Journal: bioRxiv

Article Title: ERCC1-XPF Interacts with Topoisomerase IIβ to Facilitate the Repair of Activity-induced DNA Breaks

doi: 10.1101/2020.01.03.892703

Figure Lengend Snippet: Genome-wide mapping of DSBs in tRA-treated and Ercc1 −/− MEFs. (A) . Genome-wide enrichment (read count; R.C.) of DNA DSBs (normalized per million mapped reads) on -/+2Kb flanking the TSS in untreated (Untr.), (B) . tRA-treated (tRA) and (C) . Ercc1 −/− MEFs. (D) . BLESS signals quantified by qPCR on the tRA-inducible Cfh , Rarb and Hs3st1 gene promoters, the tRA non-inducible Chordc1 gene promoter, as well as on a non-transcribed intergenic region (-) region in untreated MEFs (Untr.) or MEFs treated with tRA (tRA) or tRA and merbarone (tRA/merb). (E) . Enrichment of ERCC1, TOP IIβ and CTCF proteins on DSB-containing genomic fragments derived from primary MEFs. (F) . Venn’s logic diagrams representing the number of transcription-associated DNA DSBs on XPF-bound genomic sites in untreated (Untr.) and tRA-treated MEFs. (G) . Probability of XPF recruitment on genes by means of log^2RNA*log^2Breaks variable from the RNA-Seq and BLISS data. ( H ). BLESS signals quantified by qPCR on Rarb2 gene promoter in tRA-treated wt. and Ercc1 −/− MEFs. ( I ). BLESS signals quantified by qPCR on the promoters of PrlR and Dio1 genes known to be expressed in the P15 Ercc1 −/− and wt. livers and on the promoter of non-expressed Neun gene. ( J ). BLESS signals quantified by qPCR on the promoter of Dhfr and Prnp genes known to be expressed in the P15 Ercc1 −/− and wt. cerebella and on the promoter of the non-expressed Alb gene. Error bars indicate S.E.M. among n > three biological replicates. Asterisk indicates the significance set at p-value: *≤0.05, **≤0.01, ***≤0.001 (two-tailed Student’s t-test).

Article Snippet: Cells were rinsed with PBS, exposed to UVC irradiation (10 J/m2 ), MMC (10 μg/mL) (AppliChem), tRA (10 μM) (Sigma-Aldrich) or merbarone (2 μM) (Sigma-Aldrich) and cultured at 37°C for 1 to 16h prior to subsequent experiments.

Techniques: Genome Wide, Real-time Polymerase Chain Reaction, Derivative Assay, RNA Sequencing Assay, Two Tailed Test

Journal: bioRxiv

Article Title: ERCC1-XPF Interacts with Topoisomerase IIβ to Facilitate the Repair of Activity-induced DNA Breaks

doi: 10.1101/2020.01.03.892703

Figure Lengend Snippet: Transcription-associated DNA damage events require TOP IIβ. (A). Immunofluorescence detection of γH2AX and 53BP1 (white arrowheads) in wt. tRA-treated MEFs cultured in the presence of tRA, merbarone (Merb.) or tRA and merbarone (as indicated). The graph represents the number of γH2AX + 53BP1 + cells under the conditions shown in the x-axis. (B) . bXPF ChIP signals on the tRA-inducible Cfh , Rarb and Hs3st1 gene promoters, the tRA non-inducible Chordc1 gene promoter, as well as on a non-transcribed intergenic region (-) region in MEFs treated with tRA, merbarone (merb.) or tRA and merbarone. The ChIP signals are shown as fold enrichment of the percentage of input for bXPF over the percentage of input for BirA. ( C ). Immunofluorescence detection of γH2AX and 53BP1 in wt. untreated MEFs and wt. MEFs treated with etoposide. The graph represents the number of γH2AX + 53BP1 + cells in etoposide-treated and corresponding control cells. ( D ). bXPF ChIP signals on the tRA-inducible Cfh , Rarb and Hs3st1 gene promoters, the tRA non-inducible Chordc1 gene promoter, as well as on a non-transcribed intergenic region (-) region in MEFs treated with etoposide. (E) . Cumulative number of DNA DSBs per chromosome in untreated (Untr.), tRA-treated (tRA) or Ercc1 −/− MEFs (as indicated); for each color, the light-and dark-colored lines indicate the two BLISS replicates for each experimental condition. The red dotted line represents the average of the two biological replicates in untreated (Untr.) control MEFs. (F) . Number of DNA DSBs per million mapped reads on gene promoters in untreated (Untr.), tRA-treated (tRA) or Ercc1 −/− MEFs (as indicated); for each color, the light- and dark-colored bars indicate the two BLISS replicates for each experimental condition. The red dotted line represents the average of the two biological replicates in untreated (Untr.) control MEFs. (G) . Number of DNA DSBs per million mapped reads on gene bodies in untreated (Untr.), tRA-treated (tRA) or Ercc1 −/− MEFs (as indicated); for each color, the light- and dark-colored bars indicate the two BLISS replicates for each experimental condition. The red dotted line represents the average of the two biological replicates in untreated (Untr.) control MEFs. ( H ). Number of DNA DSBs per million mapped reads on intergenic regions in untreated (Untr.), tRA-treated (tRA) or Ercc1 −/− MEFs (as indicated); for each color, the light- and dark-colored bars indicate the two BLISS replicates for each experimental condition. Error bars indicate S.E.M. among n > three biological replicates. Asterisk indicates the significance set at p-value: *≤0.05, **≤0.01, ***≤0.001 (two-tailed Student’s t-test). For Figure 6E-H , a triple asterisk indicates the significance set at p-value ≤ 10 −15 (Mann Whitney test). Grey line is set at 10 μm scale.

Article Snippet: Cells were rinsed with PBS, exposed to UVC irradiation (10 J/m2 ), MMC (10 μg/mL) (AppliChem), tRA (10 μM) (Sigma-Aldrich) or merbarone (2 μM) (Sigma-Aldrich) and cultured at 37°C for 1 to 16h prior to subsequent experiments.

Techniques: Immunofluorescence, Cell Culture, Chromatin Immunoprecipitation, Two Tailed Test, MANN-WHITNEY

Journal: Molecules

Article Title: In Vitro Action of Flavonoids in the Canine Malignant Histiocytic Cell Line DH82

doi: 10.3390/molecules181215448

Figure Lengend Snippet: Chemical structure of flavonoids and topoisomerase inhibitors. ( A ) General structure of flavonoids; ( B ) general structure of chalcones; ( C ) quercetin; ( D ) myricetin; ( E ) fisetin; ( F ) epigallocatechin gallate (EGCG); ( G ) genistein; ( H ) luteolin; ( I ) apigenin; ( J ) trans- chalcone; ( L ) 4-methoxychalcone; ( M ) etoposide; ( N ) merbarone.

Article Snippet: Materials The topoisomerase inhibitors merbarone, etoposide, quercetin, trans -chalcone, and 4-methoxychalcone were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques:

Journal: Molecules

Article Title: In Vitro Action of Flavonoids in the Canine Malignant Histiocytic Cell Line DH82

doi: 10.3390/molecules181215448

Figure Lengend Snippet: Inhibition of the viability of DH82 cells induced by flavonoids and topoisomerase after 24, 48 and 72 h of treatment. ( A ) Quercetin; ( B ) myricetin; ( C ) fisetin; ( D ) epigallocatechin gallate (EGCG); ( E ) genistein; ( F ) luteolin; ( G ) apigenin; ( H ) trans-chalcone; ( I ) 4-methoxychalcone; ( J ) etoposide; ( L ) merbarone. Symbols represent the mean of at least three independent experiments and the error bar indicates the standard deviation. Different letters indicate statistically significant results ( p

Article Snippet: Materials The topoisomerase inhibitors merbarone, etoposide, quercetin, trans -chalcone, and 4-methoxychalcone were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Inhibition, Standard Deviation

Journal: Molecules

Article Title: In Vitro Action of Flavonoids in the Canine Malignant Histiocytic Cell Line DH82

doi: 10.3390/molecules181215448

Figure Lengend Snippet: Effect of flavonoids and topoisomerase inhibitors on the expression of topoisomerase IIa and IIb genes. The cells were treated with flavonoids at a concentration of 12.5 µg/mL for 6 h. The concentration of etoposide and merbarone was 2.5 µg/mL. The results are expressed as the mean ± standard error of at least three independent experiments. Different letters indicate statistically significant results ( p

Article Snippet: Materials The topoisomerase inhibitors merbarone, etoposide, quercetin, trans -chalcone, and 4-methoxychalcone were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Transient dsDNA breaks during pre-replication complex assembly

doi: 10.1093/nar/gkp617

Figure Lengend Snippet: Origin association of replication initiator proteins during G 1 phase. ( A ) Immunostaining showing the nuclear expression of ORC2, ORC4, Cdt1 and Cdc45 upon release from M phase. ORC2, known to be stably expressed throughout the cell cycle, represents the loading control. ( B–C ) ChIP assay showing the association of ORC2 (i), ORC4 (ii), Cdt1 (iii) and Cdc45 (iv) with the hOrs8 (B) and lamin B2 (C) origins with (grey bars) or without (black bars) merbarone treatment. Abundance of immunoprecipitated origin DNA is expressed in molecules per 2 × 10 7 cells and the error bars represent three experiments and 1 SD. The horizontal bars represent background DNA immunoprecipitated by NRS.

Article Snippet: Treatments with the topo II inhibitor merbarone (100 μM) (Calbiochem) and the DNA-PK inhibitor NU-7026 (10 μM) (Calbiochem) were performed for 1 h at 37°C.

Techniques: Immunostaining, Expressing, Stable Transfection, Chromatin Immunoprecipitation, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Transient dsDNA breaks during pre-replication complex assembly

doi: 10.1093/nar/gkp617

Figure Lengend Snippet: DNA-break formation during G 1 phase. ( A ) Schematic diagram explaining the three different scenarios of the DNA-break labelling assay. No break formation (left) should lead to background levels of DNA immunoprecipitated by the anti-biotin antibody. In the case of a break in proximity to the origin, but outside of the core region (middle), increased amounts of the PCR products upstream (Set 1), downstream (Set 2) and flanking (Ori) the origin region should be produced, while an intra-origin break (right) should result in increased Set 1 and Set 2, but decreased Ori products. ( B ) Generation of putative DNA breaks in hOrs8 (i) and lamin B2 (ii) during G 1 phase. Encircled are the two consecutive DNA breaks formed at the two chromosomal loci, two intra-origin breaks in hOrs8, and one inter-origin and one intra-origin in lamin B2. Values are expressed as ratios of the individual DNA abundances at each timepoint to the maximal abundance observed during the course of the assay, following normalization with the non-origin containing regions. Error bars represent 1 SD of three separate experiments. ( C ) Effect of merbarone on DNA-break formation at the hOrs8 (i) and lamin B2 (ii) replication origins. Error bars represent 1 SD of three separate experiments. ( D ) DNA-break labelling assay at the hOrs8 (i) and lamin B2 (ii) replication origins following NU-7026 treatment. Error bars represent 1 SD of three separate experiments.

Article Snippet: Treatments with the topo II inhibitor merbarone (100 μM) (Calbiochem) and the DNA-PK inhibitor NU-7026 (10 μM) (Calbiochem) were performed for 1 h at 37°C.

Techniques: Immunoprecipitation, Polymerase Chain Reaction, Produced

Journal: Nature Communications

Article Title: Topoisomerase II? promotes activation of RNA polymerase I transcription by facilitating pre-initiation complex formation

doi: 10.1038/ncomms2599

Figure Lengend Snippet: Effects of Top2 inhibition on Pol I transcription. ( a ) Schematic of pulse-chase labelling of cells with 3 H-uridine to determine effects of Top2 inhibition on rRNA synthesis by Pol I (used in b and c ) ( b ) Pre-rRNA (47/45S), 28S and 18S rRNA transcript levels in actively growing U2OS cells were unaffected by treatment with Top2 inhibitor merbarone (Me) for up to 15 h. 3 H-uridine was added 1 h after Me; non-radioactive uridine was added after an additional 2 h. Total RNA was extracted after 0, 4 and 12 h, separated by formaldehyde agarose gel electrophoresis and transferred to membrane. Newly synthesized pre-rRNA and rRNA transcripts were detected by autoradiography (representative experiment; upper panel); total 18S and 28S rRNAs were detected by ethidium bromide staining (lower panel). Relative efficiencies of rRNA synthesis and transcript processing are shown (bar graphs: −Me, dark-blue bars;+Me, light-red bars) for 47S/45S pre-rRNA (top), 28S rRNA (middle) and 18S rRNA (bottom), expressed as percentage of highest value (set at 100%). s.d. is shown, n =3; * P

Article Snippet: For Top2 inhibition, Top2 poison etoposide (100 μM final concentration; Merck) or catalytic inhibitors ICRF-193 (50 μM) and merbarone (100 μM; Merck) were added (except ).

Techniques: Inhibition, Pulse Chase, Agarose Gel Electrophoresis, Synthesized, Autoradiography, Staining

Journal: Nature Communications

Article Title: Topoisomerase II? promotes activation of RNA polymerase I transcription by facilitating pre-initiation complex formation

doi: 10.1038/ncomms2599

Figure Lengend Snippet: Top2α facilitates formation of PICs in Pol I transcription activation. ( a , b ) Top2 inhibition reduces activation of Pol I transcription in serum-refed cells. H1299 cells (p53 null) ( a ) or U2OS cells ( b ) were serum-starved for 20 h and (starting at –1 h) incubated with Top2 inhibitors etoposide (Et) or merbarone (Me), respectively, or without inhibitors, for 1 h. At 0 h, serum was added to the cells. RNA was extracted 15, 30, 60 and 90 min after serum addition. Pre-rRNA levels were analysed by S1 nuclease protection (complementary assay, 3 H-uridine labelling of RNA, Supplementary Fig. S6a,b ); s.d. is shown. ( c ) Top2α depletion reduces activation of Pol I transcription in serum-refed cells. HTETOP cells were incubated for 48 h +/− Tet, with serum starvation for the last 20 h. Serum was added back and Pol I transcription assayed as in a (complementary assay, Supplementary Fig. S6c ). ( d , e ) Inhibition of Top2 or depletion of Top2α reduces the accumulation of PIC components SL1, UBF and Pol I at the rDNA promoter in serum-refed cells. ChIP assays were performed on chromatin from cells treated as in a and c , respectively, using antibodies for Top2α, TAF I 63 (TAF1B; SL1), UBF and A135 (Pol I), and normalized to control IgG samples. s.d. is shown, n =3; *** P

Article Snippet: For Top2 inhibition, Top2 poison etoposide (100 μM final concentration; Merck) or catalytic inhibitors ICRF-193 (50 μM) and merbarone (100 μM; Merck) were added (except ).

Techniques: Activation Assay, Inhibition, Incubation, Chromatin Immunoprecipitation