Regulation of intron retention by DNA methylation and nucleosome occupancy — University of Technology
  1. Schedule
  2. Splicing/alternate splicing – RNA processing
  3. Symposium II
  4. Regulation of intron retention by DNA methylation and nucleosome occupancy

Regulation of intron retention by DNA methylation and nucleosome occupancy (14238)

Justin Wong 1 2 , Dadi Gao 1 2 3 , Trung Nguyen 1 2 , Amy Au 1 2 , Natalia Pinello 1 2 , William Ritchie 1 2 3 , John EJ Rasko 1 2 4
  1. Gene and Stem Cell Therapy Program, Centenary Institute, Camperdown, NSW, Australia
  2. Sydney Medical School, University of Sydney, Camperdown, NSW, Australia
  3. Bioinformatics Laboratory, Centenary Institute, Camperdown, NSW, Australia
  4. Cell and Molecular Therapies, RPA Hospital, Camperdown, NSW, Australia

Background

Retention of introns in mature mRNAs is emerging as a key physiological process in normal cells. We showed in normal human and mouse granulocytes that intron retention is a widespread mechanism triggering nonsense-mediated decay to reduce protein levels1. This process is crucial for granulocyte maturation since enforced re-expression of a nuclear lamina gene, Lmnb1, which displayed the highest levels of intron retention, decreased granulocyte count, increased nuclear volume and altered nuclear morphology1. The mechanisms regulating intron retention is as yet undefined. While epigenetic mechanisms including DNA methylation, nucleosome occupancy and histone modifications have been associated with exon skipping and inclusion, the link between these changes and intron retention is unknown. Since splicing occurs co-transcriptionally, epigenetic changes can influence the rate of RNA polymerase II elongation and splicing efficiency. For example, DNA methylation recruits Mecp2, which may act as a "road block" to decrease transcriptional rate and improve splice-site recognition. Here, we sought to explore the role of epigenetic changes in the regulation of intron retention. 

Hypothesis

Decreased DNA methylation and nucleosome occupancy levels across exon-intron boundaries promote intron retention.

Methods

We have identified differential intron retention in mouse granulocytes and their precursors (promyelocytes) using mRNA sequencing. Whole Genome Bisulfite Sequencing (WGBS), targeted bisulfite sequencing and Nucleosome Occupancy and Methylome Sequencing (NOMeSeq) were performed to determine differential methylation and nucleosome occupancy levels across exon-intron boundaries of intron-retaining transcripts in granulocytes and promyelocytes. Chromatin immunoprecipitation-qPCR was performed to determine if changes in DNA methylation levels altered recruitment of Mecp2.

Results

WGBS and targeted bisulfite sequencing revealed significant loss of DNA methylation by 10-80% near the boundaries of introns (±100bp from the splice site) that were preferentially retained in granulocytes compared to promyelocytes (P<0.05). Loss of DNA methylation was also associated with the loss of Mecp2 binding in granulocyte-specific intron-retaining genes including S100a8, Ngp and Gadd45a (P<0.05). NOMeSeq further revealed a lack of nucleosome occupancy near the preceding exon-intron boundary when an intron was retained.

Discussion

Epigenetic mechanisms involving loss of DNA methylation and nucleosome occupancy is associated with increased intron retention in mRNAs. They may promote intron retention by recruiting proteins such as Mecp2 to alter splicing efficiency.

  1. Wong et al.(2013) Cell,154:583-95
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