Shalini Oberdoerffer, Ph.D.
Dr. Oberdoerffer identified a novel role for gene-body DNA methylation in pre-mRNA splicing decisions. Work in her laboratory revealed that intragenic 5-methylcytosine and CTCF mediate reciprocal effects on the inclusion of alternative exons in spliced mRNA. Binding of CTCF within transcribed genes leads to local RNA polymerase II pausing, thereby kinetically favoring spliceosome assembly at weak upstream splice sites. In contrast, overlapping 5-methylcytosine evicts CTCF from genic DNA, thus ablating associated effects on upstream exon inclusion. Current efforts in the laboratory are focused on the mechanisms contributing to dynamic DNA methylation during lymphocyte development and the net impact on pre-mRNA splicing.
Unlike the genes of lower eukaryotes, in which protein-coding sequences are typically uninterrupted, genes of higher metazoans are characterized by a large number of coding exons separated by long stretches of non-coding introns. As genes are transcribed into mRNA, introns are excised by the megadalton spliceosome complex, which recognizes short consensus sequences at intron-exon boundaries. While introns were initially dubbed as junk DNA, exon-intron architecture has emerged as a critical platform for transcriptome diversification via alternative pre-mRNA splicing. Cassette exons thus represent an important aspect of proteome complexity in higher organisms, and current estimates indicate that greater than 90% of human genes engage in alternative splicing. However, the evolutionary drive for transcriptome expansion has posed the spliceosome with an increasingly difficult task as intron lengths have increased and splice site strengths have weakened. Alternative pre-mRNA splicing adds an additional layer of complexity as splice site recognition must be diversified in a context-dependent manner. To accomplish regulated transcript production within a multi-variable framework, pre-mRNA splicing is coordinated at multiple levels. In addition to regulation via RNA-binding protein recognition of cis-elements encoded within pre-mRNA, the rate of RNA polymerase II (pol II) transcription elongation and chromatin structure contribute to splice site recognition. Rather than operating independently, these processes are highly integrated as a result of co-transcriptional pre-mRNA splicing.
Chromatin and Splicing. Research in our laboratory focuses on the relationship between intragenic chromatin structure and alternative pre-mRNA splicing. This association is built on the observation that splicing in higher eukaryotes occurs while the nascent transcript remains tethered to the DNA template. Co-trascriptional splicing allows for kinetic regulation of splice site choice and weak exons are more likely to be excluded from spliced mRNA in response to a rapid pol II elongation rate. Similarly, an open chromatin structure characterized by histone acetylation is also associated with inclusion of weak exons in spliced mRNA. Chromatin can further function as an adaptor that recruits RNA binding proteins to their required sites of action through interaction with chromatin-binding proteins. Accordingly, chromatin modifications are asymmetrically distributed across the transcribed genome and exonic DNA shows a higher rate of nucleosome occupancy, specific histone modifications and DNA methylation relative to intronic DNA. These observations raised the possibility that the chromatin structure of transcribed genes may aid the spliceosome in the process of exon definition.
Our work specifically focuses on the role of gene body DNA methylation in alternative pre-mRNA splicing decisions. DNA methylation is particularly enriched at exons, and is depleted at introns, intronless genes and pseudoexons. This distribution pattern is conserved across eukaryotic species, implicating a fundamental role for DNA methylation in the process of exon definition. However, overall DNA methylation levels are not strictly associated with exon inclusion or exclusion, thereby suggesting an indirect role in splicing regulation. We recently confirmed such a role for DNA methylation through our identification of a splicing switch dependent on reciprocal 5-methylcytosine and the zinc-finger protein, CTCF. The interaction of CTCF with DNA promoted local RNA polymerase II pausing and increased inclusion of weak upstream exons both in a model gene and genome-wide. In contrast, overlapping DNA methylation blocked CTCF binding, abrogated polymerase pausing and led to exon exclusion. This work highlights dynamic exchange of DNA methylation as a novel determinant of developmentally regulated alternative pre-mRNA splicing. Based on these and related results, we are interested in examining the mechanism supporting variable methylation during development and whether DNA methylation is a universal adaptor of alternative splicing that operates through a network of DNA binding proteins.
Selected Recent Publications
- Methods Mol. Biol. 1126: 97-113, 2014. [ Journal Article ]
- Transcription. 3: 106-9, 2012. [ Journal Article ]
- Biochim Biophys Acta. 1819: 673-83, 2012. [ Journal Article ]
Heterogeneous nuclear ribonucleoprotein L-like (hnRNPLL) and elongation factor, RNA polymerase II, 2 (ELL2) are regulators of mRNA processing in plasma cells.Proc. Natl. Acad. Sci. U.S.A. 109: 16252-7, 2012. [ Journal Article ]
- Nature. 479: 74-9, 2011. [ Journal Article ]
In 2005, Dr. Shalini Oberdoerffer obtained her Ph.D. in immunology under the supervision of Dr. Jean-Pierre Kinet at Harvard Medical School. She then joined the laboratory of Dr. Anjana Rao at the Immune Disease Institute, Harvard Medical School, where she studied global shifts in alternative pre-mRNA splicing during the process of lymphocyte development. Dr. Oberdoerffer joined the Center for Cancer Research in 2010, where she studies the relationship between chromatin modifications and alternative pre-mRNA splicing in the context of the immune system.
|Daniel Arango Tamayo Ph.D.||Postdoctoral Fellow (Visiting)|
|Masaki Hosogane Ph.D.||Postdoctoral Fellow (Visiting)|
|Mariana Mandler Ph.D.||Postdoctoral Fellow (CRTA)|
|Kyster Nanan Ph.D.||Postdoctoral Fellow (Visiting)|
|Maria Fernanda Prigge B.S.||Research Biologist|
|David M. Sturgill, Ph.D.||Staff Scientist|