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Methods in Molecular Biology 2607 Miguel R. Branco Alexandre de Mendoza Soler Editors Transposable Elements 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 biomedical protocol publishing. Each protocol is provided in readily-reproducible step-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. Transposable Elements Methods and Protocols Edited by Miguel R. Branco Faculty of Medicine and Dentistry, Blizard Institute, Queen Mary University of London, London, UK Alexandre de Mendoza Soler School of Biological and Behavioural Sciences, Queen Mary University of London, London, UK Editors MiguelR.Branco AlexandredeMendozaSoler FacultyofMedicineandDentistry SchoolofBiologicalandBehaviouralSciences BlizardInstitute,QueenMaryUniversityofLondon QueenMaryUniversityofLondon London,UK London,UK ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2882-9 ISBN978-1-0716-2883-6 (eBook) https://doi.org/10.1007/978-1-0716-2883-6 ©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 Genomes are not static. This is true not just in evolutionary timescales, but also within a single generation. From T-cell receptor gene recombination in mammals to large-scale genome rearrangement in ciliates and horizontal gene transfer events across bacteria, genomes show themselves to be surprisingly mutable. These and many other processes involve,inonewayoranother,afascinatinggroupofgenomicentitiestermedtransposable elements (TEs). TEs have the capacity to self-mobilize and re-insert themselves in other genomic regions, and as such constitute a rich source of genetic variation, although they also represent a potential harm to the host. Both the abundance and types of TEs vary immensely across species, with extreme cases having 80% of their genome being derived fromTEs.AndwhileinmostspeciesthevastmajorityofTEshavelosttheirabilitytomove, remnants of TE sequences can also have a dramatic impact on the host organism. Indeed, TEsareincrediblymultifacetedwhenitcomestotheireffects.TEscangiverisetonewgenes and gene regulatory elements, generate disease-causing mutations, drive recombination, andtriggerinflammatoryresponses,amongothers.Theycanactinvirtuallyanyphaseofthe lifecycle,fromembryonicdevelopmenttoaging. But how do we find TEs? How do we classify them? Track their mobility? How do we dissect the mechanisms that regulate their activity? How can we study their impact on the hostorganism? When Barbara McClintock discovered TEs in the early 1950s, the only experimental toolsheusedwascytogenetics.Throughcarefulobservation,ingeniousmaizebreeding,and remarkable deduction skills, she inferred the existence of mobile elements that controlled color phenotypes in maize kernels. Arguably, the paucity of techniques that could deepen herobservationsatthetimewaspartlytoblamefor theslowprogressthatfollowed.Itwas not until molecular biology picked up momentum that a form of TEs was discovered in bacteriainthelate1960s,theninDrosophilafollowingtheboomingeneticsresearch,and eventuallyinmammalsinthe1980s. It is not surprising that boosts in TE research coincide with technological break- throughs, and the advent of high-throughput sequencing (HTS) in the late 2000s was a prime example of that. HTS provided a far broader and deeper view of TEs than was ever possible before and drove a fast expansion of the field. HTS is now an essential and ubiquitous tool to study virtually every aspect of TE genomics and epigenomics, and it has continued to evolve, especially with the latest long-read technologies and single-cell approaches. As a result, the development of powerful, robust, and efficient bioinformatic toolshasalsobecomeapriority,astheamountandcomplexityofdatageneratedcontinueto grow at breakneck speed. Together with developments in proteomics, structural biology, and genetic editing, these technical advancements have made the last decade an incredibly fertileperiodfor noveldiscoveriesinTEbiology. The collection of methods and protocols presented in this book is a small selection of state-of-the-artapproachesforstudyingTEs,whichreflectingreatparttherecenttechno- logicaladvancementsinthefield.OurselectionreflectsthecurrentemphasisonHTS-based approachesandaccompanyingbioinformatictoolsbutalsocoversmethodsforstudyingTE v vi Preface proteincomplexes,theiractivity,andtheirfunctionalimpactonthehost.Wehaveattempted to order the chapters in this book according to the central dogma of molecular biology: fromDNA,toRNA,thenprotein,andsubsequentlyactivityandfunction. The first chapters revolve around the vast amounts of genomic data that are generated onaroutinebasis.Forinstance,genomesfornewspeciesaresequencedatanever-increasing speed, including those of species that are distantly related to well-curated model systems. Therefore, it is expected that the need to carefully annotate novel TE landscapes will only increaseinthefuture,fueledbylarge-scalesequencingconsortia(e.g.,DarwinTreeofLife, Vertebrate 10K) or individual lab efforts. In this sense, long-read sequencing is rapidly changing our views on repeat annotation, as new genomes tend to capture much more of the repeat landscape than short-read-based assemblies ever could. In Chapter 1, Fernando Rodriguez and Irina R. Arkhipova explain their pipeline and best guidance on how to annotate the TEs in a new genome. Even though new genomes are made more routinely available,gettingphylogeneticallydensecoverageofTElandscapesinalllineagesisstillout ofreachformanylaboratories.Therefore,methodsbasedonrawreadssuchasdnaPipeTE, which allow the quantification and classification of TE landscapes even without a high- quality reference assembly, are also going to be important in the future, when TEs are studiedatamicro-evolutionaryscale.InChapter2,Cle´mentGoubertpresentsthispipeline andexplainsthebestpracticesonhowtorunit.Overallthesetoolsrepresenttherudiments inwhichTEexplorationandclassificationneedstostartinanynewspecies. Unlike mostothereukaryoticgenomic sequences,TEsareableto jumpacrossspecies, evenbetweendistantlineages.Thisremarkableevolutionarytrendislikelygoingtobemore prevalent than initially thought, so in Chapter 3 James D. Galbraith, Atma M. Ivancevic, Zhipeng Qu, and David L. Adelson describe a pipeline to rigorously annotate these Hori- zontalTransferofTransposonsevents. GeneticvariationduetoTEsisalsogoingtoheavilyimpactmicro-evolutionarystudies, and most importantly studies of genetic variation within populations. Most notoriously, human-based approaches are going to have a large impact on biomedical genomics. In Chapter4,XunChen,GuillaumeBourque,andCle´mentGoubertdescribeapipelinetoget a comprehensive annotation of polymorphic TE insertions in human sequencing data. UnlikesimplemutationslikeSNPs,lookingatTEsequencevariationwillnecessarilychange the way we think about genomes. Long stretches of sequence will be present in some individuals, while absent in others. Therefore, the concept of the “pangenome,” far from thesimplisticviewofauniquestablehaploidreferencegenome,willlikelybecomepredom- inant. Under this light, in Chapter 5, Cristian Groza, Guillaume Bourque, and Cle´ment GoubertshowamethodonhowtoanalyzethissortofdataforhumanTEpolymorphisms. TE variation and its impact on evolution can be used as a case example for science communication. In Chapter 6, Miriam Merenciano, Marta Coronado-Zamora, and Josefa Gonza´lezpresenttheirpipelinetoPCR-validateTEpolymorphismsinfruitflypopulations/ species. This approach has allowed them to engage high-school students into a “Citizen Science” project, in which students can participate in actual research about TE variation. Thisisagreatexampleofhowthisfieldisopeningtoawider,morediversepublic. BeyondcharacterizingthegenomicpresenceandvariationofTEs,acentralaspectthat hasfueledinterestinthesegenomicelementsistheirabilitytoregulatetranscription,arole originally suggested by Barbara McClintock herself. Yet studying transcription and regulation of TEs entails many difficulties. The repetitive nature of TEs implies that short-read sequencing technologies struggle to capture these events in an unequivocal manner. A perfect example are human LINE-1 elements; with half a million of copies in the genome, Preface vii manyofthemarehighlysimilar toeachother,makingtheassignmentofshortreadstoany givencopymuchharder thantomostproteincodinggenes.InChapter7, WilsonMcKer- rowdescribestheL1EMpipeline,whichdiscriminatesbetweenactiveLINE-1transcription, actually driven by the LINE-1 promoter, from passive cotranscription, which results from thesecopiesbeingfoundnearbyothergenes. Similar mappability challenges are faced when trying to dissect the epigenetic and transcription factor landscapes of evolutionarily young TEs. Such information is key to understanding how tissue-specific TE expression is achieved, and how it becomes deregu- latedindiseasestates.OneapproachtostudyDNAmethylationathumanLINE-1elements is presented by Claude Philippe and Gael Cristofari in Chapter 8, which involves targeted bisulfitesequencingofthe5′endsofLINE-1susingshortreads.Thisfocusestheanalysison the transcriptional promoter of the element and captures information from both reference andnon-referenceinsertions.ToprofileDNAmethylationdeeperintoTEs,onerecentand powerful solution is the use of Oxford Nanopore Technologies (ONT) platforms, which deliverlongreadstogetherwithDNAmethylationinformation.InChapter9,NathanSmits andGeoffreyFaulknerdescribetheirexperimentalandcomputationalapproachtoanalyzing TEmethylationfromONTdata.ArpitaSarkar,SophieLanciano,andGaelCristofaripresent a more targeted approach in Chapter 10, where Cas9 is used to specifically enrich LINE-1 elements and detect methylation by ONT. Beyond DNA methylation, other aspects of TE regulation(histonemarks,transcriptionfactorbinding)arehardertoassessusinglong-read technologies because routine methods require fragmentation of the chromatin. To over- come this, Darren Taylor and Miguel R. Branco describe in Chapter 11 a strategy to infer proteinenrichmentatTEsbyleveraging3DspatialinformationfromHiChIPdata. TE activity is also critically regulated at the posttranslational level. Much like viruses, TEs are in a constant arms race whereby host proteins can be hijacked to catalyze retrotransposition, but can also restrict it. As new roles for TEs are uncovered, some of these protein interactions may actually directly serve the interests of the host. Our current understanding of TE interactomes is far more limited than that of TE genomics, as it requirescarefullyoptimizedbiochemicalapproaches.LucianoH.DiStefano,JohnLaCava, and colleagues describe in Chapter 12 a comprehensive set of techniques to characterize both the protein and RNA components of human LINE-1 macromolecules. How these interactionschangebasedonsequencedifferencesbetweenLINE-1copieswilldramatically influencetheircapacityforretrotransposition.Indeed,onlyaminorityofLINE-1elements are highly active in the human genome. Assays for measuring TE activity are therefore an important tool, and these have long relied on specially designed reporter constructs. In Chapter 13, Marta Garcia-Canadas, Jose Garcia Perez, and colleagues describe a suite of assaystomeasureLINE-1andSINEretrotranspositionthathavebeenoptimizedformouse andhumanembryonicstemcells,whereendogenousTEexpressionishigh. Retrotransposition can have severely deleterious consequences for the host, as evi- dencedbyahostofhumandiseasesthatdisplaycasesdrivenbyTEinsertions.Thisincludes somatic retrotransposition, which can drive cancer and is far more difficult to detect than germline insertions. Katarzyna Siudeja presents in Chapter 14 short- and long-read approaches to detecting somatic TE insertions in Drosophila, where such events have been linkedtoneoplasiaandaging. ButthewaysbywhichTEsimpactthehostorganismcanbemanifold.Tounequivocally link their presence, expression, and/or activity to specific phenotypes, tools for genetically orepigeneticallymanipulatingthemarerequired.TheadventofCRISPRhasundoubtedly facilitated and accelerated such studies, although other approaches (e.g., TALEs) still have viii Preface theiradvantages.InChapter15,VivienM.Weber,AurelienJ.Doucet,andGaelCristofari describe an elegant approach to insert TEs into specific genomic regions using CRISPR, allowingforaveryspecificevaluationoftheconsequencesofretrotranspositioninisogenic lines. Epigenetic editing in TEs has particular advantages, as multiple copies of the same familycanbeeasilytargetedinthesameexperiment.JoannaM.JachowiczpresentsaTALE- based approach for epigenetically activating or silencing repetitive elements in Chapter 16. For a more targeted evaluation, genetic editing can be used to excise single TE copies and test their effect on cells/organisms. David M. Simpson, Conor R. Kelly, and Edward B.ChuongsharetheirCRISPR-basedapproachtogeneticallyeditTEsinChapter17. Wehopethissetofprotocolsisequallyusefulforboththeexpertsinthefield,aswellas for those starting to explore the wonderful world of TEs. Maybe you are looking for guidance on best practices or ways to improve your current protocols. Maybe you want to venture into a new facet of TE biology that requires implementing novel tools. Or maybe you didnotcare much about TEsuntil they “jumped”at youfrom your data, and you are about to embark on a novel research journey. Whatever the case, we hope this book will facilitateyourendeavorstofurtherenrichourknowledgeofTEs. London,UK MiguelR.Branco Alexandre deMendozaSoler Contents Preface ..................................................................... v Contributors................................................................. xi 1 AnOverviewofBestPracticesforTransposableElementIdentification, Classification,andAnnotationinEukaryoticGenomes ....... ....... ........ 1 FernandoRodriguezandIrinaR.Arkhipova 2 Assembly-FreeDetectionandQuantificationofTransposableElements withdnaPipeTE .... ....... ........ ....... ....... ........ ....... ........ 25 Cle´mentGoubert 3 DetectingHorizontalTransferofTransposons ...... ........ ....... ........ 45 JamesD.Galbraith,AtmaM.Ivancevic,ZhipengQu, andDavidL.Adelson 4 GenotypingofTransposableElementInsertionsSegregatinginHuman PopulationsUsingShort-ReadRealignments..... ... ...... .. ...... .... ..... 63 XunChen,GuillaumeBourque,andCle´mentGoubert 5 APangenomeApproachtoDetectandGenotypeTEInsertion Polymorphisms..... ....... ........ ....... ....... ........ ....... ........ 85 CristianGroza,GuillaumeBourque,andCle´mentGoubert 6 ExperimentalValidationofTransposableElementInsertions UsingthePolymeraseChainReaction(PCR)........ ........ ....... ........ 95 MiriamMerenciano,MartaCoronado-Zamora,andJosefaGonza´lez 7 QuantificationofLINE-1RNAExpressionfromBulkRNA-seq UsingL1EM.. ..... ...... ... ...... ....... ....... ........ ....... ........ 115 WilsonMcKerrow 8 Genome-WideYoungL1MethylationProfilingbybs-ATLAS-seq .... ........ 127 ClaudePhilippeandGaelCristofari 9 NanoporeSequencingtoIdentifyTransposableElementInsertions andTheirEpigeneticModifications ......... ....... ........ ....... ........ 151 NathanSmitsandGeoffreyJ.Faulkner 10 TargetedNanoporeResequencingandMethylationAnalysisofLINE-1 Retrotransposons ......... ....... .. ....... ....... ........ ....... ........ 173 ArpitaSarkar,SophieLanciano,andGaelCristofari 11 InferringProtein-DNABindingProfilesatInterspersedRepeats UsingHiChIPandPAtChER ....... ....... ....... ........ ....... ........ 199 DarrenTaylorandMiguelR.Branco 12 Affinity-BasedInteractomeAnalysisofEndogenousLINE-1 Macromolecules .... ....... ........ ....... ...... ... ...... ....... ........ 215 LucianoH.DiStefano,LeilaJ.Saba,MehrnooshOghbaie, HuaJiang,WilsonMcKerrow,MariaBenitez-Guijarro, MartinS.Taylor,andJohnLaCava ix