Analysis of the tissue-specific promoter of the MUC1 gene.
The 5'-sequences flanking the human MUC1 gene have been analyzed for their ability to direct expression of a reporter gene (the chloramphenicol acetyltransferase gene (CAT)) in cell lines that normally express or do not express the MUC1 gene. A construct containing 2.9 kilobase pairs of MUC1 5'-flanking sequence sequence showed expression of CAT in breast and pancreatic cell lines but not in the non-epithelial cell lines HT-1080, SK23, and HTB96. Deletion analysis showed that maximum expression was obtained in ZR-75 (breast cancer line) and HPAF (pancreatic cancer line) with only 743 base pairs of 5'-flanking sequence. Sequences within 1.6 kilobase pairs of the transcriptional start site showed enhancing activity in a vector carrying an enhancerless SV40 promoter. Analysis of proximal 5'-sequences in a promoterless CAT vector carrying the SV40 enhancer showed that sequences between -60 and -150 were crucial for tissue-specific expression. An Sp1 site at -99/-90 and an E box (E-MUC1) at -84/-64 in this region were shown by mutational analysis to play a role in the regulation of transcription. Gel shift analysis with oligonucleotides and nuclear extracts of ZR-75 showed protein binding to both of these sites. Sp1 binding activity was similar in ZR-75 and HT1080 cells, whereas binding of factors to the E-MUC1 oligonucleotide revealed quantitative and qualitative differences between epithelial and non-epithelial cells.
Imperial Cancer Research Fund, London, United Kingdom.
A
A
Kovarik
N
N
Peat
D
D
Wilson
S J
SJ
Gendler
J
J
Taylor-Papadimitriou
D001483:Base Sequence; D001940:Breast; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D018396:Mucin-1; D009077:Mucins / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009928:Organ Specificity; D010179:Pancreas; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism; D016385:TATA Box; D014162:Transfection
D008562:Membrane Glycoproteins; D018396:Mucin-1; D009077:Mucins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; D015500:Chloramphenicol O-Acetyltransferase