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J. Biol. Chem., Vol. 279, Issue 7, 5278-5287, February 13, 2004

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Identification of a gadd45 3' Enhancer That Mediates SMAD3- and SMAD4-dependent Transcriptional Induction by Transforming Growth Factor ^*

Michael B. Major and David A. Jones¶

From the Departments of Oncological Sciences and Medicinal Chemistry, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112

GADD45 regulates cell growth, differentiation, and cell death following cellular exposure to diverse stimuli, including DNA damage and transforming growth factor- (TGF). We examined how cells transduce the TGF signal from the cell surface to the gadd45 genomic locus and describe how GADD45 contributes to TGF biology. Following an alignment of gadd45 genomic sequences from multiple organisms, we discovered a novel TGF-responsive enhancer encompassing the third intron of the gadd45 gene. Using three different experimental approaches, we found that SMAD3 and SMAD4, but not SMAD2, mediate transcription from this enhancer. Three lines of evidence support our conclusions. First, overexpression of SMAD3 and SMAD4 activated the transcriptional activity from this enhancer. Second, silencing of SMAD protein levels using short interfering RNAs revealed that TGF-induced activation of the endogenous gadd45 gene required SMAD3 and SMAD4 but not SMAD2. In contrast, we found that the regulation of plasminogen activator inhibitor type I depended upon all three SMAD proteins. Last, SMAD3 and SMAD4 reconstitution in SMAD-deficient cancer cells restored TGF induction of gadd45. Finally, we assessed the function of GADD45 within the TGF response and found that GADD45-deficient cells arrested in G₂ following TGF treatment. These data support a role for SMAD3 and SMAD4 in activating gadd45 through its third intron to facilitate G₂ progression following TGF treatment.

Received for publication, October 21, 2003 , and in revised form, November 19, 2003.

^* This work was supported by a predoctoral fellowship from the Pharmaceutical Research and Manufacturers of America Foundation (to M. B. M.), a grant from the Huntsman Cancer Foundation, a grant from the Willard L. Eccles Foundation, and University of Utah Core Facility Technical Support Grant CA42104. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains an additional figure and table.

^¶ To whom all correspondence should be addressed: Huntsman Cancer Institute, 2000 Circle of Hope, Salt Lake City, UT 84112-5550. Tel.: 801-585-6107; Fax: 801-585-0900; E-mail: david.jones{at}hci.utah.edu.

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