SnapShot: Chromosome Conformation Capture
Ofi r Hakim and Tom Misteli
National Cancer Institute, NIH, Bethesda, MD 20892, USA
Chromatin-associated factors Gene
Biotin dCTP fill in
Endonuclea
digestion
Protein
Protein
Sonication
Immunoprecipitation
Immunoprecipitation biotinilated linkers
Contact library
PCR with speci c primers PCR with universal primers
Multiplexed amplification
Digestion with four ba cutter
Ligation
Inver PCR
Sonicate Pull down
PCR with speci c primers
Mmel digestion
Pull downonclick
B B B
B
B B
beat it 歌词
B
B B
B
B B B
B B
B
B B
B
B
DETECTION
LIGATION
CUTTING
CROSSLINK
COMPUTATIONAL ANALYSIS
REVERSE CROSSLINKS chIA-PeT
royan
Principle
Contacts between two defi ned regions 3,17
All against all 4,18
All contacts with a
point of interest 14
All against all 10
Contacts between two defi ned regions associated with a given protein 8
All contacts associated with a given protein 6Coverage Commonly < 1Mb Commonly < 1Mb Genome-wide Genome-wide Commonly < 1Mb Genome-wide Detection Locus-specifi c PCR HT -quencing HT -quencing HT -quencing
Locus-specifi c qPCR
HT -quencing
Limitations Low throughput and
coverage
Limited coverage Limited to one
viewpoint
Rely on one chromatin-associated factor, disregarding other contacts
Examples
Determine interaction between a known promoter and enhancer
Determine
comprehensively higher-order chromosome structure in a defi ned region
All genes and genomic elements associated with a known LCR
All intra- and
华尔街英语interchromosomal associations
Determine the role of specifi c transcription factors in the
interaction between a known promoter and enhancer
Map chromatin
interaction network of a known transcription factor
Derivatives
PCR with TaqMan probes 7 or melting curve analysis 1
Circular chromosome conformation capture 20, open-ended chromosome conformation capture 19, inver 3C 12, associated chromosome trap (ACT)11, affi nity enrichment of bait-ligated junctions 2
success的用法Yeast 5,15, tethered conformation capture 9
ChIA-PET combined
3C-ChIP-cloning (6C),16
enhanced 4C (e4C)
13
jiduNational Cancer Institute, NIH, Bethesda, MD 20892, USA
奥运英语The organization of the genome in the nuclear space is nonrandom and affects genome functions, including transcription, replication, and repair. Specific genomic regions, from the same or different chromosomes, frequently physically associate with each other and with nuclear structures, giving ri to an intricately compartmentalized nucleus. Examples of genome interactions are the association of an enhancer with a promoter or the clustering of genes such as rDNA genes in the nu
中分适合什么脸型cleolus. Genome interactions have traditionally been studied using fluorescence in situ hybridization (FISH), which allows visualization of the spatial relationship between distinct genes or genome regions. Limitations of this method are that only known interactions can be interrogated, only very few loci can be probed in an experiment, and resolution is limited to the optics of the microscope.
The family of chromosome conformation capture techniques is a t of biochemical approaches to determine the physical interaction of genome regions. C-technology approaches invariably involve five steps: (1) formaldehyde fixation to crosslink chromatin at sites of physical interaction, (2) cleavage of chromatin by restriction enzyme or sonication, (3) ligation under dilute conditions favoring ligation between DNA ends captured on the same complex over ligations from random collisions, (4) detection of ligation junctions using variable molecular biology steps depending on the variant of the methods, and (5) computational analysis to determine interaction frequencies captured in the ligation of the crosslinked chromatin.
C-technologies (3C, 4C, 5C, Hi-C) differ in their manner of detection and scope of what interactions they can probe. The 3C method tests the interaction between two known sites in the genome, 4C allows probing of unknown interactors of a known bait quence, 5C identifies all regions of interaction within a given genome domain, and Hi-C probes all occurring interactions in an unbiad
fashion genome-wide. Additional variants (ChIA-PET, ChIP-Loop) incorporate a protein precipitation step, allowing identification of genome interactions that involve a specific protein of interest. The choice of method strongly depends on the specific nature and scope of the biological question, but also on the availability of resources, including the amount of starting material and quencing capacity. Many derivatives of the standard C-techniques have been developed, often inspired by the specific biological question addresd or with the goal of improving specificity or reducing background.
C-technologies are population-bad methods. They produce relative contact probabilities rather than absolute contact frequencies. The population-bad nature is due to the fact that each genomic locus gives one pair-wi ligation junction in one cell. To allow high coverage and quantitative appraisal of contact profiles, thousands to millions of genome equivalents (cells) containing multiple ligation junctions must be included and combined in each experiment. Correlations between C contacts and DNA FISH have indi-cated that an interchromosomal association that occurs in 3%–5% of cells in a population will typically be detected as positive in most C methods. More frequent associations generally result in stronger signals; however, the strength of signal may also reflect the affinity of the physical interactions and not its frequency.
食分
A critical step in data analysis is to determine whether an interaction, detected as a ligation junction, is specific. The contact frequency decreas exponentially and is inverly related to the linear genomic distance up to a few Mb away from the reference point. Therefore, the frequency of a specific contact in the vicinity of a locus is expected to be higher than the background of random collisions. A good indicator of specificity beyond the Mb range is the detection of a given interaction as clusters of signals from adjacent restriction fragments.
The resolution of C methods is determined by the nature of the restriction enzyme(s) ud and, in the ca of methods that u quencing for detection, also by the number of quencing reads. The frequency of recognition quences of a four ba-pair (bp) endonuclea is, in principle, sixteen times higher than the frequency of recognition quence of a six bp cutter. The u of a four bp cutter is expected to increa the resolution of contacts in the Mb range, where multiple ligation events are captured for specific contacts and the background collisions. Beyond this range, however, where clusters of restriction fragments define contact regions in the range of tens to hundreds of kb, the advantage of using a four bp cutter is expected to be diminished. Although many genome-wide assays have ud dedicated microarrays, hi-throughput quencing is becoming the method of choice for global detection of ligation junctions. Sequencing depth is a technical barrier for resolution
in some approaches such as Hi-C and ChIA-PET. PCR-bad technologies overcome this limitation by amplifying a subt of contacts, with the tradeoff of reduced coverage. The pairwi nature of ligation products impos a power of two relationship between the increa in resolution and the increa in required quencing depth. Genomic coverage per quencing depth depends also on the size of the inspected genome. For example, similar quencing power provides tens of kb contact resolution in yeast, but only Mb resolution in the human genome.
RefeRences
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National Cancer Institute, NIH, Bethesda, MD 20892, USA
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