TET-catalyzed oxidation of intragenic 5-methylcytosine regulates CTCF-dependent alternative splicing

Intragenic 5‐methylcytosine and CTCF mediate opposing effects on pre‐mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5‐methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF‐binding sites were unclear. Here, we reveal the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF‐mediated alternative splicing through conversion of 5‐methylcytosine to its oxidation derivatives. 5‐hydroxymethylcytosine and 5‐carboxylcytosine are enriched at an intragenic CTCF‐binding sites in the CD45 model gene and are associated with alternative exon inclusion. Reduced TET levels culminate in increased 5‐methylcytosine, resulting in CTCF eviction and exon exclusion. In vitro analyses establish the oxidation derivatives are not sufficient to stimulate splicing, but efficiently promote CTCF association. We further show genomewide that reciprocal exchange of 5‐hydroxymethylcytosine and 5‐methylcytosine at downstream CTCF‐binding sites is a general feature of alternative splicing in naïve and activated CD4 + T cells. These findings significantly expand our current concept of the pre‐mRNA “splicing code” to include dynamic intragenic DNA methylation catalyzed by the TET proteins. Synopsis Variations in methylcytosine oxidation state are controlled by the activity of the TET enzymes. TET1/2 activity at intragenic regions allows for enhanced CTCF binding and stimulates the inclusion of alternative exons, thus adding further complexity to the splicing code. Overlapping methylation at CTCF‐binding sites allows for regulation of alternative pre‐mRNA splicing via variations in TET activity. The TET proteins directly promote alternative exon inclusion by facilitating CTCF‐associated pol II pausing downstream of weak splice sites. TET‐catalyzed 5hmC and 5caC are enriched at CTCF‐binding sites in cells and CTCF directly interacts with 5caC‐containing DNA in vitro . Reduced TET activity results in 5mC‐coupled CTCF eviction and associated exclusion of weak exons from spliced mRNA. Graphical Abstract Variations in methylcytosine oxidation state are controlled by the activity of the TET enzymes. TET1/2 activity at intragenic regions allows for enhanced CTCF binding and stimulates the inclusion of alternative exons, thus adding further complexity to the splicing code.