Transcriptional Activity of the Islet β Cell Factor Pdx1 Is Augmented by Lysine Methylation Catalyzed by the Methyltransferase Set7/9*

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.

[1]  S. Masetto.  the progression of , 2022 .

[2]  A. El-Osta,et al.  Deep sequencing reveals novel Set7 networks , 2014, Cellular and Molecular Life Sciences.

[3]  M. Roe,et al.  Characterization of mice expressing Ins1 gene promoter driven CreERT recombinase for conditional gene deletion in pancreatic β-cells , 2014, Islets.

[4]  Alexander S. Garruss,et al.  SET for life: biochemical activities and biological functions of SET domain-containing proteins. , 2013, Trends in biochemical sciences.

[5]  J. Rutter,et al.  Per-Arnt-Sim Kinase Regulates Pancreatic Duodenal Homeobox-1 Protein Stability via Phosphorylation of Glycogen Synthase Kinase 3β in Pancreatic β-Cells* , 2013, The Journal of Biological Chemistry.

[6]  S. Tersey,et al.  Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease , 2012, Journal of visualized experiments : JoVE.

[7]  J. Hecksher-Sørensen,et al.  Pdx1 Is Post-Translationally Modified In vivo and Serine 61 Is the Principal Site of Phosphorylation , 2012, PloS one.

[8]  J. Schug,et al.  Research resource: the pdx1 cistrome of pancreatic islets. , 2012, Molecular endocrinology.

[9]  R. Kulkarni,et al.  Glucose activates free fatty acid receptor 1 gene transcription via phosphatidylinositol-3-kinase-dependent O-GlcNAcylation of pancreas-duodenum homeobox-1 , 2012, Proceedings of the National Academy of Sciences.

[10]  Shiv k. Sharma,et al.  Discovery of novel alkylated (bis)urea and (bis)thiourea polyamine analogues with potent antimalarial activities. , 2011, Journal of medicinal chemistry.

[11]  W. Greene,et al.  Activation of HIV Transcription by the Viral Tat Protein Requires a Demethylation Step Mediated by Lysine-specific Demethylase 1 (LSD1/KDM1) , 2011, PLoS pathogens.

[12]  Albert Jeltsch,et al.  Specificity analysis-based identification of new methylation targets of the SET7/9 protein lysine methyltransferase. , 2011, Chemistry & biology.

[13]  Mihaela E. Sardiu,et al.  Highly Reproducible Label Free Quantitative Proteomic Analysis of RNA Polymerase Complexes* , 2010, Molecular & Cellular Proteomics.

[14]  B. Maier,et al.  Inhibition of Deoxyhypusine Synthase Enhances Islet β Cell Function and Survival in the Setting of Endoplasmic Reticulum Stress and Type 2 Diabetes* , 2010, The Journal of Biological Chemistry.

[15]  David N. Groff,et al.  Pcif1 modulates Pdx1 protein stability and pancreatic β cell function and survival in mice. , 2010, The Journal of clinical investigation.

[16]  G. Rutter,et al.  Pancreatic and duodenal homeobox 1 (PDX1) phosphorylation at serine-269 is HIPK2-dependent and affects PDX1 subnuclear localization , 2010, Biochemical and biophysical research communications.

[17]  Shiv k. Sharma,et al.  (Bis)urea and (bis)thiourea inhibitors of lysine-specific demethylase 1 as epigenetic modulators. , 2010, Journal of medicinal chemistry.

[18]  I. Talianidis,et al.  Lysine methylation regulates E2F1-induced cell death. , 2010, Molecular cell.

[19]  C. Dinarello,et al.  The unique hypusine modification of eIF5A promotes islet beta cell inflammation and dysfunction in mice. , 2010, The Journal of clinical investigation.

[20]  E. Ferrannini,et al.  Progression to Diabetes in Relatives of Type 1 Diabetic Patients: Mechanisms and Mode of Onset , 2009, Diabetes.

[21]  D. Baltimore,et al.  Regulation of NF-κB activity through lysine monomethylation of p65 , 2009, Proceedings of the National Academy of Sciences.

[22]  U. Jhala,et al.  Glucose Regulates Steady-state Levels of PDX1 via the Reciprocal Actions of GSK3 and AKT Kinases* , 2009, The Journal of Biological Chemistry.

[23]  N. Kelleher,et al.  Negative regulation of NF‐κB action by Set9‐mediated lysine methylation of the RelA subunit , 2009, The EMBO journal.

[24]  B. Maier,et al.  Peroxisome Proliferator-Activated Receptor γ Activation Restores Islet Function in Diabetic Mice through Reduction of Endoplasmic Reticulum Stress and Maintenance of Euchromatin Structure , 2009, Molecular and Cellular Biology.

[25]  B. Maier,et al.  Methyltransferase Set7/9 Maintains Transcription and Euchromatin Structure at Islet-Enriched Genes , 2009, Diabetes.

[26]  E. Li,et al.  The lysine demethylase LSD1 (KDM1) is required for maintenance of global DNA methylation , 2009, Nature Genetics.

[27]  B. Ren,et al.  Role of the Histone H3 Lysine 4 Methyltransferase, SET7/9, in the Regulation of NF-κB-dependent Inflammatory Genes , 2008, Journal of Biological Chemistry.

[28]  Xiaodong Cheng,et al.  Regulation of estrogen receptor alpha by the SET7 lysine methyltransferase. , 2008, Molecular cell.

[29]  R. Mirmira,et al.  Pdx1 and BETA2/NeuroD1 Participate in a Transcriptional Complex That Mediates Short-range DNA Looping at the Insulin Gene* , 2008, Journal of Biological Chemistry.

[30]  Min Gyu Lee,et al.  p53 is regulated by the lysine demethylase LSD1 , 2007, Nature.

[31]  R. Mirmira,et al.  A feat of metabolic proportions: Pdx1 orchestrates islet development and function in the maintenance of glucose homeostasis. , 2007, Molecular genetics and metabolism.

[32]  B. Chait,et al.  Methylation of a histone mimic within the histone methyltransferase G9a regulates protein complex assembly. , 2007, Molecular cell.

[33]  Lars Selander,et al.  Phosphorylation Marks IPF1/PDX1 Protein for Degradation by Glycogen Synthase Kinase 3-dependent Mechanisms* , 2006, Journal of Biological Chemistry.

[34]  Jean-François Couture,et al.  Structural basis for the methylation site specificity of SET7/9 , 2006, Nature Structural &Molecular Biology.

[35]  R. Mirmira,et al.  Pdx-1 Links Histone H3-Lys-4 Methylation to RNA Polymerase II Elongation during Activation of Insulin Transcription* , 2005, Journal of Biological Chemistry.

[36]  R. Mirmira,et al.  The C-terminal domain of the beta cell homeodomain factor Nkx6.1 enhances sequence-selective DNA binding at the insulin promoter. , 2005, Biochemistry.

[37]  G. Weir,et al.  Mechanism of insulin Gene Regulation by the Pancreatic Transcription Factor Pdx-1 , 2005, Journal of Biological Chemistry.

[38]  Brian D Strahl,et al.  Role of protein methylation in regulation of transcription. , 2005, Endocrine reviews.

[39]  Masafumi Matsuda,et al.  beta-Cell function in subjects spanning the range from normal glucose tolerance to overt diabetes: a new analysis. , 2005, The Journal of clinical endocrinology and metabolism.

[40]  Paul Tempst,et al.  Regulation of p53 activity through lysine methylation , 2004, Nature.

[41]  A. Mosley,et al.  Glucose regulation of insulin gene expression requires the recruitment of p300 by the beta-cell-specific transcription factor Pdx-1. , 2004, Molecular endocrinology.

[42]  Elisabeth Scheer,et al.  Gene-specific modulation of TAF10 function by SET9-mediated methylation. , 2004, Molecular cell.

[43]  R. Mirmira,et al.  Covalent Histone Modifications Underlie the Developmental Regulation of Insulin Gene Transcription in Pancreatic β Cells* , 2003, Journal of Biological Chemistry.

[44]  James D. Johnson,et al.  Increased islet apoptosis in Pdx1+/- mice. , 2003, The Journal of clinical investigation.

[45]  H. Maegawa,et al.  Sumoylation of Pdx1 is associated with its nuclear localization and insulin gene activation. , 2003, American journal of physiology. Endocrinology and metabolism.

[46]  Geoff Kelly,et al.  Structure and catalytic mechanism of the human histone methyltransferase SET7/9 , 2003, Nature.

[47]  Jeong Ho Chang,et al.  Mechanism of histone lysine methyl transfer revealed by the structure of SET7/9—AdoMet , 2003, The EMBO journal.

[48]  A. Mosley,et al.  Adenoviral Gene Transfer into β-Cell Lines , 2003 .

[49]  A. Mosley,et al.  Adenoviral gene transfer into beta-cell lines. , 2003, Methods in molecular medicine.

[50]  J. C. James,et al.  Quantitative assessment of gene targeting in vitro and in vivo by the pancreatic transcription factor, Pdx1. Importance of chromatin structure in directing promoter binding. , 2002, The Journal of biological chemistry.

[51]  D. Piston,et al.  Reduction in Pancreatic Transcription Factor PDX-1 Impairs Glucose-stimulated Insulin Secretion* , 2002, The Journal of Biological Chemistry.

[52]  D. Reinberg,et al.  Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding histone tail modifications required for heterochromatin formation. , 2002, Genes & development.

[53]  H. Watada,et al.  β-Cell Differentiation Factor Nkx6.1 Contains Distinct DNA Binding Interference and Transcriptional Repression Domains* , 2000, The Journal of Biological Chemistry.

[54]  M. German,et al.  The Homeodomain of PDX-1 Mediates Multiple Protein-Protein Interactions in the Formation of a Transcriptional Activation Complex on the Insulin Promoter , 2000, Molecular and Cellular Biology.

[55]  William L. Clarke,et al.  Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence , 1997, Nature Genetics.

[56]  B. Hogan,et al.  PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. , 1996, Development.

[57]  H. Edlund,et al.  Insulin-promoter-factor 1 is required for pancreas development in mice , 1994, Nature.

[58]  W. Rutter,et al.  The insulin and islet amyloid polypeptide genes contain similar cell-specific promoter elements that bind identical beta-cell nuclear complexes , 1992, Molecular and cellular biology.