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Volume 272, Number 39, Issue of September 26, 1997 pp. 24657-24665
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, July 22, 1997)

Andrea H. Lauber , Thomas J. Barrett ^¶ , Malayannan Subramaniam ^¶ , Mark Schuchard and Thomas C. Spelsberg ^¶

From  Mayo Medical Ventures, Mayo Clinic, Rochester, Minnesota 55905, the ^¶ Department of Biochemistry and Molecular Biology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, and the  Electrophoresis Department, Sigma Chemical Co., St. Louis, Missouri 63118

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-myc gene 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 -sheet structure at the N-terminal domain. Southern blot analyses using nuclear matrix DNA suggests that there are dual MAR sites in the c-myc promoter, 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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