Project name: Trypanosoma brucei brucei

Description: Genome-wide transcription studies are revealing increasing examples of ‘dispersed promoters’ that unlike ‘focused promoters’, lack well-conserved sequence motifs and tight regulation. Dispersed promoters are nevertheless marked by well-defined chromatin structures, suggesting that specific sequence elements must exist in these unregulated promoters.Here, we have analyzed regions of transcription initiation in the eukaryotic parasite Trypanosoma brucei, in which RNA polymerase II transcription initiation and occurs over broad regions without distinct promoter motifs and lacks regulation. Using a combination of site-specific and genome-wide assays, we identified GT-rich promoters that can drive unidirectional transcription and promote the targeted deposition of the histone variant H2A.Z in a genomic context dependent manner. In addition, upon mapping nucleosome occupancy at high resolution, we find nucleosome positioning to correlate with RNA pol II enrichment and gene expression, pointing to a role in RNA maturation. Nucleosome positioning may thus represent a previously unrecognized layer of gene regulation in trypanosomes.Our findings show that even highly dispersed, unregulated promoters contain specific DNA elements that are able to induce transcription and changes in chromatin structure.Overall design: 1) The essential RNA pol II subunit RPB9 was tagged with the TY epitope tag and ChIP was performed to generate a genome-wide map of RNA pol II stalling. 2) To map transcription initiation sites small total RNA < 200 nt was purified from T. brucei and enriched for primary transcripts containing a 5´-triphosphate by depletion of 5´-monophsphate-containing RNA using a Terminator 5´-Phosphate-Dependent Exonuclease (TEX). To identify undigested monophosphate-containing RNA contaminants, the sample was split and libraries were prepared from 5´-Polyphosphatase treated and untreated material. 3) Examination of nucleosome positioning in T. brucei bloodstream-form (BF) Wt and GT-rich promoter driven targeted H2A.Z depositioning in genetically modified T. brucei bloodstream-form cells expressing T7 RNA polymerase and Tet repressor (SM). We introduced a 210 bp GT-rich promoter sequence, its reverse complement and a 416 bp GT-rich promoter sequence in the center of a divergent transcription start region in T. brucei. The sequences are located at the genomic location Tb427_01_v4:284987-285196 and Tb427_01_v4:284987-285402, resp.

Data type: Other

Sample scope: Multiisolate

Relevance: Medical

Last updated: 2017-04-21