A systematic analysis of Trypanosoma brucei chromatin factors identifies novel protein interaction networks associated with sites of transcription initiation and termination
Preprint posted on 10 February 2021 https://doi.org/10.1101/2021.02.09.430399
Article now published in Genome Research at http://dx.doi.org/10.1101/gr.275368.121
A whole new world of chromatin regulation in Trypanosoma brucei. @dpstaneva, @robbiecarloni and others reveal new networks that control transcription in this early-branching eukaryote.
Selected by Tamara Sternlieb
Categories: cell biology, microbiology, molecular biology
Some context
Gene expression in eukaryotic organisms is regulated at several levels, the first of which is the chromatin state and its effect on transcription. Heterochromatin is compacted and repressive for transcription, while euchromatin is relaxed and transcriptionally active. These chromatin states are regulated by several modifications on DNA and on histones, the proteins around which DNA is wrapped. The dynamics of these modifications and their effect on transcription are well studied in eukaryotic model organisms, such as yeast, plants and animals. However, the Eukarya domain also includes early-branching organisms, such as protozoa parasites like Trypanosoma brucei, which have evolved for over 500 million years, diverging from the most studied organisms.
T. brucei is the causative agent of sleeping sickness in humans and nagana in cattle. Unusually for a eukaryote, most trypanosome genes are transcribed in polycistronic units, which are then resolved by trans-splicing. RNA Polymerase II begins transcription at divergent Transcription Start Regions (TSR) on the chromatin, and it typically terminates at convergent transcription regions called Transcription Termination Regions (TTR). Less frequently, transcription units are arranged head to tail. Transcription termination in such units often coincides with genes transcribed by RNA Polymerase I or RNA Polymerase III. All these regions show distinct histone variant and histone modification enrichment.
Although the general consensus is that trypanosome gene expression is mostly regulated post-transcriptionally via control of RNA stability and translation, trypanosome genomes encode for several chromatin regulator sequences, which haven’t been thoroughly studied and whose function is unknown.
Key Findings
The authors interrogated the TriTrypDB trypanosome genome database, searching for writers, readers and erasers of chromatin post-translational modifications (PTMs), specifically histone acetylation and methylation. They identified 68 proteins, of which some had been explored previously. They proceeded with a three-step analysis that revealed the intracellular localization, chromatin enrichment and protein interaction networks of the chromatin-associated factors.
They endogenously tagged the candidate proteins with YFP and first evaluated their cellular localization. Then they performed ChIP-seq to evaluate their chromatin association and enrichment in convergent and divergent TSRs and TTRs. They used genomic annotations and H2A.Z histone ChIP enrichement to identify these regions.
15 of the candidate proteins were specifically enriched at TSRs, of which 10 had not been shown to act at this region before. These proteins revealed two enrichment patterns, which the authors named Class I and Class II TSR-associated factors, characterized by sharp peaks and a broader pattern around TSRs, respectively. Class I and Class II factors may play different roles in RNAPII transcription, as initiators and facilitators of RNA Polymerase processivity, respectively. Using Immunoprecipitation and mass spectrometry, they identified interacting proteins that may participate in the RNAPII regulation networks, including factors characterized in other eukaryotes and new players.
The ChIP-seq analysis also revealed 8 proteins which displayed specific enrichment over a subset of RNAPII TTRs. They were also enriched at small nuclear RNA and tRNA genes, which are transcribed by RNAPIII. The authors’ hypothesis is that these proteins block the passage of RNAPII over genes transcribed by RNAPIII and could facilitate transcription of snRNA and tRNA genes.
While TSR-associated factors showed several reciprocal interactions, none of the eight TTR associated proteins interact with each other. The analysis of their interaction networks confirmed that these might have different functions, including altering nucleosome density, resolving R-loops and dual roles promoting RNAPII Splice Leader and RNAPIII transcription or termination of RNAPII transcription.
How I believe this moves the field forward
This is an extensive and thorough exploration of the localization and function of known and unknown proteins potentially regulating T. brucei chromatin organization and regulation. I believe the analysis of association networks for each protein opens up many new research questions to explore and will prime new discoveries that further our understanding of gene expression control in trypanosomes.
Chromatin regulation has been given a very high profile in model eukaryotic organisms and thus has been profoundly studied. Since trypanosome transcription is polycistronic, the levels of transcription regulation have been quite a challenge to explore in these organisms and RNA post-transcriptional modulation took up the protagonist role. I admire this study which shows that we shouldn’t underestimate chromatin regulation in early branching organisms.
Open questions
You mention there is some colocalization of TSRs associated factors and TTRs factors on the chromatin, but the significance of this colocalization is unknown. Do you have any new findings or hypothesis regarding these?
Authors response:
We currently don’t know why TSR and TTR factors might be enriched in the same locations. One speculations could be that the TTR factors are necessary to terminate any spurious transcription originating at TSRs, possibly in the wrong direction. That would of course not apply to bidirectional promoters which lead to the production of stable transcripts in both directions. RNAPII TSR factors might also be important for expression of RNAPIII-transcribed tRNAs and snRNAs.
On another note, while we call some proteins ”TTR factors”, we are not sure whether they are important for RNAPII transcription termination or for regulating RNAPIII transcription of tRNAs and snRNAs.
Some of the proteins that presented some form of chromatin enrichment show both nuclear and cytoplasmic localizations when tagged. Do you believe there could be some form of nuclear import/export regulation to their function?
Authors response:
We think it is possible that some proteins are regulated by import in/export out of the nucleus under different conditions, such as developmental transitions. Additionally, it is possible that some proteins have dual independent functions in the nucleus and the cytoplasm. However, at present we do not have data to support or reject these hypotheses.
Posted on: 11 March 2021 , updated on: 26 March 2021
doi: https://doi.org/10.1242/prelights.27646
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