Transcriptional Activity of the Islet β Cell Factor Pdx1 Is Augmented by Lysine Methylation Catalyzed by the Methyltransferase Set7/9*
| Autor: | Raghavendra G. Mirmira, Aarthi V. Maganti, Megan L. Sampley, Sabire Özcan, Patrick M. Woster, Sarah A. Tersey, Boobalan Pachaiyappan, Chad S. Hunter, Bernhard Maier, Amber L. Mosley, Roland Stein |
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| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
endocrine system
Methyltransferase endocrine system diseases Transcription Genetic Immunoblotting Molecular Sequence Data Biology Biochemistry Histone-Lysine N-Methyltransferase digestive system Methylation Mice Transcription (biology) Tandem Mass Spectrometry Cell Line Tumor Insulin-Secreting Cells Protein methylation Animals Humans Gene Regulation Amino Acid Sequence Molecular Biology Transcription factor Homeodomain Proteins Mice Knockout Reverse Transcriptase Polymerase Chain Reaction Lysine HEK 293 cells Cell Biology Molecular biology Mice Inbred C57BL HEK293 Cells DNA methylation NIH 3T3 Cells Trans-Activators Protein Binding |
| Popis: | Background: Pdx1 interacts with the methyltransferase Set7/9 to transactivate β cell genes. Results: Methylation of Pdx1 residue Lys-131 by Set7/9 augments Pdx1 activity. Conclusion: The ability of Pdx1 to regulate genes in β cells is partially dependent upon its methylation by Set7/9. Significance: This study reveals a previously unappreciated role for Lys methylation in the maintenance of Pdx1 activity and β cell function. 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 (SetΔβ). SetΔβ 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. |
| Databáze: | OpenAIRE |
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