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 the stages of lymphocyte development. We previously showed that the RNA binding protein, hnRNPLL, influences exclusion of exons 4 and 6 from 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. The resulting manuscript was published in Nature in November 2011. Our current work is focused on analysis of whether and how methylation is targeted to exons, and the mechanism by which variable intragenic methylation is achieved to promote alternative splicing of methyl-sensitive exons. To pursue these aims, we are continuing to work in the CD45 model system, both in vivo and in minigenes that have been adapted to achieve variable splicing outcomes. We have additionally begun to examine genome-wide sequencing data to examine the global impact of dynamic DNA methylation on pre-mRNA splicing decisions.
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