D. Wang et Hs. Sul, UPSTREAM STIMULATORY FACTOR-BINDING TO THE E-BOX AT -65 IS REQUIRED FOR INSULIN REGULATION OF THE FATTY-ACID SYNTHASE PROMOTER, The Journal of biological chemistry, 272(42), 1997, pp. 26367-26374
Fatty acid synthase (FAS) plays a central role in de novo lipogenesis
in mammals. We have shown that FAS transcription rate is induced drama
tically when fasted animals are refed with a high carbohydrate diet or
when streptozotocin-diabetic mice are given insulin, We also reported
that FAS gene transcription was up regulated by insulin through the p
roximal promoter region from -71 to -50 and that upstream stimulatory
factors (USFs), including USF1 and USF2, interact with this region in
vitro, In the present study, by using site-directed mutagenesis of the
-71/-50 region and correlating functional assays of the mutated promo
ter with USF binding activities, we demonstrate that the -65/-60 E-box
motif (5'-CATGTG-3') is functionally required for insulin regulation
and that USFs are in vivo components of the insulin response complex,
Mutation of the -65/-60 E-box sequence abolished insulin response in b
oth transiently and stably transfected 3T3-L1 adipocytes in the -2.1 k
b promoter context, which contains all the necessary regulatory elemen
ts of the promoter based on our previous transgenic mice studies, and
in the minimal -67 promoter context, Gel mobility shift assays demonst
rated that USFs can no longer bind to the -71/-50 promoter region when
the E-box is mutated. Cotransfection of USF1 and USF2 expression vect
ors with the FAS promoter-luciferase reporter constructs increased ins
ulin-stimulated FAS promoter activity, Moreover, cotransfection of dom
inant negative USF1 and USF2 mutants lacking the DNA binding domain in
hibited the insulin stimulation of the FAS promoter activity, On the o
ther hand, site-directed mutagenesis of the -65/-60 E-box surrounding
sequences within the overlapped tandem copies of sterol regulatory ele
ment-binding protein (SREBP) binding sites prevented SREBP from bindin
g to -71/-50 promoter region in vitro but had no effect on insulin reg
ulation of the FAS promoter in vivo, When rat liver nuclear extracts w
ere used in gel mobility shift assays, only USF-containing protein-DNA
complexes that can be supershifted by specific USF antibodies were ob
served, These results demonstrate that upstream stimulatory factor bin
ding to the E-box at -65 is required for insulin regulation of the fat
ty acid synthase promoter.