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Volume 272, Number 8, Issue of February 21, 1997 pp. 4828-4835
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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A B-related Binding Site Is an Integral Part of the mts1 Gene Composite Enhancer Element Located in the First Intron of the Gene

(Received for publication, July 30, 1996, and in revised form, November 20, 1996)

From the Danish Cancer Society, Department of Molecular Cancer Biology, Strandboulevarden 49, DK-2100 Copenhagen, Denmark and the ^¶ Institute for Gene Biology, Russian Academy of Sciences, Vavilov Str. 34, Moscow, Russia

The transcription of the mts1 gene correlates with the metastatic potential of mouse adenocarcinomas. Here we describe strong enhancer whose location coincides with the DNase I hypersensitivity area in the first intron of the mts1 gene. The investigation of the transcriptional activity of a series of plasmids bearing deletions in the first intron sequences revealed that the observed enhancer has a composite structure. The enhancer activity is partially formed by the B-related element: GGGGTTTTTCCAC. This sequence element was able to form several sequence-specific complexes with nuclear proteins extracted from both Mts1-expressing CSML100 and Mts1-non-expressing CSML0 adenocarcinoma cells. Two of these complexes were identified as NF-B/Rel-specific p50·p50 homo- and p50·p65 heterodimers. The third complex was formed by the 200-kDa protein. Even though the synthetic B-responsible promoter was active in mouse adenocarcinoma cells, a mutation preventing NF-B binding had no effect on the mts1 natural enhancer activity. On the contrary, the mutation in the B-related element, which abolished the binding of the 200-kDa protein, led to the functional inactivation of this site in the mts1 first intron. The mts1 B-like element activated transcription from its own mts1 gene promoter, as well as from the heterologous promoter in both CSML0 and CSML100 cells. However, in vivo occupancy of this site was observed only in Mts1-expressing CSML100 cells, suggesting the involvement of the described element in positive control of mts1 transcription.

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