BioProject

While intron retention (IR) is now recognized as a widespread and conserved mechanism of gene expression control, its regulation is poorly understood. More...

While intron retention (IR) is now recognized as a widespread and conserved mechanism of gene expression control, its regulation is poorly understood. Here, we identify significantly reduced DNA methylation levels near splice junctions flanking retained introns compared to non-retained introns in diverse primary cells and cell lines. Further, we identify increased IR following inhibition of DNA methylation indicating that reduced DNA methylation promotes IR. We demonstrate reduced occupancy of MeCP2 near the splice junctions of retained introns, mirroring the reduced DNA methylation at these sites. Accordingly, MeCP2 depletion in tissues/cells enhances IR. By analyzing the MeCP2 interactome, we demonstrate that decreased MeCP2 binding near splice junctions facilitates IR via reduced recruitment of the splicing factor, Tra2b, and increased RNA polymerase II stalling. Our study identifies a dynamic interplay between DNA methylation, MeCP2 and splicing factors including Tra2b in IR regulation and provides novel insights into the mechanisms governing splicing. Overall design: For methylome analysis, genomic DNA was extracted from FACS-purified primary mouse promyelocytes and granulocytes using the Purelink Genomic DNA kit (Thermo Fisher Scientific). DNA methylation at cytosine residues was assayed using MethylC-seq Whole Genome Bisulfite Sequencing (WGBS) at Macrogen. The DNA was fragmented to 300–500 bp via sonication using a Covaris S2 system (Covaris). End repair was performed with the End-It DNA End-Repair Kit (Epicenter). Paired-end universal library adaptors (Illumina) were ligated to the sonicated DNA according to the manufacturer’s instructions. Ligated products were purified with AMPure XP beads (Beckman, Brea, CA, USA). Adaptor-ligated DNA was bisulfite-treated using the EpiTect Bisulfite Kit (QIAGEN) as per the manufacturer’s instructions. PCR-amplification was performed using PfuTurboCx Hotstart DNA polymerase (Agilent, Santa Clara, CA, USA) with the following PCR conditions (2 min at 95 °C, 4 cycles of 15 s at 98 °C, 30 s at 60 °C, 4 min at 72 °C and then 10 min at 72 °C). The subsequent products were purified using the MinElute gel purification kit (QIAGEN). Sequencing was performed on an Hi-seq 2000 (Illumina). For MeCP2 Chip-seq, chromatin immunoprecipitation was performed using the Magna ChIP HiSens kit (Milipore 17-10460) following the manufacturer's instructions. Sample concentration and size distribution of immunoprecipitated DNA were measured with Qubit dsDNA HS Assay Kits (Invitrogen) and High Sensitivity DNA Analysis KIt (Agilent Technologies), respectively. Approximately 20 ng of the input and ChIP DNA samples were sent to Macrogen for ChIP-seq library preparation and sequencing on an Illumina Hiseq2000. Less...