Identification of core sequences involved in metabolism-dependent nuclear protein binding to the rat insulin-like growth factor I gene.
In the liver, most insulin-like growth factor I (IGF-I) transcripts originate in exon 1, where important cis-regulatory regions are located downstream from the major transcription initiation sites. Within these regions, we have attempted to identify sequences which are involved in the decrease in IGF-I gene transcription associated with diabetes mellitus. The function of different genomic templates was assessed by in vitro transcription, which revealed a consistent 50-80% decrease in the activity of nuclear extracts from streptozotocin-diabetic as compared with normal rats. The disparity in transcriptional activity between normal and diabetic nuclear extracts was reduced with templates containing 11-bp mutations within DNase I protected regions III or V (+42 and +129 bp, respectively, from the major transcription initiation site), but a mutation between regions IV and V had little effect. Within region III, gel mobility shift analysis and methylation interference studies indicated that DNA-protein interactions involve a GCGC core sequence. In region V, gel mobility shift studies and uracil interference analysis revealed interactions involving a TTAT core. While gel mobility shift analysis and transient transfection studies indicate that the GCGC core sequence in region III recognizes C/EBP, the AT-rich sequence in region V is likely to recognize a protein with homeodomain characteristics. Identification of the nuclear factor(s) interacting with regions III and V, downstream from exon 1 initiation sites, will be important for understanding the mechanism of reduced IGF-I gene transcription due to diabetes mellitus.
Zhu
J L
JL
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Pao
C I
CI
Hunter
E
E
Lin
K W
KW
Wu
G J
GJ
Phillips
L S
LS
D000818:Animals; D001483:Base Sequence / Q000235:genetics; D001665:Binding Sites / Q000502:physiology; D002457:Cell Extracts / Q000502:physiology; D002467:Cell Nucleus / Q000737:chemistry; D003921:Diabetes Mellitus, Experimental / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000235:genetics* / Q000378:metabolism; D008297:Male; D009154:Mutation / Q000502:physiology; D009687:Nuclear Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012016:Reference Values; D014158:Transcription, Genetic / Q000502:physiology
D002457:Cell Extracts; D009687:Nuclear Proteins; D007334:Insulin-Like Growth Factor I