A systematic analysis of Trypanosoma brucei chromatin factors identifies novel protein interaction networks associated with sites of transcription initiation and termination

Abstract

Nucleosomes composed of histones are the fundamental units around which DNA is wrapped to form chromatin. Transcriptionally active euchromatin or repressive heterochromatin is regulated in part by the addition or removal of histone post-translational modifications (PTMs) by 'writer' and 'eraser' enzymes, respectively. Nucleosomal PTMs are recognised by a variety of 'reader' proteins which alter gene expression accordingly. The histone tails of the evolutionarily divergent eukaryotic parasite Trypanosoma brucei have atypical sequences and PTMs distinct from those often considered universally conserved. Here we identify 68 predicted readers, writers and erasers of histone acetylation and methylation encoded in the T. brucei genome and, by epitope tagging, systemically localize 63 of them in the parasite's bloodstream form. ChIP-seq demonstrated that fifteen candidate proteins associate with regions of RNAPII transcription initiation. Eight other proteins exhibit a distinct distribution with specific peaks at a subset of RNAPII transcription termination regions marked by RNAPIII- transcribed tRNA and snRNA genes. Proteomic analyses identified distinct protein interaction networks comprising known chromatin regulators and novel trypanosome-specific components. Notably, several SET-domain and Bromo-domain protein networks suggest parallels to RNAPII promoter-associated complexes in conventional eukaryotes. Further, we identify likely components of TbSWR1 and TbNuA4 complexes whose enrichment coincides with the SWR1-C exchange substrate H2A.Z at RNAPII transcriptional start regions. The systematic approach employed provides detail of the composition and organization of the chromatin regulatory machinery in Trypanosoma brucei and establishes a route to explore divergence from eukaryotic norms in an evolutionarily ancient but experimentally accessible eukaryote.

Genetic Interaction Between Site-Specific Epigenetic Marks and Roles of H4v in Transcription Termination in Trypanosoma brucei

“…The data suggest that H4v is the major signal to stop transcription termination at TTSs, and TTS marks may involve in RNA processing events occurring after transcription. TFIIS2-2 (transcription elongation factor) is enriched at TTSs and interacts with PAF1 complex in T. brucei (Staneva et al, 2021). PAF1 contributes to chromatin remodeling, transcription elongation, and polyadenylation in yeast and mouse cells (Penheiter et al, 2005;Yang et al, 2016).…”

“…TFIIS2-2 (transcription elongation factor) is enriched at TTSs and interacts with PAF1 complex in T. brucei ( Staneva et al, 2021 ). PAF1 contributes to chromatin remodeling, transcription elongation, and polyadenylation in yeast and mouse cells ( Penheiter et al, 2005 ; Yang et al, 2016 ).…”

“…If a cell deposited a functional pair of H3-H4 PTMs capable of stable nucleosome formation at TTSs, these cells could emerge and survive ( Figure 9A ). Several H3v and H4v PTMs are now identified ( Kraus et al, 2020 ), although their functions are not yet known, as well as chromatin proteins associated at TTSs, which include readers and writers of histone PTMs (BDF7, PHD2, PHD4, and DOT1A; Staneva et al, 2021 ). H4 (or H3) with a specific PTM may associate with H3v (or H4v) for depositing to TTSs.…”

Site-specific epigenetic marks in Trypanosoma brucei transcription termination, antigenic variation, and proliferation

“…8A). Several of H3v and H4v PTM are now identified 8 , although their functions are not yet known, as well as chromatin proteins associated at TTSs, which include readers and writers of histone PTMs (BDF7, PHD2, PHD4, DOT1A) 51 . H4 (or H3) with a specific PTM may associate with H3v (or H4v) for depositing to TTSs.…”

“…Polyadenylation machinery may interact with TTS chromatin to distinguish coding region from non-coding within precursor RNAs, preventing polyadenylation of antisense transcripts. TFIIS2-2 (transcription elongation factor) is enriched at TTSs and interacts with PAF1 complex in T. brucei 51 . PAF1 contributes to chromatin remodeling, transcription elongation and polyadenylation in yeast and mouse cells 57,58 .…”