Discussion
Kinetoplastids such as
Trypanosoma brucei possess unusual mechanisms for regulating gene expression relative to other eukaryotes. Fungi (e.g. yeasts), flies and mammals all exhibit specialized chromatin on repetitive elements at centromeres (i.e. heterochromatin), adjacent to telomeres and at promoters. In general, analysis of writers, readers, and erasers of histone post-translational modifications in kinetoplastids is at a relatively early stage. Moreover, little is known about the nature of chromatin that is assembled on repetitive elements in
T. brucei or other kinetoplastids. We previously surveyed 65 putative chromatin-associated factors in
Trypanosoma brucei [1]. Our analyses revealed that the predicted histone methyltransferase SET27 and the Chromodomain protein CRD1 are tightly concentrated at RNAPII transcription start regions (TSRs). We have found that SET27 and CRD1, together with four previously uncharacterized constituents (CSD1, PHD6, PWWP1, PBP1) form what we refer to as the SET27 promoter-associated regulatory complex (SPARC), which is specifically enriched at TSRs [2]. SET27 loss leads to aberrant RNAPII recruitment to promoter sites, accumulation of polyadenylated transcripts upstream of normal transcription start sites, and conversion of some normally unidirectional promoters to bidirectional promoters. Our analyses uncover a novel chromatin-associated complex required to establish accurate promoter position and directionality. We are currently undertaking detailed analyses of SPARC components to determine their activities and, ultimately, the overall structure of this complex. To probe the composition of chromatin that is associated with repetitive elements at various locations in the
T. brucei genome we have expressed synthetic nuclear TALE-YFP proteins designed to bind terminal telomere (TTAGGG)n repeats (Tel-TALE) or other distinct types of repetitive sequences. Proteomic analyses of Tel-TALE protein affinity selected from bloodstream form cell extracts provides proof-of-principle in that many known telomere-associated proteins are identified as being significantly enriched. Thus, locus-specific proteomics provides a potentially useful tool for investigating specific chromatin contexts in trypanosomes. Ongoing analyses of synthetic TALE-YFP proteins designed to bind centromeric CIR147 repeats, VSG gene expression site 70 bp repeats, and small chromosome associated 177bp repeats will be discussed.
1. Staneva DP, Carloni R, Auchynnikava T, Tong P, Rappsilber J, Jeyaprakash AA, Matthews KR, Allshire RC. (2021) A systematic analysis of Trypanosoma brucei chromatin factors identifies novel protein interaction networks associated with sites of transcription initiation and termination. Genome Research 31:2138-2154. 2. Staneva DP, Bresson S, Auchynnikava T, Spanos C, Rappsilber J, Jeyaprakash AA, Tollervey D, Matthews KR, Allshire RC. (2022) The SPARC complex defines RNAPII promoters in Trypanosoma brucei. eLife 11:e83135.
