Epigenetic regulation of alternative pre-mRNA splicing

Alternative splicing of pre-mRNA is a process that can generate multiple protein-coding isoforms through combinatorial use of splice sites, leading to regulated expansion of the transcriptome. The large-scale genomic sequencing studies have revealed that alternative splicing is a major mechanism of transcriptome expansion, yet the regulatory mechanisms governing alternative splicing remain unclear. Studies have largely focused on cis-elements encoded within pre-mRNA and trans-acting splicing factors, which bind to nascent transcripts. It is now widely accepted that pre-mRNA processing occurs co-transcriptionally and recent evidences show that differential marks on the DNA binding histone proteins can affect alternative splicing outcome. However, it is unclear how these events are modulated at alternative exons. It has further been demonstrated that DNA methylation is enriched at exons relative to introns. These studies raise the possibility that DNA structure may influence alternative splicing of pre-mRNA.

We recently demonstrated a mechanistic link between DNA methylation and alternative splicing through modulation of a DNA binding protein, CTCF.  We found that CTCF binds at exon 5 of PTPRC DNA.  PTPRC encodes the CD45 protein, which is a well- characterized model of alternative splicing. Alternative splicing of CD45 exons 4-6 is associated with distinct stages of lymphocytes development. Through a variety of experimental techniques, reduced CTCF binding was uniformly associated with loss of pol II pausing at exon 5 DNA and with reduced inclusion of exon 5 in CD45 transcripts.  In contrast, DNA methylation inhibits CTCF binding and promotes exclusion of alternative exons, thereby establishing a mechanistic basis for alternative splicing regulation through reciprocal CTCF and DNA methylation. Global genomic analysis demonstrated that intragenic CTCF binding is a commonly utilized mechanism to support inclusion of upstream weak exons in spliced mRNA.

Our current work focuses on two additional epigenetic modifications at CD45 DNA that are associated with alternative splicing: the presence of 5-hydroxymethylcytosine in immature lymphocytes and H3K9me3 in mature lymphocytes. Through RNAi mediated depletion of the upstream enzymes responsible for these modifications, we achieved opposing effects on exon 5 inclusion.

Aberrant DNA methylation and other epigenetic changes and global changes in pre-mRNA alternative splicing have been individually linked to many cancers. The goal of my future research will be to identify whether these variables are connected, and whether they function as drivers of tumorigenesis. 

In conclusion, this study provides a mechanistic link between epigenetic marks such as DNA methylation and regulation of alternative splicing and highlights a role for the modification of DNA in pre-mRNA processing decisions.