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. 2018 Dec 3;7:e27706. doi: 10.7554/eLife.27706

Figure 3. SOX4 directly regulates EDN1 mRNA expression and promotes production of the mature ET-1 peptide.

(A) Gene Ontology (GO) analysis on core-SOX4 target genes. Top terms are shown. (B) Heatmap of gene expression changes upon conditional SOX4 activation in HMLE cells for genes associated with the GO-term Blood vessel morphogenesis (HMLE regulated and bound). Genes with a SOX4-binding site within 20 kb of a TSS in MDA-MB-231 cells are annotated in red (C) Normalized ChIP-seq tracks for SOX4, H3K27me3, H3K4me3, POL2, and H3K27ac surrounding the EDN1 locus in HMLE-S4 cells. For histone marks and POL2 both DOX-treated and -untreated profiles are represented (-/+). (D) Normalized RNA-seq profile for the EDN1 gene in untreated and 4-OHT-treated ERSOX4 HMLE cells (-/+). (E) qRT-PCR analysis of EDN1 expression in 4-OHT-treated ER and ERSOX4 HMLE cells. Data represented as mean ± SD, normalized to β2M of three independent biological replicates (**p-value<0.01, Student’s t-test). (F) Luciferase assay in HEK293T cells transiently transfected with the full-length EDN-1 promoter (FL) as well as luciferase constructs in which the conserved sites have been mutated (−70 and −350). Results obtained from three independent biological replicates wherein three independent technical replicates were used per condition (ns: non-significant; ****p-value<0.001, Student’s t-test). (G) Pulldown assay in Flag-Sox4 transfected HEK293T cells using biotinylated DNA-probes. Probes matching an optimal SOX4-binding sequence, and the −70 bp and −350 bp of the EDN1 promotor sequence are indicated and mutated versions thereof (- indicates empty beads). Representative data obtained from three independent biological replicates. (H) Enzyme-linked immunosorbent assay (ELISA) analysis of ET-1 expression in the overnight conditioned media of 4-OHT treated and untreated HMLE-S4 cells. Results obtained from three independent biological replicates (*p-value<0.05, Student’s t-test).

Figure 3.

Figure 3—figure supplement 1. Analysis of patient survival and cellular processes associated with the core-SOX4 target gene signature.

Figure 3—figure supplement 1.

(A) GO-term analysis for core-SOX4 target genes based on location in cell for gene-product (B) Selected genes coding for secreted factors (C–D) GO-term analysis performed in cytoscape using ClueGO of the 411 positively regulated and 239 negatively regulated genes. Visualized GO-terms are significantly enriched (p<0.05).
Figure 3—figure supplement 2. SOX4 activates the EDN1-luciferase promoter and SOX4 expression correlates with EDN1 in breast cancer patients.

Figure 3—figure supplement 2.

(A) Schematic representation of the EDN1 promoter. The magnification shows the SOX4 and H3K27ac bound locus. Represented are the phastcon score for mammalian conservation in 46 vertebrates (upper track), with conserved SOX4-binding sites indicated underneath in green (−350 bp and −70 bp from TSS). Represented below is a schematic image of the EDN1-luciferase promoter used in this study. (B) Luciferase assay in HEK293T cells transiently transfected with the EDN1-luciferase promoter, renilla-luciferase in combinantion with either empty vector pcDNA3 (EV), flag-SOX4-pcDNA3 (SOX4), dominant negative SOX4 (DN, aa1-135), or SOX4 + DN. Data represented as mean ± S.E.M. normalized to renilla relative to EV (three independent biological replicates, statistical significance determined by ANOVA) (C) qRT-PCR quantification of SOX4 and EDN1 expression in SCR control and SOX4 KD HCC1954 cells. Data represented as mean ± SD of three independent biological replicates (***p-value<0.001, Student’s t-test).