Abstract
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 kappaB-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-kappaB/Rel-specific p50.p50 homo- and p50.p65 heterodimers. The third complex was formed by the 200-kDa protein. Even though the synthetic kappaB-responsible promoter was active in mouse adenocarcinoma cells, a mutation preventing NF-kappaB binding had no effect on the mts1 natural enhancer activity. On the contrary, the mutation in the kappaB-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 kappaB-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.
Highlights
The mts1 gene encodes a Ca2ϩ-binding protein belonging to the S100 subfamily [1,2,3,4]
At the initial step of the search for cis-elements regulating mts1 transcription, we compared the abilities of the mts1 first intron and 5Ј-flanking sequences to enhance transcription in mouse adenocarcinoma CSML0 and CSML100 cells
The activity of these plasmids was studied in CSML100 and CSML0 adenocarcinoma cells compared with the activity of SV40 promoter/enhancer, taken as 100%
Summary
The mts gene encodes a Ca2ϩ-binding protein belonging to the S100 subfamily [1,2,3,4]. Mts transcription was shown to be inducible in several cell systems [3, 4, 10].1 It was shown in direct transfection experiments that constitutive overexpression of the Mts protein in a benign rat epithelial cell line [11] and in src-transformed rat fibroblasts [9] influenced in vitro cell invasion and promoted tumor progression. The expression of the mts gene correlated with the metastatic phenotype of mouse and human carcinoma cells [12, 13]. Transcriptional regulation of the mts gene has been studied in the mouse metastatic adenocarcinoma cell line CSML100, where this gene is highly expressed. The constructs that contained the CAT gene, under the control of the mts regulatory sequences, revealed some level of transcriptional activity when they were transiently transfected into mouse adenocarcinoma cells CSML0, even though the endogenous mts transcription was not detectable in those cells [14]. It was speculated that DNA methylation is involved in transcriptional control of the mts gene [14, 16]
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