RNA/DNA Hybrid Interactome Identifies DXH9 as a Molecular Player in Transcriptional Termination and R-Loop-Associated DNA Damage

Abstract

R-loops comprise an RNA/DNA hybrid and displaced single-stranded DNA. They play important biological roles and are implicated in pathology. Even so, proteins recognizing these structures are largely undefined. Using affinity purification with the S9.6 antibody coupled to mass spectrometry, we defined the RNA/DNA hybrid interactome in HeLa cells. This consists of known R-loop-associated factors SRSF1, FACT, and Top1, and yet uncharacterized interactors, including helicases, RNA processing, DNA repair, and chromatin factors. We validate specific examples of these interactors and characterize their involvement in R-loop biology. A top candidate DHX9 helicase promotes R-loop suppression and transcriptional termination. DHX9 interacts with PARP1, and both proteins prevent R-loop-associated DNA damage. DHX9 and other interactome helicases are overexpressed in cancer, linking R-loop-mediated DNA damage and disease. Our RNA/DNA hybrid interactome provides a powerful resource to study R-loop biology in health and disease.

Keywords: DHX9; DNA damage; PARP1; R-loop; RNA/DNA hybrid; interactome; topoisomerase; transcription.

Graphical abstract

Figure 1

Design of RNA/DNA Hybrid IP Method in HeLa Cells (A) RNA/DNA hybrid IP workflow in HeLa cells. (B) RNA/DNA hybrid slot blot with S9.6 antibody. (C) Top: silver stain of RNA/DNA hybrid IP. No antibody and isotype-matched IgG2a antibody were used as controls. Bottom: western blot of RNA/DNA hybrid IP using indicated antibodies. Arrows indicate hypophosphorylated (IIa) and hyperphosphorylated (IIo) forms of Pol II. Triple amounts of input and IP samples were loaded for SETX. (D) Top: silver stain of RNA/DNA hybrid IP following benzonase treatment. Bottom: western blot of RNA/DNA hybrid IP, probed with Top1 and H3 antibodies. (E) Silver stain of RNA/DNA hybrid IP in the presence of RNA/DNA hybrid competitor. (F) Western blot for Top1 of RNA/DNA hybrid IP with indicated synthetic competitors. (C–E) ^∗, indicates the heavy chain of S9.6 and IgG2a antibodies. ^∗∗, indicates BSA, used to block protein A dynabeads. See also Figure S1.

Figure 2

Characterization of the RNA/DNA Hybrid Interactome (A) Volcano plot displaying MS results of three biological replicates of RNA/DNA hybrid IP. Averaged log₂ ratios between RNA/DNA hybrid IP and control IP (with addition of 1.3 μM synthetic RNA/DNA hybrid competitor) are plotted against their Benjamini-Hochberg-corrected −log₁₀ p values across all replicates using a moderated t test. Proteins significantly enriched in RNA/DNA hybrid IP are in orange (p < 0.01) and constitute the RNA/DNA hybrid interactome. Proteins identified with p > 0.01 are in gray. Dashed lines indicate the significance cutoffs (log₂ enrichment > 2 and –log₁₀ > 2). (B) Protein classes overrepresented in RNA/DNA hybrid interactome (corrected p < 0.05, Fisher’s exact test). Representative proteins are given in brackets. (C) Top: overlap between mRNA interactome and RNA/DNA hybrid interactome in HeLa cells. Bottom: common proteins enriched in both RNA/DNA hybrid and mRNA interactomes (I) and proteins unique to the RNA/DNA hybrid interactome (II). The x axis indicates statistical significance of overrepresentation. (D) The chromatin probability analysis of 7,635 HeLa proteins (HeLa proteome) and RNA/DNA hybrid interactome. (E) Enrichment for proteins known to bind both RNA and DNA in the RNA/DNA hybrid interactome compared to the HeLa proteome (p < 1.6 × 10⁻²⁸, Fisher’s exact test). (F) Validation of RNA/DNA hybrid interactors using western blot, probed with indicated antibodies. Drosha, NPC, and Lamin B1 are negative controls. (−) IP lane corresponds to control IP with IgG2a antibody. See also Figure S2.

Figure 3

Validation of New RNA/DNA Hybrid Interactome Candidates (A) Workflow of RNA/DNA hybrid IP with RNase H digestion. (B and C) HeLa genomic DNA input was either treated (+) or not (−) with RNase H before enrichment for RNA/DNA hybrids with the S9.6 antibody. Genomic RNA/DNA hybrids were incubated with nuclear extracts depleted for RNA/DNA hybrids with RNase A, followed by S9.6 IP. RNA/DNA hybrid slot blot (B) and western blot of RNA/DNA hybrid IP, probed with indicated antibodies (C). (D–I) Genomic DNA from HeLa cells transfected with control (siCtrl) or indicated siRNAs was treated with RNase H. siTop1 (D), siDHX9 #1 (E), siWHSC1 (F), siSAFB2 (G), siDNA-PK (H), siPARP1 (I) were used. Top: RNA/DNA hybrid slot blot. Bottom: quantification of S9.6 signal. Values are normalized to the siCtrl and represent the means ± SEMs, n ≥ 3. See also Figure S3.

Figure 4

DHX9 Promotes Transcriptional Termination (A and B) Western blot probed with DHX9 antibody (A) and RNA/DNA hybrid slot blot (B) of RNA/DNA hybrid IP from cells treated (+) or not (−) with actinomycin D. (C and D) Western blot probed with DHX9 and tubulin antibodies (C) and RNA/DNA hybrid slot blot (D) of IPs carried out with DHX9 and tubulin antibodies. (E) IF analysis of DHX9 (green). DAPI (blue) depicts nuclei. Scale bar, 10 μm. (F) Western blot of HeLa protein extracts, treated with control, DHX9 #1, and SETX siRNAs and probed with indicated antibodies. hnRNPUL1 is a loading control. (G) Diagram of β-actin (left) and γ-actin (right) genes depicting exons (black), UTRs (white), transcriptional start site (TSS), termination region (gray), and qPCR amplicons. (H) DIP in HeLa cells, treated with control, DHX9 #1 and SETX siRNAs, on β-actin (left) and γ-actin (right) genes. Values are normalized to in1. (I) Read-through transcription analysis of β-actin (left) and γ-actin (right) genes in HeLa cells treated with control, DHX9 #1, and SETX siRNAs, using RT-qPCR. Values are normalized to β-actin in3 and γ-actin in1. (H–I) Bars, means ± SEMs, n ≥ 3. See also Figure S4.

Figure 5

DHX9 Depletion Triggers R-Loop Accumulation in Response to CPT (A, B, and F) Diagram of β-actin gene (A, top), DIP (A, bottom), DHX9 ChIP (B), and PARP1 ChIP (F) in HeLa cells, treated with CPT for the indicated time, on β-actin gene. Values are relative to in1 in the DMSO sample. (C) DHX9 ChIP in HEK293T cells, transfected with FLAG (−RNase H1) or RNase H1 (+RNase H1) and treated with CPT for 60 min. Values are relative to in1 −RNase H1 in the DMSO sample. (D) DIP in HeLa cells, transfected with control (shades of blue) or DHX9 #1 (shades of red) siRNA and treated with CPT for indicated time, on the β-actin gene. Values are relative to in1 for each siRNA. The p-value is calculated for the siDHX9 versus the siCtrl sample. (E) Western blot of IgG2a (negative control), PARP1, and DHX9 IPs in HeLa cells treated with CPT and probed with indicated antibodies. Left: input, right: IP. (G) DIP in HeLa cells, treated with DMSO (shades of blue) or Olaparib (shades of green) before addition of CPT for 60 min, on the β-actin gene. Values are relative to in1 for DMSO and Olaparib. The pvalue is calculated for the Olaparib + CPT versus the DMSO + CPT samples. (A–D, F, and G) Bars, means ± SEMs, n ≥ 3. See also Figures S5 and S6.

Figure 6

DHX9 Prevents R-Loop-Associated DNA Damage in Response to CPT (A) IF analysis of HeLa cells transfected with control or DHX9 #1 siRNA and treated with DRB before the addition of CPT for 60 min and stained for γH2AX (red) and DAPI (blue). Left: representative images. Bars, 10 μm. Right: γH2AX fluorescence intensity per nucleus from a representative experiment (≥300 nuclei were analyzed per condition). The horizontal red bars represent the means, and each dot is one nucleus. (B) The same as in (A), but instead of DRB, cells were transfected with FLAG (−RNase H1) or RNase H1 plasmid. ^∗∗∗p < 0.001, ns, not significant (one-way ANOVA Tukey’s multiple comparisons test). See also Figure S7.

Figure 7

RNA/DNA Hybrid Interactome Helicases Are Amplified in Human Cancers (A) Enrichment of DEAD and DEAH/RHA helicases in the RNA/DNA hybrid interactome. (B) Gain (red) and loss (blue) of RNA/DNA hybrid-interacting DEAD/H helicases in cancer. The y axis shows the percentage of the total tested cancer samples based on copy number variations (COSMIC database). (C) Transcriptional expression changes of RNA/DNA hybrid-interacting DEAD/H helicases in cancer. The y axis shows the number of cancer studies (ONCOMINE database). (D) Model showing the role of the RNA/DNA hybrid interactome and the top candidate DHX9 in regulating R-loop balance in health and disease.