Insulin-responsive nuclear proteins facilitate Sp1 interactions with the insulin-like growth factor-I gene.
The diabetes-induced decrease in insulin-like growth factor-I transcription appears to be mediated by footprint region V in exon 1. Since region V contains both an Sp1 site and an AT-rich element that recognizes an insulin-responsive binding protein (IRBP), we tested the hypothesis that Sp1 interactions are facilitated by an IRBP. Binding of nuclear extracts to region V probes was reduced by mutational or chemical interference with the AT-rich element. Blocking the AT site also reduced interactions of Sp1 with region V in vitro and blunted transactivation of region V reporter constructs by Sp1 in vivo. Sp1 binding was enhanced by small quantities of hepatic nuclear extracts, but enhancement was reduced by the AT mutation and abolished by a 5-base pair insertion between the AT-rich and GC-rich sites, and transactivation by Sp1 in vivo was diminished by inserting bases between the AT-rich and GC-rich elements. However, treating cells with insulin increased the ability of nuclear extracts to enhance Sp1 binding. These findings indicate that the presence of the AT-rich element is essential for the actions of Sp1 in vitro and in vivo, and the combination of both spacing requirements and insulin responsiveness suggests that IRBP may interact directly with Sp1.
Kaytor
E N
EN
Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Zhu
J L
JL
Pao
C I
CI
Phillips
L S
LS
D020856:AT Rich Sequence / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D016384:Consensus Sequence / Q000502:physiology; D004247:DNA / Q000378:metabolism; D020862:GC Rich Sequence / Q000502:physiology; D022781:Hepatocytes / Q000378:metabolism; D007328:Insulin / Q000502:physiology*; D007334:Insulin-Like Growth Factor I / Q000235:genetics* / Q000378:metabolism; D008297:Male; D009687:Nuclear Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016329:Sp1 Transcription Factor / Q000378:metabolism*
D007328:Insulin; D009687:Nuclear Proteins; D016329:Sp1 Transcription Factor; D007334:Insulin-Like Growth Factor I; D004247:DNA