Fig. 6: RNF40 supports the glycolysis-related gene expression program in BLBC cells.

A Downregulated genes with no loss of promoter-proximal H3K27ac significantly enrich for 2 hallmark gene sets (p-adj<0.05), enriched as well in RNF40^high-BLBC patients (p-adj<0.05). Source of curated hallmark gene set collection: https://www.gsea-msigdb.org/gsea/msigdb/, source of BLBC patient transcriptomic data: https://portal.gdc.cancer.gov/. Pathway enrichment was performed using Enrichr (https://maayanlab.cloud/Enrichr/). B GSEA profiles of the “HALLMARK_GLYCOLYSIS” gene set significantly enriched in siControl-treated HCC1806 cells and RNF40^high-expressing BLBC patients. C Volcano plot of genes enriched in siControl-treated HCC1806 cells and RNF40^high-expressing BLBC patients (downregulated genes in RNF40-silenced HCC1806 cells: log2FC ≤ −0.7, p-val < 0.05, basemean≥15, BLBC patients: log2FC ≤ −0.6, p-val<0.05, basemean≥5, x-axis shows the log2FC positive values). Gene members of the “HALLMARK_GLYCOLYSIS” (H_GLYCOLYSIS) and of the “CORDENONSI_YAP_CONSERVED_SIGNATURE” (C6_YAP_CORD.) gene set are represented in red and purple, respectively. D RT-qPCR of selected glycolysis genes from the “HALLMARK_GLYCOLYSIS” gene set, significantly downregulated in siRNF40-treated HCC1806 cells. E Aggregate profile of the TSS-associated H3K27ac occupancy of glycolytic genes in siControl- and siRNF40-treated HCC1806 cells. F Dot plot of the genome-wide TSS-associated H3K27ac changes in HCC1806 cells upon RNF40 silencing. Green dots represent differentially changed H3K27ac-occupied TSS regions. Red and purple dots represent gene members of the “HALLMARK_GLYCOLYSIS” and of the “CORDENONSI_YAP_CONSERVED_SIGNATURE” gene set, respectively (regions with H3K27ac loss: FC ≤ 0.87, regions with H3K27 gain: FC ≥ 1.13, basal H3K27ac peak concentration≥2). G ChIP-RT-qPCR of H3K27ac (upper panel) at selected TSS-proximal regions (represented as dashed lines, lower panel) of selected glycolytic genes (ME1, ENO1, DLAT). H ChIP-RT-qPCR of RNApol-II at specific promoter-proximal regions of selected glycolysis genes (DLAT, ME1, ENO1) and YAP1-target genes (AXL, PDLIM2, TK1) (represented as dashed lines in Fig. S1G). I X-Y plot (left panel) and the respective bar graph (right panel) of the normalized expression of the glycolysis and YAP1-target genes in untreated and CDK9i-treated HCC1806 cells at 6 and 48 h of treatment. J RT-qPCR of glycolysis and YAP1-target genes in siControl- and siRNF40-treated HCC1806 cells, without or with NELFE silencing. Statistical test: Student t-test (D, G, H, I). One-way Anova (J). ns = not significant, ^*p-val<0.05, ^***p-val < 0.005. J: ^#p-val < 0.05, ^##p-val < 0.01, ^###p-val < 0.005 for comparison between siControl- and siRFN40-treated HCC1806 cells. Error bars: Standard error of the mean (SEM). ChIP-sequencing of H3K27ac was performed in one replicate per condition (n = 1). All the rest of the experiments were performed in biological triplicates per condition. RPGC: reads per genome coverage.