Journal: Oncogene

Article Title: Epigenomic mapping identifies an enhancer repertoire that regulates cell identity in bladder cancer through distinct transcription factor networks

doi: 10.1038/s41388-023-02662-1

Figure Lengend Snippet: A Cell viability in RT112 and SCaBER under siRNA treatment against FOXA1. B Venn diagram comparing differentially expressed genes in RT112 and SCaBER FOXA1 KD. C GSEA plot of Msig Hallmark GSEA Analysis of genes differentially regulated in RT112 and SCaBER cell lines upon FOXA1 siRNA (2 independent siRNA, 2 replicates). D Heatmap of genes in Hallmark interferon gamma response genes that are differentially regulated in FOXA1 KD vs Ct (min Fold Change = 1,5). E Heatmap of Top Luminal TFs expression in RT112 and SCaBER cell lines upon FOXA1 KD. F PCA projection of TCGA Tumours and CRispR mutant clones on the Basal/Luminal signatures. G GSVA analysis of FOXA1 CRispR mutant clones on Urothelial differentiation signature from Eriksson et al. H GSVA analysis of FOXA1 CRispR mutant clones on Basal TFs identified in Fig. I Overrepresentation analysis of DEG in FOXA1 mutant vs Controls. J Volcano plot of Deseq2 RNA-seq analysis comparing pooled CRispR mutant FOXA1 clones in SD48 and RT112 versus controls. K Transient overexpression of HA-FOXA1 in mutant FOXA1 CRispR clones, wildtype RT112 and SCaBER. qPCR expression of ZBED2 after transfection of HA-FOXA1 relative to control plasmid, 4 days post transfection including 24 h of Puromycin selection ( n = 3 for CrispR clones, n = 2 for WT RT112 and SCaBER). Significance was calculated using 2way ANOVA test ( p -value < 0.05 = *).

Article Snippet: The human bladder cancer-derived cell lines RT112, 5637, KK47, and SCaBER were obtained from DSMZ (Heidelberg, Germany).

Techniques: Expressing, CRISPR, Mutagenesis, Clone Assay, RNA Sequencing Assay, Over Expression, Transfection, Plasmid Preparation, Selection

Journal: Oncogene

Article Title: Epigenomic mapping identifies an enhancer repertoire that regulates cell identity in bladder cancer through distinct transcription factor networks

doi: 10.1038/s41388-023-02662-1

Figure Lengend Snippet: A TCGA expression of ZBED2 by Subtypes. B TCGA expression Heatmap of ZBED2 and FOXA1 and TCGA correlation between ZBED2 and FOXA1. C Expression of FOXA1 and ZBED2 in single-cell transcriptomics from bladder cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE), highlighting the nearly mutually exclusive expression of these genes. D Genome browser view of ZBED2 and FOXA1 loci in SD48 and 5637 cell lines. E GSEA analysis (Hallmark) of ZBED2 correlated genes in basal cells population of GSM4307111 scRNA-seq Tumour. F GSEA analysis (Hallmark) of gene expression upon siZBED2 KD in RT112 (siZBED2-1 and siZBED2-2). G 3’seq STAT2 and CD274 (PD-L1) expression in RT112 and SCaBER after siZBED2 and siFOXA1.

Article Snippet: The human bladder cancer-derived cell lines RT112, 5637, KK47, and SCaBER were obtained from DSMZ (Heidelberg, Germany).

Techniques: Expressing, Single-cell Transcriptomics

Journal: Nucleic Acids Research

Article Title: STAG2 loss-of-function affects short-range genomic contacts and modulates the basal-luminal transcriptional program of bladder cancer cells

doi: 10.1093/nar/gkab864

Figure Lengend Snippet: STAG1 and STAG2 show both overlapping and unique distributions over genomic elements and chromatin states in RT112 cells. ( A ) ChIP-Seq read density heatmaps for STAG1, STAG2, and SMC1 at common, STAG1-enriched (STAG1 > STAG2), and STAG2-enriched (STAG2 > STAG1) cohesin positions within a peak-centered 6kb window. ( B ) Read density distribution for STAG1 and STAG2 at common, STAG1-enriched, and STAG2-enriched positions within a peak-centered 6kb window. ( C ) Bar-plot diagram showing the distribution of common, STAG1-enriched, and STAG2-enriched cohesin positions over genomic elements. ( D ) Distribution of cohesin-bound genomic sites throughout chromatin states identified in RT112 cells by ChromHMM and based on combinations of histone modifications and CTCF (see for definition of chromatin states). ( E ) Peak-centered enrichment plot for CTCF over the three categories of cohesin-bound positions showing relative depletion in STAG2-enriched sites.

Article Snippet: RT112 bladder cancer cells used at CNIO and Institut Curie were from the same original stock; HEK293T cells (transformed human embryonic kidney) were from the ATCC.

Techniques: ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: STAG2 loss-of-function affects short-range genomic contacts and modulates the basal-luminal transcriptional program of bladder cancer cells

doi: 10.1093/nar/gkab864

Figure Lengend Snippet: STAG2 loss in RT112 cells does not interfere with A/B compartments or TAD boundaries. ( A ) Western blot analysis of control (shNT) and STAG2-silenced RT112 cells showing efficient depletion of STAG2 at the protein level. ( B ) Reproducibility, measured by a stratum-adjusted correlation coefficient (SCC), between pair-wise comparisons of Hi-C datasets. ( C ) Hi-C matrices for chr2 at 500 kb resolution in cells transduced with control or STAG2-targeting shRNAs. The darker red reflects a greater frequency of interaction. ( D ) Compartment tracks for chr2 at 100 kb resolution as determined by the values of the first principal component (PC1) in control and STAG2-silenced cells. ( E ) Expression, as defined by RNA-Seq (log 2 FPKM), of genes within compartments A and B. As expected, genes assigned to compartment A are more transcriptionally active than genes in compartment B. t -test: *** P < 0.001. ( F ) Compartmentalization saddle plots: average intra-chromosomal interaction frequencies between 200kb bins, normalized by expected interaction frequency based on genomic distance. Bins are sorted by their PC1 value derived from control cells Hi-C data. Preferential B-B interactions are in the upper left corner, and preferential A-A interactions are in the lower right corner. Numbers in corners represent the strength of AA interactions as compared to AB interactions and BB interactions over BA interactions. ( G ) Scatterplot of PC1 values of the eigenvectors of intrachromosomal interaction matrices for control and STAG2-silenced cells. The Venn diagrams show the overlap in terms of compartment-switching bins between sh1 and sh2. The number of genes mapped to genomic bins switching compartments is also indicated. Only one GO term is significantly enriched (FDR < 0.01) among genes switching from A to B: GO:0050907 (detection of chemical stimulus involved in sensory perception). ( H ) Effect of STAG2-depletion on the number of TADs per chromosome. Boxplot notches represent the confidence interval around the median. The number of total TADs is indicated below the boxplots. ( I ) Histograms depicting the strength of the TAD borders detected in control and STAG2-silenced cells, according to the TADbit score. ( J ) Average insulation profile around TAD boundaries (±600 kb) in control and STAG2-silenced cells. ( K ) Density plot depicting the distribution of TAD sizes identified in control and STAG2-silenced cells. ( L ) Hi-C normalized interaction matrices for chr2 at 100kb resolution comparing TAD organization in control and STAG2-silenced cells. ( M ) Effect of STAG2-depletion on conservation of TAD borders. Boxplot notches represent the confidence interval around the median.

Article Snippet: RT112 bladder cancer cells used at CNIO and Institut Curie were from the same original stock; HEK293T cells (transformed human embryonic kidney) were from the ATCC.

Techniques: Western Blot, Control, Hi-C, Transduction, Expressing, RNA Sequencing, Derivative Assay, Insulation

Journal: Nucleic Acids Research

Article Title: STAG2 loss-of-function affects short-range genomic contacts and modulates the basal-luminal transcriptional program of bladder cancer cells

doi: 10.1093/nar/gkab864

Figure Lengend Snippet: STAG2 depletion leads to deregulation of the basal/luminal transcriptional programs in RT112. ( A ) Scatter plots of expression values (FPKM) of genes in control versus STAG2-silenced cells. Statistically significant differentially expressed genes are highlighted in dark (FDR < 0.05) or light red ( P < 0.05). ( B ) Scatter plot showing a positive and significant correlation between gene expression changes in sh1 and sh2 (left). Venn diagrams displaying the overlap between sh1 and sh2 in terms of significant up- and down-regulated genes. ( C ) GSEA enrichment plots of gene sets associated with the luminal and basal subtypes of muscle-invasive UBC showing significant deregulation in STAG2-silenced RT112 cells. ( D ) Distribution of STAG2 expression (FPKM) in the UROMOL cohort of 476 UBC samples , highlighting the thresholds of the first and fourth quartiles (119 samples per group). We defined ‘STAG2 high’ cases as those with expression values in the fourth quartile, and ‘STAG2 low’ cases as those with STAG2 levels in the first quartile. ( E ) GSEA enrichment plots for genes down-regulated in STAG2-silenced cells in ‘STAG2 high’ versus ‘STAG2 low’ tumor samples. ( F , G ) Heatmaps displaying relative expression values (Z-score of FPKM) of genes significantly down-regulated in RT112 cells with sh1 (F) or sh2 (G) and in ‘STAG2 low’ versus ‘STAG2 high’ tumor samples.

Article Snippet: RT112 bladder cancer cells used at CNIO and Institut Curie were from the same original stock; HEK293T cells (transformed human embryonic kidney) were from the ATCC.

Techniques: Expressing, Control, Gene Expression

Journal: Nucleic Acids Research

Article Title: STAG2 loss-of-function affects short-range genomic contacts and modulates the basal-luminal transcriptional program of bladder cancer cells

doi: 10.1093/nar/gkab864

Figure Lengend Snippet: STAG2 silencing is accompanied by rewiring of DNA contacts. ( A ) MD plots depicting the fold change in interaction frequency of DNA contacts in control (shNT) versus STAG2-silenced cells in relation to the linear distance between interacting regions for chromosome 15. Interactions showing statistically significant differences (adjusted P < 0.05) are highlighted in red (gained) or blue (lost). ( B ) Scatter plot comparing changes in interaction frequency between cells transduced with sh1 versus cells transduced with sh2. Gained and lost interactions are highlighted in red and blue, respectively. ( C ) Interaction frequency of lost and gained contacts in shNT and STAG2-silenced cells. ( D ) Distance between peaks of lost and gained DNA contacts. Gained interactions span longer distances than lost interactions. ( E ) Top four scoring motifs enriched in the subsets of interactions defined in C. For a more extensive list of significantly enriched motifs, see . ( F ) Distribution of control, lost, and gained interactions over compartments and ( G ) chromatin states in RT112 cells (see ). ( H ) Left: chromatin contact network generated from the 20kb resolution Hi-C interaction map of control cells, showing in pink the nodes involved in contacts that are lost upon STAG2 silencing. Right: chromatin assortativity of nodes that lose contacts as the network is filtered eliminating contacts spanning short distances.

Article Snippet: RT112 bladder cancer cells used at CNIO and Institut Curie were from the same original stock; HEK293T cells (transformed human embryonic kidney) were from the ATCC.

Techniques: Control, Transduction, Generated, Hi-C