METHODS IN ENZYMOLOGY Editors-in-Chief JOHN N. ABELSON and MELVIN I. SIMON Division of Biology California Institute of Technology Pasadena, California Founding Editors SIDNEY P. COLOWICK and NATHAN O. KAPLAN AcademicPressisanimprintofElsevier 525BStreet,Suite1900,SanDiego,CA92101-4495,USA 225WymanStreet,Waltham,MA02451,USA TheBoulevard,LangfordLane,Kidlington,Oxford,OX51GB,UK Firstedition2012 Copyright#2012,ElsevierInc.AllRightsReserved. Nopartofthispublicationmaybereproduced,storedinaretrievalsystemortransmittedin anyformorbyanymeanselectronic,mechanical,photocopying,recordingorotherwise withoutthepriorwrittenpermissionofthepublisher PermissionsmaybesoughtdirectlyfromElsevier’sScience&TechnologyRights DepartmentinOxford,UK:phone(+44)(0)1865843830;fax(+44)(0)1865853333; email:permissions@elsevier.com.Alternativelyyoucansubmityourrequestonlineby visitingtheElsevierwebsiteathttp://elsevier.com/locate/permissions,andselecting ObtainingpermissiontouseElseviermaterial Notice Noresponsibilityisassumedbythepublisherforanyinjuryand/ordamagetopersonsor propertyasamatterofproductsliability,negligenceorotherwise,orfromanyuseor operationofanymethods,products,instructionsorideascontainedinthematerialherein. Becauseofrapidadvancesinthemedicalsciences,inparticular,independentverificationof diagnosesanddrugdosagesshouldbemade ForinformationonallAcademicPresspublications visitourwebsiteatstore.elsevier.com ISBN:978-0-12-391940-3 ISSN:0076-6879 PrintedandboundinUnitedStatesofAmerica 12 13 14 11 10 9 8 7 6 5 4 3 2 1 CONTRIBUTORS SamarAlami LaboratoryofBiochemistryandMolecularBiology,NationalCancerInstitute,National InstitutesofHealth,Bethesda,Maryland,USA BrittanyN.Albaugh DepartmentofBiomolecularChemistry,SchoolofMedicineandPublicHealth,and WisconsinInstitutesofDiscovery,UniversityofWisconsin,Madison,Wisconsin,USA MarkT.Bedford DepartmentofMolecularCarcinogenesis,TheUniversityofTexasMDAnderson CancerCenter,Smithville,Texas,USA SheChen NationalInstituteofBiologicalSciences,Beijing,PRChina DonghangCheng DepartmentofMolecularCarcinogenesis,TheUniversityofTexasMDAndersonCancer Center,Smithville,Texas,USA MairE.A.Churchill DepartmentofPharmacology,UniversityofColorado,SchoolofMedicine,Aurora, Colorado,USA JohnM.Denu DepartmentofBiomolecularChemistry,SchoolofMedicineandPublicHealth;Wisconsin InstitutesofDiscovery,andIntegratedProgramofBiochemistry,UniversityofWisconsin, Madison,Wisconsin,USA DouglasC.DonhamIII DepartmentofPharmacology,UniversityofColorado,SchoolofMedicine,Aurora, Colorado,USA StephenM.Fuchs DepartmentofBiology,TuftsUniversity,Medford,Massachusetts,USA BenjaminA.Garcia DepartmentofBiochemistryandBiophysics,PerelmanSchoolofMedicine,Universityof Pennsylvania,Philadelphia,Pennsylvania,USA DariaA.Gaykalova DepartmentofPharmacology,UMDNJ-RobertWoodJohnsonMedicalSchool,Piscataway, NewJersey,USA AaronR.Hieb DepartmentofBiochemistryandMolecularBiology,andHowardHughesMedicalInstitute, ColoradoStateUniversity,FortCollins,Colorado,USA xi xii Contributors Fu-KaiHsieh DepartmentofPharmacology,UMDNJ-RobertWoodJohnsonMedicalSchool, Piscataway,NewJersey,USA KrzysztofKrajewski DepartmentofBiochemistry&Biophysics,UniversityofNorthCarolinaatChapelHill, ChapelHill,NorthCarolina,USA OlgaI.Kulaeva DepartmentofPharmacology,UMDNJ-RobertWoodJohnsonMedicalSchool, Piscataway,NewJersey,USA,andFacultyofBiology,MoscowStateUniversity,Moscow, Russia ShuLin DepartmentofBiochemistryandBiophysics,PerelmanSchoolofMedicine,Universityof Pennsylvania,Philadelphia,Pennsylvania,USA KarolinLuger DepartmentofBiochemistryandMolecularBiology,andHowardHughesMedicalInstitute, ColoradoStateUniversity,FortCollins,Colorado,USA EdLuk LaboratoryofBiochemistryandMolecularBiology,NationalCancerInstitute,National InstitutesofHealth,Bethesda,Maryland,USA KathrynMalecek DepartmentofBiochemistryandMolecularBiology,UniversityofChicago,Chicago, Illinois,USA GakuMizuguchi HowardHughesMedicalInstitute,JaneliaFarmResearchCampus,Ashburn,Virginia,USA NikolaiA.Pestov DepartmentofPharmacology,UMDNJ-RobertWoodJohnsonMedicalSchool, Piscataway,NewJersey,USA,andFacultyofBiology,MoscowStateUniversity,Moscow, Russia ScottB.Rothbart LinebergerComprehensiveCancerCenter,andDepartmentofBiochemistry&Biophysics, UniversityofNorthCarolinaatChapelHill,ChapelHill,NorthCarolina,USA AlexanderRuthenburg DepartmentofBiochemistryandMolecularBiology,andDepartmentofMolecularGenetics andCellBiology,UniversityofChicago,Chicago,Illinois,USA JeanK.Scorgie DepartmentofPharmacology,UniversityofColorado,SchoolofMedicine,Aurora, Colorado,USA LiShen HowardHughesMedicalInstitute,andDepartmentofBiochemistryandBiophysics, LinebergerComprehensiveCancerCenter,UniversityofNorthCarolinaatChapelHill, ChapelHill,NorthCarolina,USA Contributors xiii KevanM.Shokat DepartmentofCellularandMolecularPharmacology,HowardHughesMedicalInstitute, UniversityofCalifornia,SanFrancisco,California,USA MatthewD.Simon DepartmentofMolecularBiophysicsandBiochemistry,ChemicalBiologyInstitute,Yale University,NewHaven,Connecticut,USA BrianD.Strahl LinebergerComprehensiveCancerCenter,andDepartmentofBiochemistry&Biophysics, UniversityofNorthCarolinaatChapelHill,ChapelHill,NorthCarolina,USA VasilyM.Studitsky DepartmentofPharmacology,UMDNJ-RobertWoodJohnsonMedicalSchool, Piscataway,NewJersey,USA,andFacultyofBiology,MoscowStateUniversity,Moscow, Russia VidyasiriVemulapalli DepartmentofMolecularCarcinogenesis,TheUniversityofTexasMDAndersonCancer Center,Smithville,Texas,USA MichielVermeulen MolecularCancerResearch,UniversityMedicalCenterUtrecht,3584CG,Utrecht,The Netherlands EliseK.Wagner DepartmentofBiomolecularChemistry,SchoolofMedicineandPublicHealth;Wisconsin InstitutesofDiscovery,andIntegratedProgramofBiochemistry,UniversityofWisconsin, Madison,Wisconsin,USA DuaneD.Winkler DepartmentofBiochemistryandMolecularBiology,ColoradoStateUniversity,Fort Collins,Colorado,USA Wei-HuaWu LaboratoryofBiochemistryandMolecularBiology,NationalCancerInstitute,National InstitutesofHealth,Bethesda,Maryland,USA MoXu NationalInstituteofBiologicalSciences,Beijing,PRChina YiZhang HowardHughesMedicalInstitute,andDepartmentofBiochemistryandBiophysics, LinebergerComprehensiveCancerCenter,UniversityofNorthCarolinaatChapelHill, ChapelHill,NorthCarolina,USA BingZhu NationalInstituteofBiologicalSciences,Beijing,PRChina PREFACE In 2004, when we edited the last three-volume series on Chromatin and Chromatin Remodeling Enzymes, remarkable progress had been made accessing the enzyme complexes that serve to remodel or modify histones andDNA,givingmolecularinsightsintowhatmightbeconsidered“epige- neticlandscapes”thatleadtostable,andpotentiallyheritable,statesofgene expression.Clearly,thatwasonlythetipoftheiceberg.Overthepast8years, interestinchromatinbiology,andepigeneticsingeneral,hasskyrocketed,in part due to numerous disease links to dysfunction in most, if not all, of the general classes of chromatin proteins, leading to the misactivation or mis- silencing of gene targets. Moreover, chromatin protein motifs such as bromodomainsthatrecognizehistoneacetyl-lysinemarkshavenowproven to be effective drug targets in several types of human disorders, lending supporttothegeneralviewthatmodulatingnonenzymatic,protein–protein interactions in chromatin-associated proteins can lead to therapeutically useful outcomes in humans. Researchers, in some cases armed now with molecular insights gained from X-ray and NMR structures, are beginning to unravel the “rules” of effector recruitment to the chromatin template. As well, the complexities of epigenetic landscapes have now been exploredinthecontextofwholegenomes,bycombiningRNAandchro- matinimmunoprecipitationwithdeepsequencingapproaches.Importantly, thisworkisbeingdoneexaminingnormalanddiseasestates,stimulatedbya flurry of recent findings of missense mutations in epigenetic-modifying activities,includingthehistoneproteinsthemselves.Challengesremainsuch as determining “cause versus effect” in bringing about pathological disease states, even to the point of knowing whether histones and/or methylated DNAarethephysiologicallyrelevantsubstratesformanyoftheseactivities. In addition, there remains a wide gap between reductionist, biochemical (vertical)approachesandthenow-popular“omic”(horizontal)approaches, providing an excellent opportunity for clever new strategies to help “connect the dots” underlying these different scientific styles. Withtheunquestionedimportanceofchromatinstructureandfunction in human biology and disease, a wealth of new researchers are entering the field, bringing with them new expertise, methods, and approaches. This updated, two-volume series aims to bring many of these advances to the community, including exposure to genome-wide approaches, xv xvi Preface single-molecule microscopy methods, and peptide array-based assays. We are most grateful to our colleagues for their efforts in contributing to these volumes. The popularity of the field has also spawned numerous other sources of chromatin methods that the reader may refer to. If the speed of discovery and technology development in prior years is any guide, we anticipate that this volume set will soon require updating. However, weareconfidentthateachoftheseriesdevotedtochromatinovertheyears continues to provide an enduring and evolving set of tools for many new- comers who seek to address the most fundamental problems in chromatin biology. In closing, we wish to make a special note in appreciation of Jonathan Widom, who passed away in 2011. Jon was the foremost practitioner of chromatin biophysics in our era and contributed on many occasions to MethodsinEnzymology.Weowehimadebtofgratitudeforleavingalegacy ofkeyconceptsinnucleosomedynamics,robustbiophysicaltechniques,and invaluablereagentsthatheandhislaboratoryprovidedtothechromatinfield for decades. It is fitting that the closing chapter of these volumes honors his most recent contribution—a new method for mapping nucleosome positions in vivo at base pair resolution. Jon will be greatly missed. CARL WU C. DAVID ALLIS METHODS IN ENZYMOLOGY VOLUME I. Preparation and Assay of Enzymes Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME II. Preparation and Assay of Enzymes Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME III. Preparation and Assay of Substrates Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME IV. Special Techniques for the Enzymologist Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME V. Preparation and Assay of Enzymes Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VI. Preparation and Assay of Enzymes (Continued) Preparation and Assay of Substrates Special Techniques Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VII. Cumulative Subject Index Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VIII. Complex Carbohydrates Edited by ELIZABETH F. NEUFELD AND VICTOR GINSBURG VOLUME IX. Carbohydrate Metabolism Edited by WILLIS A. WOOD VOLUME X. Oxidation and Phosphorylation Edited by RONALD W. ESTABROOK AND MAYNARD E. PULLMAN VOLUME XI. Enzyme Structure Edited by C. H. W. HIRS VOLUME XII. Nucleic Acids (Parts A and B) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XIII. Citric Acid Cycle Edited by J. M. LOWENSTEIN VOLUME XIV. Lipids Edited by J. M. LOWENSTEIN xvii xviii MethodsinEnzymology VOLUME XV. Steroids and Terpenoids Edited by RAYMOND B. CLAYTON VOLUME XVI. Fast Reactions Edited by KENNETH KUSTIN VOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B) Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME XIX. Proteolytic Enzymes Edited by GERTRUDE E. PERLMANN AND LASZLO LORAND VOLUME XX. Nucleic Acids and Protein Synthesis (Part C) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXI. Nucleic Acids (Part D) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXII. Enzyme Purification and Related Techniques Edited by WILLIAM B. JAKOBY VOLUME XXIII. Photosynthesis (Part A) Edited by ANTHONY SAN PIETRO VOLUME XXIV. Photosynthesis and Nitrogen Fixation (Part B) Edited by ANTHONY SAN PIETRO VOLUME XXV. Enzyme Structure (Part B) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVI. Enzyme Structure (Part C) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVII. Enzyme Structure (Part D) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVIII. Complex Carbohydrates (Part B) Edited by VICTOR GINSBURG VOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXXI. Biomembranes (Part A) Edited by SIDNEY FLEISCHER AND LESTER PACKER MethodsinEnzymology xix VOLUME XXXII. Biomembranes (Part B) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXIII. Cumulative Subject Index Volumes I-XXX Edited by MARTHA G. DENNIS AND EDWARD A. DENNIS VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B) Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XXXV. Lipids (Part B) Edited by JOHN M. LOWENSTEIN VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones) Edited by BERT W. O’MALLEY AND JOEL G. HARDMAN VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones) Edited by BERT W. O’MALLEY AND JOEL G. HARDMAN VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides) Edited by JOEL G. HARDMAN AND BERT W. O’MALLEY VOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems) Edited by JOEL G. HARDMAN AND BERT W. O’MALLEY VOLUME XL. Hormone Action (Part E: Nuclear Structure and Function) Edited by BERT W. O’MALLEY AND JOEL G. HARDMAN VOLUME XLI. Carbohydrate Metabolism (Part B) Edited by W. A. WOOD VOLUME XLII. Carbohydrate Metabolism (Part C) Edited by W. A. WOOD VOLUME XLIII. Antibiotics Edited by JOHN H. HASH VOLUME XLIV. Immobilized Enzymes Edited by KLAUS MOSBACH VOLUME XLV. Proteolytic Enzymes (Part B) Edited by LASZLO LORAND VOLUME XLVI. Affinity Labeling Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XLVII. Enzyme Structure (Part E) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF