The African trypanosome Trypanosoma brucei evades the immune system of the mammalian host by periodically switching its surface coat which is made up of Variant Surface Glycoprotein (VSG). T. brucei shows monoallelic expression of one VSG out of a repertoire of ~1200 genes with the active VSG gene expressed from one of ~15 telomeric expression sites (ESs). The mechanism behind the monoallelic exclusion of ESs is unclear. NLP was identified as a novel and essential AT-hook protein binding transcriptionally silent simple sequence repeats in T. brucei. I depleted NLP using RNAi in various T. brucei reporter lines containing an eGFP in different transcriptionally silent areas of the genome and monitored derepression of eGFP using flow cytometry. After NLP knock-down, I observed 45-65 fold derepression of silent ESs, and up to 5 fold derepression of other transcriptionally silent areas. Using chromatin immunoprecipitation (ChIP) I found an enrichment of NLP in certain non-transcribed loci including the rDNA spacers. I also found that blocking NLP synthesis results in a rapid fall in levels of the active VSG transcript. Lastly, I discovered using tandem affinity purification (TAP) followed by mass spectrometry that NLP is a part of a novel TbISWI complex in T. brucei, which also includes two previously unidentified protein partners. The high mobility group B (HMGB) protein family constitutes a major abundant class of non-histone chromatin associated DNA-binding proteins which play a role in chromatin architecture in a wide range of eukaryotes. In T. brucei, the HMGB protein TDP1, which contains two HMG boxes and one DEK C terminal DNA-binding domain, was first identified as binding to VSG ES promoter oligomer sequences. I report that TDP1 is an essential nuclear protein enriched in the nucleolus and expression site body, and is involved in facilitating transcription. Blocking TDP1 synthesis using RNAi mediated knock-down results in approximately 40-90% reduction in transcription of RNA polymerase I transcribed genes. Using ChIP, I find that TDP1 is enriched in the rDNA and on the active VSG ES in bloodstream form T. brucei. Additionally, the relative proportion of TDP1 binding the procyclin promoter compared with the upstream spacer and downstream EP1 genes is greater in procyclic form compared with bloodstream form T. brucei. Lastly, I performed TAP experiments with TDP1 and found that TDP1 interacts with the core histones. These results indicate that TDP1 is an architectural chromatin protein important for transcription control in T. brucei.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:547456 |
| Date | January 2011 |
| Creators | Narayanan, Mani Shankar |
| Contributors | Rudenko, Gloria |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:62fdd635-7b92-493a-ac60-fbda5d0f05ac |
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