It is presently estimated that greater than 90% of human genes undergo alternative pre-mRNA splicing, and aberrant splicing has been linked to a variety of human pathologies. Alternative splicing decisions are determined by the ability of weak splice sites to effectively compete with strong splice sites for detection by the spliceosome. There is substantial evidence indicating that spliceosome assembly occurs on nascent RNA co-transcriptionally. RNA polymerase II (pol II) carboxy terminal domain (CTD) associated splicing factors detect enhancer or silencer sequences encoded within the newly synthesized transcript to promote or inhibit spliceosome assembly, respectively. Variations in splicing factor expression can thereby result in alternative splicing. Similarly, the rate of pol II transcription elongation can influence alternative splicing, wherein a slow transcription rate favors spliceosome assembly at weak splice sites. A surprising result of recent genome-wide chromatin-immunoprecipation-sequencing (ChIP-seq) studies is the non-random distribution of several epigenetic marks in exons relative to introns. In particular, exons display elevated nucleosome density, DNA methylation and specific histone marks relative to introns. Collectively, these studies raise the possibility that epigenetic modifications may be maintained on DNA to aid the spliceosome in the process of exon definition, and that differential chromatin assembly may represent a critical aspect of alternative splicing regulation. 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). We found that the zinc-finger DNA-binding protein, CTCF, binds to CD45 exon 5 and supports exon 5 inclusion in CD45 transcripts by promoting local pausing pol II, leading to favorable spatiotemporal conditions for spliceosome assembly. We further explored the mechanism by which exon 5 becomes excluded from CD45 transcripts during the process of lymphocyte maturation. Previous studies have shown that CTCF binding to DNA is inhibited by the presence of 5-methylcytosine. We determined that mature lymphocytes acquire DNA methylation of exon 5, leading to exclusion of exon 5 from spliced mRNA (Nature, in press). The goal of this project, going forward, is to investigate the mechanism supporting de novo methylation of exon 5 during normal lymphocyte development.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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