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

Abstract

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.

Epigenomics meets splicing through the TETs and CTCF

“…We further showed that TET-catalyzed oxidation of 5mC was required for CD45 exon inclusion in na€ ıve lymphocytes, and determined that declining TET1 and TET2 levels following lymphocyte activation were responsible for emerging 5mC at the CTCF binding site. 5 These dynamic methylation changes were relatively restricted to the CTCF dependent exon, as were the observed changes in splicing, thus demonstrating the predicted spatial and temporal regulation of the epigenetic splicing switch.…”

“…The resulting integrated analysis revealed strong adherence to the previously established model for alternative exons with downstream CTCF binding: locations with increased 5hmC and decreased 5mC (CTCF binding) upon T cell activation were associated with upstream exon inclusion, whereas locations with decreased 5hmC and increased 5mC (CTCF eviction) were associated with upstream exon exclusion. 5 These genome-wide results establish the TET proteins as global regulators of CTCFdependent splicing, and provide a basis for dynamic regulation of epigenetic splicing switches through variations in TET activity. Notably, our study uncovered an additional layer of complexity in CTCF-dependent splicing.…”

“…While levels are generally low within genomic DNA, 5caC was readily detected within CTCF binding sites in the CD45 gene and genome-wide in thymine-DNA glycosylase (TDG) knockout embryonic stem cells, which accumulate 5caC. 5,6 While the direct significance of these findings to splicing regulation in vivo remains unclear, they raise a number of intriguing questions. First, enhanced interaction of CTCF with 5caC-containing DNA suggests that 5caC adopts unique structural and/or electrophysical properties.…”

Epigenomic reprogramming in inorganic arsenic-mediated gene expression patterns during carcinogenesis

“…Epigenetic modifications are thought to regulate AS by two non-mutually exclusive means: 1) by affecting the kinetics of polymerase elongation and 2) through recruitment of splicing regulatory factors. Two types of epigenetic marks, DNA methylation (125)(126)(127)(128) and histone PTMs (120,(129)(130)(131)(132), have been implicated in the selection of exoncandidates. Exposure to iAs significantly alters DNA methylation (133) and histone PTMs (48-50, 62, 134-136), and so it is reasonable to expect that this exposure can induce changes in alternative splicing.…”

“…Other DNA-binding proteins with zinc finger motifs inhibited by iAs are the methylcytosine dioxygenases (TET1/2), needed to oxidize 5-methylcytosine to 5-hydroxymethylcytosine and 5-carboxylcytosine (148). Inactivation of TET1/2 by iAs allows 5-methylcytosine to accumulate at CTCF target sites and prevents CTCF from binding to its target sites, resulting in exon exclusion (126). In summary, if iAs blocks the binding of PARP1 or CTCF to DNA, the chromatin-associated functioning of these proteins is altered, which includes splicing decisions.…”