Fig. 4: MTHFD2 KO induces aberrant centromere overexpression, strong methylation defects and increased structural variation.

a Western blot of HCT116 MTHFD2 knock-out (KO1, KO2) and wild-type (WT) cells. Vinculin is used as loading control. b Immunofluorescence of MTHFD2 WT, KO1 and KO2 cells. MTHFD2 is shown in green and DAPI in blue; non-confocal mode, scale bar 10 μm. c Percentage of centromeric intersects normalized by the total mapped reads in WT and KO1 cells (n = 6); unpaired two-tailed Wilcoxon test. d Relative mRNA expression of 20 chromosomal centromeres in KO1 cells normalized to WT cells. The dashed line indicates fold change = 1; means + s.e.m. (n = 5), one-sample two-tailed t-test (P values for each centromere are indicated on the top). e Comparison of the log₂ mean intensity of nuclear levels of histone marks H4K20me1, H3K9me3, and H3K27me3 in WT and KO1 cells; unpaired two-tailed Wilcoxon test. Representative images are shown above; non-confocal mode, scale bar 10 μm. f–h Fold enrichment of H4K20me1 (f), H3K9me3 (g), and H3K27me3 (h) signal normalized to IgG in centromeric regions of 4 independent chromosomes in WT and KO1 cells; means + s.d. (n = 3), one-sample two-tailed t-test. i Whole-genome scheme showing the hypermethylated CpG sites in red and hypomethylated CpG sites in blue. Regions shown in pale purple correspond to the centromeres and peri-centromeres. The height of the bars is proportional to the degree of hyper- or hypomethylation. j Chromosomes 6 and 14 showing the peri-centromeric alterations found in KO1 and KO2 cells in red. Number of alterations (insertions, deletions, duplications or inversions) found in WT and KO1 cells in the whole-genome (k) or at the centromeric regions (l). Source data are provided as a Source Data file. The n indicates the number of biological sample replicates for c, d, f–h, and number of individual cells for e. For e, individual cells from 4 biological replicates were pooled.