Evidence that the mouse insulin receptor substrate-1 belongs to the gene family on which the promoter is activated by estrogen receptor alpha through its interaction with Sp1.
In the present study, the molecular mechanism underlying the up-regulatory effect of estradiol (E2) on mouse insulin receptor substrate-1 (IRS-1) promoter was investigated in CHO cells on which the same promoter had first been functionally characterized. The mouse IRS-1 promoter bears four consensus half Estrogen Responsive Elements (ERE) sequences and thirteen AP-1- and ten Sp1-binding elements. We performed molecular dissection of this promoter gene providing 3' different deleted constructs, containing the same AP-1 rich region with a progressively increased number of ERE half sites located downstream. None of these constructs was responsive to E2, while a downstream region (nt -1420 to -160) rich in GC elements was induced by E2. However, the latter region lost its intrinsic E2 responsiveness when the whole IRS-1 promoter was mutated for deletion in all four ERE half sites. Deletion analysis of the ERE half sites demonstrated that only ERE located at the position -1500 to -1495, close to the GC-rich region, was able to maintain the induced activatory effect of E2 on the IRS-1 gene. Electrophoretic mobility shift and chromatin immunoprecipitation assays identified the region containing the half ERE/Sp1 (nt -1500 to -1477) as the one conferring E2 responsiveness to the whole promoter. This effect occurs through the functional interaction between E2/ERalpha and Sp1.
Panno
M L
ML
Department of Cellular Biology, University of Calabria, Italy.
Mauro
L
L
Marsico
S
S
Bellizzi
D
D
Rizza
P
P
Morelli
C
C
Salerno
M
M
Giordano
F
F
Andò
S
S
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D047628:Estrogen Receptor alpha / Q000378:metabolism*; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016329:Sp1 Transcription Factor / Q000378:metabolism*
D017931:DNA Primers; D047628:Estrogen Receptor alpha; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D010750:Phosphoproteins; D016329:Sp1 Transcription Factor