Fig 2: DNA:RNA heteroduplex formation, caused by DHX9 repression, impairs transcription at the CCND1 promoter. (A) Representative western blot upon treatment with DMSO (vehicle), 5 µM etoposide (ETO), and combination of etoposide and KU 55933 (10 µM) (ETO + KU). Histograms represent DHX9 (B), cleaved PARP1 (C) (n.d. not detectable), Sam68 (D), and CCND1 (E) protein levels normalized to β-actin, relative to DMSO. (F) qPCR analysis of the promoter region of CCND1 gene on S9.6 immunoprecipitated chromatin (DRIP experiment). (G) RT-qPCR analysis of pncCCND1_B in DNA:RNA immunoprecipitated heteroduplexes (CARIP experiment). Statistical analysis was performed by ANOVA with Bonferroni correction ANOVA. (B–G) p-value (*, # < 0.05, ** < 0.01, ***, ### < 0.005). (H) Schematic representation of the CCND1 promoter region. Acetylation of histone proteins is required for the transcription of CCND1 gene by EWS-FLI1. This oncoprotein interacts with the DNA/RNA helicase DHX9, facilitating RNAPII recruitment and CCND1 transcription. Etoposide treatment enhances the transcription of the pncCCND1_B, whereas it inhibits the expression of CCND1 through the formation of an inhibitory complex due to the formation of RNA:DNA hybrid structures.

Fig 3: c-Myc binds to the Sam68 promoter region. (A) sqPCR and (B) qPCR analyses of ChIP experiments performed in LNCaP cells using c-MYC antibody and IgG (IgG), or no antibody (no ab), as negative control. Associated DNA is expressed as % of input (B). Schematic representation of the indicated gene promoters and of the 16q22 intergenic region is shown in (A) (left panel). c-MYC binding sites (solid box), TSS and primers position for PCR analyses (arrows) are indicated. (C) The bar graph represents the luciferase assay performed in HEK293T cells transfected with wt or mutated (Mut1, Mut2 and Mut1-2) Sam68 promoter reporters in combination, or not (empty vector, EV), with c-MYC-pCDNA3 vector (c-MYC). A schematic representation of wt and c-MYC binding site mutants (Mut1, Mut2 and Mut1-2) of the Sam68 promoter is also shown (left panel). (B and C) Data represent mean ± SD of three biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant (Student's t-test).

Fig 4: Sam68 and HDAC1 participate in the DNA damage response. (A) Immunofluorescence analysis was performed on TC-71 cells treated with either DMSO, 5 µM etoposide (ETO), or etoposide with pre-treatment of 10 µM of KU55933 (ETO + KU). (B) Western blot analysis of co-immunoprecipitation experiments performed from nuclear extracts of TC-71 cells treated with DMSO (vehicle), 5 µM etoposide (ETO), or etoposide with pre-treatment of 10 µM of KU 55,933 (ETO + KU). HDAC1 protein was immunoprecipitated with a specific antibody; CBP protein was used as the positive control. Sam68 was detected in the immunoprecipitated proteins. (C) Histograms represent the densitometric analysis of Sam68-HDAC1 interaction in each condition relative to DMSO, from three independent experiments. Statistical analysis was performed by ANOVA with the Bonferroni post hoc test. p-value (*, # < 0.05, ** < 0.01, n.s. > 0.05).

Fig 5: Etoposide induces the formation of DNA:RNA hybrids at CCND1 promoter. (A) Western blot analyses were performed on 15 μg of total extracts of TC-71 cells treated for 16 h with either DMSO (vehicle) or 5 µM etoposide (ETO). DHX9, Sam68, CCND1, and γH2A. X protein levels were analyzed. GAPDH was used as loading control. (B) Histograms represent Sam68 binding to pncCCND1_B from cross-linked and immunoprecipitation experiments. Values are expressed as input percentage and normalized to IgGs signal. (C) qPCR analysis of S9.6 immunoprecipitated chromatin from DNA:RNA heteroduplex in the promoter region of the CCND1 gene. As represented in the illustration, the S9.6 antibody recognizes DNA:RNA hybrids. (D) EWS-FLI1 recruitment on the CCND1 promoter was evaluated by ChIP experiments. Histograms represent input percentage from three independent experiments. (E) Western blot analysis to monitor recombinant expression of GFP or GFPDHX9 in TC-71 cells, treated with either DMSO or etoposide 5 μM. Ten μg of total extract was loaded in each lane. (F) qPCR analysis of S9.6 immunoprecipitated chromatin from DNA:RNA heteroduplexes in the promoter region of the CCND1 gene. Histograms represent input percentage. (G) RT-qPCR analysis of CCND1 expression in TC-71 cells transfected with either GFP or GFP-DHX9, and treated with DMSO or etoposide. Histograms represent three independent experiments (±S.D.). Statistical analysis was performed by ANOVA with Bonferroni post hoc test. p-value (*, # < 0.05, **, ## < 0.01, ***, ### < 0.005).