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Originally published In Press as doi:10.1074/jbc.M207177200 on August 12, 2002
J. Biol. Chem., Vol. 277, Issue 43, 40235-40246, October 25, 2002
Mechanism of Gonadotropin Gene Expression
IDENTIFICATION OF A NOVEL NEGATIVE REGULATORY ELEMENT AT THE
TRANSCRIPTION START SITE OF THE GLYCOPROTEIN HORMONE -SUBUNIT
GENE*
Wanfen
Xiong §,
William E.
Tapprich¶, and
G.
Stanley
Cox
From the Department of Biochemistry and Molecular
Biology, University of Nebraska Medical Center, Omaha, Nebraska
68198-4525 and the ¶ Biology Department, University of Nebraska at
Omaha, Omaha, Nebraska 68182
Regulation of the glycoprotein hormone
-subunit (GPH ) gene has been studied extensively in pituitary and
placental cell lines, but little is known of the transcriptional
regulators important for its ectopic expression. To investigate the
molecular basis for ectopic expression, it was critical to
define cis-regulatory elements and their cognate
trans-acting factors that modulate promoter activity in
epithelial cell types that do not normally express GPH. DNA-mediated
transient expression of promoter-reporter constructs was used to
identify a novel negative regulatory element located at the GPH gene
transcription start site. Truncation or site-directed mutagenesis of
this element produced up to a 10-fold increase in promoter activity.
Electrophoretic mobility shift analysis detected a protein that binds
specifically to a DNA motif encompassing the cap site. Based on
competitive DNA binding studies with mutated oligonucleotides, it was
determined that bases from 5 to 2 and +4 to +11 are critical for
protein binding. The DNA sequence flanking the transcription start site from 9 to +11 is an imperfect palindrome; consequently, this motif is
referred to as the cap site diad element (CSDE) and the cognate factor
as the cap site-binding protein (CSBP). CSBP activity was present at
different levels in nuclear extracts prepared from a variety of cell
types. Significantly, the ratio of activities exhibited by the GPH
promoter with a mutated CSDE compared with the promoter with a
wild-type CSDE was dependent on the transfected cell line and its
content of CSBP. These results indicate that a negative regulatory
element centered at the GPH gene cap site and its cognate
DNA-binding protein make a significant contribution to the production
of -subunit in a variety of tumor tissues. A detailed understanding
of this cis/trans pair may further suggest a
mechanism to explain, at least in part, how this gene becomes activated in nonendocrine tumors.
*
This work was supported by Grant 3103 from the Council for
Tobacco Research U.S.A., Inc. and Grants 98-12 and 03-08 from the State
of Nebraska Department of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Dept. of Surgery, University of Nebraska Medical
Center, Omaha, NE 68198-7690.
To whom correspondence and reprint requests should be
addressed: Dept. of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 984525 Nebraska Medical Center, Omaha, NE
68198-4525. Tel.: 402-559-6651; Fax: 402-559-6650; E-mail: gscox@unmc.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
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