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A DOT1B/RNaseH2 protein complex is involved in R-loop processing, genomic integrity and antigenic variation in Trypanosoma brucei

ABSTRACT: The parasite Trypanosoma brucei periodically changes the expression of its protective variant surface glycoproteins (VSGs) to evade the host’s immune system in a process known as antigenic variation. One route to change VSG expression is by transcriptional activation of a previously silent VSG expression site (ES), a subtelomeric region containing the VSG genes. Homologous recombination of a different VSG from a large reservoir into the active ES represents another route. The conserved histone methyltransferase DOT1B is involved in transcriptional silencing of inactive ES and influences ES switching kinetics. The molecular machinery that enables DOT1B to execute these regulatory functions remains elusive, however. To better understand DOT1B-mediated regulatory processes, we purified DOT1B-associated proteins using complementary biochemical approaches. We identified several novel DOT1B interactors. One of these was the trimeric Ribonuclease H2 complex, previously shown to resolve RNA-DNA hybrids, maintain genome integrity, and play a role in antigenic variation. Our study revealed that DOT1B depletion results in an increase in RNA-DNA hybrids, accumulation of DNA damage and recombination-based ES switching events. Surprisingly, the observed VSG deregulation was similar to Ribonuclease H2 mutants. We here propose that the epigenetic regulator DOT1B forms a complex with RNase H2 that acts in resolving R-loops to ensure genome integrity and contribute to the tightly regulated process of antigenic variation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: F Butter

LAB HEAD: Falk Butter

PROVIDER: PXD017838 | Pride | 2021-11-01

REPOSITORIES: Pride

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			Action	DRS
	20161226_QEP1_FBU_FB_trypanosomes_BSF_DOT1B_1.raw	Raw
	20161226_QEP1_FBU_FB_trypanosomes_BSF_DOT1B_2.raw	Raw
	20161226_QEP1_FBU_FB_trypanosomes_BSF_DOT1B_3.raw	Raw
	20161226_QEP1_FBU_FB_trypanosomes_BSF_DOT1B_4.raw	Raw
	20161226_QEP1_FBU_FB_trypanosomes_BSF_wt_1_161228115520.raw	Raw

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Project description:Variant Surface Glycoproteins (VSG) coat parasitic African trypanosomes and underpin antigenic variation and immune evasion. This VSG is a super-abundant virulence factor that is subject to post-transcriptional gene expression controls mediated via the VSG 3’-untranslated region (3’-UTR). To identify positive VSG regulators in bloodstream form cells, we used prior genome-scale screening data to prioritise mRNA binding protein (mRBPs) knockdowns that phenocopy VSG mRNA knockdown, displaying both loss-of-fitness and pre-cytokinesis accumulation. The top three candidate VSG regulators were CFB2 (cyclin F-box protein 2, Tb927.1.4650), MKT1 (Tb927.6.4770) and PBP1 (polyadenylate binding-protein binding-protein, Tb927.8.4540). Notably, CFB2 was recently found to regulate VSG transcript stability, and all three proteins were found to associate with each other. We used data-independent acquisition for accurate label-free quantification and deep proteome coverage to quantify expression profiles following depletion of each mRBP. Only CFB2 knockdown significantly reduced VSG expression and the expression of a reporter under the control of a VSG 3’-untranslated region (3’-UTR). CFB2 knockdown also triggered depletion of cytoplasmic ribosomal proteins, consistent with translation arrest observed when VSG synthesis is blocked. In contrast, PBP1 knockdown triggered depletion of CFB2, MKT1, and other components of the PBP1-complex. Finally, all three knockdowns triggered depletion of cytokinesis initiation factors, consistent with the cytokinesis defect that was confirmed here for all three knockdowns. Thus, genome-scale knockdown datasets facilitate the triage and prioritisation of candidate regulators. Quantitative proteomic analysis confirms 3’-UTR dependent positive control of VSG expression by CFB2 and interactions with additional mRBPs. Our results also reveal connections between VSG expression control by CFB2, ribosomal protein expression, and cytokinesis.

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A DOT1B/RNaseH2 protein complex is involved in R-loop processing, genomic integrity and antigenic variation in Trypanosoma brucei

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