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J. Biol. Chem., Vol. 269, Issue 31, 19848-19858, 08, 1994
GK Scott, JC Daniel, X Xiong, RA Maki, D Kabat and CC Benz
Promoter elements accounting for HER2 (c-erbB-2/neu) overexpression were
searched for in several human breast cancer cell lines (MDA-453, BT-474,
ZR-75-1, MCF-7) known to express constitutively a 30-fold range in HER2
transcripts per gene copy. HER2 overexpressing cells showed a single
prominent DNase I hypersensitive site near a conserved and hitherto
unrecognized ets response element (GAGGAA), located 38 bases down-stream
from the CAAT box and directly 5' of the TATA box in the human HER2
promoter. Transient transfection of HER2 promoter constructs (0.125, 0.5,
and 2.0 kilobase pairs (kb)) demonstrated that the most proximal promoter
region (0.125 kb) was capable of conferring up to 30- fold enhanced
activity in HER2-overexpressing cell lines relative to low HER2-expressing
control lines. Site-directed mutagenesis of the ets response element
(GAGGAA-->GAGAGA) caused a > or = 60% reduction in promoter activity
affecting at least 0.5 kb of upstream HER2 regulatory sequence. Gel-shift
assays with nuclear extracts and oligonucleotide sequences spanning the
0.125-kb promoter region detected an ETS- immunoreactive complex, present
most abundantly in cells overexpressing HER2, whose high-affinity binding
depended on the GAGGAA response element. Methylation interference confirmed
the ETS-specific pattern of protein binding by this complex to guanine
bases in the ets response element. UV cross-linking and immunoprecipitation
implicate a approximately 60-kDa ETS protein, and candidate ETS genes
expressed in these breast cancer cells include GABP alpha, elk-1, elf-1,
and PEA3.
Binding of an ETS-related protein within the DNase I hypersensitive site of the HER2/neu promoter in human breast cancer cells
Cancer Research Institute, University of California, San Francisco 94143-0128.
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