Structural and functional characterization of two human V-ATPase subunit gene promoters.
The vacuolar-ATPase (V-ATPase) is a multi-subunit enzyme that couples ATP hydrolysis to proton pumping across membranes. V-ATPase genes are considered to be housekeeping genes and are expressed in human neoplastic tissue and in cell lines. We have isolated and characterized several genomic clones containing the 5'-end of the human V-ATPase genes. DNA sequence analysis of the promoters of two V-ATPase subunit genes, encoding C (ATP6C) and c (ATP6F), reveals GC-rich regions in the region of the first exon. Neither TATA- nor CCAAT-boxes were found in these promoters, but both GC-boxes and E-boxes were identified. Transient transfection analysis, using a series of 5' nested deletions of promoter-luciferase constructs in human cancer cells, demonstrated that a positive cis-acting regulatory region was present in these TATA-less promoters. The regions between -79 and -40 of the ATP6C promoter and between -245 and -99 of the ATP6F promoter were identified as being likely to be extremely important for basal promoter activity. Electrophoretic mobility shift assays (EMSA) of these cis-regulatory regions revealed the basal promoter to be highly complex, with cooperative binding of several transcription factors, including Sp family members. These data identify the critical regulatory regions for both the ATP6C and ATP6F basal promoters and stress the functional importance of multiple protein complexes, involving the Sp family of transcription factors, in regulating gene expression.
Izumi
Hiroto
H
Department of Molecular Biology, University of Occupational and Environmental Health, School of Medicine, 1-1 Iseigaoka, Yahatanishi-ku, 807-8555, Kitakyushu, Japan.
Ise
Tomoko
T
Murakami
Tadashi
T
Torigoe
Takayuki
T
Ishiguchi
Hiroshi
H
Uramoto
Hidetaka
H
Yoshida
Yoichiro
Y
Yoshida
Takeshi
T
Tanabe
Mizuho
M
Kohno
Kimitoshi
K
D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics; D015183:Restriction Mapping; D014162:Transfection; D025262:Vacuolar Proton-Translocating ATPases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D021122:Protein Subunits; D025262:Vacuolar Proton-Translocating ATPases; C506979:ATP6V0B protein, human