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Methods in Molecular Biology 2577 Izuho Hatada · Takuro Horii Editors Epigenomics Methods and Protocols M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Epigenomics Methods and Protocols Edited by Izuho Hatada and Takuro Horii Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, Japan Editors IzuhoHatada TakuroHorii InstituteforMolecularandCellular InstituteforMolecularandCellularRegulation Regulation GunmaUniversity GunmaUniversity Maebashi,Gunma,Japan Maebashi,Gunma,Japan ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2723-5 ISBN978-1-0716-2724-2 (eBook) https://doi.org/10.1007/978-1-0716-2724-2 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface Epigeneticmodificationsturngenesonandoffanddifferbetweenindividuals,tissues,and even cells, contributing to their unique characteristics. Diseases can also stem from epige- neticmedicationsthatcauseabnormalactivationorsilencingofgenes.Theadventofnext- generationsequencing(NGS)technologyattheturnofthiscenturyhasmadeitpossibleto analyze epigenetic modifications across the entire genome, inaugurating the dawn of the epigenomics era. Ongoing research using NGS technology continues to show that epigenetic changes are at the basis of many human diseases. However, it was not until the early 2010s that a versatile method emerged to identify the epigenomic changes that cause a specific disease. Methods that enable the switching on and off of specific genes artificially alsoholdgreatpromisefordeterminingwhichgene(s)isresponsibleforacertaindiseaseand couldalsohaveawiderangeofmedicalandbiotechnologicalapplications. Theclusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)/Cas9system frombacteriahasbeensuccessfullyadaptedforRNA-guidedgenomeeditinginawiderange of organisms. Cas9 cleaves DNA upon recognition of a target sequence by the gRNA. Conversely, dCas9, a nuclease-defective Cas9, binds specifically to the target sequence without cleaving it. Therefore, dCas9 can recruit epigenetic enzymes to a specific gene andintroducechangesinasingleepigeneticmark.Thisnewtechnologyprovidesaplatform for the specific manipulation of the epigenome, a capability that could only have been dreamedofafewyearsago. Thisbookprovidesacomprehensivecollectionofprotocolsforepigenomicresearch.It consistsoftwomainparts.Thefirstpartdescribesthemethodsusedtoanalyzeepigenomic modifications,suchasDNAmethylation(Chapters1,2,and3),histonemodifications,and chromatinstructure(Chapters4,5,6,7,8,9,10,and11).Theseconddescribesmethods formanipulatingtheepigenome,suchasplatformsforepigenomeediting(Chapters12,13, 14, and 15), inducible systems for epigenome editing (Chapter 16), and epigenetically modifiedanimals(Chapters17,18,19,and20). Wethankalloftheauthorsfor theiroutstandingcontributionstothisbook.Onbehalf of all of us, we hope that you will find the information in this book useful and that it will furtherexpandtheutilityandscopeofepigenomicsresearch. Maebashi,Gunma,Japan IzuhoHatada TakuroHorii v Contents Preface ..................................................................... v Contributors................................................................. ix PART I ANALYSIS OF THE EPIGENOME: DNA METHYLATION 1 DNAMethylationAnalysisUsingBisulfitePyrosequencing .......... ........ 3 KenHigashimoto,SatoshiHara,andHidenobuSoejima 2 Post-bisulfiteAdaptorTaggingBasedonanssDNALigation Technique(tPBAT) ........ ........ ....... ....... ........ ....... ........ 21 FumihitoMiura,YukikoShibata,MikiMiura, andTakashiIto 3 ReducedRepresentationBisulfiteSequencing(RRBS)...... .. ....... ........ 39 KazuhikoNakabayashi,MichihiroYamamura, KeitaHaseagawa,andKenichiroHata PART II ANALYSIS OF THE EPIGENOME: HISTONE MODIFICATION AND CHROMATIN STRUCTURE 4 ChromatinImmunoprecipitationSequencing(ChIP-seq)forDetecting HistoneModificationsandModifiers........ ....... .... .... ...... ...... ... 55 ShinjiroHino,TetsuyaSato,andMitsuyoshiNakao 5 ATAC-SeqAnalysisofAccessibleChromatin:FromExperimentalSteps toDataAnalysis .... ....... ........ ....... ....... ........ ....... ........ 65 MayuTatara,TaiyoIkeda,SatoshiH.Namekawa, andSoMaezawa 6 Low-InputCUT&RUNforMouseOocytesandPreimplantation Embryos........... ....... ........ ....... ....... ....... ....... ..... .... 83 RyoyaHayashiandAzusaInoue 7 ImagingChromatinAccessibilitybyAssayofTransposase-Accessible ChromatinwithVisualization ....... ....... ....... ........ ....... ........ 93 YusukeMiyanari 8 STREAMING-TagSystem:TechnologytoEnableVisualization ofTranscriptionalActivityandSubnuclearLocalizationofSpecific EndogenousGenes......... ........ ....... ....... ........ ....... ........ 103 HiroakiOhishiandHiroshiOchiai 9 BioinformaticsPipelinesforIdentificationofSuper-Enhancers and3DChromatinContacts ..... ... ....... ....... ........ ....... ........ 123 AkihikoSakashita,ChikaraTakeuchi,SoMaezawa, andSatoshiH.Namekawa 10 AMethodforDetectionofSomaticLINE-1InsertionsattheSingle-Cell LevelfromPostmortemHumanBrain....... ....... ........ ....... ........ 147 MikiBundoandKazuyaIwamoto vii viii Contents 11 SolubilizationofMouseSpermChromatinforSequencingAnalyses UsingaChaperonProtein .......... ....... ....... ........ ....... ........ 161 YukoFukuda,KeishiShintomi,KosukeYamaguchi, YasuhiroFujiwara,andYukiOkada PART III MANIPULATION OF THE EPIGENOME: PLATFORMS FOR EPIGENOME EDITING 12 EfficientTargetedDNAMethylationwithdCas9-Coupled DNMT3A-DNMT3LMethyltransferase..... ....... ........ ....... ........ 177 PavelBashtrykov,NivethikaRajaram,andAlbertJeltsch 13 RegulationofGeneExpressionUsingdCas9-SunTagPlatforms...... ........ 189 SumiyoMorita,TakuroHorii,andIzuhoHatada 14 ConcatenatedCoiled-CoilTagforHighlyEfficient,SmallMolecule-Inducible UpregulationofEndogenousMammalianGenes .... ........ .... ... ...... .. 197 TinaLebarandRomanJerala 15 Design,Construction,andValidationofTargetedGeneActivation withTREESysteminHumanCells......... ....... ........ ....... ........ 211 AtsushiKunii,TakashiYamamoto,andTetsushiSakuma PART IV MANIPULATION OF THE EPIGENOME: INDUCIBLE SYSTEM FOR EPIGENOME EDITING 16 ASplitCRISPR–Cpf1PlatformforInducibleGeneActivation ....... ........ 229 TakahiroOtabe,YutaNihongaki,andMoritoshiSato PART V MANIPULATION OF THE EPIGENOME: EPIGENETICALLY MODIFIED ANIMALS 17 TargetedDNAMethylationinMouseEarlyEmbryos........ ....... ........ 243 TaigaYamazaki,YuHatano,NoritadaKobayashi, andKazuoYamagata 18 GenerationofEpigeneticDiseaseModelMicebyTargeted DemethylationoftheEpigenome.... ....... ....... .... .... ... .... ........ 255 TakuroHorii,SumiyoMorita,andIzuhoHatada 19 InVivoTissue-SpecificDNADemethylationinMouseLiver ThroughaHydrodynamicTailVeinInjection ....... ........ ....... ........ 269 KoshiHashimotoandNozomiHanzawa 20 TargetedManipulationofHistoneModificationinMedakaEmbryos ......... 279 HirotoS.Fukushima,HiroyukiTakeda, andRyoheiNakamura Index ...................................................................... 295 Contributors PAVELBASHTRYKOV • InstituteofBiochemistryandTechnicalBiochemistry,Departmentof Biochemistry,UniversityofStuttgart,Stuttgart,Germany MIKIBUNDO • DepartmentofMolecularBrainScience,GraduateSchoolofMedicalSciences, KumamotoUniversity,Kumamoto,Japan YASUHIROFUJIWARA • InstituteforQuantitativeBiosciences,TheUniversityofTokyo,Tokyo, Japan YUKOFUKUDA • InstituteforQuantitativeBiosciences,TheUniversityofTokyo,Tokyo,Japan HIROTOS.FUKUSHIMA • DepartmentofBiologicalSciences,GraduateSchoolofScience,The UniversityofTokyo,Tokyo,Japan NOZOMIHANZAWA • DepartmentofDiabetesandEndocrinology,NationalDisasterMedical Center,Tachikawa,Tokyo,Japan SATOSHIHARA • DivisionofMolecularGeneticsandEpigenetics,Departmentof BiomolecularSciences,FacultyofMedicine,SagaUniversity,Saga,Japan KEITAHASEAGAWA • DepartmentofObstetricsandGynecology,KeioUniversitySchoolof Medicine,Shinjuku,Tokyo,Japan KOSHI HASHIMOTO • DepartmentofDiabetes,EndocrinologyandHematology,Dokkyo MedicalUniversitySaitamaMedicalCenter,Koshigaya,Saitama,Japan KENICHIROHATA • DepartmentofMaternal-FetalBiology,ResearchInstitute,National Center forChildHealthandDevelopment,Setagaya,Tokyo,Japan IZUHO HATADA • LaboratoryofGenomeScience,BiosignalGenomeResourceCenter, InstituteforMolecularandCellularRegulation,GunmaUniversity,Maebashi,Gunma, Japan YUHATANO • FacultyofBiology-OrientedScienceandTechnology,KindaiUniversity, Kinokawa,Wakayama,Japan RYOYAHAYASHI • LaboratoryforEpigenomeInheritance,RIKENCenter forIntegrative MedicalSciences,Yokohama,Kanagawa,Japan;TokyoMetropolitanUniversity,Hachioji, Tokyo,Japan KENHIGASHIMOTO • DivisionofMolecularGeneticsandEpigenetics,Departmentof BiomolecularSciences,FacultyofMedicine,SagaUniversity,Saga,Japan SHINJIROHINO • DepartmentofMedicalCellBiology,InstituteofMolecularEmbryologyand Genetics,KumamotoUniversity,Kumamoto,Japan TAKUROHORII • LaboratoryofGenomeScience,BiosignalGenomeResourceCenter,Institute forMolecularandCellularRegulation,GunmaUniversity,Maebashi,Gunma,Japan TAIYOIKEDA • DepartmentofAppliedBiologicalScience,TokyoUniversityofScience,Chiba, Japan AZUSAINOUE • LaboratoryforEpigenomeInheritance,RIKENCenter forIntegrative MedicalSciences,Yokohama,Kanagawa,Japan;TokyoMetropolitanUniversity,Hachioji, Tokyo,Japan TAKASHIITO • DepartmentofBiochemistry,KyushuUniversityGraduateSchoolofMedical Sciences,Fukuoka,Japan KAZUYAIWAMOTO • DepartmentofMolecularBrainScience,GraduateSchoolofMedical Sciences,KumamotoUniversity,Kumamoto,Japan ix x Contributors ALBERTJELTSCH • InstituteofBiochemistryandTechnicalBiochemistry,Departmentof Biochemistry,UniversityofStuttgart,Stuttgart,Germany ROMANJERALA • DepartmentofSyntheticBiologyandImmunology,NationalInstituteof Chemistry,Ljubljana,Slovenia NORITADAKOBAYASHI • DivisionofBiomedicalResearch,KitasatoUniversityMedical Center,KitasatoUniversity,Kitamoto,Saitama,Japan ATSUSHIKUNII • DivisionofIntegratedSciencesforLife,GraduateSchoolofIntegrated SciencesforLife,HiroshimaUniversity,Hiroshima,Japan TINALEBAR • DepartmentofSyntheticBiologyandImmunology,NationalInstituteof Chemistry,Ljubljana,Slovenia SOMAEZAWA • DepartmentofAppliedBiologicalScience,TokyoUniversityofScience,Chiba, Japan FUMIHITO MIURA • DepartmentofBiochemistry,KyushuUniversityGraduateSchoolof MedicalSciences,Fukuoka,Japan MIKIMIURA • DepartmentofBiochemistry,KyushuUniversityGraduateSchoolofMedical Sciences,Fukuoka,Japan YUSUKEMIYANARI • NanoLSI,CancerResearchInstitute,KanazawaUniversity, Kanazawa,Japan SUMIYOMORITA • LaboratoryofGenomeScience,BiosignalGenomeResourceCenter, InstituteforMolecularandCellularRegulation,GunmaUniversity,Maebashi,Gunma, Japan KAZUHIKONAKABAYASHI • DepartmentofMaternal-FetalBiology,ResearchInstitute, NationalCenter forChildHealthandDevelopment,Setagaya,Tokyo,Japan RYOHEINAKAMURA • DepartmentofBiologicalSciences,GraduateSchoolofScience,The UniversityofTokyo,Tokyo,Japan MITSUYOSHINAKAO • DepartmentofMedicalCellBiology,InstituteofMolecularEmbryology andGenetics,KumamotoUniversity,Kumamoto,Japan SATOSHIH.NAMEKAWA • DepartmentofMicrobiologyandMolecularGenetics,Universityof CaliforniaDavis,Davis,CA,USA YUTANIHONGAKI • GraduateSchoolofArtsandSciences,TheUniversityofTokyo,Tokyo, Japan;DepartmentofCellBiology,JohnsHopkinsUniversitySchoolofMedicine, Baltimore,MD,USA HIROSHIOCHIAI • GraduateSchoolofIntegratedSciencesforLife,HiroshimaUniversity, Higashi-Hiroshima,Japan HIROAKIOHISHI • GraduateSchoolofIntegratedSciencesforLife,HiroshimaUniversity, Higashi-Hiroshima,Japan YUKIOKADA • InstituteforQuantitativeBiosciences,TheUniversityofTokyo,Tokyo,Japan TAKAHIROOTABE • GraduateSchoolofArtsandSciences,TheUniversityofTokyo,Tokyo, Japan;KanagawaInstituteofIndustrialScienceandTechnology,Kanagawa,Japan NIVETHIKARAJARAM • InstituteofBiochemistryandTechnicalBiochemistry,Departmentof Biochemistry,UniversityofStuttgart,Stuttgart,Germany AKIHIKO SAKASHITA • DepartmentofMolecularBiology,KeioUniversitySchoolofMedicine, Tokyo,Japan TETSUSHISAKUMA • DivisionofIntegratedSciencesforLife,GraduateSchoolofIntegrated SciencesforLife,HiroshimaUniversity,Hiroshima,Japan MORITOSHISATO • GraduateSchoolofArtsandSciences,TheUniversityofTokyo,Tokyo, Japan;KanagawaInstituteofIndustrialScienceandTechnology,Kanagawa,Japan TETSUYASATO • ResearchLaboratory,H.U.GroupResearchInstituteG.K.,Tokyo,Japan