Tumor-associated changes in tDNA methylation promote differences in tRNA expression. A Heatmaps showing the average β-value for each tDNA in the different tissues from TCGA set of normal (left) and tumoral (middle) samples and in cancer cell lines (right). β-values span from 0 (green, unmethylated) to 1 (red, hypermethylated). Grey indicates missing values. B Heatmap showing the Spearman’s ρ of the correlation between tDNA gene methylation and the expression of their cognate tRNA in all TCGA tumor samples (left) and separated by tissue of origin (right). Yellow and blue denote positive and negative correlation, respectively. C ChIP-qPCR experiments indicate an increased binding of GTF3C1 (left) and POLR3A (right) to tRNA-Arg-TCT-4-1 (top) and tRNA-Ile-AAT-8-1 (below) genes upon treatment with 5’-azacytidine (AZA) in DND41 and SW48. Data represent the mean ± SD of three biological replicates analyzed using an unpaired two-tailed Student’s t-test. * p < 0.05; ** p < 0.01; *** p < 0.001. D qRT-PCR shows a recovery of tRNA-Arg-TCT-4-1 (left) and tRNA-Ile-AAT-8-1 (right) expression in DND41 and SW48 cell lines after the use of AZA. All qRT-PCR data represent the mean ± SD of biological triplicates analyzed using the unpaired two-tailed Student’s t-test. * p < 0.05; ** p < 0.01.