EMBO/soda-nel huggingface dataset:
Antithetical NFATc1–Sox2 and p53–miR200 signaling networks govern pancreatic cancer cell plasticity (2014)
Figure 5

NFATc1 promotes Sox2 transcription in PDAC

A Sox2 mRNA expression of pancreatic lysates from KNC, KPNC, and KPC mice, analyzed by real‐time PCR in three independent experiments (means ± SD).
B, C Western blot analysis shows Sox2 expression in pancreatic lysates of (B) KNC mice with indicated tumor grading and in (C) KPNC and KPC mice.
D Protein expression levels after siRNA‐mediated p53 depletion in KNC tumor cells.
E, F NFATc1 and Sox2 protein expression after (E) NFATc1 depletion or (F) upon CsA (1 μM, 24 h) treatment in Panc1 cells.
G Sox2 and E‐cadherin mRNA expression after NFATc1 depletion in L3.6 cells.
H Sequence alignment of 5′ and 3′ enhancer regions show highly conserved NFATc1 binding in different species; putative NFAT binding sides within the enhancer regions of the Sox2 promoter are highlighted in red.
I, J ChIP assays show NFATc1 binding to Sox2 enhancer and promoter in KNC tumor cells (I) and RNA polymerase II binding to Sox2 promoter upon CsA (1 μM, 24 h) treatment in L3.6 cells (J).
K Co‐immunoprecipitation of endogenous HA‐NFATc1 and Sox2 was performed in KPNC cells.
L ChIP experiments show NFATc1 binding along with H3K27 acetylation (H3K27ac) mark at the selected Snai1 enhancer region and RNA polymerase II binding at Snai1 promoter in the presence or absence of Sox2 in KPNC tumor cells. Representative results from at least three independent experiments are shown (means ± SD; P 0.05).
M Relative mRNA expression levels of EMT‐related marker genes upon Sox2 depletion in KPNC tumor cells (P 0.05).
N Zeb1 and Snail mRNA levels in L3.6 cells after shRNA‐mediated depletion of Sox2 and transient overexpression of NFATc1 shows that loss of Sox2 significantly rescues NFATc1‐mediated induction of EMT genes (*P 0.05).

Source data are available online for this figure.
Antithetical NFATc1–Sox2 and p53–miR200 signaling networks govern pancreatic cancer cell plasticity (2014)
Figure 6

The NFATc1–Sox2 complex promotes EMT and cancer stem cell‐like properties in PDAC

NFATc1 and Sox2 protein expression in L3.6 cells (adherent versus spheres).
Relative mRNA expression of NFATc1 and Sox2 in L3.6 cells as well as in KPNC mice tumor cells (adherent versus spheres). Graph represents data from three independent experiments (means ± SD). Asterisks display significance (*P 0.05).
Cell sorting was performed to collect CD44 high and low populations in KPNC spheres (left panel), which were analyzed for NFATc1 and Sox2 mRNA expression (right panel).
Sox2 mRNA expression upon CsA (1 μM) treatment in L3.6 cells (adherent versus spheres) (*P 0.05).
Bright‐field microscopy images as well as quantification of KPNC spheres after transient NFATc1 depletion.

Source data are available online for this figure.
Antithetical NFATc1–Sox2 and p53–miR200 signaling networks govern pancreatic cancer cell plasticity (2014)
Figure 7

p53 constrains NFATc1‐dependent promotion of EMT phenotype and cancer stem cell‐like properties through induction of miRNA‐200c

A Expression analysis of indicated miRNAs upon adenoviral re‐expression of p53 in KP∆NC tumor cells, detected by real‐time PCR (P 0.05).
B Protein expression of EMT marker genes after adenoviral re‐expression of GFP‐tagged p53 in KP∆NC tumor cells.
C, D Protein (C) and mRNA (D) expression levels of EMT and stemness marker genes upon miRNA‐34a and miRNA‐200c overexpression using specific mimics in KPNC tumor cells. Representative results from at least three independent experiments are shown (means ± SD; P 0.05).
E Western blot analysis of EMT marker expression in KNC cells following depletion of miR‐200c.
F ChIP experiments showing NFATc1 binding along with H3K27 acetylation mark at the selected Snai1 enhancer region as well as Sox2 and RNA polymerase II binding at Snai1 promoter after miR‐200c overexpression in KPNC tumor cells. Means ± SD are shown from one out of three independent experiments.
G ChIP analysis in KP∆NC tumor cells after adenoviral GFP‐tagged p53 overexpression. NFATc1 and H3K27ac binding are illustrated on Snai1 enhancer, and Sox2 and RNA polymerase II binding are demonstrated on Snai1 promoter. Means ± SD are shown from one out of three independent experiments.
H Quantification of KP∆NC spheres after adenoviral GFP‐tagged p53 overexpression.

Source data are available online for this figure.
BAZ2A-mediated repression via H3K14ac-marked enhancers promotes prostate cancer stem cells (2021)
Figure 1

Figure 1

BAZ2A-BRD is an epigenetic reader of H3K14ac that mediates the BAZ2A association with H3K14ac-chromatin domains in PC3 cells

A. Heatmap profiles of BAZ2A bound regions in PC3 cells and the corresponding signals of input, BAZ2A-BRD mutant (Y1775F), H3K14ac and H3K27ac. Data were ranked to BAZ2A-binding in PC3 cells.

B. Validation of ChIPseq results by anti-HA ChIP-qPCR of PC3 wild-type (WT) and a PC3 cell line that expresses endogenous BAZ2A with a FLAG-HA tag (PC3-H/F-BAZ2A). GAPDH promoter represents a region not bound by BAZ2A. Average values of three independent experiments. Data were normalized to input and to AOX1 levels. Statistical significance (P-values) was calculated using two-tailed t-test (*<0.05, **< 0.001, ns: not significant). Error bars represent SD.

C. Wiggle tracks displaying BAZ2A-bound regions, H3K14ac, H3K27ac, and input at AOX1, EVX2, and GAPDH.

D. Pearson correlation heat map of BAZ2A-bound regions and histone marks. Data for BAZ2A, H3K4me3, H3K27me3, H3K27ac, H3K14ac are from this work. H3K9me3, H3K9me2, H3K4me1 were obtained from ENCODE.

E. Images of histone peptide arrays probed with 10nM of recombinant BAZ2A-BRD wild-type (GST-BAZ2A-BRDwt) and mutants (GST-BAZ2A-BRDY1830F or GST-BAZ2A-BRDN1873L). Visualization of binding was performed by incubation with anti-GST antibodies and imaged on Odyssey Infrared Imaging System. Blue circles mark peptides recognized by BAZ2A-BRD (i.e. H3K14ac), whereas black circles show some of the acetylated peptides not recognized by BAZ2A-BRD (H3K27ac and H3K9ac). Right panel shows heatmap of the relative binding intensity of BAZ2A-BRD against modified histone peptides. Binding intensity was calculated as average of fold change from peptides signal over background controls of 2 different arrays.

F. BAZ2A read coverage quantification at ±1Kb from peak summit of BAZ2A-bound regions identified in PC3 cells and the corresponding reads for BAZ2A-BRD mutant (Y1775F) in PC3 cells. Statistical significance (P-values) was calculated using two-tailed t-test (***< 0.001).

G. Regions bound by BAZ2A in PC3 cells are enriched in H3K14ac. Read coverage quantification for H3K14ac levels at regions bound by BAZ2A in PC3 cells (BAZ2Awt and BAZ2A-BRD mutant (Y1775F)) at ±1Kb from BAZ2A peak summits. Statistical significance (P-values) was calculated using two-tailed t-test (***< 0.001).

Data information: Boxplots in Fig. 1 show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range.
BAZ2A-mediated repression via H3K14ac-marked enhancers promotes prostate cancer stem cells (2021)
Figure 2

Figure 2

EP300-mediated acetylation of H4K14 mediates the repression of a set of genes with BAZ2A-bound promoter

A. Scatter plot showing the expression of EP300 and BAZ2A in two large cohorts of metastatic and primary PCa. Data were from (Cancer Genome Atlas Research, 2015; Robinson et al., 2015).

B. EP300 acetylates H3K14 in PC3 cells. Western blot showing H3K14ac levels in PC3 cells treated for three days without and with the selective catalytic EP300 inhibitor A-485 (10 μM). Histone H3 serves as protein loading control.

C. Genomic annotations of BAZ2A-bound regions in PC3 cells.

D. Volcano plot showing fold change (log2 values) in transcript levels of PC3 cells upon BAZ2A knockdown. Gene expression values of two replicates were averaged and selected for 1.5 fold changes and P <0.05.

E. Table showing expression changes of genes with BAZ2A-bound promoter upregulated upon BAZ2A-KD in PC3 cells in metastatic PCa vs. normal prostate tissue and in PC3 cells depleted of EP300 via siRNA.

F. qRT-PCR showing increased expression levels of genes with BAZ2A-bound promoter and downregulated in metastatic tumors (AOX1, MANB2, DMPK, and DDB1 - see E) in PC3 cells treated with siRNA-EP300. Data were from three independent experiments. Values were normalized to GAPDH mRNA. Statistical significance (P-values) was calculated using two-tailed t-test (*<0.05, ****<0.0001). Error bars represent SD.
BAZ2A-mediated repression via H3K14ac-marked enhancers promotes prostate cancer stem cells (2021)
Figure 3

Figure 3

The association of BAZ2A with a class of inactive enhancers marked by H3K14ac represses genes implicated in aggressive PCa

A. BAZ2A binds to a class of inactive enhancers marked by H3K14ac. Heat maps of BAZ2A, H3K14ac, H3K27ac, H3K4me1, and DNAseq signal at annotated intergenic enhancers are shown. Enhancer regions were clustered in 4 groups based on the presence (+) or absence (-) of H3K27ac and H3K4me1. C1 (H3K27ac+ / H3K4me1+), C2 (H3K27ac+ / H3K4me1-), C3 (H3K27ac- / H3K4me1+), and C4 (H3K27ac- / H3K4me1-).

B. Inactive enhancers (C2) are enriched in H3K14ac and BAZ2A levels. Read coverage at enhancer classes C1 and C2 for BAZ2A, H3K14ac, H3K27ac, H3K4me1 and H3K27me3 at ±1Kb from annotated enhancer regions. Statistical significance (P-values) was calculated using two-tailed t-test (***< 0.001). Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range.

C. Pie charts showing the number of genes in the nearest linear genome proximity to BAZ2A-bound C2 enhancers and their expression changes upon BAZ2A-KD in PC3 cells.

D. Wiggle tracks showing RNA levels in PC3 cells treated with siRNA-BAZ2A and occupancy of BAZ2A, H3K14ac, H3K27ac, and H3K4me1 at C2 enhancer and its nearest gene CXCR4.

E. Top ten biological process gene ontology (GO) terms as determined using DAVID 6.8 for genes in the nearest linear proximity to BAZ2A-bound C2-enhancers that are differentially expressed upon BAZ2A-KD in PC3 cells.

F. Heatmap showing expression changes of BAZ2A-bound C2-enhancer genes that were both significantly upregulated upon BAZ2A-KD and lower expressed in Gleason 9 and 10 tumours (advanced) compared to Gleason 6 tumours (indolent). Values from tumours were from (Cancer Genome Atlas Research, 2015).

G. GO terms associated with the genes listed in F. Genes labeled in bold were analyzed in H.

H. qRT-PCR showing increased expression levels of BAZ2A-bound C2-enhancer genes in PC3 cells treated with siRNA-EP300. Data were from three independent experiments. Downregulation of EP300 expression is shown in Fig. 2F. Values were normalized to GAPDH mRNA. Statistical significance (P-values) was calculated using two-tailed t-test (*<0.05, **<0.001). Error bars represent SD.
BAZ2A-mediated repression via H3K14ac-marked enhancers promotes prostate cancer stem cells (2021)
Figure 4

Figure 4

BAZ2A-bromodomain is required for PCa stem cells

A. RNA-seq showing differential gene expression between PC3-CSCs and the parental PC3 cells (P<0.05, Log2 fold change 1).

B. Top ten biological process gene ontology (GO) terms as determined using DAVID 6.8 for genes downregulated and upregulated in PC3-CSCs relative to parental PC3 cells.

C. qRT-PCR of genes differentially expressed in PC3-CSCs compared to PC3 cells showing the upregulation of CSC markers (ALDH1A2, LGR5, CD24) and downregulation of genes related to developmental processes (HOXA1, HOXA4, SOX7). Data are from two independent experiments. mRNA levels were normalized to GAPDH.

D. BAZ2A is required for the dedifferentiation of PC3 into PC3-CSCs. Left panel: Representative images from two independent experiments showing the impairment of PC3 cells to form tumorspheres upon stable depletion of BAZ2A. Right panel: BAZ2A mRNA levels in three independent PC3 cell lines stably expressing shRNA-BAZ2A were measured by qRT-PCR and normalized to GAPDH mRNA from 2 independent experiments.

E. Left panel: qRT-PCR showing BAZ2A mRNA levels in PC3 cells depleted of endogenous BAZ2A with siRNA specifically targeting human BAZ2A sequences and expressing mouse BAZ2Awt and BAZ2A-BRD mutant (BAZ2AY1775F). Values were normalized to MRPS7 mRNA. Data are from 3 independent experiments. Right panel: Quantification of tumorspheres from 3 independent experiments. Statistical significance (P-values) was calculated using two-tailed t-test (* <0.05; **< 0.01). Error bars represent SD.
BAZ2A-mediated repression via H3K14ac-marked enhancers promotes prostate cancer stem cells (2021)
Figure 5

Figure 5

Pharmacological targeting of BAZ2A-BRD impairs the transition of PCa cells into a dedifferentiated, cancer stem-like state.

A. Heatmaps showing the relative BAZ2A-BRD binding intensity at modified histones peptides upon incubation with BAZ2A-BRDi BAZ2-ICR or GSK2801 displayed in Fig. EV4A. Binding intensity was calculated as average of fold change from peptides signal over background controls of 2 different arrays.

B. Cell proliferation curves of PC3 cells treated with siRNA-BAZ2A. Error bars represent the standard deviation from three independent experiments.

C. Cell proliferation curves of PC3 cells treated with 5μM BAZ2-ICR and GSK2801. Error bars represent the standard deviation from three independent experiments.

D. Representative images of tumorspheres generated from PC3 cells treated with BAZ2-ICR (1μM) or GSK2801 (1μM).

E. Half-maximal effective concentration (EC50) of BAZ2-ICR. Measurements were performed 5 days after treatment of PC3 cells and initiation of tumorspheres using the indicated BAZ2-ICR concentration. The amount of tumorspheres was assessed by measurement of cell viability. Error bars represent SD. Values are from three independent experiments.

F. Representative images showing tumorspheres derived from the metastatic PCa cell line DU-145 treated with DMSO or BAZ2-ICR (1μM) inhibitor for 5 days.
BAZ2A-mediated repression via H3K14ac-marked enhancers promotes prostate cancer stem cells (2021)
Figure 6

Figure 6

Pharmacological targeting of BAZ2A-BRD impairs the initiation of PCa driven by Pten-loss in a model of PCa organoids

A. Representative bright field images of organoids derived from mouse prostate cells treated with DMSO, BAZ2-ICR (5μM), GSK2801 (5μM) or GSK126 (5μM) inhibitors followed by transduction with shRNA-Control or shRNA-Pten.

B. Quantification of shRNA-Control and shRNA-Pten organoid morphology upon treatment with DMSO, EZH2 (GSK126) and BAZ2A-BRD inhibitors (BAZ2-ICR and GSK2801).

C. Representative images of Ki67-immunostained mouse prostate organoid sections expressing shRNA-control and shRNA-Pten and treated with DMSO or BAZ2-ICR (5μM).

D. Quantification of Ki67+ cells in mouse prostate organoids shown in C. **< 0.01, ****P < 0.0001, two-tailed Student's t test; ns, not significant. The median values are represented by a line.

E. Quantification of mouse prostate organoids per field expressing shRNA-control and shRNA-Pten and treated with DMSO, GSK126 (5μM), BAZ2-ICR (5μM), or GSK2801 (5μM). Values were normalized to shRNA-control samples. Average values of two independent experiments.

Expanded View Figure Legends
The androgen receptor depends on ligand-binding domain dimerization for transcriptional activation. (2021)
Figure 1

Figure 1. Disrupting AR LBD dimerization.

(A) Crystal structure of the human AR LBD core dimer based on PDB 5JJM (1 monomer in grey and 1 monomer in pink). W752, located at the interface of the AR LBD dimer, is involved in hydrogen bond formation with T756 of the neighboring LBD and thereby stabilizes the interface. R752 disrupt this stabilization by steric hindrance and charge repulsion. The side chains of both W and R are shown at position 752. A close-up of the LBD-LBD interface is given. The ligand (DHT) is depicted as spheres.

(B) Acceptor photobleaching FRET after transfection of Hep3B cells with labelled WT LBD or labelled W752R mutated LBD. Representative confocal images of Hep3B cells transiently expressing WT or W752R AR in the presence of 10 nM DHT are shown below the bars. Scale bar = 10 µm. The bar graphs show means ± SEM (biological replicates, n= 54 (WT LBD) and n = 65 (W752R LBD), unpaired two-tailed Student's t-test, ***P < 0.001).

(C) Representative pictures of the AGD of 13-week-old WT male (upper left), WT female (lower left), ARLmon/Y (upper right) and AR-/Y (lower right) mice.

(D) Upper panel: a representative picture of the urogenital tract of a WT male and an ARLmon/Y mouse. Lower panel: a representative picture of the testis of a WT male and an ARLmon/Y mouse. Scale bar = 1 cm.

(E-F) Serum levels of T (E) and LH (F) in WT males, ARLmon/Y and AR-/Y mice at the age of 13 weeks. The bar graphs show means ± SEM (biological replicates, n = 8, one-way ANOVA with Tukey's multiple comparisons test, **P < 0.01, ***P < 0.001, ns = not significant).
The androgen receptor depends on ligand-binding domain dimerization for transcriptional activation. (2021)
Figure 2

Figure 2. Transcriptome analysis of testes.

(A-B) Volcano plots visualizing the differentially expressed genes in ARLmon/Y (A) and AR-/Y (B) testes compared to testes from WT males. The dotted horizontal lines represent a q-value of 0.05, while the dotted vertical lines represent a FC of 1.5. Genes that are downregulated compared to WT are shown using blue dots, while red dots represent the upregulated genes. Hsd17b3 and Star are indicated as yellow and green stars, respectively.

(C) Box plots showing testicular expression from the bulk RNA-seq data (biological replicates, n = 5) of SC- and LC-specific genes extracted from the single-cell RNA-seq data described by Green et al (Green et al, 2018). Box plots are composed of a box from the 25th to 75th percentile with the median as a line and whiskers calculated via the Tukey method. Expression levels are normalized to average expression in WT for each gene. Two-way ANOVA with Tukey's multiple comparisons test, ***P < 0.001.

(D) RT-qPCR analyses of Rhox5 and Insl3 of testes from 13-week old WT males, ARLmon/Y and AR-/Y. Expression levels are normalized to WT. The bar graphs show means ± SEM (biological replicates, n = 6, one-way ANOVA with Tukey's multiple comparisons test, ***P < 0.001, ns = not significant)
The androgen receptor depends on ligand-binding domain dimerization for transcriptional activation. (2021)
Figure 3

Figure 3. Steroidogenesis in ARLmon/Y and AR-/Y mice.

(A) Expression levels of genes involved in steroidogenesis normalized to WT. The bar graphs show means ± SEM (data derived from RNA-seq analysis of testes, biological replicates, n = 5, one-way ANOVA with Tukey's multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant).

(B) Serum levels of A-dione in WT males, ARLmon/Y and AR-/Y mice at the age of 13 weeks. The bar graphs show means ± SEM (biological replicates, n = 8, one-way ANOVA with Tukey's multiple comparisons test, ***P < 0.001, ns = not significant, n.d. = not detected; below detection limit of 0.175 nmol/L).

(C) Expression levels of Hsd17b3 normalized to WT. The bar graphs show means ± SEM (data derived from RNA-seq analysis of testes, biological replicates, n = 5, one-way ANOVA with Tukey's multiple comparisons test, **P < 0.01, ***P < 0.001).

(D) Reporter gene assays in Hela cells co-transfected with the mouse Insl3 promotor together with the WT or mutant mouse AR. The bar graphs show means ± SEM (biological replicates, n = 4, unpaired two-tailed Student's t-test, **P < 0.01, ns = not significant).

(E) Schematic overview of the feedback mechanism between testes and pituitary in the transcriptional regulation of steroidogenic genes.
The androgen receptor depends on ligand-binding domain dimerization for transcriptional activation. (2021)
Figure 4

Figure 4. Evaluation of the renal androgen response after disrupting AR LBD dimerization.

(A) The experimental set-up to study the androgen response in kidney.

(B) PCA on the renal transcriptomes of WT ORX, WT ORX + T, ARLmon/Y ORX and ARLmon/Y ORX + T.

(C) Heatmap for the genes that are differentially expressed in kidney between WT ORX and WT ORX + T (q < 0.05; FC > 1.5) and corresponding levels in ARLmon/Y ORX and ARLmon/Y ORX + T.

(D-F) mRNA expression levels extracted from RNA-seq data for Fkbp5 (D), Odc1 (E) and Kap (F) in kidneys used for RNA-seq. Expression levels are normalized to WT ORX. The bar graphs show means ± SEM (biological replicates, n = 5, one-way ANOVA with Tukey's multiple comparisons test, **P < 0.01, ***P < 0.001, ns = not significant).
The androgen receptor depends on ligand-binding domain dimerization for transcriptional activation. (2021)
Figure 5

Figure 5. In vitro analysis of the W752R mutation.

(A) Transactivation assay in HEK293 cells with an integrated 4xSLP-HRE2 E1B 12 TATA Luc reporter and transiently transfected with human WT or W752R AR followed by stimulation with increasing DHT concentrations. The bar graphs show means ± SEM (biological replicates, n = 4, two-way ANOVA with Sidak's multiple comparisons test, *** < P 0.001, ns = not significant).

(B) Specific ligand binding assay in COS cells with overload of cold ligand and increasing concentrations of radioactive labeled DHT. Values are normalized to protein expression. Individual data points are shown with nonlinear regression curve fit (n = 2).

(C) Western blot on cytoplasmic, nuclear and whole cell extracts derived from Hela cells transfected with human WT or W752R AR followed by stimulation with increasing concentrations of DHT. Both panels represent the same blot. The blot was cut in two parts and antibodies against HSP90 and Lamin A/C were used to confirm cellular fractionation of cytoplasmic and nuclear proteins. For AR visualization (upper panel), longer exposure time was used. # = residual AR expression (lower panel).

(D) EMSA using nuclear extracts of COS-7 cells transfected with AR WT or AR W752R and incubated with radiolabeled ARE, more specifically TAT-GRE (Denayer et al., 2010). Shift (S) occured through binding of the AR dimer on the ARE. Supershift (SS) occurred after addition of an AR-antibody. S = shift and U = unbound ARE. Western blot on AR, depicted on the right, shows similar expression levels for WT and mutated receptors.
The androgen receptor depends on ligand-binding domain dimerization for transcriptional activation. (2021)
Figure 6

Figure 6. Chromatin binding of the ARLmon.

(A) Western blot on whole cell extracts and chromatin fractions extracted from kidneys of WT males and ARLmon/Y mice. Antibodies against GAPDH and Histone 3 were used to confirm cellular fractionation. Relative intensities are indicated, whereby the upper bands (AR) are normalized to the lower bands (GAPDH or Histone 3). Both panels represent the same blot. The blot was cut in two parts and a longer exposure time was used for AR visualization (upper panel).

(B) AR occupancy (blue) and levels of the histone marks H3K27ac (green) and H3K27me3 (red) at ARBS within the regulatory regions of Fkbp5, Kap (both androgen-regulated in kidney) and Tox3 (androgen-regulated in prostate but not in kidney and hence used as a negative control) were assessed by ChIP-qPCR in kidneys from 13-week-old ARLmon/Y mice and WT males. The bar graphs show means ± SEM (biological replicates, n = 6, one-sample t-test with Benjamini-Hochberg correction for multiple testing was used to determine whether a mean Log2 FC was statistically different from 0, *P < 0.05, **P < 0.01, ***P < 0.001). Fold enrichments are shown in Appendix Figure S6C-E.

(C) Representative pictures of immunofluorescence AR staining (green) in kidneys of castrated WT males and ARLmon/Y mice supplemented with vehicle or T. Nuclei are shown in blue. Scale bar = 20 µm.