A DOT1B/Ribonuclease H2 Protein Complex Is Involved in R-Loop Processing, Genomic Integrity, and Antigenic Variation in Trypanosoma brucei

Eisenhuth, Nicole; Vellmer, Tim; Rauh, Elisa T; Butter, Falk; Janzen, Christian J.

doi:10.1128/mbio.01352-21

Cited by 8 publications

(16 citation statements)

References 109 publications

(153 reference statements)

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“…Moreover, R-loop accumulation is associated with increased local chromatin accessibility 92 , activation of gene expression 93, 94 , and modulation of transcription termination 95, 96 . In T. brucei R-loops have been implicated in transcription initiation and mRNA maturation 97, 98 , telomere function 99 , and host immune evasion by antigenic variation 100–102 . Reflecting the myriad functions of R-loops, a wide range of proteins have been shown to interact with RNA-DNA hybrids in mammals and T. brucei 103, 104 .…”

Section: Resultsmentioning

confidence: 99%

R-loops acted on by RNase H1 are a determinant of chromosome length-associated DNA replication timing and genome stability inLeishmania

Damasceno,

Briggs,

Krasilnikova

et al. 2024

Preprint

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Genomes in eukaryotes normally undergo DNA replication in a choreographed temporal order, resulting in early and late replicating chromosome compartments. Leishmania, a human protozoan parasite, displays an unconventional DNA replication program in which the timing of DNA replication completion is chromosome size-dependent: larger chromosomes complete replication later then smaller ones. Here we show that both R-loops and RNase H1, a ribonuclease that resolves RNA-DNA hybrids, accumulate in Leishmania major chromosomes in a pattern that reflects their replication timing. Furthermore, we demonstrate that such differential organisation of R-loops, RNase H1 and DNA replication timing across the parasite chromosomes correlates with size-dependent differences in chromatin accessibility, G quadruplex distribution and sequence content. Using conditional gene excision, we show that loss of RNase H1 leads to transient growth perturbation and permanently abrogates the differences in DNA replication timing across chromosomes, as well as altering levels of aneuploidy and increasing chromosome instability in a size-dependent manner. This work provides a link between R-loop homeostasis and DNA replication timing in a eukaryotic parasite and demonstrates that orchestration of DNA replication dictates levels of genome plasticity in Leishmania.

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Section: Resultsmentioning

confidence: 99%

R-loops acted on by RNase H1 are a determinant of chromosome length-associated DNA replication timing and genome stability inLeishmania

Damasceno,

Briggs,

Krasilnikova

et al. 2024

Preprint

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show abstract

“…( 22 )), loss of RNase H2A causes pronounced DNA damage accumulation at transcription start sites ( 49 ). In addition, immunoprecipitation of the histone methyltransferases DOT1A or DOT1B reveals interaction with RNase H2 ( 32 , 51 ), with DOT1A-RNase H2 activity potentially resolving R-loops at transcription termination sites containing RNA Pol-III genes ( 32 ). We did not detect either DOT1A or DOT1B in the DRIP-MS data, perhaps suggesting the methyltransferases do not directly interact with RNA–DNA hybrids.…”

Section: Resultsmentioning

confidence: 99%

“…Despite DRIP-MS not detecting direct RNase H2A or DOT1B interaction with RNA–DNA hybrids, there is clear evidence of functional interaction between these proteins and their involvement in antigenic variation: loss of DOT1B or RNase H2A results in similar changes in R-loop levels, DNA damage levels and Variant Surface Glycoprotein (VSG) expression alterations ( 49 , 51 ). In fact, a connection between R-loops and VSG expression may also extend to the histone variants recovered by DRIP-MS, given similarities in changes to VSG transcription control after loss of either RNase H ( 49 , 50 ) or H3.V and/or H4.V ( 30 , 119 , 120 ).…”

Section: Resultsmentioning

confidence: 99%

Immunoprecipitation of RNA–DNA hybrid interacting proteins in Trypanosoma brucei reveals conserved and novel activities, including in the control of surface antigen expression needed for immune evasion by antigenic variation

Girasol,

Briggs,

Marques

et al. 2023

Nucleic Acids Research

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RNA–DNA hybrids are epigenetic features of genomes that provide a diverse and growing range of activities. Understanding of these functions has been informed by characterising the proteins that interact with the hybrids, but all such analyses have so far focused on mammals, meaning it is unclear if a similar spectrum of RNA–DNA hybrid interactors is found in other eukaryotes. The African trypanosome is a single-cell eukaryotic parasite of the Discoba grouping and displays substantial divergence in several aspects of core biology from its mammalian host. Here, we show that DNA–RNA hybrid immunoprecipitation coupled with mass spectrometry recovers 602 putative interactors in T. brucei mammal- and insect-infective cells, some providing activities also found in mammals and some lineage-specific. We demonstrate that loss of three factors, two putative helicases and a RAD51 paralogue, alters T. brucei nuclear RNA–DNA hybrid and DNA damage levels. Moreover, loss of each factor affects the operation of the parasite immune survival mechanism of antigenic variation. Thus, our work reveals the broad range of activities contributed by RNA–DNA hybrids to T. brucei biology, including new functions in host immune evasion as well as activities likely fundamental to eukaryotic genome function.

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“…mRNA-seq-based data demonstrate that DOT1A and DOT1B mRNA is constantly detected throughout the cell cycle and are both slightly increased in G2, suggesting that DOT1A and/or DOT1B methyltransferase activities, but not their expression, are cell cycle-regulated. Recently, RNase H2, which hydrolyzes RNA in DNA/RNA hybrids, was identified as a DOT1A and DOT1B binding partner 61 , 62 . RNase H2 interacts with PCNA and is mainly active during S phase to resolve R-loops for initiating transcription and regulating antigenic variation in T. brucei 63 .…”

Section: Discussionmentioning

confidence: 99%

Two DOT1 enzymes cooperatively mediate efficient ubiquitin-independent histone H3 lysine 76 tri-methylation in kinetoplastids

Frisbie,

Hashimoto,

Xie

et al. 2024

Nat Commun

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In higher eukaryotes, a single DOT1 histone H3 lysine 79 (H3K79) methyltransferase processively produces H3K79me2/me3 through histone H2B mono-ubiquitin interaction, while the kinetoplastid Trypanosoma brucei di-methyltransferase DOT1A and tri-methyltransferase DOT1B efficiently methylate the homologous H3K76 without H2B mono-ubiquitination. Based on structural and biochemical analyses of DOT1A, we identify key residues in the methyltransferase motifs VI and X for efficient ubiquitin-independent H3K76 methylation in kinetoplastids. Substitution of a basic to an acidic residue within motif VI (Gx6K) is essential to stabilize the DOT1A enzyme-substrate complex, while substitution of the motif X sequence VYGE by CAKS renders a rigid active-site loop flexible, implying a distinct mechanism of substrate recognition. We further reveal distinct methylation kinetics and substrate preferences of DOT1A (H3K76me0) and DOT1B (DOT1A products H3K76me1/me2) in vitro, determined by a Ser and Ala residue within motif IV, respectively, enabling DOT1A and DOT1B to mediate efficient H3K76 tri-methylation non-processively but cooperatively, and suggesting why kinetoplastids have evolved two DOT1 enzymes.

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A DOT1B/Ribonuclease H2 Protein Complex Is Involved in R-Loop Processing, Genomic Integrity, and Antigenic Variation in Trypanosoma brucei

Cited by 8 publications

References 109 publications

R-loops acted on by RNase H1 are a determinant of chromosome length-associated DNA replication timing and genome stability inLeishmania

R-loops acted on by RNase H1 are a determinant of chromosome length-associated DNA replication timing and genome stability inLeishmania

Immunoprecipitation of RNA–DNA hybrid interacting proteins in Trypanosoma brucei reveals conserved and novel activities, including in the control of surface antigen expression needed for immune evasion by antigenic variation

Two DOT1 enzymes cooperatively mediate efficient ubiquitin-independent histone H3 lysine 76 tri-methylation in kinetoplastids

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