Fig. 6: Intersection of the Atlas data with fetal brain Hi-C data reveals FOXM1-ANXA2R axis that impacts survival of GB patients and regulates glioma invasion.
A Graphical representation of chromatin contacts from Hi-C data. B Heatmap showing normalized average expression of 17 differentially expressed genes in (FDR < 0.01) selected in the contact analysis. C Barplot showing significance of enhancer-promoter co-activation for five genes computed as the Pearson correlation of H3K27ac enrichments in putative enhancers and H3K4me3 enrichments in corresponding promoters. D FOXM1 expression in gliomas. The adjusted p values computed with edgeR Bioconductor correspond to expression differences. Logo represents FOXM1 DNA-binding motif. Boxplots show the quartiles of the dataset, lines indicate median and the whiskers the largest or smallest value. E, F Scatterplots showing co-expression of FOXM1 and ANXA2R as log-transformed FPKMs in our (E) and TCGA (F) datasets. Pearson correlation coefficients shown above the plots. G Kaplan–Meier survival curves plotted for patients stratified by ANXA2R or FOXM1 expression; differences assessed with the log-rank test. H ANXA2R and FOXM1 expression in normal human astrocytes (NHA), patient-derived (WG4 and IPIN) and established glioma cells (LN229, LN18, U87) determined by RT-qPCR; data normalized to GAPDH mRNA; p values calculated on the raw data with two-sided t test. I Capture-C profile representing interactions around the viewpoint in the ANXA2R promoter (green arrow). Region enrichments indicate the putative enhancer (purple arrow). Fragment-based raw data are visualized as grey dots, whereas blue dots represent smoothed data. J Significant enrichment of FOXM1 binding at the ANXA2R promoter and enhancer, and CCNB1 and PKL1 promoters (positive controls). No significant FOXM1 enrichment detected at H19, myoglobin and CCND1 promoters (negative controls) or with a neutral IgG antibody. Results calculated as % of input, mean ± SD (n = 5), significance with ratio paired t test, two-sided. K, L Knockdown of FOXM1 in WG4 glioma cells verified by qPCR and Western blotting; densitometry of immunoblots determined from three experiments, mean ± SD, (K) two-sided paired t test, (L) two-sided t test. M Reduced ANXA2R expression in siFOXM1-transfected cells determined with qPCR; mean ± SD, n = 4, two-sided paired t test. N Knockdown of FOXM1 reduces migration and invasion of glioma. Cell migration after scratch quantified in four fields before and 24 h after scratch. Results shown as mean ± SD, n = 4. Invasion determined with Matrigel and Transwell® inserts. Images from five fields acquired using fluorescence microscopy, cell nuclei counted with ImageJ software. Results shown as mean ± SD, n = 4 (in duplicate). p values calculated using paired t test, two-sided.
