Processing of CD45 pre-mRNA is a well-established model system to study the regulatory mechanisms of alternative splicing. CD45 is a trans-membrane protein tyrosine phosphatase that initiates signaling through antigen receptors by dephosphorylating the inhibitory tyrosine on Src family kinases. Variable exclusion of exons 4-6 of CD45 transcripts is tightly correlated with stages of lymphocyte development. We previously showed that the RNA binding protein, hnRNPLL, influences exclusion of exons 4 and 6 of CD45 transcripts, but not exon 5. Considering the growing evidence for DNA-mediated regulation of spliceosome assembly, we explored the hypothesis that exon 5 inclusion is mediated by the epigenetic structure of the CD45 gene (PTPRC). Through our studies in primary lymphocytes and cell lines, we found that the methylation-sensitive DNA binding protein, CCCTC-binding factor (CTCF), promotes inclusion of weak exons in spliced mRNA by mediating pol II pausing. Binding of CTCF to exon 5 of the gene encoding CD45 is associated with pol II accumulation and inclusion of the weak exon in mature transcripts. In contrast, methylation of CD45 exon 5 DNA on 5-cytosine ablates CTCF binding, abolishes local pol II pausing and results in exclusion of exon 5 from CD45 transcripts. Combined RNA-seq and CTCF ChIP-seq analysis in CTCF depleted cells indicated that intragenic CTCF is a global regulator of alternative pre-mRNA splicing. CTCF binding specifically promotes inclusion of weak upstream exons in spliced mRNA, supporting a model in which CTCF-mediated pol II pausing provides favorable spatiotemporal conditions for spliceosome assembly at weak exons. These findings provide a framework for epigenetic regulation of splicing outcome through reciprocal heritable changes in DNA methylation and CTCF binding, and established the first mechanistic link between DNA methylation and pre-mRNA splicing. The resulting manuscript was published in Nature in November 2011. Our current and future goals include a deeper analysis of developmentally regulated intragenic de novo methylation. Specifically, we are examining the mechanism supporting de novo methylation of CD45 exon 5 in activated lymphocytes and whether this is related to additional changes at the chromatin or RNA level.
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