A Y-box consensus sequence is required for basal expression of the human multidrug resistance (mdr1) gene.

M.E Goldsmith,M.J Madden,C.S Morrow,K.H Cowan

doi:10.1016/s0021-9258(18)53398-6

Abstract

Basal transcription of the human multidrug resistance (mdr1) promoter was studied by chloramphenicol acetyltransferase (CAT) reporter fusion gene analysis in two parental and doxorubicin-resistant human tumor cell lines. Deletion of mdr1 DNA sequences to -89 relative to the start of transcription (at +1) had little effect on expression. Deletion of nucleotide sequences from -89 to -70, however, resulted in a 5-10-fold reduction in mdrCAT expression. DNase I footprint analysis demonstrated that the region from -85 to -70 was protected from nuclease digestion using nuclear extracts from these cell lines. The sequence between -82 and -73 is perfectly homologous with the 10-base pair Y-box consensus sequence found in the promoters of all major histocompatibility complex class-II (MHC II) genes. The Y-box sequence in MHC II genes is required for accurate and efficient transcription and contains the sequence CCAAT in the reverse orientation (Dorn, A., Durand, B., Marfing, C., Le Meur, M., Benoist, C., and Mathis, D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6249-6253). Mutations in the reverse CCAAT sequence of the Y-box consensus substantially reduced expression of an mdrCAT vector and eliminated nucleoprotein binding in an electrophoretic mobility shift assay. These results suggest that proteins which bind to the putative Y-box consensus sequence are critical for basal transcriptional regulation of the human mdr1 gene.

Highlights

Basal transcription of the human multidrug resistancepromoter was studied by chloramphenicol acetyltransferase (CAT) reporter fusion gene analysis in two parental and doxorubicin-resistant human tumor cell lines.Deletion of mdrl DNA sequences to -89 relative to the start of transcription had little effect on expression
Previous transient mdrCAT expression studies from our laboratory demonstrated that deletionof mdrl promoter sequences from -4741 to -137 relative to the staortf transcription at +1 had little effect on mdrl promoter activity.' In order to examine basal mdrl promoter elements, a series of mdrCAT expression vectors were designed aroundprotein binding regions indicated by DNase I footprint analysis
This series of expression vectors was transfected into two paired parental and multidrug-resistant cell lines in order to determine if there was a correlation between the positionof bound nuclear proteins and mdrgl ene expression

Summary

RESULTS

Previous transient mdrCAT expression studies from our laboratory demonstrated that deletionof mdrl promoter sequences from -4741 to -137 relative to the staortf transcription at +1 had little effect on mdrl promoter activity.' In order to examine basal mdrl promoter elements, a series of mdrCAT expression vectors were designed aroundprotein binding regions indicated by DNase I footprint analysis. This series of expression vectors was transfected into two paired parental and multidrug-resistant cell lines in order to determine if there was a correlation between the positionof bound nuclear proteins and mdrgl ene expression. The tissuespecific pattern of expression of the mouse mdrlb gene is similar to that of the human mdrl gene while that of the mouse mdrla is different [2,31]

DISCUSSION

A Y-box ConsenSseuqsuence inHthuemmadnrl

ANTISENSE