! ! ! ! ! ! Epigenetic!control!of!transcription!in!the!African! Trypanosome!Trypanosoma*brucei.! ! ! ! ! ! by! Louise!Elizabeth!Kerry! ! ! ! ! ! Thesis!submitted!in!partial!fulfillment!of!the!requirements!for!the!degree!of! Doctor!of!Philosophy!(Ph.!D.)!in!Molecular!and!Cellular!Biosciences.! Department!of!Life!Sciences,!Division!of!Cell!and!Molecular!Biology,! Imperial!College!London! ! ! ! ! ! ! Declaration!! ! Declaration!of!originality! I! declare! that! all! of! the! work! presented! in! this! thesis! is! my! own,! and! that! all! else,! information,! data,! results,! figures! and! ideas! from! another! source! or! from! collaborations! have!been!appropriately!referenced!or!acknowledged.! ! ! ! Louise!Elizabeth!Kerry!! November!2017! ! ! Copyright!declaration! The! copyright! of! this! thesis! rests! with! the! author! and! is! made! available! under! a! Creative! Commons!Attribution!NonOCommercial!No!Derivatives!license.!Researches!are!free!to!copy,! distribute!or!transmit!the!thesis!on!the!condition!that!they!attribute!it,!that!they!do!not!use! it!for!commercial!purposes!and!that!they!do!not!alter,!transform!or!build!upon!it.!!For!any! reuse! or! redistribution,! researchers! must! make! clear! to! others! the! license! terms! of! this! work.! ! ! ! ! ! ! 1! Abstract! Abstract! ! Trypanosoma! brucei! relies! on! an! essential! Variant! Surface! Glycoprotein! (VSG)! coat! for! survival!in!the!mammalian!bloodstream.!A!single!VSG!gene!is!transcribed!by!RNA!polymerase!I!(Pol!I)! in! a! strictly! monoallelic! fashion! from! one! of! multiple! telomeric! VSG! expression! sites! (ES).! The! epigenetic! mechanisms! that! maintain! monoallelic! expression! of! a! single! VSG! remain! unclear.! The! goal!of!this!research!was!to!advance!our!understanding!of!the!epigenetic!factors!that!contribute!to! regulation!of!Pol!I!transcription!in!T.!brucei!and!to!evaluate!epigenetic!drug!targets!as!novel!antiO parasitic!agents.!!! The! distribution! of! the! histone! H3K4me3! modification! at! Pol! I! transcribed! loci! was! determined!by!ChIPOqPCR.!The!H3K4me3!modification!was!not!enriched!within!the!VSG!ESs!or!the! Pol!I!rDNA!transcription!unit,!suggesting!that!this!modification!does!not!correlate!with!active!Pol!I! promoters,!as!is!the!case!in!higher!eukaryotes.!!ChIPOqPCR!was!also!used!to!ascertain!the!epigenetic! state!of!the!preOactive!ESs!present!in!T.!brucei!DDR!cells!that!express!two!VSGs!from!two!active!ESs.! Characterisation!of!the!DDR!parasites!showed!that!both!ESs!appear!to!be!in!a!preOactive!state,!and! that!the!singularity!of!expression!site!body!contributes!to!monoallelic!expression!of!the!VSG!genes.! As! VSG! ESs! are! appealing! targets! for! antiOtrypanosomal! chemotherapies,! I! evaluated! the! potential!of!histone!methyltransferase!inhibitors!(HKMTI)!and!Pol!I!inhibitors!as!novel!trypanocidal! agents.! ! The! HKMTI! and! Pol! I! inhibitors! cause! parasite! cell! death! in! a! timeO! and! doseOdependant! manner!with!favourable!selectivity!compared!with!mammalian!cells.!The!Pol!I!inhibitors!are!toxic!to! trypanosomes!as!a!result!of!specifically!blocking!T.!brucei!Pol!I!transcription.!Whereas,!cell!death!in! the!HKMTI!treated!parasites!appears!to!be!due!to!offOtarget!effects.!Ultimately,!this!research!could! contribute!to!the!development!of!novel!treatments!for!Human!African!Trypanosomiasis.!!!!!! 2! Contents!! ! Contents! ! Table!of!Contents! Declaration!of!originality!............................................................................................................!1! Copyright!declaration!.................................................................................................................!1! Abstract!.....................................................................................................................................!2! Table!of!Contents!.......................................................................................................................!3! List!of!Tables!..............................................................................................................................!5! List!of!Figures!.............................................................................................................................!5! Acknowledgements!....................................................................................................................!8! Abbreviations!.............................................................................................................................!9! Chapter!One!....................................................................................................................!11! Introduction!..............................................................................................................................!11! 1.1!O!Introduction!to!Human!African!Trypanosomiasis!................................................................!11! 1.2!O!Current!treatments!for!Human!African!Trypanosomiasis!...................................................!13! 1.3!–!Biology!of!the!African!Trypanosomes!Trypanosoma!brucei!...............................................!16! 1.4!O!Trypanosoma!brucei!life!cycle!.............................................................................................!17! 1.5!O!Antigenic!Variation!in!Trypanosome!brucei!.........................................................................!18! 1.6!O!Genome!organisation!and!polycistronic!transcription!in!Trypanosoma!brucei!...................!22! 1.7O!Introduction!to!chromatin!and!epigenetics!..........................................................................!24! 1.8O!Chromatin!structure!and!epigenetic!marks!of!RNA!Polymerase!II!transcription!units!in!T.! brucei!...........................................................................................................................................!25! 1.9!O!Monoallelic!expression!of!VSG!and!the!role!of!RNA!Polymerase!I!......................................!28! 1.10!O!Epigenetics!and!transcriptional!control!of!the!VSG!ES!in!bloodstream!form!T.!brucei!......!31! 1.11!O!The!role!of!the!Expression!Site!Body!in!monoallelic!expression!........................................!33! 1.12!O!Repurposing!of!drugs!and!target!classes!for!treatment!of!neglected!tropical!diseases!....!35! 1.13!O!Thesis!aims!........................................................................................................................!37! Chapter!Two!....................................................................................................................!38! Material!and!Methods!..............................................................................................................!38! 2.1!O!Trypanosome!culturing!and!strains!.....................................................................................!38! 2.2!O!Cloning!and!constructs!........................................................................................................!45! 2.3!O!Transfection!of!BSF!T.!brucei!...............................................................................................!47! 2.4!O!Chemical!inhibitors!..............................................................................................................!47! 2.5!O!Reverse!Transcription!Quantitative!Polymerase!Chain!Reaction!(RTOqPCR)!.......................!47! 2.6!O!Chromatin!Immunoprecipitation!(ChIP)!and!quantitative!PCR!(qPCR)!................................!48! 2.7!O!Trypanosome!proliferation!assay!........................................................................................!49! 2.8!O!Wash!out!assay!....................................................................................................................!49! 2.9!O!ESB!reassembly!assay!..........................................................................................................!49! 2.10!O!In!vitro!cytotoxicity!assay!..................................................................................................!50! 2.11!O!Flow!cytometry!of!endogenous!fluorescence!and!antibody!stained!T.!brucei!..................!50! 2.12!O!Dilution!assay!.....................................................................................................................!52! 2.13!O!Immunofluorescence!microscopy!.....................................................................................!52! 3! Contents!! ! 2.14!O!Immunoblot!analysis!.........................................................................................................!53! 2.15!O!Peptide!Competition!assay!................................................................................................!54! Chapter!Three!..................................................................................................................!55! Distribution!of!the!H3K4me3!epigenetic!mark!in!bloodstream!form!Trypanosoma*brucei! polymerase!I!transcribed!loci.!...................................................................................................!55! 3.1!O!Introduction!.........................................................................................................................!55! 3.2!O!Aims!and!experimental!outline!............................................................................................!58! 3.3!O!Validation!of!the!VSG!surface!coat!expressed!in!HNI!221+!and!HNI!VO2+!BSF!T.!brucei!cell! lines.!.............................................................................................................................................!59! 3.4!O!Evaluation!of!specificity!of!two!commercially!available!antiOH3K4me3!antibodies!............!61!