title: Transcriptional activity of the islet β cell factor Pdx1 is augmented by lysine methylation catalyzed by the methyltransferase Set7/9.
abstract: The transcription factor Pdx1 is crucial to islet β cell function and regulates target genes in part through interaction with coregulatory factors. Set7/9 is a Lys methyltransferase that interacts with Pdx1. Here we tested the hypothesis that Lys methylation of Pdx1 by Set7/9 augments Pdx1 transcriptional activity. Using mass spectrometry and mutational analysis of purified proteins, we found that Set7/9 methylates the N-terminal residues Lys-123 and Lys-131 of Pdx1. Methylation of these residues occurred only in the context of intact, full-length Pdx1, suggesting a specific requirement of secondary and/or tertiary structural elements for catalysis by Set7/9. Immunoprecipitation assays and mass spectrometric analysis using β cells verified Lys methylation of endogenous Pdx1. Cell-based luciferase reporter assays using wild-type and mutant transgenes revealed a requirement of Pdx1 residue Lys-131, but not Lys-123, for transcriptional augmentation by Set7/9. Lys-131 was not required for high-affinity interactions with DNA in vitro, suggesting that its methylation likely enhances post-DNA binding events. To define the role of Set7/9 in β cell function, we generated mutant mice in which the gene encoding Set7/9 was conditionally deleted in β cells (Set(Δ)β). Set(Δ)β mice exhibited glucose intolerance similar to Pdx1-deficient mice, and their isolated islets showed impaired glucose-stimulated insulin secretion with reductions in expression of Pdx1 target genes. Our results suggest a previously unappreciated role for Set7/9-mediated methylation in the maintenance of Pdx1 activity and β cell function.
authors.affiliation: From the Department of Cellular and Integrative Physiology.
authors.forename: Aarthi V
authors.initials: AV
authors.lastname: Maganti
authors.affiliation: Department of Pediatrics and the Herman B. Wells Center for Pediatric Research.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Maier
authors.affiliation: Department of Pediatrics and the Herman B. Wells Center for Pediatric Research.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Tersey
authors.affiliation: the Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536.
authors.forename: Megan L
authors.initials: ML
authors.lastname: Sampley
authors.affiliation: Department of Biochemistry and Molecular Biology, and.
authors.forename: Amber L
authors.initials: AL
authors.lastname: Mosley
authors.affiliation: the Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536.
authors.forename: Sabire
authors.initials: S
authors.lastname: Özcan
authors.affiliation: the Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, and.
authors.forename: Boobalan
authors.initials: B
authors.lastname: Pachaiyappan
authors.affiliation: the Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, and.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Woster
authors.affiliation: the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
authors.forename: Chad S
authors.initials: CS
authors.lastname: Hunter
authors.affiliation: the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
authors.forename: Roland
authors.initials: R
authors.lastname: Stein
authors.affiliation: From the Department of Cellular and Integrative Physiology, Department of Pediatrics and the Herman B. Wells Center for Pediatric Research, Department of Biochemistry and Molecular Biology, and Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202, rmirmira@iu.edu.
authors.forename: Raghavendra G
authors.initials: RG
authors.lastname: Mirmira
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D057809:HEK293 Cells; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008745:Methylation; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D018398:Homeodomain Proteins; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D011495:Histone-Lysine N-Methyltransferase; D008239:Lysine
keywords: Diabetes; Gene Knockout; Pdx1; Protein Methylation; Set7/9; Transcription; pancreatic Islet