Identification and genomic distribution of select combinatorial post-translational modification patterns of Trypanosoma brucei histones

Maree, Johannes Petrus (2020-04)

Thesis (PhD)--Stellenbosch University, 2020.

Thesis

ENGLISH ABSTRACT:Trypanosoma brucei is a dixenous parasitic protozoan, and the causative agent of African Sleeping sickness, or Human African Trypanosomiasis. Trypanosomes diverged very early on from the main eukaryotic lineage and display some very unusual genomic features. Pol II transcribed genes are arranged in long, non-overlapping polycistronic transcription units (PTUs) containing up to a hundred functionally unrelated genes. These PTUs are separated by strand switching regions (SSRs) which can either be divergent (dSSR) or convergent (cSSR), depending on the direction of transcription. The entire housekeeping portion on the genome is constitutively transcribed with pol II initiating from poorly defined promoters. Putative transcription start sites are epigenetically demarcated by histone post-translational modifications (PTMs) and variant deposition, with transcription initiating bi-directionally from divergent SSRs as well as internal stop/start sites (ISS) at PTU head-to-tail regions. Over the past two decades it has become increasingly clear that in Trypanosoma the epigenome has a vital role in gene regulation and transcriptional control. T. brucei lacks pol II promoter and other regulatory sequences and employs epigenetic marks to demarcate and control genomic function. More than a decade has passed since the last survey of T. brucei histone PTMs, and improvements in mass spectrometry (MS) technology and software now allows us to map PTMs with great accuracy. Application of these technologies revealed numerous histone PTMs in T. brucei, with some forming intricate combinatorial PTM patterns. The C-terminal tail of histone H2A was found to be hyperacetylated. Half of all H2A peptides observed were hyperacetylated, with up to 5 acetylated lysines occurring simultaneously. On histone H3 the lysine 10 residue was found to be trimethylated, present as a binary PTM with hydroxylated H3P40. H3HyPro40 and H2AHyPro26 were the only hydroxylated residues detected, and further analysis supports the validity of this PTM in T. brucei. Investigation of the genome-wide distribution of hyperacetylated H2A PTMs via MNase-ChIP-seq revealed that hyperacetylation of the C-terminal tail in specific forms served distinct functions and displayed different genomic localization. It was further determined that one of these states of hyperacetylation, H2AK125ac, was enriched at dSSRs and ISSs. The H2AK125ac pattern was seen to co-localize with H2A.V and RPB9, indicators of putative pol II transcription start sites. Immunofluorescence microscopy (IF) microscopy showed that different H2A hyperacetylation patterns localized to distinct nuclear foci, further emphasizing distinct functionality. The data and findings of this study provides further insight into the epigenetic mechanisms employed by T. brucei and hopefully leads to development of targeted epigenetic therapies.

AFRIKAANSE OPSOMMING: Trypanosoma brucei is 'n twee-fasige parasitiese protosoa, en die oorsaak van Afrika-slaapsiekte, of Menslike Afrika Trypanosomiasis. Trypanosome het baie vroeg van die hoof eukariotiese stam vertak en vertoon 'n paar baie ongewone genomiese kenmerke. Pol II-transkribeerde gene word rangskik in lang, nie-oorvleuelende polisistroniese transkripsie-eenhede (PTU's) wat tot honderd funksioneel onverwante gene bevat. Hierdie PTU's word geskei deur ketting-wisselareas (SSRs) wat óf divergent (dSSR) of konvergent (cSSR) kan wees, afhangende van die rigting van transkripsie. Die hele huishoudelike geen kompliment van die genoom word voltyds getranskribeer deur pol II wat by swak gedefinieerde promotors begin. Transkripsie begin areas word epigeneties afgebaken deur histoon PTMs en variant neervoeging, en transkripsie begin in twee rigting by divergente SSRs sowel as by interne stop / begin plekke (ISS) by PTU kop-na-stert gebiede. Oor die afgelope twee dekades het dit al hoe duidelikker geword dat die trypanosomale epigenoom 'n deurslaggewende rol speel in transkripsionele beheer en geenregulering. T. brucei ontbreek pol II promotor en ander regulerende DNA elemente en gebruik epigenetiese merke om die genomiese funksie af te baken en te beheer. Meer as 'n dekade is verby sedert die laaste opname van T. brucei histoon PTMs. Verbetering in MS-tegnologie en sagteware laat ons nou toe om PTM's met groot akkuraatheid te karteer. Toepassing van hierdie tegnologieë het talle histoon PTM's in T. brucei getoon, met die vorming van ingewikkelde kombinatoriese PTM patrone. Die C-terminale stert van H2A is bevind om gehiper-asetileer te wees. Die helfte van alle H2Apeptiede waargeneem is ge-asetileerd, met tot 5 ge-asetileerde lisiene wat gelyktydig voorkom. Op histoon H3 was die lisien 10 aminosuur getrimetileer, teenwoordig as 'n binêre PTM, saam met gehidroksileerde H3P40. H3HyPro40 en H2AHyPro26 is die enigste gehidroksileerde residu's wat waargeneem was, en verdere analise ondersteun die geldigheid van hierdie PTM in T. brucei. Genoom-wye verspreiding van gehiper-asetileerde H2A PTMs via MNase-ChIP-seq het getoon dat die hiper-asetilering van die C-terminale stert, in spesifieke vorme, verskillende lokalisering en funksies vertoon. Daar is verder bepaal dat een van hierdie vorme, H2AK125ac, by dSSRs en ISS's verryk was. Die H2AK125ac-patroon was gesien om saam met H2A.V en RPB9 voor te kom, aanwysers van pol II transkripsie begin plekke. IF mikroskopie het getoon dat verskillende H2A-hiper-hiperasetilasiepatrone gelokaliseer word na afsonderlike kern fokus plekke, wat unieke funksionaliteit beklemtoon. Die data en bevindings van hierdie studie lewer verdere insig tot die epigenetiese meganismes wat deur T. brucei gebruik word, en lei hopelik tot die ontwikkeling van geteikende epigenetiese terapieë.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/107919
This item appears in the following collections: