Journal: Nucleic Acids Research

Article Title: RPA hyperphosphorylation hinders the resolution of R-loops and G-quadruplex-associated R-loops during RAS-driven senescence

doi: 10.1093/nar/gkag331

Figure Lengend Snippet: Genome-wide analysis reveals loss of RNase H1 activity at RPA32-hyperphosphorylated R-loop–enriched regions in pre-RIS cells. ( A ) Schematic representation of the ChIP-seq and DRIP-seq experiments performed in the indicated cells at the indicated time after 4-OHT treatment. ( B ) Metaplots of RNase H1 ChIP-seq levels in the indicated cells, with respect to TSS and TES. ( C ) Histogram representing the percentage of genome coverage of γH2AX and RNase H1 enriched peaks in the indicated cells. ( D ) Volcano plot showing RNase H1 peaks that are significantly enriched or depleted in the comparison between the RPA70 + RAS and the H2B + RAS cells. ( E ) Histogram showing the percentage of γH2AX + genomic regions overlapping with areas of RNase H1 binding loss in H2B + RAS cells compared with the RPA70 + RAS condition, and vice versa. ( F ) Metaplot showing the log2 ratio of γH2AX signal in H2B + RAS with respect to RPA70 + RAS cells within 6kb around the RNase H1 peaks lost in H2B + RAS cells. ( G ) Histogram and metaplot showing the percentage of γH2AX + genomic regions in H2B + RAS cells overlapping with genomic regions exhibiting increased TrAEL-seq read density in IMR90 RAS (≥1.5-fold enrichment after 3 days of RAS expression relative to day 0), consistent with replication fork pausing or altered fork dynamics (GSE299123). ( H ) Histogram showing the percentage of TrAEL-seq–enriched regions in HRAS-expressing cells overlapping with genomic loci characterized by R-loop accumulation in BJ + RAS pre-RIS cells. ( I ) Histogram showing that RNase H1 peaks lost in H2B + RAS cells are associated with a depletion of RPA32 binding compared to RPA70 + RAS cells. ( J ) Metaplot of log2 ratio of RPA32 phosphorylation rate (pRPA32/RPA32) signal in H2B + RAS with respect to RPA70 + RAS cells within 6kb around the RNase H1 peaks lost in H2B + RAS cells. ( K ) Venn diagram showing that 25.3% of R-loops exclusive to the pre-RIS condition do not exhibit overlap (1 nucleotide) of RNase H1 binding in H2B + RAS cells, while 74.6% are characterized by RNase H1 association. ( L ) Venn diagram showing that 25.3% of R-loops exclusive to the pre-RIS condition that do not exhibit overlap (≥1 nucleotide) with RNase H1 binding in H2B + RAS cells show overlap with 38.3% of G4 regions. ( M ) Metaplot showing the hyperphosphorylation of RPA32 in H2B + RAS cells with respect to RPA70 + RAS within 6 kb around the center of the peaks of R-loops exclusive to the RIS condition and characterized by RNase H1 binding. For reference, a heatmap depicting the TrAEL-seq signal in IMR90 cells expressing RAS at the indicated time points is shown. ( N, O ) Distribution of R-loops, G4s, γH2AX and pRPA32 phosphorylation rate in RIS (BJ + RAS) and RIS-escape achieved by RPA70 overexpression (RPA70 + RAS). QPS are indicated. In C, E, G, H, I data are expressed as mean ± SD; two independent sequencing each coming from three biological replicates. * P < 0.05, ** P < 0.01. Pairwise t-test was applied to indicated comparisons.

Article Snippet: DNA SMART ChIP-Seq Kit (Takara) was used for library generation of samples subjected to Illumina sequencing.

Techniques: Genome Wide, Activity Assay, ChIP-sequencing, Comparison, Binding Assay, Expressing, Phospho-proteomics, Over Expression, Sequencing

Journal: Nucleic Acids Research

Article Title: RPA hyperphosphorylation hinders the resolution of R-loops and G-quadruplex-associated R-loops during RAS-driven senescence

doi: 10.1093/nar/gkag331

Figure Lengend Snippet: RAS-induced senescence is characterized by the accumulation of irreparable DNA damage, DNA:RNA hybrids and unsuccessful loading of BRCA1. A. Immunoblot analysis of the indicated proteins in BJ-hTERT/RAS-ER cells, expressing the indicated transgenes and treated or not as indicated with 4-OHT (1µM). % of SA-β-gal positivity is indicated. B. Quantification of SA-β-gal- positive cells and S-phase entering cells (BrdU assay, BrdU pulse of 3 h) in BJ-hTERT/RAS-ER Hygro cells or BJ-hTERT/RAS-ER HDAC4 cells, treated (+RAS) or not (-RAS) with 4-OHT for the indicated days (D2 = day2, D8 = day8) to induce HRAS G12V expression. Mean ± SD; n = 4. * P < 0.05, ** P < 0.01 and *** P < 0.001 (Dunn’s Multiple Comparison Test with respect to -RAS D2). Pairwise t-test was applied to indicated comparisons. C. Representative microscopic images of SA-β-gal stained BJ-hTERT/RAS-ER Hygro cells or BJ-hTERT/RAS-ER HDAC4 cells at the indicated time (days) after 4OHT treatment (Bar = 40 μm). D. Dot blot analysis using S9.6 antibody to detect R-loops and AE-2 antibody to detect dsDNA. Around 100 and 500 ng of nucleic acids extracted from the indicated cells grown for 2 days in presence or absence of 4-OHT were treated or not with 10u of RNase H and spotted on nitrocellulose film. E. Quantification of Dot blot shown in D. Mean ± SD; n = 3. F. Schematic representation of the ChIP-seq and DRIP-seq experiments performed in the indicated cells at the indicated time after 4-OHT treatment. G. Histogram representing the percentage of genome coverage of RNA PolII Ser5, γH2AX, BRCA1, R-loops and G4s enriched peaks in the indicated cells. Mean ± SD; two independent sequencing each coming from three biological replicates. * P < 0.05, ** P < 0.01 and *** P < 0.001. (Dunn’s Multiple Comparison Test with respect to -RAS D2). Pairwise t-test was applied to indicated comparisons. H. P -value and q-value of pathway enrichment analysis performed by using MSigDB (RRID:SCR_016863) on genes associated to DNA:RNA hybrids found exclusive of BJ + RAS condition.

Article Snippet: DNA SMART ChIP-Seq Kit (Takara) was used for library generation of samples subjected to Illumina sequencing.

Techniques: Western Blot, Expressing, BrdU Staining, Comparison, Staining, Dot Blot, ChIP-sequencing, Sequencing

Journal: Nucleic Acids Research

Article Title: RPA hyperphosphorylation hinders the resolution of R-loops and G-quadruplex-associated R-loops during RAS-driven senescence

doi: 10.1093/nar/gkag331

Figure Lengend Snippet: The co-occurrence of R-loops and G-quadruplex structures characterized the RIS condition. ( A ) Metaplot and heatmap of DRIP-seq signal 6kb around the 11 439 genomic regions with significant enrichment of DNA:RNA hybrids in BJ + RAS cells compared with BJ + RAS/HDAC4 cells. ( B ) Metaplot of RNA PolII Ser5, γH2AX, BRCA1 and G4 ChIP-seq signals within 6 kb around the 11 439 R-loops characterizing the pre-RIS condition. ( C ) Metaplot of log2 ratio of γH2AX signal in BJ + RAS with respect to BJ + RAS/HDAC4 cells within 6 kb around the 11 439 R-loops characterizing the RIS condition. ( D ) Histogram reporting the percentage of RIS-specific R-loops co-localizing with G4s within the indicated genomic interval. ( E ) Histogram reporting the genomic distribution of R-loops and “G-loop like” structures (defined as containing a G4 within 3 kb from DNA:RNA hybrids detected in DRIP-seq). ( F ) Histograms reporting the percentage of RIS-specific R-loops with G4 (defined as containing a G4 within 3kb from DNA:RNA hybrids detected in DRIP-seq) or without G4 co-localizing with G4s withint the indicated genomic interval in the indicated cellular conditions. ( G ) Distribution of R-loops, RNA PolII Ser5 (RNP2 Ser5), G4s, γH2AX, and BRCA1 on chromosome 12 and 16 in the indicated cells expressing or not RAS and HDAC4, as indicated. The highlighted region represents the genomic locus where a significant enrichment of the DRIP-seq signal is detected in BJ + RAS cells. At the PDXDC1 locus, enrichment encompasses multiple distinct regions within the highlighted interval, which spans approximately 15 kb. In B and F, data are expressed as mean ± SD; two independent sequencing each coming from 3 biological replicates. * P < 0.05, ** P < 0.01. Pairwise t-test was applied to indicated comparisons.

Article Snippet: DNA SMART ChIP-Seq Kit (Takara) was used for library generation of samples subjected to Illumina sequencing.

Techniques: ChIP-sequencing, Expressing, Sequencing

Journal: Nucleic Acids Research

Article Title: RPA hyperphosphorylation hinders the resolution of R-loops and G-quadruplex-associated R-loops during RAS-driven senescence

doi: 10.1093/nar/gkag331

Figure Lengend Snippet: G-loops exhibit resistance to RNase H1-mediated cleavage, suggesting a structural configuration that impairs enzymatic accessibility. ( A ) Motif discovery analysis reporting the three most frequent motifs found enriched in G4 ChIP-seq in BJ + RAS cells and co-localizing with R-loops within 3 kb. Each motif was analyzed using G4Hunter to identify the optimal sequence containing a putative QPS. The G4Hunter score is reported along with the predicted tetrads. ( B ) Plot reporting the distances in BJ + RAS of the G4s (corresponding to the three motifs found in Fig. ) with respect to the closest R-loop, divided according to their co-occurrence on either the template or non-template strand. ( C ) Heatmap showing the signal intensity of G4 structures and DRIP-seq in BJ + RAS cells, relative to the indicated genomic coordinates and the presence of QPSs marked by red arrows. In the case of the KRAS locus, these correspond to the well-characterized G4 regions referred to as ‘near’, ‘mid’, and ‘far’. ( D ) Metaplot of DRIP-seq and G4, RNA PolII Ser5 and BRCA1 ChIP-seq signals in BJ + RAS and BJ + RAS/HDAC4 cells within 6kb of “G-loop like” structures (R-loops co-occurring with G4 in non-template strand) identified in BJ + RAS cells. ( E ) The in vitro assay was performed by incubating equimolar amounts (800 pmol) of Cy5.5-labeled R-loops, G-loops containing the G4 motif “A” (ref. Fig. ) or DNA:RNA duplex (Cy5.5 labeling at 5′ of RNA) with increasing amounts of RNase H1. Native gel electrophoresis was performed to separate the various species of nucleic acids, which were schematized on the side. The fluorescence of the Cy5.5 labeled RNAs was acquired with the fluorescence reader and then the gel was stained with EtBr to detect the DNA duplexes with the transilluminator. ( F ) Histogram representing the ribonuclease efficiency of RNase H1 against synthetic R-loop and G-loop substrates (increasing amounts: 80, 160, and 400 pmol). Lanes corresponding to gel electrophoresis (in E) are indicated. Structures containing G4 in displaced DNA strand are in black (lanes 8–12). ( G ) Histogram representing the increase in fluorescence of Thioflavin T in the presence of substrates containing G4. Fluorescence emission was collected at 485 nm after excitation at 425 nm and expressed as increase of fluorescence quantum yield at 485 nm in the presence of G4-forming sequences in displaced DNA strand (lanes 8–12, increasing amounts: 80, 160, and 400 pmol). ( H ) Dot blot analysis using BG4 antibody to detect G4. Increasing amount of the indicated synthetic DNA:RNA structures (DNA:RNA heteroduplex not containing G4, R-loop not containing G4, G-loop with motif A, G-loop with 32R G4) were spotted on nitrocellulose film. RNA was Cy5.5 labeled at 5′. Cy5.5 fluorescence was used as loading control. ( I ) Fluorescence anisotropy binding assays showing the interaction between catalytically inactive RNase H1 (deadRNase H1) and the indicated nucleic acid substrates. Binding curves were fitted to determine the apparent dissociation constants ( K d ) for each substrate. In F and G, data are expressed as mean ± SD; n = 4. * P < 0.05, ** P < 0.01, *** P < 0.05. Pairwise t-test was applied to indicated comparisons.

Article Snippet: DNA SMART ChIP-Seq Kit (Takara) was used for library generation of samples subjected to Illumina sequencing.

Techniques: ChIP-sequencing, Sequencing, In Vitro, Labeling, Nucleic Acid Electrophoresis, Fluorescence, Staining, Dot Blot, Control, Binding Assay