title: Creb1-Mecp2-(m)CpG complex transactivates postnatal murine neuronal glucose transporter isoform 3 expression.
abstract: The murine neuronal facilitative glucose transporter isoform 3 (Glut3) is developmentally regulated, peaking in expression at postnatal day (PN)14. In the present study, we characterized a canonical CpG island spanning the 5'-flanking region of the glut3 gene. Methylation-specific PCR and bisulfite sequencing identified methylation of this CpG ((m)CpG) island of the glut3 gene, frequency of methylation increasing 2.5-fold with a 1.6-fold increase in DNA methyl transferase 3a concentrations noted with advancing postnatal age (PN14 vs PN3). 5'-flanking region of glut3-luciferase reporter transient transfection in HT22 hippocampal neurons demonstrated that (m)CpGs inhibit glut3 transcription. Contrary to this biological function, glut3 expression rises synchronously with (m)CpGs in PN14 vs PN3 neurons. Chromatin immunoprecipitation (IP) revealed that methyl-CpG binding protein 2 (Mecp2) bound the glut3-(m)CpGs. Depending on association with specific coregulators, Mecp2, a dual regulator of gene transcription, may repress or activate a downstream gene. Sequential chromatin IP uncovered the glut3-(m)CpGs to bind Mecp2 exponentially upon recruitment of Creb1 rather than histone deacetylase 1. Co-IP and coimmunolocalization confirmed that Creb1 associated with Mecp2 and cotransfection with glut3-(m)CpG in HT22 cells enhanced glut3 transcription. Separate 5-aza-2'-deoxycytidine pretreatment or in combination with trichostatin A reduced (m)CpG and specific small interference RNAs targeting Mecp2 and Creb1 separately or together depleting Mecp2 and/or Creb1 binding of glut3-(m)CpGs reduced glut3 expression in HT22 cells. We conclude that Glut3 is a methylation-sensitive neuronal gene that recruits Mecp2. Recruitment of Creb1-Mecp2 by glut3-(m)CpG contributes towards transactivation, formulating an escape from (m)CpG-induced gene suppression, and thereby promoting developmental neuronal glut3 gene transcription and expression.
authors.affiliation: Department of Pediatrics, Division of Neonatology and Developmental Biology, Neonatal Research Center, David Geffen School of Medicine University of California LosAngeles, Los Angeles, California 90095-1752, USA.
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Chen
authors.forename: Bo-Chul
authors.initials: BC
authors.lastname: Shin
authors.forename: Shanthie
authors.initials: S
authors.lastname: Thamotharan
authors.forename: Sherin U
authors.initials: SU
authors.lastname: Devaskar
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D018899:CpG Islands / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D019175:DNA Methylation / Q000502:physiology; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D051274:Glucose Transporter Type 3 / Q000502:physiology*; D051783:Methyl-CpG-Binding Protein 2 / Q000502:physiology*; D051379:Mice; D015534:Trans-Activators
chemical_list: C492004:Creb1 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D051274:Glucose Transporter Type 3; C497393:Mecp2 protein, mouse; D051783:Methyl-CpG-Binding Protein 2; C495689:Slc2a3 protein, mouse; D015534:Trans-Activators