GT ‐rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes

Abstract

Genome‐wide transcription studies are revealing an increasing number 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 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 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.

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

“…Unidirectional transcription resulting in productive mRNAs is typically ensured since antisense transcription is susceptible to early termination linked to rapid degradation. Mapping TSSs using tri-phosphate RNA-seq has previously suggested bi-directional activity, with strong strand bias, of Pol II initiation sites in T. brucei [68,69]. The RNA-seq data presented here is consistent with bi-directional activity of TSS, early termination and stimulation of divergent antisense transcription in the PNUTS knockdown leading to the activation of genes within the dSSR (Figs 4C and 5A and S10 Fig).…”

How to create coats for all seasons: elucidating antigenic variation in African trypanosomes

“…The position of individual nucleosomes across all of the core chromosomes has now been determined in both bloodstream-and procyclic-form T. brucei [23,24]. This has allowed us to determine the chromatin state at different types of transcription units.…”

“…Pol II promoters in T. brucei are diffuse and lack distinctive sequence elements [25]. Instead, Pol II promoters appear to be predominantly functionally defined by discrete chromatin structure around the strand switch regions where Pol II transcription initiates [24]. In addition, chromatin structure appears to play a role in the repression of the silent VSG genes, and positioned nucleosomes are found flanking the silent VSG gene arrays [23].…”

Fluorescence‐based assay for accurate measurement of transcriptional activity in trypanosomatid parasites

“…The mechanisms that control transcription in these parasites have been broadly studied by ChIP-seq (chromatin immunoprecipitation followed by next-generation sequencing) to identify potential transcription factors that interact with strandswitch regions of chromatin (13,19). Other methodologies can be applied, as deep sequencing RNA (RNAseq) and in vitro transcription with radiolabeled nucleotide (20).…”