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.
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 beta-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.
Lauber
A H
AH
Mayo Medical Ventures, Mayo Clinic, Rochester, Minnesota 55905, USA.
Barrett
T J
TJ
Subramaniam
M
M
Schuchard
M
M
Spelsberg
T C
TC
D000818:Animals; D030161:Avian Proteins; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002645:Chickens; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D016259:Genes, myc; D006801:Humans; D008969:Molecular Sequence Data; D015530:Nuclear Matrix / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011980:Receptors, Progesterone / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D030161:Avian Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C085646:RBF-1 protein, Gallus gallus; D011980:Receptors, Progesterone; D004247:DNA