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. 2019 Sep 26;47(17):9180-9197.
doi: 10.1093/nar/gkz644.

Trypanosoma brucei ribonuclease H2A is an essential R-loop processing enzyme whose loss causes DNA damage during transcription initiation and antigenic variation

Emma Briggs  1 , Kathryn Crouch  1 , Leandro Lemgruber  1 , Graham Hamilton  2 , Craig Lapsley  1 , Richard McCulloch  1
Affiliations

Trypanosoma brucei ribonuclease H2A is an essential R-loop processing enzyme whose loss causes DNA damage during transcription initiation and antigenic variation

Emma Briggs et al. Nucleic Acids Res. .

Abstract

Ribonucleotides represent a threat to DNA genome stability and transmission. Two types of Ribonuclease H (RNase H) excise ribonucleotides when they form part of the DNA strand, or hydrolyse RNA when it base-pairs with DNA in structures termed R-loops. Loss of either RNase H is lethal in mammals, whereas yeast survives the absence of both enzymes. RNase H1 loss is tolerated by the parasite Trypanosoma brucei but no work has examined the function of RNase H2. Here we show that loss of T. brucei RNase H2 (TbRH2A) leads to growth and cell cycle arrest that is concomitant with accumulation of nuclear damage at sites of RNA polymerase (Pol) II transcription initiation, revealing a novel and critical role for RNase H2. Differential gene expression analysis reveals limited overall changes in RNA levels for RNA Pol II genes after TbRH2A loss, but increased perturbation of nucleotide metabolic genes. Finally, we show that TbRH2A loss causes R-loop and DNA damage accumulation in telomeric RNA Pol I transcription sites, also leading to altered gene expression. Thus, we demonstrate separation of function between two nuclear T. brucei RNase H enzymes during RNA Pol II transcription, but overlap in function during RNA Pol I-mediated gene expression during host immune evasion.

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Figures

Figure 1.
Figure 1.
RNase H2A is essential for bloodstream form T. brucei viability. (A) Levels of TbRH2A transcripts in tetracycline (tet) induced (+) and uninduced (–) cells after 36 h of culture, relative to 2T1 cells (levels set at 100%), determined by RT-qPCR; error bars show SD of two independent experiments. (B) Cumulative growth curves of tet+ and tet- TbRH2A RNAi cultures, showing cell densities over 72 h (uninduced cells were diluted at 24 and 48 h). (C) Bar graph showing, at multiple time points, the percentage of tet-induced cells in the population that correspond to the following cell types, defined by DAPI staining of the nucleus (N) and kinetoplast (K): 1N1K, 1N2K, 2N2K, 1NXK (>2 K foci), YNXK (>2 K foci and aberrant N number or morphology), and other (do not conform to the above). Tet- shows the average of uninduced samples from all time-points. >200 cells were examined at each time point and error bars depict SD from three independent experiments. (D) Example images of induced (tet+) and un-induced (tet–) cells after 30 h of growth; scale bar, 5 μm. (E) Profiles of propidium iodide (PI) stained uninduced (tet–, pink) and RNAi induced (tet+, blue) populations after 6, 12, 24 and 36 h growth; y-axes show cell counts, and x-axes shows PI-area fluorescence. (F) Graph showing the percentage of cells in each expected cell cycle stage (G1, S and G2-M), or cells with genome content >4N, based on measuring proportion of the population with 2N, 2N-4N, 4N and >4N content; tet– shows the average of all tet– time points. In (B), (C) and (F), error bars show SD of three independent experiments.
Figure 2.
Figure 2.
TbRH2A-depleted parasites accumulate DNA damage yet continue to synthesise DNA. (A) High-resolution imaging of DAPI (blue) and γH2A (green) immunofluorescence with (tet+) and without (tet–) RNAi induction, at various time points; scale bar, 5 μm. Images to the right show increased magnification of the boxed nuclear DNA. (B) Bar graphs showing the percentage of tet+ and tet– populations positively staining for γH2A after 12, 24 and 36 h growth; error bars show SD of three independent experiments. (C) Western blot detection of γH2A in whole cell protein extracts after 12, 24 and 36 h growth of tet+ and tet– cells; EF1α staining is shown as a loading control. (D) Example SR-SIM images of DAPI, γH2A and EdU staining are shown, along with 3D reconstructions (model). TbRH2A RNAi cells were cultured in the presence (tet+) or absence (tet–) of tet for 36 h before imaging; scale bar = 1 μm. (E) Bar graph showing the percentage of tet+ and tet– populations positively staining for EdU incorporation after 12, 24 and 36 h of growth; error bars show the SD for three independent experiments (further examples of immunofluorescence images of EdU and γH2A staining are shown in Supplementary Figure S3).
Figure 3.
Figure 3.
Altered RNA–DNA hybrid distribution and increased DNA damage at sites of RNA Pol II transcription initiation after TbRH2A depletion. (A) Average DRIP-seq signal is shown as metaplots plotted for TbRH2A RNAi uninduced (tet–, blue) and RNAi induced (tet+, orange) data sets over divergent (left), head-to-tail (middle) and convergent (right) SSRs (±1 kb). In all cases 5′ and 3′ denote SSR boundaries defined by flanking transcript coordinates. Transcription direction is shown above the plots by dashed arrows. Standard error is shown as shaded regions. (B) Example screenshots of DRIP-seq signal in tet+ and tet– cells at individual SSRs in each class; CDS (thick black), UTR (thin black lines) and snRNA/tRNA genes (red) are shown below the DRIP-seq tracks. (C) Metaplots of γH2A ChIP-seq signal in TbRH2A RNAi induced samples relative to uninduced is shown after 24 h (pink) and 36 h (green) of RNAi induction; average signal is plotted across SSRs as for (A). (D) γH2A ChIP-seq signal in induced relative to uninduced cells is also shown plotted across chromosome 8 after 24 h (pink) and 36 h (green) of growth; γH2A ChIP-seq in RH1 null mutant cells relative to wild-type cells is shown below (red) for comparison (scale 1–3 fold-change). Upper track shows genes on sense (black) and antisense (red) strands, and arrows highlight transcription direction; the lowest track shows tandem repeat sequences.
Figure 4.
Figure 4.
Gene expression is altered upon depletion of TbRH2A. (A) Volcano plots displaying differential expression of genes after TbRH2A knockdown. Each data point represents a gene. Genes were deemed significantly differentially expressed when RNA-seq indicated an adjusted P value <0.05 of gene-specific RNA in induced cells relative to uninduced. X-axes show the log2 fold-change between un-induced and induced after 24 h (left) and 36 h (right) of culture, and y-axes shows log2 adjusted p value. Data was generated with two independent replicates for each condition and time point. Significantly differentially expressed genes are shown in red or blue (denoting VSG); all other genes, including VSGs, are shown in black. (B) Number of genes significantly up-regulated in tet-induced TbRH2A RNAi cells relative to uninduced, after 24 (left) and 36 h (right) of growth, are shown annotated as VSGs, ESAGs, procyclin, RHS-associated, and other genes; total numbers are shown below each chart.
Figure 5.
Figure 5.
R-loop and yH2A levels increase across VSG BESs in cells depleted of TbRH2A. (A) Localisation of R-loops is shown in BES1 (active site of VSG transcription in WT cells) and BES3 (one normally transcriptionally silent site), comparing DRIP-seq signal in TbRH2A RNAi cells grown for 24 hr in the absence (tet–; blue) or presence (tet+; orange) of RNAi induction, and compared with WT cells (pink). BES features are shown as follows: promoter (aqua), ESAGs (blue, numbered), 70-bp repeats (purple) and VSGs (red); pseudogenes are indicated (ψ), and the end of the available BES sequence is denoted by a black circle. (B) DRIP-qPCR using primers _targeting the sequences of ESAG6, ESAG8, VSG221 (BES1) and VSG121 (BES3), with or without E. coli RNase HI (EcRHI) treatment, showing the percentage of amplification in the IP sample relative to input from tet induced (tet+) and uninduced (tet–) cells grown for 36 h. Error bars display SEM for three replicates. (C) yH2A ChIP-seq signal enrichment is shown mapped to BES1 and BES3 as a ratio of reads in tet-induced samples relative to uninduced (each first normalized to the cognate input sample) after 24 (purple) and 36 (green) h growth; γH2A ChIP-seq signal (normalised to input) is shown in WT cells for comparison (pink). (D) γH2A ChIP-qPCR, as in (B): data is shown for tet induced (tet+) and uninduced (tet–) cells after 36 hr of growth. Error bars display SD for two replicates.
Figure 6.
Figure 6.
Depletion of TbRH2A results in increased transcription of silent VSGs. (A) Graph of RNA levels for five VSGs, determined by RT-qPCR, in tet-induced TbRH2A RNAi cells, plotted as fold-change relative to levels of the cognate VSG RNA in uninduced cells after both 24 and 36 h of culture; VSG221 (pink) is in the active BES (BES1) of WT cells, while VSG121 (yellow), VSG800 (blue), VSGT3 (green) and VSG13 (grey) are in silent BESs; error bars show SD for three independent experiments. (B) Graph depicting the number of VSG genes whose RNA levels display significant upregulation in RNAi-induced RNA-seq samples relative to uninduced, both 24 and 36 h after growth; the total number is sub-categorised depending on whether the VSGs have been localized to the BES, are intact genes in the subtelomeric arrays (array), are in minichromosomes (MC), are pseudogenes (pseudo), or are in a metacyclic VSG ES (MES). (C) Normalized RNA-seq read depth abundance (y-axes) is plotted for two independent replicates (overlaid orange and blue) in TbRH2A RNAi parasites after 24 and 36 hr of growth, with (tet+) or without (tet–) RNAi induction. Read depth is shown relative to gene position (x-axes) for BES1 and BES3. (D) As in (C), showing RNA-seq read depth abundance (y-axes) across a selection of non-BES VSG CDS (x-axes), after 36 h of growth.
Figure 7.
Figure 7.
Depletion of TbRH2A induces switching of the VSG coat. (A) Co-immunofluorescence imaging of VSG221 (pink) and VSG121 (yellow) surface expression. Example images are shown of cells after 24 h of culture without (tet–) or with (tet+) RNAi induction (Scale bar, 5 μm). (B) Graph of the percentage of uninduced (tet–, all time points) and RNAi-induced cells (after 12, 24 and 36 h of culture with tet) expressing only VSG221 (pink) or only VSG121 (yellow) on their surface, as well as cell with both (orange) or neither (grey) of the two VSGs on their surface, as determined by co-immunofluorescence imaging with anti-VSG221 and VSG121 antiserum. >200 cells were analysed for each time point and three experimental replicates (error bars denote SEM). The table below shows the average percentage of the three replicates in each case.
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