EMBO/soda-nel huggingface dataset:
The Torpedo Effect in Bacillus subtilis: RNase J1 Resolves Stalled Transcription Complexes (2019)
Figure 1

Figure 1.Global changes in ΔrnjA compared to wt.

A, B. Comparisons of RNAseq and ChIPseq data. Wt strain (A) and ∆rnjA (B). The x-axis shows the relative RNAP occupancy at a given gene (normalized ChIPseq coverage). The y-axis shows the relative abundance of the transcript (normalized RNAseq coverage; each dot is one gene). The genes are color-coded, ranging from yellow (low RNA abundance) to red (high RNA abundance) in wt. The ∆rnjA strain has higher RNAP occupancy mainly among the genes with less abundant transcripts. The color coding in (B) reveals no dramatic overall changes in the vertical direction (RNA abundance). If anything, some of the low abundance transcripts decreased further in level in the mutant strain. The main difference (mostly among the low abundance transcripts) is their shift in the horizontal direction to the right (towards higher occupancy with RNAP). Data represent mean values of three independent experiments.

C. Relative expression of all sigma factors in wt (LK1371) versus ΔrnjA (LK1381). Wt levels of each sigma factor were set as 1 (indicated with the horizontal line).

D. Sigma-dependent genes and correlation with expression of sigma factors (significantly changed) in ΔrnjA (LK1381). The most down-regulated sigma factor was sigD and almost all sigD-dependent genes were downregulated. A similar trend is visible for the rest of sigma factors and their respective dependent genes. The x-axis shows expression of each sigma factor, the y-axis shows expression of genes for each sigma regulon. The violet (dark blue) line: regression line.
The Torpedo Effect in Bacillus subtilis: RNase J1 Resolves Stalled Transcription Complexes (2019)
Figure 2

Figure 2. Class I, II, III, and IV genes.

Average gene profiles of normalized RNA-seq and RNAP ChIPseq coverages from wt (solid lines) and ∆rnjA (dashed lines) strains were plotted for gene classes I-IV (n = 762, 315, 553 and 1654 genes, respectively). Open reading frames were rescaled to 1 kb, upstream and downstream regions of 0.2 kb were also included in the plots. Data represent mean values of 3 independent experiments. For definition of class I-IV genes, see text and Supplementary Materials and Methods in Appendix (page S20). The pie-chart shows the overall distribution of class I-IV and other genes.
The Torpedo Effect in Bacillus subtilis: RNase J1 Resolves Stalled Transcription Complexes (2019)
Figure 3

Figure 3. RNAP and RNase J1 may interact in the cell.

A. SIM of exponential B. subtilis cells. RNase J1 was fused to GFP (green), RNAP to mCherry (red). The graph shows relative fluorescence intensities at the cell midsection (along the long axis); SIM of the cell is below. Yellow indicates colocalization of the two proteins.

B. Pull-down with RNase J1-8xHis tag and RNAP-10xHis tag and detection of the proteins with antibodies. Lanes 1, 2 - RNase J1-8xHis was used to pull down RNAP; lanes 3, 4 - RNAP-10xHis was used to pull down RNase J1; lanes 5, 6 - purified proteins were used as markers; lanes 7, 8 - strains without His-tagged proteins were used as negative controls to demonstrate the specificity of the interaction. M, molecular size marker; EX, exponential phase; STA, stationary phase.

C. Pull-down with RNase J1-8xHis tag from stationary phase cells - the same conditions as in (B). The samples then either were (lane 9) or were not (lane 10) treated with RNase A to detect whether the interaction was via RNA. Lanes 11, 12 - purified proteins were used as markers. The experiment was performed three times (biological replicates) with the same result.
The Torpedo Effect in Bacillus subtilis: RNase J1 Resolves Stalled Transcription Complexes (2019)
Figure 4

Figure 4. RNase J1 disassembles stalled transcription complexes (TC).

A. Schematic representation of the flow of the experiment. TCs were assembled on DNA-RNA scaffolds (DNA was biotinylated); RNA was labeled at 3' with radioactive UTPs (asterisks). The RNA length (including label) was 33 nt.

B. Representative primary data - polyacrylamide gel (the experiment was performed 3x with the same results). Lane 1, the full length (33 nt) labeled RNA; lane 2, the same as lane 1 but it included also incubation with RNase J1 (J1), 17-18 nt long fragments (RNase J1 stopped by RNAP; indicated with asterisks) and <5 nt fragments (RNA released from TC into buffer - indicative of TC disassembly) are shown; lane 3, the same as lane 1 but included also incubation with RNase R (R); lanes 4 and 5, TCs were denatured by heat prior to RNase addition to demonstrate the activity and cleavage patterns of both enzymes; lanes 6 and 7, the same as lanes 2 and 3 but the buffer was washed off (TCs were retained by streptavidin beads) to demonstrate which RNA fragments were associated with TC; lanes 8, 9 (M1, M2) Mw marker generated by treating the 30 nt RNA with alkali and formamide (M1-4 min treatment, M2-7 min treatment). As reported in (Costanzo et al., 2016) [and references therein], the cleavage by alkali or formamide leaves the phosphate group of the attacked phosphodiester bond bound at 3', initially in the 2',3' cyclic form (upper band in the band couples). This successively opens (lower band in the band couples) yielding a double-banded pattern for short oligoribonucleotides.
The Torpedo Effect in Bacillus subtilis: RNase J1 Resolves Stalled Transcription Complexes (2019)
Figure 6

Figure 6. RNase J1 specifically dissociates RNAP from DNA in stalled TCs.

A. TCs were assembled on DNA-RNA scaffolds (DNA was biotinylated and attached to streptavidin coated magnetic beads); TCs were then divided into three tubes and challenged with buffer ("-", mock treatment) or RNase J1 of Xrn1. Dissociation of RNAP from DNA was monitored by detecting RNAP with anti-β antibody in two fractions: B - beads (RNAP still bound to DNA), R - released (free in buffer). The gel strip shows representative primary data (Western blot). The graph shows averages (the bars) of two experiments (biological replicates) and the error bars show the range. The combined signal for B+R for each treatment was set as 100 %.

B. Primary data - polyacrylamide gel. The experimental setup was the same as in Fig. 5 but with RNAP from E. coli. Lane 1, the full length (33 nt) labeled RNA; lane 2, the same as lane 1 but it included also incubation with RNase J1 (J1), lane 3, the same as lane 1 but included also incubation with Xrn1.

C. Quantitation of three independent experiments. "Full length" indicates the remaining undigested RNA. "OUTSIDE", "INSIDE" are fragments as explained in Fig. 5. The bars represent 100 % (all fragments). The black-grey-white boxes indicate the percentage of each fragment group (in %). The error bars indicate ± SEM for each group of fragments
The Torpedo Effect in Bacillus subtilis: RNase J1 Resolves Stalled Transcription Complexes (2019)
Figure 7

Figure 7. Effect of RNase J1, Rho and HelD in UV sensitivity assays.

A. Exponential cells of indicated strains (below the bars) were plated onto LB agar and either were, or were not UV-irradiated. After overnight incubation, CFU were counted. UV sensitivity of the mutant strains (KO) was then calculated as the ratio between irradiated vs non-irradiated cells and normalized to this ratio from the wt strain As a consequence, the wt ratio is 1. The experiment was conducted 4x (biological replicates) and the bars show the geometric mean. The p-values, shown above the bars, were computed using two-tailed unpaired t-test for logarithms of the ratios. The error bars show ±SEM (computed on the log scale).

B. Relative expression (mRNA) of helD, mfd, rho, greA, nusA, nusB, and nusG in the ∆rnjA strain (normalized to wt [set as 1]).
Prostate cancer cell-intrinsic interferon signaling regulates dormancy and metastatic growth in bone (2020)
Figure 2

Figure 2 Intrinsic type I IFN pathways are specifically suppressed in proliferating PCa cells from bone

(A-C) Bulk RNA-seq analysis of DE genes (DEG) significantly suppressed (FDR < 0.05; generalized linear model extraction (GLM) by edgeR) in proliferating RM1 bone-derived (BD) cells compared to parental RM1 and lung metastases. Tumor-intrinsic type I IFN signaling is suppressed in proliferating bone lesions (n = 1) compared to parental RM1 (n = 2) cells as shown by (A) camera analysis of Hallmark gene set responses associated with bone metastasis (bar length is the -log10FDR; false discovery rate). Color indicates mean log2fold change (FC) of all genes in gene set. Bar width is relative gene set size. Dashed line shows FDR = 0.05; (B) barcode plot showing enrichment of the Hallmark IFN-α response gene set in genes suppressed in bone metastases, using the signed log-likelihood ratio statistic (EdgeR). Bars indicate value of the statistic for each gene in the gene set; (C) log2FC values of key downregulated IRGs, with genes retained in dormant cells and classical downstream IFN signaling targets indicated. Altered IRGs directly involved in IFN-α/β production (light blue) compared to downstream IFN targets (dark blue) are segregated. p values represented as * < 0.05, ** < 0.005 and *** < 0.0005; GLM by edgeR.

(D) Schematic of proliferating RM1 cell isolation from bone and lung metastases following IC injection for preliminary RNA-seq and orthogonal validation by qRT-PCR.

(E) Heatmap of mean normalized voom expression of IRGs suppressed in RM1 bone metastases (n = 1) compared to lung metastases (n = 1) and parental cells (n = 2).

(F) qRT-PCR validation of Irf7 and Irf9 downregulation in RM1 cells from bone metastases (RMI BD) compared to parental RM1 cells, lung metastases (RM1 lung) and naïve bone marrow (BM) (n = 3 mice per group). p values represented as ** < 0.005 and *** < 0.0005 (Student's t test).

(G) qRT-PCR of Irf7 and Irf9 downregulation in parental RM1 cells, RM1 cells from bone metastases (RM1 BD) in WT and Ifnar1 deficient (-/-) mice, with naïve BM from WT and Ifnar1 (-/-) animals for reference. (n = 3 mice per group; n = 1 for -/- BM control). p values represented as * < 0.05 and ** < 0.005 (Student's t test).

Data information: All error bars ± SEM.
Prostate cancer cell-intrinsic interferon signaling regulates dormancy and metastatic growth in bone (2020)
Figure 3

Figure 3 Loss of tumor-intrinsic type I IFN is inducible by bone marrow cells and is reversed by HDACi

(A) Stability of Irf7 and Irf9 mRNA suppression by qRT-PCR in ex vivo bone-derived cells (RM1 BD Irf-; n = 7) in culture compared to a bone-derived line that showed initial loss during early passage (RM1 BD REV (EP); n = 3) then reverted to parental expression levels at late passage (RM1 BD REV (LP); n = 4) compared to parental RM1 (n = 3).

(B) ELISA of IFN-α production by RM1 parental (n = 3), RM1 bone-derived Irf low (RM1 BD Irf-; n = 4) and RM1 BD REV cells (n = 3) subsequent to poly I:C stimulation.

(C) qRT-PCR analysis of Irf7 and Irf9 expression in RM1 BD Irf- cells ± 48 h treatment with MS275 (1 μM) (n = 7-9).

(D) Schematic of contact and transwell co-culture systems.

(e) qRT-PCR analysis of Irf7 and Irf9 expression in parental RM1 cells (n = 4) 48 h post-contact culture with naïve BM (n = 6).

(F) qRT-PCR analysis of Irf9 expression in parental RM1 cells ± 48 h co-culture with naïve BM under contact (non-transwell; NT) and transwell (0.4 μm filters that prevent cell contact) conditions (n = 6-8 per condition).

(G) qRT-PCR analysis of Irf9 expression in parental RM1 cells ± 48 h contact co-culture with naïve BM ± MS275 (1 μM) (n = 3-6 per condition).

Data information: p values represented as * < 0.05, ** < 0.005 and *** < 0.0005 (Student's t test). All error bars ± SEM.
Prostate cancer cell-intrinsic interferon signaling regulates dormancy and metastatic growth in bone (2020)
Figure 4

Figure 4 Molecular restoration of tumor-intrinsic type I IFN suppression inhibits bone-metastatic outgrowth and modulates osteoimmunity

(A) Bone metastasis-free survival in WT C57BL/6 mice harboring RM1 parental, RM1 BD Irf- and RM1 BD REV (LP) tumors (n = 5 per group; **p = 0.0043 by log-rank (Mantel-Cox) test).

(B) Schematic of enforced Irf7 expression in RM1 BD Irf- cells under exogenous promoter control.

(C) ELISA of IFN-α production by RM1 parental, RM1 BD Irf- base vector (BV) and RM1 BD Irf7 over-expressing (OE) with 24 h poly I:C stimulation (n = 4).

(D) Bone metastasis-free survival in WT C57BL/6 mice subsequent to IC inoculation of RM1 BD Irf- BV (n = 9) and RM1 BD Irf7 OE (n = 7) cells (**p = 0.0028 by log-rank (Mantel-Cox) test).

(E) Bone metastasis-free survival in Ifnar1 -/- mice harboring RM1 BD Irf- BV and RM1 BD Irf7 OE tumors (n = 4 per group) and representative bioluminescent imaging of tumor burden in leg bones (also shown in Appendix Fig S4F).

(F) Immunohistochemical (IHC) staining for cerulean (anti-eCFP; green) on naïve WT bone, RM1 parental, RM1 BD Irf- BV and RM1 BD Irf7 OE tumor-bearing bones derived from WT animals; and RM1 BD Irf7 OE tumor-bearing bones derived from Ifnar1 -/- animals at survival assay endpoints. Blue represents DAPI nuclear staining. Scale bar, 100 μm.

(G-I) FACS analysis of dormant (claret+) RM1 BD Irf- and RM1 BD Irf7 OE cells from bone at (G) day 11 post-IC injection (n = 4 per group) and (h) day 17 (n = 4-6 mice group/time point) with (I) D17 active (claret-) colony quantitation (mean +1) determined by multiphoton imaging and IMARIS interrogation (n = 9 bones from 3 mice per condition). Median shown. Upper and low box hinges denote first and third quartiles. Whiskers mark value limits.

(J) FACS analysis of IFN-γ+ and TNF-α+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from tumor-bearing mice) activation by RM1 parental, RM1 BD Irf- BV and RM1 BD Irf7 OE cells (n = 6 per condition/group). (K) Mean quantitation of TRAP-stained osteoclasts differentiated with M-CSF ± RANKL in co-culture with RM1 BD Irf- BV and RM1 BD Irf7 OE cells with representative wells shown (n = 3 per condition M-CSF; n = 6 per condition M-CSF + RANKL; also shown in Appendix Fig S4J). Scale bar represents 100 μm.

Data information: p values represented as * < 0.05, ** < 0.005 and *** < 0.0005 (Student's t test). All error bars ± SEM, except where stated.
Prostate cancer cell-intrinsic interferon signaling regulates dormancy and metastatic growth in bone (2020)
Figure 5

Figure 5 Bone metastasis is inhibited by therapeutic induction of cell-intrinsic (HDACi) and systemic type I IFN

(A) ELISA of IFN-α production by RM1 BD Irf- cells ± single agent MS275 and poly I:C treatment or 48 h pre-treatment with MS275 (pMS) prior to poly I:C stimulation (n = 6).

(B) Mean fluorescence intensity of H2-Kb staining on RM1 BD Irf- cells by FACS ± MS275, poly I:C or combination treatment (n = 3).

(C) FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from RM1 BD Irf- tumor-bearing mice) activation upon re-stimulation with RM1 cells and MS275, poly I:C or combination treatment, with NAC (no antigen-presenting cells) and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1) with (D) representative FACS plots shown.

(E) Bone metastasis-free survival in WT C57BL/6 mice harboring RM1 BD Irf- cells ± MS275, poly I:C or combination treatment (n = 6 per group; log-rank (Mantel-Cox) test).

(F) Mean total tumor burden in bone (femurs, tibias, spine and humerus) at endpoint by bioluminescent intensity score (log2 p/sec/cm2/sr) with values < 4.0x104 representing zero burden (n = 24 bones per group; 6 mice per group) and representative bioluminescent imaging of tumor burden in spine (all shown in Appendix Figure S5E).

Data information: p values represented as * < 0.05, ** < 0.005 and *** < 0.0005 (Student's t test in A-C and F; log-rank (Mantel-Cox) test in E). All error bars ± SEM.
Prostate cancer cell-intrinsic interferon signaling regulates dormancy and metastatic growth in bone (2020)
Figure 6

Figure 6 HDACi and systemic IFN induction provides long-term immune protection from bone-metastatic colonization and outgrowth

(A, B) FACS analysis of (A) peripheral blood (PB) T and NK lymphocyte representation and activation status and (B) FoxP3+ CD4+ and CD4+ effector memory T cell status at days 4 and 10 post-IC tumor cell inoculation across treatment settings (n = 4-6 per group).

(C, D) Bioluminescent imaging (C) of a combination group mouse with tumor clearance from day 7 to 41 (full experimental cohort shown in Appendix Fig S5E) post-IC inoculation with (D) associated specific CD8+ memory T cell response. This is represented by FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from tumor-bearing mice) activation upon re-stimulation with RM1 BD Irf- cells and MS275, poly I:C or combination treatments in vitro, with NAC and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1). Representative FACS plots shown on right.

Data information: p values represented as * < 0.05, ** < 0.005 and *** < 0.0005 (Student's t test). All error bars ± SEM.
Prostate cancer cell-intrinsic interferon signaling regulates dormancy and metastatic growth in bone (2020)
Figure 7

Figure 7 IFN signaling is decreased in bone-metastatic PCa

(A-C) Compositional analysis of RNA-seq data (FDR < 0.05; GLM by edgeR) from bone metastases (n = 9) and primary tumors (n = 12) revealed (A) immune cells and (B) tumor cells were relative (% of total) across all samples, with a mean tumor purity >85% as determined by ARMET. Median shown. Upper and low box hinges denote first and third quartiles. Upper and lower whiskers mark the values ± 1.5IQR from the hinge. Data points beyond the whiskers are shown individually. p values not significant by Wilcoxon rank sum test (R). Hallmark gene set responses associated with bone metastasis by camera analysis shows (C) enrichment of type I (IFN-α) IFN responses in suppressed genes (barcode plot).

(D) Heatmap of type I IFN DE IRGs (normalized log2CPM; counts per million) significantly suppressed in bone metastases (n = 9) compared to primary (n = 12) and benign (n = 3) tumors, grouped by type I IFN specific, and type II and III IFN co-regulation status. Scale truncated at ± 5 relative log2CPM. GLM by edgeR.

(E) IHC for IRF9 expression in primary prostate tumors and bone metastases with matched samples indicated. IRF9 indicated by brown (DAB) staining. Scale bar represents 100 μm. Full-face slides shown in Appendix Fig S7C.

(F) Heatmap of HLA genes (normalized log2CPM) suppressed (p <0.05; FDR <0.05) in bone metastases (n = 9), primary tumors (n = 12) and benign tumors (n = 3). Scale truncated at ±5 relative log2CPM. GLM by edgeR.

(G) CIBERSORT analysis of absolute leukocyte proportions (arbitrary units, AU) in mRNA samples of bone metastases (n = 9), primary tumors (n = 12) and benign tumors (n = 3). p values by Mann-Whitney U.

(H) Kaplan Meier Curve of human prostate (n = 499 from 498 TCGA samples) biochemical recurrence based on primary tumor alterations in a core 8-IRG signature (Appendix Fig S7D). Patient groups stratified by mRNA z-Score (RNA-seq V2 RSEM) ± 1.5. Hazard ratio 1.674 (95% confidence interval (CI) 2.707 to 1.035); *p = 0.03 by log-rank test.

Expanded View figure legends
Genome-wide cooperation of EMT-transcription factor ZEB1 with YAP and AP-1 in breast cancer (2020)
Figure 1

Figure 1. ZEB1 DNA-binding sites overlap with YAP- and JUN-binding sites.

(A) The three top enriched DNA-binding motifs identified by HOMER known motif analysis on 200 bp regions centred on the ZEB1 peak summits. An extended list can be found in Appendix Table S1.

(B) Enrichment profiles of TEAD4 and AP-1 consensus motifs in 600 bp regions centred on the ZEB1 peak summits. The two grey dashed lines highlight the 200 bp regions around the ZEB1 peak summits which were used for HOMER known motif analysis shown in (A).

(C) Overlap between ZEB1 and JUN DNA-binding sites. Sites are counted as overlapping when their peak summit positions are not more than 200 bp apart. P-value from permutation test.

(D) Enrichment profile of JUN peak summit positions in 1 kb regions centred on the ZEB1 peak summits. The two grey dashed lines highlight the region of +/- 200 bp around the ZEB1 peak summit positions where JUN peaks are counted as overlapping with ZEB1 peaks.

(E) Heatmap representing the signal enrichment of JUN ChIP-seq reads in 2 kb regions centred on the ZEB1 peak summits.

(F) Circle plot displaying the genome-wide distribution of ZEB1 (outer middle circle, blue), JUN (inner middle circle, green) and YAP (inner circle, red) peaks across all chromosomes (outer circle). Peak density is visualised as a heatmap with more intense colours indicating a higher number of peaks in a specific location.

(G) ZEB1, JUN and YAP DNA-binding overlaps at 1,993 genomic sites in MDA-MB-231 cells. Triple overlap is assumed when both JUN and YAP peak summit positions are within 200 bp of a ZEB1 peak summit position. P-value from permutation test.
Genome-wide cooperation of EMT-transcription factor ZEB1 with YAP and AP-1 in breast cancer (2020)
Figure 2

Figure 2. ZEB1 interacts with JUN and FOSL1.

(A) In situ proximity ligation assay (PLA) of ZEB1 with JUN, FOSL1 and TAZ shows close proximity of ZEB1 with JUN and FOSL1 but not TAZ in the nucleus of MDA-MB-231 cells indicated by red fluorescent dots. As negative control ZEB1 was transiently knocked down by siRNA. In the left panel representative microscopic images are shown. Scale bar = 20 µm. Quantification of the PLA is shown on the right. n=3; one representative experiment with means +/- s.e.m. of at least 900 cells per condition is shown; ***P ≤ 0.001; 2way-ANOVA plus Tukey´s post-test.

(B) Co-immunoprecipitation (Co-IP) of endogenous ZEB1 and FOSL1 or JUN in MDA-MB-231 cells shows co-precipitation of ZEB1 with FOSL1 and JUN.
Genome-wide cooperation of EMT-transcription factor ZEB1 with YAP and AP-1 in breast cancer (2020)
Figure 3

Figure 3. Characterisation of a ZEB1-only and a ZEB1/YAP/JUN peak set.

(A) Definition of two different ZEB1 peak subsets based on the presence or absence of overlapping YAP and JUN peaks. The two illustrative genome browser images show one example of a ZEB1-only peak and one example of a ZEB1/YAP/JUN peak (at the EFEMP1 gene and within the SHROOM3 gene, respectively). Control (ctrl) is the input for ZEB1 and IgG for the YAP and JUN ChIP-seqs. ChIP-seqs and respective controls were scaled accordingly.

(B) Circle plot displaying the genome-wide distribution of ZEB1-only (middle circle, blue) and ZEB1/YAP/JUN peaks (inner circle, orange) across all chromosomes (outer circle). Peak density is visualised as a heatmap with more intense colours indicating higher accumulation of peaks at a specific location.

(C) Localisation of ZEB1 peak summits relative to the closest TSS.

(D) Distribution of ZEB1-only and ZEB1/YAP/JUN peaks in respect to different genomic features. Promoters were defined as 1.5 kb upstream to 0.5 kb downstream of the TSS. Promoter peaks were annotated to their respective genes and integrated with transcriptome data of ZEB1 knockdown compared to control MDA-MB-231 cells.

(E) Hypergeometric testing showed that the association of ZEB1-only promoter peaks with genes repressed in the presence of ZEB1 is stronger than with activated genes, while ZEB1/YAP/JUN promoter peaks are only associated with activated genes. Odds ratio (OR): measure of association between categorical variables with OR > 1 indicating a positive association. P-value from Fisher's exact test.

(F) GO term analysis of the ZEB1-only repressed and ZEB1/YAP/JUN activated gene sets using Enrichr. Odds ratios are given in brackets.