Saturday, April 20, 2024
4/20/2024
DESCRIPTION (provided by applicant): This proposal aims to study the reprogramming of the Trypanosoma brucei epigenome that accompanies movement of this trypanosome from the insect to the human host. T. brucei causes African sleeping sickness or trypanosomiasis, and occurs in extensive parts of sub-Saharan Africa. Hundreds of thousands of individuals suffer from trypanosomiasis. The epigenome of an organism represents heritable information that is encoded outside of the nucleotide sequence of its genome, and includes nucleosome positions, histone isotypes, reversible histone modifications, and DNA modifications. The epigenome is an essential regulatory interface to the control of DNA function, and epigenetic misregulation is responsible for many serious human diseased states. The epigenome thus represents a potent therapeutic target, and several compounds that interfere with proteins that recognize or that confer specific histone modifications have either received FDA approval or are undergoing clinical trials. These compounds hold tremendous promise for the treatment of diseases such as cutaneous T-cell lymphoma or multiple linked leukemia, the prevalent leukemia in human infants. The genome-wide mapping of nucleosome positions and the elucidation of the modification types and positions of the histone H3 tail in T. brucei are planned in this proposal. Nucleosome positions will be mapped by paired end sequencing of 147 bp nucleosome fragments isolated from procyclic fly form (PF) and from human blood form (BF) cultures. The sequenced fragments will be aligned to the T. brucei genome, and the nucleosome positions mapped relative to the polycistronic transcription units and silent genomic loci. It was suggested that in the absence of a defined transcription start sequence in T. brucei, the correct positioning of an RNA polymerase at the start of a polycistronic unit was conferred by local chromatin structure and histone modifications. It is therefore expected that nucleosome positions and histone modifications will play a major role in understanding epigenetic control of transcription. Intriguingly, the T. brucei H3 tail lacks K9, generally associated with transcriptional repression n the tri-methylated state. In this study the type and position of modifications of the H3 tail, as wll as the genome-wide distribution of select modifications, will be determined by LC-MS/MS and by ChIP-seq. Particular attention will be paid to modifications associated with silent genomic loci and with silent Expression Sites (ES). A single Variable Surface Glycoprotein (VSG) is mono-allelically expressed from one of 15 ESs. Switching of the ES and the expressed VSG allows continual evasion of the human immune system. It is likely that insight into the epigenomic reprogramming of T. brucei associated with human infection will identify novel epigenetic targets for future drug development.
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