词条 | Chromatin immunoprecipitation | ||||||||
释义 |
Briefly, the conventional method is as follows:
Typical ChIPThere are mainly two types of ChIP, primarily differing in the starting chromatin preparation. The first uses reversibly cross-linked chromatin sheared by sonication called cross-linked ChIP (XChIP). Native ChIP (NChIP) uses native chromatin sheared by micrococcal nuclease digestion. Cross-linked ChIP (XChIP)Cross-linked ChIP is mainly suited for mapping the DNA target of transcription factors or other chromatin-associated proteins, and uses reversibly cross-linked chromatin as starting material. The agent for reversible cross-linking could be formaldehyde[2] or UV light.[3] Then the cross-linked chromatin is usually sheared by sonication, providing fragments of 300 - 1000 base pairs (bp) in length. Mild formaldehyde crosslinking followed by nuclease digestion has been used to shear the chromatin.[4] Chromatin fragments of 400 - 500bp have proven to be suitable for ChIP assays as they cover two to three nucleosomes. Cell debris in the sheared lysate is then cleared by sedimentation and protein–DNA complexes are selectively immunoprecipitated using specific antibodies to the protein(s) of interest. The antibodies are commonly coupled to agarose, sepharose or magnetic beads. Alternatively, chromatin-antibody complexes can be selectively retained and eluted by inert polymer discs.[5][6] The immunoprecipitated complexes (i.e., the bead–antibody–protein–target DNA sequence complex) are then collected and washed to remove non-specifically bound chromatin, the protein–DNA cross-link is reversed and proteins are removed by digestion with proteinase K. An epitope-tagged version of the protein of interest, or in vivo biotinylation [7] can be used instead of antibodies to the native protein of interest. The DNA associated with the complex is then purified and identified by polymerase chain reaction (PCR), microarrays (ChIP-on-chip), molecular cloning and sequencing, or direct high-throughput sequencing (ChIP-Seq). Native ChIP (NChIP)Native ChIP is mainly suited for mapping the DNA target of histone modifiers. Generally, native chromatin is used as starting chromatin. As histones wrap around DNA to form nucleosomes, they are naturally linked. Then the chromatin is sheared by micrococcal nuclease digestion, which cuts DNA at the length of the linker, leaving nucleosomes intact and providing DNA fragments of one nucleosome (200bp) to five nucleosomes (1000bp) in length. Thereafter, methods similar to XChIP are used for clearing the cell debris, immunoprecipitating the protein of interest, removing protein from the immunoprecipated complex, and purifying and analyzing the complex-associated DNA. Comparison of XChIP and NChIPThe major advantage for NChIP is antibody specificity. It is important to note that most antibodies to modified histones are raised against unfixed, synthetic peptide antigens and that the epitopes they need to recognize in the XChIP may be disrupted or destroyed by formaldehyde cross-linking, particularly as the cross-links are likely to involve lysine e-amino groups in the N-terminals, disrupting the epitopes. This is likely to explain the consistently low efficiency of XChIP protocols compare to NChIP. But XChIP and NChIP have different aims and advantages relative to each other. XChIP is for mapping target sites of transcription factors and other chromatin associated proteins; NChIP is for mapping target sites of histone modifiers (see Table 1). Table 1 Advantages and disadvantages of NChIP and XChIP
History and New ChIP methodsIn 1984 John T. Lis and David Gilmour, at the time a graduate student in the Lis lab, used UV irradiation, a zero-length protein-nucleic acid crosslinking agent, to covalently cross-link proteins bound to DNA in living bacterial cells. Following lysis of cross-linked cells and immunoprecipitation of bacterial RNA polymerase, DNA associated with enriched RNA polymerase was hybridized to probes corresponding to different regions of known genes to determine the in vivo distribution and density of RNA polymerase at these genes. A year later they used the same methodology to study distribution of eukaryotic RNA polymerase II on fruit fly heat shock genes. These reports are considered the pioneering studies in the field of chromatin immunoprecipitation.[8][9] XChIP was further modified and developed by Alexander Varshavsky and co-workers, who examined distribution of histone H4 on heat shock genes using formaldehyde cross-linking.[10][11] This technique was extensively developed and refined thereafter.[12] NChIP approach was first described by Hebbes et al., 1988,[13] and also been developed and refined quickly.[14] The typical ChIP assay usually take 4–5 days, and require 106~ 107 cells at least. Now new techniques on ChIP could be achieved as few as 100~1000 cells and complete within one day.
ChIP has also been applied for genome wide analysis by combining with microarray technology (ChIP-on-chip) or second generation DNA-sequencing technology (Chip-Sequencing). ChIP can also combine with paired-end tags sequencing in Chromatin Interaction Analysis using Paired End Tag sequencing (ChIA-PET), a technique developed for large-scale, de novo analysis of higher-order chromatin structures.[24][25][26] Limitations
See also
References1. ^{{cite journal| author = Collas, Philippe.| title = The Current State of Chromatin Immunoprecipitation| journal = Molecular Biotechnology| volume =45| issue =1| pages =87–100|date=January 2010| pmid = 20077036| doi = 10.1007/s12033-009-9239-8}} 2. ^{{cite journal|author=Jackson, Vaughn|title=Studies on histone organization in the nucleosome using formaldehyde as a reversible cross-linking agent|journal=Cell|volume=15|issue=3|pages=945–54|date=November 1978|pmid=569554|doi=10.1016/0092-8674(78)90278-7|url=http://linkinghub.elsevier.com/retrieve/pii/0092-8674(78)90278-7|issn=|accessdate=2010-03-13}} 3. ^{{cite journal|vauthors=Gilmour DS, Lis JT |title=In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster|journal=Molecular and Cellular Biology|volume=5|issue=8|pages=2009–18|date=August 1985|pmid=3018544|pmc=366919|doi=10.1128/mcb.5.8.2009|issn=}} 4. ^{{cite journal|vauthors=Bauer UM, Daujat S, Nielsen SJ, Nightingale K, Kouzarides T |title=Methylation at arginine 17 of histone H3 is linked to gene activation|journal=EMBO Reports|volume=3|issue=1|pages=39–44|date=January 2002|pmid=11751582|pmc=1083932|doi=10.1093/embo-reports/kvf013|url=|issn=}} 5. ^{{Cite journal|last=Beynon|first=Amy L.|last2=Parkes|first2=Lindsay J.|last3=Turner|first3=Matthew L.|last4=Knight|first4=Steve|last5=Conlan|first5=Steve|last6=Francis|first6=Lewis|last7=Stocks|first7=Ben|date=September 2014|title=Chromatrap® 96: a new solid-state platform for high-throughput ChIP|journal=Nature Methods|language=en|volume=11|issue=9|pages=i-ii|doi=10.1038/nmeth.f.372|issn=1548-7091}} 6. ^1 {{Cite web|url=https://www.chromatrap.com/technology/|title=Chromatrap|last=|first=|date=|website=|archive-url=|archive-date=|dead-url=|access-date=}}Revolutionary solid state platform for chromatin immunopreciptation. 7. ^{{cite journal | author = Viens A|display-authors=et al | year = 2004 | title = Use of protein biotinylation in vivo for chromatin immunoprecipitation | url = | journal = Analytical Biochemistry | volume = 325 | issue = 1| pages = 68–76 | pmid = 14715286 | doi=10.1016/j.ab.2003.10.015}} 8. ^{{cite journal | pmid = 6379641 | volume=81 | issue=14 | title=Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes | pmc=345570 | author=Gilmour DS, Lis JT | journal=Proc Natl Acad Sci U S A | pages=4275–9 | doi=10.1073/pnas.81.14.4275 | year=1984}} 9. ^{{cite journal | pmid = 3018544 | pmc=366919 | volume=5 | issue=8 | title=In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster | date=August 1985 | author=Gilmour DS, Lis JT | journal=Mol. 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Biol. | pages=2009–18 | doi=10.1128/mcb.5.8.2009}} 10. ^{{cite journal| author = Varshavsky A| title = Discovery of cellular regulation by protein degradation| journal = Journal of Biological Chemistry| volume = 283| issue = 50| pages = 34469–89|date=December 2008| pmid = 18708349| doi = 10.1074/jbc.X800009200| issn =| pmc=3259866}} 11. ^{{cite journal|author1=Solomon, Mark J |author2=Larsen Pamela L |author3=Varshavsky, Alexander. | title = Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene| journal = Cell| volume = 53| issue = 6| pages = 937–47|date=June 1988| pmid = 2454748| doi =10.1016/S0092-8674(88)90469-2| url = http://linkinghub.elsevier.com/retrieve/pii/S0092-8674(88)90469-2| issn =| accessdate = 2010-03-06}} 12. ^{{cite journal| author = Orlando V| title = Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation| journal = Trends in Biochemical Sciences| volume = 25| issue = 3| pages = 99–104|date=March 2000| pmid = 10694875| doi =10.1016/S0968-0004(99)01535-2| url = http://linkinghub.elsevier.com/retrieve/pii/S0968-0004(99)01535-2| issn =| accessdate = 2010-03-14}} 13. ^{{cite journal|author1=Hebbes, Tim R |author2=Thorne, Alan W |author3=Crane-Robinson C. | title = A direct link between core histone acetylation and transcriptionally active chromatin| journal = The EMBO Journal| volume = 7| issue = 5| pages = 1395–402|date=May 1988| pmid = 3409869| pmc = 458389| doi =10.1002/j.1460-2075.1988.tb02956.x| issn =}} 14. ^{{cite journal|author1=O'Neill, Laura P |author2=Turner, Bryan M | title = Immunoprecipitation of native chromatin: NChIP| journal = Methods| volume = 31| issue = 1| pages = 76–82|date=September 2003| pmid = 12893176| doi =10.1016/S1046-2023(03)00090-2| issn =}} 15. ^{{cite journal|author1=O'Neill, Laura P|author2=VerMilyea, Matthew D|author3=Turner, Bryan M|date=July 2006|title=Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations|url=|journal=Nature Genetics|volume=38|issue=7|pages=835–41|doi=10.1038/ng1820|issn=|pmid=16767102}} 16. ^{{cite journal|author1=Nelson, Joel D |author2=Denisenko, Oleg |author3=Sova, Pavel |author4=Bomsztyk, Karol | title = Fast chromatin immunoprecipitation assay| journal = Nucleic Acids Research| volume = 34| issue = 1| pages = e2| year = 2006| pmid = 16397291| pmc = 1325209| doi = 10.1093/nar/gnj004| url =| issn =}} 17. ^{{cite journal|author1=Nelson, Joel D|author2=Denisenko, Oleg|author3=Bomsztyk, Karol|year=2006|title=Protocol for the fast chromatin immunoprecipitation (ChIP) method|url=|journal=Nature Protocols|volume=1|issue=1|pages=179–85|doi=10.1038/nprot.2006.27|issn=|pmid=17406230}} 18. ^{{cite book|year=2009|title=The fast chromatin immunoprecipitation method|url=|journal=Methods in Molecular Biology|volume=567|issue=|pages=45–57|doi=10.1007/978-1-60327-414-2_3|issn=|pmid=19588084|vauthors=Nelson J, Denisenko O, Bomsztyk K|isbn=978-1-60327-413-5}} 19. ^{{cite journal|author1=Dahl, John Arne|author2=Collas, Philippe|date=April 2007|title=Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay, unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells|url=|journal=Stem Cells|volume=25|issue=4|pages=1037–46|doi=10.1634/stemcells.2006-0430|issn=|pmid=17272500}} 20. ^{{cite journal|author1=Dahl, John Arne|author2=Collas, Philippe|year=2008|title=A rapid micro chromatin immunoprecipitation assay (microChIP)|url=|journal=Nature Protocols|volume=3|issue=6|pages=1032–45|doi=10.1038/nprot.2008.68|issn=|pmid=18536650}} 21. ^{{cite book|author1=Dahl, John Arne|author2=Collas, Philippe|year=2009|title=MicroChIP: chromatin immunoprecipitation for small cell numbers|url=|journal=Methods in Molecular Biology|volume=567|issue=|pages=59–74|doi=10.1007/978-1-60327-414-2_4|issn=|pmid=19588085|isbn=978-1-60327-413-5}} 22. ^{{cite journal|author1=Flanagin, Steve |author2=Nelson, Joel D |author3=Castner, David G |author4=Denisenko, Oleg |author5=Bomsztyk, Karol | title = Microplate-based chromatin immunoprecipitation method, Matrix ChIP: a platform to study signaling of complex genomic events| journal = Nucleic Acids Research| volume = 36| issue = 3| pages = e17|date=February 2008| pmid = 18203739| pmc = 2241906| doi = 10.1093/nar/gkn001| url =| issn =}} 23. ^{{Cite journal|last=Fanelli|first=Mirco|last2=Amatori|first2=Stefano|last3=Barozzi|first3=Iros|last4=Soncini|first4=Matias|last5=Zuffo|first5=Roberto Dal|last6=Bucci|first6=Gabriele|last7=Capra|first7=Maria|last8=Quarto|first8=Micaela|last9=Dellino|first9=Gaetano Ivan|date=2010-12-14|title=Pathology tissue–chromatin immunoprecipitation, coupled with high-throughput sequencing, allows the epigenetic profiling of patient samples|journal=Proceedings of the National Academy of Sciences|language=en|volume=107|issue=50|pages=21535–21540|doi=10.1073/pnas.1007647107|issn=0027-8424|pmid=21106756|pmc=3003125}} 24. ^{{cite book|author1=Fullwood, Melissa J|author2=Han, Yuyuan|author3=Wei, Chia-Lin|author4=Ruan, Xiaoan|author5=Ruan, Yijun|date=January 2010|title=Chromatin interaction analysis using paired-end tag sequencing|url=|journal=Current Protocols in Molecular Biology|volume=Chapter 21|issue=|pages=Unit 21.15.1–25|doi=10.1002/0471142727.mb2115s89|issn=|pmid=20069536|isbn=978-0471142720}} 25. ^{{cite journal|author1=Li, Guoliang |author2=Fullwood, Melissa J |author3=Xu, Han |author4=Mulawadi, Fabianus Hendriyan |author5=Velkov, Stoyan |author6=Vega, Vinsensius |author7=Ariyaratne, Pramila Nuwantha |author8=Mohamed, Yusoff Bin |author9=Ooi, Hong-Sain |author10=Tennakoon, Chandana |author11=Wei, Chia-Lin |author12=Ruan, Yijun |author13=Sung, Wing-Kin | title = ChIA-PET tool for comprehensive chromatin interaction analysis with paired-end tag sequencing| journal = Genome Biology| volume = 11| issue = 2| pages = R22|date=February 2010| pmid = 20181287| doi = 10.1186/gb-2010-11-2-r22| url =| issn =| pmc = 2872882}} 26. ^{{cite web|url=https://www.sciencedaily.com/releases/2009/11/091104132700.htm|title=ChIA-PET: Novel Method For 3-D Whole Genome Mapping Research |date=2009-11-08|work=ScienceDaily|publisher=Agency for Science, Technology and Research (A*STAR), Singapore.|accessdate=14 March 2010}} External links{{Commons category|Chromatin immunoprecipitation}}
4 : Protein methods|Molecular biology techniques|Protein–protein interaction assays|Immunologic tests |
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