Callitrichid ancestor before the emergence of the genus Callithrix.     

Ly-m19: The Lyb-2 region of mouse chromosome 4 controls a new surface alloantigen

Spleen cells from an SJL mouse immunized with 70'/3 cells, an established pre-B cell line, were fused with cells of the nonsecretor myeloma line NS.1. One established hybridoma cell line (clone K10.6) continuously secreted antibody that recognized a new antigenic specificity tentatively named Ly-m19. This newly found antigen is detectable on both T and B cells. Cytotoxicity assays reveal that 75 percent of the spleen and lymph-node cells, 35 percent of bone-marrow cells, and 15 percent of thymus cells reacted with antibody of clone K10.6. Strains expressing the specificity Ly-m19.1 are characterized by negative reactions and include the strains AKR, CE/J, RF/J, GR/A, SJL, P/J, BDP/J, and LG/J. All other strains so far tested are Ly-m19.2. This strain distribution pattern distinguishes Ly-m19 from any known murine lymphocyte alloantigen, but it parallels the Lyb-2 ^c haplotype. Linkage test of a set of AKXL recombinant inbred strains revealed close linkage of Ly-m19 and Lyb-2 loci on mouse chromosome 4.Abbreviations used in this paper LPS lipopolysaccharide - B6 C57BL/6 - Con-A concanavalin A - MLC mixed-lymphocyte culture The prefix m (monoclonal) is used following a suggestion by Klein and co-workers (1979).