Journal of Biological Chemistry
Volume 272, Issue 39, 26 September 1997, Pages 24657-24665
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NUCLEIC ACIDS, PROTEIN SYNTHESIS, AND MOLECULAR GENETICS
A DNA-binding Element for a Steroid Receptor-binding Factor Is Flanked by Dual Nuclear Matrix DNA Attachment Sites in the c-myc Gene Promoter*

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The receptor-binding factor (RBF) for the avian oviduct progesterone (Pg) receptor (PR) has previously been shown to be a unique 10-kDa nuclear matrix protein that generates high affinity PR-binding sites on avian DNA. This paper describes the use of Southwestern blot and DNA gel shift analyses with RBF protein to identify a minimal 54-base pair RBF-binding element in the matrix-associated region (MAR) of the Pg-regulated c-mycgene promoter. This element contains a 5′-GC-rich domain and a 3′-AT-rich domain, the latter of which has a homopurine/homopyrimidine structure. The gel shift assays required the generation of an RBF-maltose fusion protein (RBF-MBP), which specifically binds this element and is supershifted when the anti-RBF polyclonal antibody is added. Computer analysis of the full-length amino acid sequence for RBF predicts a DNA-binding motif involving a β-sheet structure at the N-terminal domain. Southern blot analyses using nuclear matrix DNA suggests that there are dual MAR sites in the c-mycpromoter, which flank an intervening domain containing the RBF element. The co-transfection of this MAR sequence, containing the RBF element and cloned into a luciferase reporter vector, together with an RBF expression vector construct, into steroid treated human MCF-7 cells, results in a decrease of the c-myc promoter activity relative to control transfections containing only the parent vector of the RBF expression construct. These data suggest that a unique chromatin/nuclear matrix structure, composed of the RBF-DNA element complex which is flanked by nuclear matrix attachment sites, serves to bind the PR and repress the c-myc promoter.

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*

This work was supported by National Institutes of Health Grant HD09140, the Mayo Foundation, and National Institutes of Health Training Grants HD07108 (to M. S.), CA90441 (to M .S.), and AR07591 (to T. B.).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.

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The first two authors contributed equally to this work.