Isoform switching and exon skipping induced by the DNA methylation inhibitor 5-Aza-2'-deoxycytidine.

DNA methylation and histone modification play crucial roles in regulation of gene expression in mammalian developments as well as human diseases, such as cancer1,2. During tumorigenesis, the promoter regions of tumor suppressor genes could undergo abnormal hypermethylation, which lead to the silencing of these genes3,4,5. Moreover, transient exposure to low doses of DNA-demethylation agents can trigger durable antitumor effects in tumors6,7. Recently, clinical trials have been focused on investigating the possible utility of methylation inhibitors in solid tumors, either alone or in combination with other demethylation drugs8,9. Thus, reactivation of tumor suppressor genes by demethylation agents has become a possible and promising approach for cancer therapy. Alternative splicing is closely associated with differentiation and development, and is a major source for protein diversity10. It enables cells to generate proteins of different coding sequences and functions from a single gene. Genome-wide approaches have revealed that tumorigenesis often involved large-scale alterations in alternative splicing11. Researchers also found that demethylation drugs could target transcribed regions, which suggest that the effects of demethylation drugs are not limited to the reactivation of promoters of silenced genes, but are prone to change exon recognition6,12,13. The demonstration that intragenic DNA methylation could affect elongation efficiency indicated that DNA methylation may facilitate exon inclusion14. A recent study further proved that intragenic DNA methylation modulated exon recognition, thus it is necessary to investigate the relationship between demethylation treatment and alternative splicing, which was generally overlooked in previous studies15. DNA methyltransferases (DNMT) inhibitors, such as 5-azacytidine (5-Aza-CR) and 5-Aza-2′-deoxycytidine (5-Aza-CdR), were approved by the FDA for the treatment of myelodysplastic syndrome16,17. Therefore, a comprehensive understanding of how these demethylation drugs affect gene reactivation and alternative splicing is necessary for understanding their therapeutic effects and exploring new cancer therapies. In this study, we treated human bladder cell line UM-UC-3 with 5-AZA-CdR for 24 hours, then monitored expression changes at 5, 9, 13 and 17 days after treatment and employed deep RNA sequencing to analyze alterations in gene expression and alternative splicing. Additionally, we measured whole-genome methylation levels by the Illumina 450K methylation array at 5 and 17 days, to correlate with gene expression changes.