CD44 alternative splicing is a sensor of intragenic DNA methylation in tumors

Abstract: DNA methylation (meDNA) is a suspected modulator of alternative splicing, while splicing in turn is involved in tumour formations nearly as frequently as DNA mutations. Yet, the impact of meDNA on tumorigenesis via its effect on splicing has not been thoroughly explored. Here, we find that HCT116 colon carcinoma cells inactivated for the DNA methylases DNMT1 and DNMT3b undergo a partial epithelial to mesenchymal transition (EMT) associated with alternative splicing of the CD44 transmembrane receptor. The skipping of CD44 variant exons is in part explained by altered expression or splicing of splicing and chromatin factors. A direct effect of meDNA on alternative splicing was sustained by transient depletion of DNMT1 and the methyl-binding genes MBD1, MBD2, and MBD3. Yet, local changes in intragenic meDNA also altered recruitment of MBD1 protein and of the chromatin factor HP1$\gamma$ known to alter transcriptional pausing and alternative splicing decisions. We further tested if meDNA level has sufficiently strong direct impact on the outcome of alternative splicing to have a predictive value in the MCF10A model for breast cancer progression and in patients with acute lymphoblastic leukemia (B ALL). We found that a small number of differentially spliced genes mostly involved in splicing and signal transduction is systematically correlated with local meDNA. Altogether, our observations suggest that, although DNA methylation has multiple avenues to alternative splicing, its indirect effect may be also mediated through alternative splicing isoforms of these sensors of meDNA.