title: Binding of activating transcription factor 6 to the A5/Core of the rat insulin II gene promoter does not mediate its transcriptional repression.
abstract: Pancreatic β-cells have a well-developed endoplasmic reticulum due to their highly specialized secretory function to produce insulin in response to glucose and nutrients. It has been previously reported that overexpression of activating transcription factor 6 (ATF6) reduces insulin gene expression in part via upregulation of small heterodimer partner. In this study, we investigated whether ATF6 directly binds to the insulin gene promoter, and whether its direct binding represses insulin gene promoter activity. A bioinformatics analysis identified a putative ATF6 binding site in the A5/Core region of the rat insulin II gene promoter. Direct binding of ATF6 was confirmed using several approaches. Electrophoretic mobility shift assays in nuclear extracts from MCF7 cells, isolated rat islets and insulin-secreting HIT-T15 cells showed ATF6 binding to the native A5/Core of the rat insulin II gene promoter. Antibody-mediated supershift analyses revealed the presence of both ATF6 isoforms, ATF6α and ATF6β, in the complex. Chromatin immunoprecipitation assays confirmed the binding of ATF6α and ATF6β to a region encompassing the A5/Core of the rat insulin II gene promoter in isolated rat islets. Overexpression of the active (cleaved) fragment of ATF6α, but not ATF6β, inhibited the activity of an insulin promoter-reporter by 50%. However, the inhibitory effect of ATF6α was insensitive to mutational inactivation or deletion of the A5/Core. Therefore, although ATF6 binds directly to the A5/Core of the rat insulin II gene promoter, this direct binding does not appear to contribute to its repressive activity.
authors.affiliation: Montreal Diabetes Research Center, Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Technopole Angus, 2901 Rachel Est, Montreal, Quebec H1W 4A4, Canada.
authors.forename: Julie
authors.initials: J
authors.lastname: Amyot
authors.forename: Isma
authors.initials: I
authors.lastname: Benterki
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Fontés
authors.forename: Derek K
authors.initials: DK
authors.lastname: Hagman
authors.forename: Mourad
authors.initials: M
authors.lastname: Ferdaoussi
authors.forename: Tracy
authors.initials: T
authors.lastname: Teodoro
authors.forename: Allen
authors.initials: A
authors.lastname: Volchuk
authors.forename: Érik
authors.initials: É
authors.lastname: Joly
authors.forename: Vincent
authors.initials: V
authors.lastname: Poitout
mesh_terms: D051702:Activating Transcription Factor 6 / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016384:Consensus Sequence / Q000235:genetics; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D007328:Insulin / Q000235:genetics*; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism; D007783:Lactones / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012322:RNA Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D016415:Sequence Alignment; D012717:Sesquiterpenes / Q000494:pharmacology; D014158:Transcription, Genetic
chemical_list: D051702:Activating Transcription Factor 6; D007328:Insulin; D007783:Lactones; D012322:RNA Precursors; D012333:RNA, Messenger; D012717:Sesquiterpenes; C046319:thapsigargicin