Circular chromosome conformation capture followed by illumina sequencing 4cseq is among the most powerful techniques to determine 3d chromatin organization. It is both a tribute to the creativity of the users and the versatility of the technology. A cautionary note on the use of chromosome conformation. Research article highresolution 4c reveals rapid p53. Scaffolding bacterial genomes and probing hostvirus. In this chapter, we present a protocol for highresolution circular chromosome conformation capture coupled to deep sequencing. The sequel to 3c offers several innovations to the basic protocol, and reveals how unknown dna regions interact with a region of interest. The chromosome conformation capture 3c methodology. It is important to catch up with the speed of technical development and make use of these tools in order to understand the epigenetic mechanisms systematically.
Interpretation of chromosome conformation capture data in. Parp1 and ctcf regulate the contacts between clockcontrolled genes and laminaassociated chromatin as well as oscillating transcription. Us8076070b2 genomewide chromosome conformation capture. In 1993, the nuclear ligation assay was published, a method that could. High resolution allows a more detailed analysis of chromosome structure, which enables detection of more subtle chromosome abnormalities. Chromosome conformation capture analysis 3c techniques.
Ctcf mediates the celltype specific spatial organization. Wo2008084405a2 circular chromosome conformation capture. Later, high resolution in situ fluorescent hybridization assays allowed identifying the. Chromosome conformation capture technology has enabled the. Analysis of hundreds of cis regulatory landscapes at high.
Highresolution chromosome analysis is suggested for patients who have a family history of a particular abnormality and for patients who have very specific clinical features. The study in this thesis had focused on the dynamics of higher order chromatin structures, particularly on. Chromosome conformation capture 3c technology and its derivatives are important tools used in this effort. Furthermore, using highresolution circular chromosome conformation capture coupled to deep sequencing 4cseq, we reveal that cohesins eviction from p53 target gene bodies in response to stress coincides with the disruption of chromatin loops that shape the spatial architecture of these loci. It is based on formaldehyde crosslinking of living cells followed by enzyme digestion, intramolecular ligation and quantitative qpcr analysis. Pdf chromosome conformation capture 3c technology is a powerful and increasingly. We characterized the celltype specific spatial organization of the kcnq5 gene locus mediated by ctcf in detail using chromosome conformation capture 3c and 3cderived techniques. Targeted highresolution chromosome conformation capture at genomewide scale. Highresolution circular chromosome conformation capture assay. Similar to the original 3c, in the initial protocol dilution hic, ligation is done in. Another assay that has been developed is called chromosome conformation capture 3c technology, which provides a tool to study the structural organisation of a genomic region.
Illumina sequencing, chromosome conformation capture assay, titration, dissection. We report the use of chromosome conformation capture. These methods quantify the number of interactions between genomic loci that are nearby in 3d space, but may be separated by many nucleotides in the linear genome. Chromosome conformation capture techniques are a set of molecular biology methods used to. Growing evidence has revealed that the multivalent functional zinc finger protein ccctcbinding factor ctcf is a master regulator of genome spatial organization, and mediates the ubiquitous chromatin loops within the genome.
High resolution studies allow more detailed analysis of chromosome structure, which may detect more subtle chromosome abnormalities not detectable by routine studies. Chromatin loops play important roles in the dynamic spatial organization of genes in the nucleus. Chromosome conformation capture 3c assay makes tremendous progress into hic which can detect genomewide long interactions on chromosomes which have celltype speci. Chromosome conformation capture 3c is used to quantify physical dna contacts in vivo at high resolution. High resolution multiplex analysis of chromosome conformation technical journal club 28. Circular chromosome conformation capture with sequencing 4cseq 4c library was prepared as described previously with minor modifications 21. Genomewide chromosome conformation capture download pdf info publication number us8076070b2. Here we describe a new method, chromatin in situ proximity chrisp, which reproducibly scores for proximities between two different chromatin fibers in 3d with a resolution of. Chromosome conformation capture carbon copy technology. The dna trapped within crosslinked protein complexes is then digested with a restriction enzyme. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. Determination of local chromatin interactions using a.
The asterisk indicates the newly formed restriction site. The chromosome conformation capture 3c technique is a method to study chromatin interactions at specific genomic loci. Highthroughput assays for measuring the threedimensional 3d configuration of dna have provided unprecedented insights into the relationship between dna 3d configuration and function. Using circular chromosome conformation capture 4c methodology, we discovered that ctcf may be a master organizer in mediating the spatial organization of the kcnq5 gene locus. Chromosomal dna must be highly compacted to fit within the tiny volume of the cell, while at the same time it must maintain a conformation that allows critical cellular processes access to the genome. A number of methods use oligonucleotide capture to enrich 3c and hic. Multiplexed chromosome conformation capture sequencing for rapid genomescale highresolution detection of longrange chromatin interactions. The size of restriction fragments determines the resolution of interaction. Production of dna minicircles less than 250 base pairs through a novel concentrated dna circularization assay enabling minicircle design with nf.
Circular chromosome conformation capture with unique molecular identifiers 3 5c. Chromatin conformation capture sequencing 129 ng capture c. Chromatin interaction analysis with pairedendtag sequencing chiapet is an emerging technology that has unique advantages in chromatin interaction analysis, and thus provides insight into the study of transcription regulation. Higher resolution hic data was generated in 2014 following the development of in. Key words circular chromosome conformation capture, 4cseq, 3d chromatin organization. Data interpretation from assays such as chiapet and hic is challenging because the data is large and cannot be easily rendered using standard genome browsers. This protocol generates a very highresolution descrip. The pioneering chromosome conformation capture 3c method provides the opportunity to study chromosomal folding in the nucleus. Using chromosome conformation capture, it is now possible to analyze in detail the interaction between enhancers, silencers, boundary elements and promoters at individual loci, but these techniques are not readily scalable. High resolution chromosome analysis is suggested for patients who have a family history of a particular abnormality and for patients who have very specific clinical features. Long noncoding rnas with enhancerlike function in human cells.
We characterized the celltype specific spatial organization of the kcnq5 gene locus mediated by ctcf in detail using chromosome conformation capture 3c and 3c. Nextgeneration sequencing ngs in biomarker discovery. Pdf multiplexed chromosome conformation capture sequencing. Highresolution mapping of the spatial organization of a bacterial chromosome tung b. Bickmore1 1mrc human genetics unit, institute of genetics and molecular medicine, university of. Available formats pdf please select a format to send. Chromosome conformation capture technologies and their impact in. Here we evaluate and compare the current 3cbased methods including 4c chromosome conformation captureonchip, 5c chromosome conformation capture carbon copy, hic, and chiapet, summarize their contribution to our current understanding of genome structure, and discuss how shape influences genome function. Different techniques have been developed and applied to uncover the intrinsic mechanism of genome architecture, especially the chromosome conformation capture 3c and 3cderived methods. Here, we describe a protocol for our 4c method, which produces a highresolution. Cohesion also participated in mediating the organization of this locus. This assay, called chromosome conformation capture 3c, laid the. The technologies rely on the principle that loci that are spatially close to one another. A simple procedure for base pair resolution chipseq.
Longrange chromatin interactions play an important role in transcription regulation. The invention relates to the use of gcc genome conformation capture technology in determining the three dimensional arrangement of an entire genome. Pdf targeted highresolution chromosome conformation. To systematically explore the epigenetic dimension of such interactions, we devised a strategy termed circular chromosome conformation capture 4c. High resolution multiplex analysis of chromosome conformation. Chromosome conformation capture 3c technology and its genomewide derivatives. Circular chromosome conformation capture followed by illumina sequencing. The resulting interchromosomal network connected, surprisingly, both active and repressive chromatin. Chromatin conformation capture carbon copy 4 puseq. Here we present a highthroughput approach capturec to analyze cis interactions.
The advent of highresolution chromosome conformation capture assays such as 5c, hic and porec has allowed for unprecedented sequencelevel inves. They proposed a methodology called capturing chromosome conformation 3c to characterize some overall physical properties at high resolution. Chromosome conformation capture an overview sciencedirect. Illingworth,1 denis paquette,2 josee dostie,2 and wendy a. A signi cant contribution to the exploration of the structure of the chromosomes has been given by dekker at al. Using circular chromosome conformation capture 4c methodology, we.
These contacts were quantified using meta3c, a derivative of the chromosome conformation capture method 3c. Highresolution circular chromosome conformation capture. Chromatin interaction analysis with pairedend tag chia. Therefore, it is a candidatebased approach, usually used to assay the interaction.
Briefly, dna molecules within a mixture of microbial species are frozen in space with a crosslinking agent. Software tools for visualizing hic data genome biology. Circular chromosome conformation capture 4c, which involves the circularization of chimeric dna fragments and the amplification of dna sequences with primers within the bait but proximal to the target sequence during ligation, can be used to screen for interactions without perception of the existence of two different complexes. Chromosome conformation capture techniques are a set of molecular biology methods used to analyze the spatial organization of chromatin in a cell. These 4c methods are 3c on chip, openended 3c, circular 3c, and. Highresolution mapping of the spatial organization of a. Initially established for yeast the 3c technique has been adapted to plants in recent years in order to study chromatin interactions and their role in transcriptional gene regulation. This is a major problem as it hampers our understanding of chromatin conformation in single cells and how these respond to environmental cues. This sets an unprecedented stage for exploring the mechanisms that link chromosome structure and biological function. Laub1,4 chromosomes must be highly compacted and organized within cells, but how this is achieved in vivo remains poorly understood. Significant efforts have recently been put into the investigation of the spatial organization and the chromatininteraction networks of genomes. Circular chromosome conformation capture 4c erasmus.