Table Of ContentSpringer Protocols
Methods in Molecular Biology 567
Chromatin
Immunoprecipitation
Assays
Methods and Protocols
Edited by
Philippe Collas
M M B TM
ETHODS IN OLECULAR IOLOGY
SeriesEditor
JohnM.Walker
SchoolofLifeSciences
UniversityofHertfordshire
Hatfield,Hertfordshire,AL109AB,UK
Forothertitlespublishedinthisseries,goto
www.springer.com/series/7651
Chromatin Immunoprecipitation
Assays
Methods and Protocols
Edited by
Philippe Collas
University of Oslo, Oslo, Norway
Editor
PhilippeCollas
DepartmentofBiochemistry
UniversityofOslo
Oslo0372
Norway
[email protected]
SeriesEditor
JohnWalker
UniversityofHertfordshire
Halfield,Herts
UK
ISSN1064-3745 e-ISSN1940-6029
ISBN978-1-60327-413-5 e-ISBN978-1-60327-414-2
DOI10.1007/978-1-60327-414-2
SpringerDordrechtHeidelbergLondonNewYork
LibraryofCongressControlNumber:2009931091
#HumanaPress,apartofSpringerScienceþBusinessMedia,LLC2009
Allrightsreserved.Thisworkmaynotbetranslatedorcopiedinwholeorinpartwithoutthewrittenpermissionofthe
publisher(HumanaPress,c/oSpringerScience+BusinessMedia,LLC,233SpringStreet,NewYork,NY10013,USA),
exceptforbriefexcerptsinconnectionwithreviewsorscholarlyanalysis.Useinconnectionwithanyformofinformation
storageandretrieval,electronicadaptation,computersoftware,orbysimilarordissimilarmethodologynowknownor
hereafterdevelopedisforbidden.
Theuseinthispublicationoftradenames,trademarks,servicemarks,andsimilarterms,eveniftheyarenotidentifiedas
such,isnottobetakenasanexpressionofopinionastowhetherornottheyaresubjecttoproprietaryrights.
Whiletheadviceandinformationinthisbookarebelievedtobetrueandaccurateatthedateofgoingtopress,neither
theauthorsnortheeditorsnorthepublishercanacceptanylegalresponsibilityforanyerrorsoromissionsthatmaybe
made.Thepublishermakesnowarranty,expressorimplied,withrespecttothematerialcontainedherein.
Coverillustration:ThebackgroundartisderivedfromFigure2inChapter7
Printedonacid-freepaper
SpringerispartofSpringerScienceþBusinessMedia(www.springer.com)
Preface
Virtually all aspects of cellular function, such as replication of DNA, separation of
chromosomes during cell division, DNA repair, or gene expression, depend on the
interaction of proteins with DNA. The nature of DNA-binding proteins is wide and
ranges from structural proteins making up the nucleosome, enzymes modulating
chromatin structure to enable, facilitate, or repress gene expression, transcription
factors, and various cofactors. The biological significance of these associations in the
context of gene expression, development, cell differentiation, and disease has immen-
sely been enhanced in the past 20 years by the advent of a technique referred to as
chromatinimmunoprecipitation,orChIP.ThepurposeoftheChIPassayistoidentify
genomic sequence(s) associated with a protein of interest, for example, your favorite
transcriptionfactor,inthegenome.ChIP,then,hasbecomethetechniqueofchoiceto
determinethegenomicenrichmentprofilesoftranscriptionfactors,post-translationally
modified histones, histone variants, or chromatin-modifying enzymes. In the ChIP
assay,theproteinofinterestisimmunoprecipitatedfromachromatinpreparationusing
specific antibodies. After stringent washes, the DNA is released and the sequences
bound by the immunoprecipitated protein are identified. Sequence identification
methods have rapidly evolved from dot- or slot-blots in the early days to polymerase
chainreaction.Subsequently,thecombinationofChIPwithDNAmicroarrayorhigh-
throughputsequencingtechnologieshasenabledtheprofilingofproteinoccupancyon
a genome-wide scale. It has also promoted the appearance of new algorithms for
mappingproteinbindingthroughoutthegenome.
ChIP, therefore, is arguably a power tool. Nevertheless, it has for a long time
remained a cumbersome procedure taking several days and requiring very large num-
bers(severalmillions)ofcells.Theselimitationshavesparkedmodificationsoftheassay
andvariationsinDNAdetectionapproachestoshortentheprocedure,simplifysample
handling,andmakeChIPamenabletosmallcellnumbers.Asaresult,theChIPassay
has become increasingly popular in several areas of molecular and cell biology. To
illustratethispoint,aPubMedsearchwiththekeyword‘‘chromatinimmunoprecipita-
tion’’bringsupfourpublicationsin1988andatotalofover6,400todate,including
1,578publicationsin2008alone(seeFig.1).
Release of this volume on Chromatin Immunoprecipitation Assays by Humana
Press is, therefore, timely. The volume is devoted to recent developments in ChIP
andrelatedprotocols,whichhaveprovenreliableintheliteratureandwhichIbelieve
will remain current and of great interest to researchers for many years to come. The
chapters describe protocols on subjects such as characterization of ChIP antibodies,
ChIP methods for small cell numbers, fast ChIP protocols, and assays adapted to
variousspeciesandcelltypes.Severalstrategiesfortheanalysisofgenome-widedata
sets are also included. The book also extends beyond ChIP assays per se to include
protocols on immunoprecipitation-based DNA methylation analyses, determination
v
vi Preface
Fig.1. YearlynumberofPubMedpublicationsrespondingtothesearchcriterion‘‘chromatinimmunoprecipitation’’.
of spatial chromatin organization of large genomic regions, as well as RNA
immunoprecipitation.
These protocols have been carefully detailed by researchers deeply involved in their
development or improvement. All of the contributors and their teams deserve many
thanksfortheirtime,effort,andgenerosity.Ithasbeenfuntoworkonthisproject,and
I wish to thank John Walker for his invitation to put together this volume, and the
entireproductionteamatHumanaPress.
PhilippeCollas
Contents
Preface............................................................ v
Contributors........................................................ ix
1. TheState-of-the-ArtofChromatinImmunoprecipitation ................... 1
PhilippeCollas
2. CharacterizationandQualityControlofAntibodiesUsed
inChIPAssays .................................................... 27
Ge´raldineGoens,DorinaRusu,LaurentBultot,Jean-JacquesGoval,
andJuanaMagdalena
3. TheFastChromatinImmunoprecipitationMethod........................ 45
JoelNelson,OlegDenisenko,andKarolBomsztyk
4. mChIP:ChromatinImmunoprecipitationforSmallCellNumbers............. 59
JohnArneDahlandPhilippeCollas
5. Fish’nChIPs:ChromatinImmunoprecipitationintheZebrafishEmbryo ....... 75
LeifC.Lindeman,LinnT.Vogt-Kielland,PeterAlestro¨m,
andPhilippeCollas
6. EpitopeTaggingofEndogenousProteinsforGenome-WideChromatin
ImmunoprecipitationAnalysis ........................................ 87
ZhengheWang
7. FlowCytometricandLaserScanningMicroscopicApproaches
inEpigeneticsResearch.............................................. 99
LorantSzekvolgyi,LaszloImre,DoanXuanQuangMinh,EvaHegedus,
ZsoltBacso,andGaborSzabo
8. SerialAnalysisofBindingElementsforTranscriptionFactors ................ 113
JiguoChen
9. ModelingandAnalysisofChIP-ChipExperiments......................... 133
RaphaelGottardo
10. UseofSNP-ArraysforChIPAssays:ComputationalAspects ................. 145
EnriqueM.Muro,JenniferA.McCann,MichaelA.Rudnicki,
andMiguelA.Andrade-Navarro
11. DamID:AMethylation-BasedChromatinProfilingApproach................ 155
MonaAbed,DoritKenyagin-Karsenti,OlgaBoico,andAmirOrian
12. ChromosomeConformationCapture(from3Cto5C)andItsChIP-Based
Modification...................................................... 171
AlexeyGavrilov,ElviraEivazova,IrynaPirozhkova,MarcLipinski,
SergeyRazin,andYegorVassetzky
13. DeterminingSpatialChromatinOrganizationofLargeGenomicRegions
Using5CTechnology............................................... 189
NynkeL.vanBerkumandJobDekker
14. AnalysisofNascentRNATranscriptsbyChromatinRNAImmunoprecipitation.. 215
AlesObrdlikandPiergiorgioPercipalle
vii
viii Contents
15. MethylDNAImmunoprecipitation .................................... 237
Jean-JacquesGovalandJuanaMagdalena
16. ImmunoprecipitationofMethylatedDNA............................... 249
AnitaL.SørensenandPhilippeCollas
17. Erratum.............................. .......................... E1
Index ................................................................ 263
Contributors
MONA ABED (cid:129) Center for Vascular and Cancer Biology, The Rappaport Faculty of
MedicineandResearchInstitute,Technion-IsraelInstituteofTechnology,Haifa,Israel
PETER ALESTRo¨M (cid:129) Department of Basic Sciences and Aquatic Medicine, Norwegian
SchoolofVeterinaryScience,Oslo,Norway
MIGUELA.ANDRADE-NAVARRO (cid:129) Max-Delbru¨ckCenterforMolecularMedicine,Berlin,
Germany
ZSOLT BACSO (cid:129) Department of Biophysics and Cell Biology, University of Debrecen,
Debrecen,Hungary
OLGA BOICO (cid:129) Center for Vascular and Cancer Biology, The Rappaport Faculty of
MedicineandResearchInstitute,Technion-IsraelInstituteofTechnology,Haifa,Israel
KAROLBOMSZTYK (cid:129) MolecularandCellularBiologyProgramandUWMedicineatLake
Union,UniversityofWashington,Seattle,WA,USA
LAURENT BULTOT (cid:129) Diagenode sa, Sart-Tilman, Li`ege, Belgium; Universite´ UCL,
Bruxelles,Belgium
JIGUO CHEN (cid:129) Department of Biological Sciences, Mississippi State University,
MississippiState,MS,USA
PHILIPPE COLLAS (cid:129) Department of Biochemistry, Institute of Basic Medical Sciences,
UniversityofOslo,Oslo,Norway
JOHN ARNE DAHL (cid:129) Department of Biochemistry, Institute of Basic Medical Sciences,
UniversityofOslo,Oslo,Norway
JOBDEKKER (cid:129) PrograminGeneFunctionandExpressionandDepartmentofBiochem-
istryandMolecularPharmacology,UniversityofMassachusettsMedicalSchool,Worce-
ster,MA,USA
OLEG DENISENKO (cid:129) UW Medicine at Lake Union, University of Washington, Seattle,
WA,USA
ELVIRAEIVAZOVA (cid:129) VanderbiltUniversity,Nashville,TN,USA
ALEXEYGAVRILOV (cid:129) CNRSUMR-8126,Universite´ Paris-Sud11,InstitutdeCance´rologie
Gustave Roussy, France; Institute of Gene Biology, Russian Academy of Sciences, Moscow,
Russia
GE´RALDINEGOENS (cid:129) Diagenodesa,Sart-Tilman,Li`ege,Belgium
RAPHAEL GOTTARDO (cid:129) Department of Statistics, University of British Columbia, Van-
couver,BC,Canada
JEAN-JACQUESGOVAL (cid:129) Diagenodesa,Sart-Tilman,Li`ege,Belgium
EVA HEGEDUS (cid:129) Department of Biophysics and Cell Biology, University of Debrecen,
Debrecen,Hungary
DORIT KENYAGIN-KARSENTI (cid:129) Center for Vascular and Cancer Biology, The Rappaport
Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology,
Haifa,Israel
LASZLO IMRE (cid:129) Department of Biophysics and Cell Biology, University of Debrecen,
Debrecen,Hungary
ix
x Contributors
LEIF C. LINDEMAN (cid:129) Department of Biochemistry, Institute of Basic Medical Sciences,
UniversityofOslo,Oslo,Norway
MARC LIPINSKI (cid:129) CNRS UMR-8126,Universite´ Paris-Sud 11, Institut de Cance´rologie
GustaveRoussy,Villejuif,France
JUANAMAGDALENA (cid:129) Diagenodesa,Sart-Tilman,Li`ege,Belgium
JENNIFER A. MCCANN (cid:129) Department of Medicine and Department of Cellular and
MolecularMedicine,FacultyofMedicine,UniversityofOttawa,Ottawa,ON,Canada
DOAN XUAN QUANG MINH (cid:129) Department of Biophysics and Cell Biology, University
ofDebrecen,Debrecen,Hungary
ENRIQUEM.MURO (cid:129) Max-Delbru¨ckCenterforMolecularMedicine,Berlin,Germany
JOEL NELSON (cid:129) Molecular and Cellular Biology Program, University of Washington,
Seattle,WA,USA
ALESOBRDLIK (cid:129) DepartmentofCellandMolecularBiology,KarolinskaInstitute,Stock-
holm,Sweden
AMIR ORIAN (cid:129) Center for Vascular and Cancer Biology, The Rappaport Faculty
of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa,
Israel
PIERGIORGIOPERCIPALLE (cid:129) DepartmentofCellandMolecularBiology,KarolinskaInsti-
tute,Stockholm,Sweden
IRYNA PIROZHKOVA (cid:129) CNRS UMR-8126, Universite´ Paris-Sud 11, Institut de Cance´ro-
logieGustaveRoussy,Villejuif,France
SERGEYRAZIN (cid:129) InstituteofGeneBiology,RussianAcademyofSciences,Moscow,Russia
MICHAEL A. RUDNICKI (cid:129) Regenerative Medicine Program and Sprott Centre for Stem
Cell Research, Ottawa Health Research Institute, and Department of Medicine and
Department of Cellular and Molecular Medicine, Faculty of Medicine, University
ofOttawa,Ottawa,ON,Canada
DORINARUSU (cid:129) Diagenodesa,Sart-Tilman,Li`ege,Belgium
ANITA L. SØRENSEN (cid:129) Department of Biochemistry, Institute of Basic Medical Sciences,
UniversityofOslo,Oslo,Norway
GABOR SZABO (cid:129) Department of Biophysics and Cell Biology, University of Debrecen,
Debrecen,Hungary
LORANT SZEKVOLGYI (cid:129) Institut Curie, Recombinaison et Instabilite´ Ge´ne´tique,
UMR7147CNRS,InstitutCurie,Universite´ PierreetMarieCurie,Paris,France
NYNKE L. VAN BERKUM (cid:129) Program in Gene Function and Expression and Department
of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical
School,Worcester,MA,USA
YEGOR VASSETZKY (cid:129) CNRS UMR-8126, Universite´ Paris-Sud 11, Institut de Cance´r-
ologieGustaveRoussy,39,rueCamille-Desmoulins,94805VillejuifCEDEX
LINN T. VOGT-KIELLAND (cid:129) Department of Basic Sciences and Aquatic Medicine,
NorwegianSchoolofVeterinaryScience,Oslo,Norway
ZHENGHE WANG (cid:129) Department of Genetics and Case Comprehensive Cancer Center,
CaseWesternReserveUniversity,Cleveland,OH,USA
