Fig 2: ARIP4 enforces transcriptional induction in reporter cells.(A) Schematic representation of the U2OS DISC reporter system. The U2OS DISC reporter (U2OS 263) cell line stably expresses YFP-tagged MS2 protein. Dox treatment (+Dox) induces the expression of the MS2 mRNA (stem-loop structure), which is recognized by the YFP-MS2 protein (green). The reporter also contains LacO repeats, which are recognized by mCherry–Lac I (red). (B) Representative images of DISC reporter cells. (Left) Without Dox, YFP-MS2 is evenly distributed in the nucleus. mCherry–Lac I (red dot) recognizes the LacO array, denoting the reporter cassette (arrowhead). (Right) Upon Dox addition, nascently transcribed MS2 mRNA is recognized by YFP-MS2, forming a bright green dot at the reporter cassette (arrowhead). (C) Representative time-lapse images of a Dox-induced reporter cell expressing mCherry-ARIP4. Arrowheads indicate the position of the reporter locus. (D) Immunofluorescence images of Dox-induced reporter cells upon depletion of ARIP4. (E) Representative Western blot of ARIP4-depleted DISC reporter cells used in (D). (F) Quantification of nascent transcription marked by YFP-MS2 accumulation (YFP−MS2+) upon ARIP4 depletion of data represented in (D). (G) Immunofluorescence images of ARIP4-depleted Dox-induced reporter cells reconstituted with gRNA-2–resistant WT and helicase-dead (DE462/463AA and K310A) mutants of V5-ARIP4-3XFLAG. (H) Quantification of nascent transcription marked by YFP-MS2 accumulation upon ARIP4 reconstitution shown in (G). (I) Immunofluorescence images of Dox-induced reporter cells treated with TOP2 inhibitors (TOP2i). Cells were pretreated with 25 μM etoposide or 50 μM merbarone for 4 hours before Dox treatment for an additional 4 hours. DMSO, dimethyl sulfoxide. (J) Quantification of nascent transcription marked by YFP-MS2 accumulation upon TOP2i. (K) Immunofluorescence images of Dox-induced reporter cells treated with indicated siRNAs. Cells were harvested at least 48 hours after siRNA treatment and 4 hours after Dox treatment. (L) Quantification of nascent transcription marked by YFP-MS2 accumulation upon indicated siRNA treatment.

Fig 3: ARIP4 is necessary for androgen-dependent break formation at promoter proximal regions of AR target genes.(A) (Top) Schematic of AR target genes KLK3 and TMPRSS2 and target regions used for break labeling ChIP-qPCR amplification. (Bottom) Break labeling assay upon 1 nM R1881 treatment at indicated time points (0 to 30 min). Biotin-dUTP incorporated into breaks formed upon androgen treatment was immunoprecipitated against immunoglobulin G (IgG) (negative control) or anti-biotin antibody. (B) Break labeling assay of ARIP4-depleted LNCaP cells. (C) Break labeling assay upon TOP2i and ARIP4 depletion. LNCaP cells were cultured in charcoal-stripped FBS for at least 48 hours and treated with merbarone (50 μM) for 4 hours prior to R1881 stimulation. (D) Break labeling assay upon flavopiridol (FP) and wash out (FP+wash) treatment as depicted in fig. S7F was conducted at indicated time points after 1 nM R1881 stimulation.

Fig 4: ARIP4 enforces androgen-dependent induction of AR target genes.(A) Representative images of different ARIP4 expression levels (scored 0 to 3) in normal prostatic tissue and prostate cancer TMA sections. (B) Quantification of ARIP4 staining scores in primary prostate tumor and paired nontumor tissue (n = 100). (C) Quantification of ARIP4 staining scores from prostatectomy and TURP TMA of various Gleason scores (total n = 160) compared to benign prostatic tissue of noncancer patients (n = 19). Data are presented as means ± SD. (D) mRNA expression of ARIP4 in ARIP4-depleted RNA-seq datasets. sgControl indicates cells expressing empty vector control, while sgARIP4-1 and sgARIP4-2 are ARIP4-depleted cells expressing CRISPR-Cas9 and two distinct sgRNAs targeting ARIP4. (E) Volcano plot of differentially expressed genes with and without androgen (R1881) (P-adjusted < 0.05) in WT (sgControl) LNCaP cells. (F) Box and whisker plots of fold induction of AR target gene expression upon 1 nM R1881 treatment with and without ARIP4.

Fig 5: ARIP4 preferentially binds and unwinds R-loops in vitro.(A) Purified ARIP4 and ARIP4 helicase mutants used in in vitro assays. (B) In vitro binding assay of increasing concentrations of ARIP4 with indicated nucleic acid structures (30 nM). (C) In vitro unwinding assay of indicated nucleic acid structures (10 nM) in the presence of ARIP4. dsDNA, dsRNA, and DNA:RNA hybrids were incubated with 400 nM ARIP4, while D-loops and R-loops were incubated with 40 nM ARIP4. Δ indicates reactions that were heat denatured as a positive control. (D) Quantification of ARIP4 unwinding activity against D-loops and R-loops by band intensities normalized to band intensity at 0 min. (E) In vitro unwinding assay of indicated nucleic acid structures (10 nM) in the presence of WT or helicase-dead (DE462/463AA and K310A) ARIP4. (F) In vitro binding assay of indicated nucleic acid structures (30 nM) in the presence of WT and helicase-dead (DE462/463AA and K310A) ARIP4.