Enzymes of Epigenetics, Part B PDF
Preview Enzymes of Epigenetics, Part B
METHODS IN ENZYMOLOGY Editors-in-Chief ANNA MARIE PYLE Departments of Molecular, Cellular and Developmental Biology and Department of Chemistry Investigator, Howard Hughes Medical Institute Yale University DAVID W. CHRISTIANSON Roy and Diana Vagelos Laboratories Department of Chemistry University of Pennsylvania Philadelphia, PA Founding Editors SIDNEY P. COLOWICK and NATHAN O. KAPLAN AcademicPressisanimprintofElsevier 50HampshireStreet,5thFloor,Cambridge,MA02139,USA 525BStreet,Suite1800,SanDiego,CA92101–4495,USA TheBoulevard,LangfordLane,Kidlington,OxfordOX51GB,UK 125LondonWall,London,EC2Y5AS,UK Firstedition2016 Copyright©2016ElsevierInc.Allrightsreserved. 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ISBN:978-0-12-805381-2 ISSN:0076-6879 ForinformationonallAcademicPresspublications visitourwebsiteathttps://www.elsevier.com/ Publisher:ZoeKruze AcquisitionEditor:ZoeKruze EditorialProjectManager:SarahLay ProductionProjectManager:MageshKumarMahalingam CoverDesigner:GregHarris TypesetbySPiGlobal,India CONTRIBUTORS S.Ackloo StructuralGenomicsConsortium,UniversityofToronto,Toronto,ON,Canada P.D.Adams CR-UKBeatsonLabs,InstituteofCancerSciences,UniversityofGlasgow,Glasgow, UnitedKingdom C.H.Arrowsmith StructuralGenomicsConsortium;PrincessMargaretCancerCentre,UniversityofToronto, Toronto,ON,Canada J.Baeza SchoolofMedicineandPublicHealth,WisconsinInstituteforDiscovery,Universityof Wisconsin,Madison,WI,UnitedStates D.Barsyte-Lovejoy StructuralGenomicsConsortium,UniversityofToronto,Toronto,ON,Canada A.Bedalov FredHutchinsonCancerResearchCenter,Seattle,WA,UnitedStates S.L.Berger PerelmanSchoolofMedicine,UniversityofPennsylvania,Philadelphia,PA,UnitedStates M.R.Birtwistle IcahnSchoolofMedicineatMountSinai,NewYork,NY,UnitedStates X.-C.Cai MemorialSloanKetteringCancerCenter,NewYork,NY,UnitedStates C.Chatterjee UniversityofWashington,Seattle,WA,UnitedStates S.Chowdhury FredHutchinsonCancerResearchCenter,Seattle,WA,UnitedStates E.M.Cornett CenterforEpigenetics,VanAndelResearchInstitute,GrandRapids,MI,UnitedStates H.Dai SirtuinDPU,GlaxoSmithKline(GSK),Collegeville,PA,UnitedStates J.M.Denu SchoolofMedicineandPublicHealth,WisconsinInstituteforDiscovery,Universityof Wisconsin;MorgridgeInstituteforResearch,Madison,WI,UnitedStates A.Dhall UniversityofWashington,Seattle,WA,UnitedStates B.M.Dickson CenterforEpigenetics,VanAndelResearchInstitute,GrandRapids,MI,UnitedStates xi xii Contributors B.D.Dill TheRockefellerUniversityProteomicsResourceCenter,TheRockefellerUniversity, NewYork,NY,UnitedStates J.L.Ellis SirtuinDPU,GlaxoSmithKline(GSK),Collegeville,PA,UnitedStates J.Fan SchoolofMedicineandPublicHealth,WisconsinInstituteforDiscovery,Universityof Wisconsin,Madison,WI,UnitedStates L.A.Farrelly IcahnSchoolofMedicineatMountSinai,NewYork,NY,UnitedStates B.A.Garcia PerelmanSchoolofMedicine,UniversityofPennsylvania,Philadelphia,PA,UnitedStates B.P.Hubbard UniversityofAlberta,Edmonton,AB,Canada O.Iwasaki TheWistarInstitute,Philadelphia,PA,UnitedStates J.L.Johnson InstituteforImmunology,PerelemanSchoolofMedicine,UniversityofPennsylvania, Philadelphia,PA,UnitedStates K.Kapilashrami MemorialSloanKetteringCancerCenter,NewYork,NY,UnitedStates K.R.Karch PerelmanSchoolofMedicine,UniversityofPennsylvania,Philadelphia,PA,UnitedStates K.-D.Kim TheWistarInstitute,Philadelphia,PA,UnitedStates S.Krishnan UniversityofMichigan,AnnArbor,MI,UnitedStates E.Lima-Fernandes StructuralGenomicsConsortium,UniversityofToronto,Toronto,ON,Canada M.Luo MemorialSloanKetteringCancerCenter,NewYork,NY,UnitedStates I.Maze IcahnSchoolofMedicineatMountSinai,NewYork,NY,UnitedStates J.L.Meier ChemicalBiologyLaboratory,NationalCancerInstitute,Frederick,MD,UnitedStates P.Mews PerelmanSchoolofMedicine,UniversityofPennsylvania,Philadelphia,PA,UnitedStates Contributors xiii H.Molina TheRockefellerUniversityProteomicsResourceCenter,TheRockefellerUniversity, NewYork,NY,UnitedStates D.C.Montgomery ChemicalBiologyLaboratory,NationalCancerInstitute,Frederick,MD,UnitedStates K.Noma TheWistarInstitute,Philadelphia,PA,UnitedStates P.Prinos StructuralGenomicsConsortium,UniversityofToronto,Toronto,ON,Canada T.S.Rai InstituteofBiomedicalandEnvironmentalHealthResearch,UniversityoftheWestof Scotland,Paisley,UnitedKingdom Z.Ramjan CenterforEpigenetics,VanAndelResearchInstitute,GrandRapids,MI,UnitedStates S.B.Rothbart CenterforEpigenetics,VanAndelResearchInstitute,GrandRapids,MI,UnitedStates S.Sidoli PerelmanSchoolofMedicine,UniversityofPennsylvania,Philadelphia,PA,UnitedStates J.A.Simon FredHutchinsonCancerResearchCenter,Seattle,WA,UnitedStates D.A.Sinclair GlennLabsfortheBiologicalMechanismsofAging,HarvardMedicalSchool,Boston,MA, UnitedStates;TheUniversityofNewSouthWales,Sydney,NSW,Australia B.D.Strahl UniversityofNorthCarolinaatChapelHill,ChapelHill,NC,UnitedStates M.M.Szewczyk StructuralGenomicsConsortium,UniversityofToronto,Toronto,ON,Canada R.C.Trievel UniversityofMichigan,AnnArbor,MI,UnitedStates G.Vahedi InstituteforImmunology,PerelemanSchoolofMedicine,UniversityofPennsylvania, Philadelphia,PA,UnitedStates R.M.Vaughan CenterforEpigenetics,VanAndelResearchInstitute,GrandRapids,MI,UnitedStates C.E.Weller UniversityofWashington,Seattle,WA,UnitedStates PREFACE These two volumesof Methods ofEnzymologycover therapidly developing fieldofEpigenetics.Thecentraldogmaofmolecularbiology,firstproposed byFrancisCrickin1956,providedaframeworkforunderstandingthetrans- fer of genetic information, which flows from DNA to RNA to protein. However, in the early 1980s, it was discovered that methylation of the DNA can change its function, hinting that genetic information transfer could be altered in other ways. In the 1990s, it was discovered that gene functioncouldindeed bealtered inmanyotherways andthemodernfield of Epigenetics was born. Epigenetics, a term first coined by Conrad Waddington in 1942 as “the branch of biology which studies the causal interactions between genesand their products, whichbring thephenotype intobeing,”isnowknownasthestudyofheritablechangesingeneexpres- sion due to internal or environmental signals that results in the change of cellular function or physiological phenotype, but that is not caused by changes in the genetic information. An example of epigenetic regulation is cell differentiation, whereby cells of an organism with identical genetic information, such as cells of the nose, eyes, and hair, carry out different cellular functions. Attheheart of epigeneticsistheregulation of chromatin,thepackaged form of DNA. The building blocks of chromatin are nucleosome core particles containing about 146bp of DNA wrapped around an octamer of histone proteins, two copies each of histones H2A, H2B, H3, and H4. ChromatinmediatesallDNA-templatedevents,includingDNAtranscrip- tion,replication,andrepair,andisregulatedbymanyproteinsandnoncoding RNAs.Theproteinsthatregulatechromatinincludeposttranslationalmod- ification (PTM) “writer” enzymes, “eraser” enzymes that remove these PTMs, and “reader” proteins that bind chemically modified histones or DNA. PTMs of DNA include methylation at the 5 position of cytosine andvariousoxidationstatesof5-methyl-cytosine.PTMsofhistonesinclude acetylation,methylation,andubiquitinationonlysineresidues;methylation andcitrullinationofarginineresidues;andphosphorylationofthreonineand serineresidues.ATP-dependentchromatin-remodelingenzymesthatfunc- tiontorepositionnucleosomeswithinchromatin;histonechaperoneproteins thatinsertorevictionofhistonevariantsinandoutofchromatin,respectively; andnoncodingRNAmoleculesalsocontributetochromatinregulation. xv xvi Preface Chromatin-regulatory proteins work together to mediate epigenetic regulation,withramificationsforcellularfunctionandphysiologicalpheno- type,andoverthelastdecade,ithasbecomeapparentthatthedysfunctionof epigeneticregulatorscandrivemanydiseasesincludingmetabolicandneu- rodegenerativedisordersandvariouscancers.Overthelastdecade,wehave seen significant progress on understanding the molecular mechanisms underlying epigenetic regulation leading to new insights into cellular function and physiological phenotype, the development of new technolo- gies,andthedevelopmentofsmall-moleculeprobestostudythesebiological processesandsmallepigeneticdrugsthatarecurrentlyinclinicaltrialstotreat disease. The remarkable progress in the field of epigenetic research that has occurred over the last two decades is highlighted in these two volumes of Methods of Enzymology, entitled Enzymes of Epigenetics. In Volume 1, Chapters 1 through 5 cover Chromatin Structure and Histones. This includes strategies for in vitro chromatin assembly (Chapter 1) and the assembly of protein–chromatin complexes (Chapter 2) for biochemical, biophysical, andstructuralstudies,preparationofrecombinantcentromericnucleosomes with and without nonhistone centromere proteins (Chapter 3), functional characterization of histone deposition by histone chaperones (Chapter 4), and methods to study nucleosome sliding by ATP-dependent chromatin- remodeling enzymes (Chapter 5). In Volume 1, Chapters 6 through 13 cover Posttranslational Histone Modification Enzymes and Complexes. This includes in vitro activity assays for MYST histone acetyltransferases and adaptation for high-throughput inhibitor screening (Chapter 6), and the preparation, biochemical analysis, andstructuredeterminationoftheclassical(Chapter7)andsirtuin(Chapter8) histone lysine deacetylases, SET domain histone methyltransferases (Chapter 9), LSD1/KDM1A (Chapter 10), and JmjC (Chapter 12). Also included are chapters on LSD1 histone demethylase assays and inhibition (Chapter 11) and preparation and analysis of native chromatin-modifying complexes(Chapter13). InVolume1,Chapters14and15coverHistoneModificationBindersand include the preparation, biochemical analysis, and structure determination of the acetyl-lysine reader bromodomains (Chapter 14) and the readers of the methyllysine mark (Chapter 15). InVolume1,Chapters16through20coverDNAModificationsandNucleic Acid Regulators. This includes quantification of oxidized 5-methylcytosine bases and TET enzyme activity (Chapter 16), characterization of how Preface xvii DNA modifications affect DNA binding by C H zinc finger proteins 2 2 (Chapter17),regulationofchromosomeendsbytheriboproteintelomerase complex (Chapter 18), detection and analysis of long noncoding RNAs (Chapter 19), and identification of centromeric RNAs involved in histone dynamicsinvivo(Chapter20). In Volume 2, Chapters 1 through 8 cover Epigenetic Technologies. This includes identification and quantification of histone PTMs using high-resolution mass spectrometry (Chapter 1), substrate specificity profil- ingofhistone-modifyingenzymesusingpeptidemicroarray(Chapter2),an open-source platform to analyze such microarrays (Chapter 3), chemical biologyapproachesforcharacterizationofepigeneticregulators(Chapter4), mapping lysine acetyltransferase–ligand interactions by activity-based capture (Chapter 5), methods for investigating histone acetylation stoichi- ometry and turnover rate (Chapter 6), semisynthesis of acetylated and sumoylated histone analogs (Chapter 7), and an IF-FISH approach for covisualization of gene loci and nuclear architecture in fission yeast (Chapter 8). In Volume 2, Chapters 9 through 11 cover Small-Molecule Epigenetic Regulators and include the preparation and analysis of sirtuin deacetylase inhibitors (Chapter 9) and activators (Chapter 10) and methyltransferase inhibitors (Chapter 11). In Volume 2, Chapters 12 through 15 cover Epigenetics and Biological Connectionsandincludeexploringthedynamicrelationshipbetweencellular metabolism and chromatin structure using SILAC-mass spec and ChIP- sequencing (Chapter 12), proteomic methods to investigate the dynamics of histone turnover in the central nervous system (Chapter 13), ChIP-seq techniques to map the epigenome of senescent cells (Chapter 14), and exploiting chromatin biology to understand immunology (Chapter 15). Iexpectthatthesevolumeswillbeausefulresourcefor investigatorsin the epigenetics field as well as those outside of the epigenetics field who would like to incorporate epigenetics into their own research programs. RONEN MARMORSTEIN Abramson Family Cancer Research Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States CHAPTER ONE Identification and Quantification of Histone PTMs Using High- Resolution Mass Spectrometry K.R. Karch, S. Sidoli, B.A. Garcia1 PerelmanSchoolofMedicine,UniversityofPennsylvania,Philadelphia,PA,UnitedStates 1Correspondingauthor:e-mailaddress:[email protected] Contents 1. Introduction 4 2. HistoneExtractionfromCells 6 2.1 MaterialsandBufferRecipes 6 2.2 CellHarvest 7 2.3 NucleiIsolation 7 2.4 AcidExtraction 8 3. Bottom-UpMassSpectrometry 9 3.1 MaterialsandBufferRecipes 10 3.2 DerivatizationandDigestion 10 3.3 Desalting 11 3.4 OnlineRP-HPLCandMSAcquisition 12 3.5 DataAnalysis 14 4. OfflineFractionationofHistoneSpecies 18 4.1 MaterialsandBufferRecipes 18 4.2 HistoneVariantPurification 18 5. Middle-DownMassSpectrometry 19 5.1 MaterialsandBufferRecipes 20 5.2 Digestion 21 5.3 WCX-HILICandMS 21 5.4 DataAnalysis 22 6. Top-DownMassSpectrometry 24 6.1 MaterialsandBufferRecipes 25 6.2 Top-DownMSUsingDirectInfusion 25 6.3 DataAnalysis 26 References 28 Abstract DNA is organized into nucleosomes, composed of 147 base pairs of DNA wrapped aroundanoctamerofhistoneproteinsincludingH2A,H2B,H3,andH4.Histonesare MethodsinEnzymology,Volume574 #2016ElsevierInc. 3 ISSN0076-6879 Allrightsreserved. http://dx.doi.org/10.1016/bs.mie.2015.12.007 4 K.R.Karchetal. critical regulators of many nuclear processes, including transcription, DNA damage repair, and higher order chromatin structure. Much of their function is mediated through extensive and dynamic posttranslational modification (PTM) by nuclear enzymes. Histone PTMs are thought to form a code, where combinations of PTMs areresponsibleforspecificbiologicalfunctions.Here,wepresentprotocolstoidentify and quantify histone PTMs using nanoflow liquid chromatography coupled to mass spectrometry (MS). We first describe how to purify histones and prepare them for MS. We then describe three MS platforms for histone PTM analysis, including bottom-up,middle-down,andtop-downapproaches,andexplaintherelativebenefits andpitfallsofeachapproach.Wealsoincludetipstoincreasethethroughputoflarge experiments. 1. INTRODUCTION DNA must be highly organized and tightly regulated within the nucleustomaintainpropergeneexpression.Thecellaccomplishesthistask byorganizingDNAintoaprotein–DNAcomplexcalledchromatin.Within chromatin,DNAiscontainedinnucleosomes,whicharecomposedof147 basepairsofDNAwrappedaroundanoctamerofhistoneproteinswithtwo copies of each core histone—H2A, H2B, H3, and H4 (Luger, Ma¨der, Richmond, Sargent, & Richmond, 1997). Linker histone H1 can bind thefreeDNAthatexistsbetweennucleosomes.Duetotheirintimateasso- ciationwithDNA,histonesaremajorregulatorsofchromatinstructureand function.Histoneproteinsareextensivelyanddynamicallyposttranslationally modifiedonspecificresiduesbyamyriadofenzymesinthenucleus.These posttranslationalmodifications(PTMs)mediatehistonefunctionbydirectly alteringthechemistryofthesurroundingchromatinorthroughtheactionof otherproteinsthatcanbindthesemodifications.Agrowingbodyofresearch supports the hypothesis that PTMs form a “histone code” and can act in tandem to illicit a specific biological response (Jenuwein & Allis, 2001). HistonePTMprofilesarecriticaltomaintainnuclearstability,andaberrant regulationofhistonePTMsisimplicatedinmanydiseasesincludingcancer. As such, the ability to identify and quantify histone PTMs in biological systems is vital for understanding nuclear processes and how disease states may arise. Histone PTM analysis had traditionally been accomplished using antibody-based approaches such as Western blots, chromatin immunopre- cipitation, and deep sequencing. These methods have been instrumental inelucidatingtherolesofmanyhistonePTMsbutsufferfromseveralcritical
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