Identification of
Glucose-responsive and Insulin-responsive Elements in Promoter of Mouse ob Gene
WANG Fang-Nian, MA Chun-Gu, ZHANG Yan-Ling, ZHANG Nai-Xian,
CHEN You-Mei, TANG Qi-Qun, SONG Hou-Yan*
( The Laboratory of Molecular Genetics, Shanghai Medical University,
Shanghai 200032, China )
Abstract Glucose and
insulin stimulate leptin gene expression in vitro and in vivo.
To identify cis-elements that are responsible for the glucose and
insulin effects, mouse 3T3-L1 adipocytes were transiently transfected with
reporter constructs with serial deletions in mouse ob gene promoter.
The cis-elements were identified with Gel mobility shift assays
(GMSA), DNase I footprint assays and PCR mediated site-directed mutation
assays. Transient transfections detected a negative cis-acting
element, a glucose-responsive element (GLRE), and an insulin-responsive element
(IRE) in the region from -1 719 bp to -1 452 bp of mouse ob gene. This
region does not contain any known GLRE or IRE. GMSA identified a DNA binding
protein which specifically binds a native probe prepared from mouse ob
gene promoter (-1 719 bp/-1 452 bp), and the binding was repressed by glucose
or insulin. DNase I footprint assays and PCR mediated site-directed mutations
assays identified that the binding motif AGCAAAA, spanning -1 698 bp to -1 692
bp of the mouse ob gene promoter, was responsible for the effects of
glucose and insulin on ob gene expression. These studies suggest that
a negative cis-acting element is located between -1 719 bp and -1 452
bp of the mouse ob gene promoter, and glucose and insulin simulate
mouse ob gene expression by repressing the binding of a transcription
factor to this element. This element, AGCAAAA, spanning -1 698 bp to -1 692 bp
is a novel GLRE and IRE.
Key words ob gene£»leptin£»glucose£»insulin£»response element
*Corresponding author£ºTel,86-21-64033738£»Fax,86-21-64033738£»e-mail, [email protected]