To probe the mechanism supporting site-specific modulation of CTCF in regulated alternative splicing, we explored the basis for signal-dependent variable methylation at an intragenic CTCF site within the CD45 gene. Data from our previous investigations identified alternative splicing of CD45 exon 5 as an ideal candidate for exploring developmental regulation of the 5mC-CTCF epigenetic splicing switch. Briefly, variable exclusion of exons 4-6 from transcripts encoding the protein tyrosine phosphatase CD45 is a robust model for signal-dependent alternative splicing in the immune system. Exclusion of exons 4 and 6 from CD45 transcripts in peripheral T and B lymphocytes results from activation-induced up-regulation of the splicing repressor, hnRNPLL. In contrast, exclusion of exon 5 from CD45 mRNA is regulated at the DNA level: naive lymphocytes include exon 5 in CD45 transcripts due to CTCF binding at exon 5 DNA, whereas activated and mature lymphocytes exclude exon 5 through 5mC-associated CTCF eviction. Focusing on this tractable CD45 switch, we investigated the mechanism leading to the emergence of 5mC at exon 5 DNA in response to activation. To our surprise, bisulfite sequencing revealed a steady level of overlapping methylation at the CTCF-binding site in both the naive (exon 5-including) and activated populations. As standard bisulfite analysis fails to discriminate between 5mC and the TET protein-catalyzed oxidation product 5-hydroxymethylcytosine (5hmC), we examined for overlapping 5hmC in the CTCF-binding populations. Antibodies specific to 5mC or 5hmC were utilized to enrich for sonicated genomic DNA fragments containing the respective modifications through methylated DNA-immunoprecipitation (MedIP). Quantitative analysis of recovered CD45 DNA in lymphocyte MedIP revealed opposing patterns: 5hmC was consistently elevated at exon 5 in CTCF-binding cells whereas 5mC was reciprocally elevated in the absence of CTCF binding. These data provided a rationale for the seemingly fixed level of DNA methylation in our exon 5-including versus excluding lymphocyte populations and pointed us to the TET proteins as potential regulators of CTCF-dependent splicing. The alpha-ketoglutarate dependent TET proteins (TET1, TET2 and TET3 in mammals) catalyze successive oxidation of 5mC to 5hmC, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). While each of the derivatives is inherently stable, levels in genomic DNA progressively decrease as oxidation state increases. In addition, 5fC and 5caC are substrates for the base excision repair enzyme, thymine DNA glycosylase (TDG), culminating in site-specific active DNA demethylation. Having detected overlapping 5hmC at a splicing associated CTCF-binding site, we explored whether the TET proteins actively promote inclusion of exon 5 in processed mRNA. To this end, we performed RNAi in a B cell line with determined overlapping 5hmC and CTCF at exon 5 DNA. Depletion of TET1 and TET2, the main family members expressed in our queried lymphocytes, led to increased 5mC and decreased CTCF at exon 5 DNA, along with reduced inclusion of exon 5 in CD45 transcripts. Importantly, TET depletion did not influence overall CD45 expression and the observed changes in DNA methylation and pre-mRNA splicing were relatively restricted to the CTCF dependent exon and were not detected at the adjacent alternative exons. Through in vitro stimulation of naive primary CD4+ T cells, we further determined that activating signals that promote exclusion of exon 5 from CD45 transcripts also result in decreased nuclear levels of TET1 and TET2. As in the RNAi results, the related decrease in 5hmC and increased 5mC were focused at exon 5 DNA and were not detected at an upstream CTCF binding site in CD45 intron 2, thus demonstrating the predicted spatial and temporal regulation of the CTCF/5mC epigenetic splicing switch. Co-detection of CTCF and oxidized 5mC derivatives (collectively referred to as 5oxiC) at CD45 exon 5 called for a re-examination of the precise determinants of pol II pausing and associated exon inclusion. To uncouple the respective contributions of CTCF and 5oxiC, we generated stable clones in CHO fibroblasts from in vitro methylated CD45 minigenes with a wildtype or mutated exon 5 CTCF binding site. We reasoned that when integrated into genomic DNA, the TET proteins would oxidize overlapping 5mC at the minigene DNA in a portion of selected stable clones. Accordingly, we successfully identified copy number matched clones derived from wildtype and mutated CD45 minigenes that showed a high degree of minigene oxidation, culminating in comparable detection of 5oxiC species. A detailed comparison of these clones showed that overlapping 5oxiC in the context of a mutated CTCF binding site was insufficient to promote pol II pausing at CD45 exon 5 and associated exon inclusion. In contrast, the unmethylated and 5oxiC-containing clones with intact CTCF binding at the minigene DNA were virtually indistinguishable on both these accounts. Together with the T and B cell data, these results establish that a main mechanism of CD45 alternative splicing in developing lymphocytes involves a failure to target the TET proteins to exon 5 DNA, resulting in increased 5mC and associated exclusion of exon 5 from CD45 mRNA through loss of CTCF. The activation-induced decline in TET levels CD4+ T cells allowed for a generalization of our findings in a physiologically relevant context. To determine whether the TET proteins globally regulate CTCF-dependent splicing events, we performed CTCF ChIP-seq, 5mC MedIP-seq, 5hmC MedIP-seq and RNA-seq in naive and activated CD4+ T cells. Note that 5hmC is consistently detected at locations that undergo further oxidation, as evidenced by the presence of 5fC and 5caC, and is thus an accurate proxy for general TET activity. Integrated analysis was performed to identify CTCF binding sites with evidence of overlapping differential methylation (increased 5mC and decreased 5hmC, or vice versa). These sites were further queried for proximity to upstream or downstream alternatively spliced exons. The resulting analysis adhered to the previously established model: CTCF binding sites with decreased 5hmC and increased 5mC upon T cell activation (sites of CTCF eviction) were associated with exclusion of upstream exons in the corresponding RNA-seq analysis. Note that despite the decrease in overall TET levels, CTCF sites with the opposing change in methylation (increased 5hmC and decreased 5mC) were also detected, and were reciprocally associated with upstream exon inclusion. These genome-wide results establish the TET proteins as global regulators of CTCF-dependent splicing through dynamic regulation of overlapping methylation. The sum of these cellular data firmly establishes a role for 5mC oxidation in promoting CTCF association with genic DNA. To further determine whether the observed association with 5hmC represents a transient intermediate in a pathway to demethylation, or reflects a direct role for a 5mC oxidized derivative in CTCF binding, we performed electrophoretic mobility shift assay (EMSA) with purified CTCF and variably modified CD45 probes. As expected, CTCF bound to unmethylated CD45 DNA, but failed to bind in the presence of overlapping 5mC. However, to our surprise, CTCF did not bind to 5hmC or 5fC-containing DNA, but instead formed a robust complex in the presence of 5caC. This observed association is unlikely to be an in vitro artifact, as despite its overall low abundance, 5caC was readily detected at several queried CTCF binding sites through 5caC MedIP and qPCR. Altogether, these results identify TET proteins as critical regulators of CTCF-dependent alternative splicing that locally antagonize overlapping 5mC and directly promote CTCF binding (5caC).
| Sinclair, Wilson R; Arango, Daniel; Shrimp, Jonathan H et al. (2017) Profiling Cytidine Acetylation with Specific Affinity and Reactivity. ACS Chem Biol 12:2922-2926 |
| Nanan, Kyster K; Ocheltree, Cody; Sturgill, David et al. (2017) Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource. Nucleic Acids Res 45:12780-12797 |
| Marina, Ryan J; Oberdoerffer, Shalini (2016) Epigenomics meets splicing through the TETs and CTCF. Cell Cycle 15:1397-9 |
| Marina, Ryan J; Sturgill, David; Bailly, Marc A et al. (2016) TET-catalyzed oxidation of intragenic 5-methylcytosine regulates CTCF-dependent alternative splicing. EMBO J 35:335-55 |
| Oberdoerffer, Shalini (2012) A conserved role for intragenic DNA methylation in alternative pre-mRNA splicing. Transcription 3:106-9 |
| Shukla, Sanjeev; Oberdoerffer, Shalini (2012) Co-transcriptional regulation of alternative pre-mRNA splicing. Biochim Biophys Acta 1819:673-83 |
