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

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

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

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 clement 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 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-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.

Original languageEnglish (US)
Pages (from-to)24657-24665
Number of pages9
JournalJournal of Biological Chemistry
Volume272
Issue number39
DOIs
StatePublished - Sep 26 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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