title: Identification by high-throughput imaging of the histone methyltransferase EHMT2 as an epigenetic regulator of VEGFA alternative splicing.
abstract: Recent evidence points to a role of chromatin in regulation of alternative pre-mRNA splicing (AS). In order to identify novel chromatin regulators of AS, we screened an RNAi library of chromatin proteins using a cell-based high-throughput in vivo assay. We identified a set of chromatin proteins that regulate AS. Using simultaneous genome-wide expression and AS analysis, we demonstrate distinct and non-overlapping functions of these chromatin modifiers on transcription and AS. Detailed mechanistic characterization of one dual function chromatin modifier, the H3K9 methyltransferase EHMT2 (G9a), identified VEGFA as a major chromatin-mediated AS target. Silencing of EHMT2, or its heterodimer partner EHMT1, affects AS by promoting exclusion of VEGFA exon 6a, but does not alter total VEGFA mRNA levels. The epigenetic regulatory mechanism of AS by EHMT2 involves an adaptor system consisting of the chromatin modulator HP1γ, which binds methylated H3K9 and recruits splicing regulator SRSF1. The epigenetic regulation of VEGFA is physiologically relevant since EHMT2 is transcriptionally induced in response to hypoxia and triggers concomitant changes in AS of VEGFA. These results characterize a novel epigenetic regulatory mechanism of AS and they demonstrate separate roles of epigenetic modifiers in transcription and alternative splicing.
authors.affiliation: National Cancer Institute, NIH, Bethesda, MD 20892, USA maayan.salton@gmail.com.
authors.forename: Maayan
authors.initials: M
authors.lastname: Salton
authors.affiliation: National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Ty C
authors.initials: TC
authors.lastname: Voss
authors.affiliation: National Cancer Institute, NIH, Bethesda, MD 20892, USA mistelit@mail.nih.gov.
authors.forename: Tom
authors.initials: T
authors.lastname: Misteli
mesh_terms: D017398:Alternative Splicing; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D044127:Epigenesis, Genetic; D057809:HEK293 Cells; D006649:Histocompatibility Antigens / Q000502:physiology*; D011495:Histone-Lysine N-Methyltransferase / Q000502:physiology*; D006801:Humans; D061986:MCF-7 Cells; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000378:metabolism; D034622:RNA Interference; D016601:RNA-Binding Proteins / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
chemical_list: C518294:CBX3 protein, human; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D006649:Histocompatibility Antigens; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D000068103:Serine-Arginine Splicing Factors; C445405:EHMT2 protein, human; D011495:Histone-Lysine N-Methyltransferase