Nucleic Acids Research, Vol 25, Issue 10 1975-1983, Copyright © 1997 by Oxford University Press
T Taira, T Narita, SM Iguchi-Ariga and H Ariga
Expression of the human heat shock protein 70 gene (hsp70) is induced by
various kinds of stress and by oncogenes. In the absence of stress, hsp70
is mainly expressed in the G1and S phases of the cell cycle, but the
elements contributing to cell cycle-dependent expression from the hsp70
promoter remain elusive. We have previously reported that two elements,
named HSP-MYCA and HSP-MYCB, located approximately 200 bp upstream (-200)
from the transcription start site (+1) of human hsp70, are important for
initiation of DNA replication at the hsp70 locus. In this report we examine
the effect of these two elements on transcriptional activity from the hsp70
promoter, especially in terms of cell cycle-dependent expression. Various
segments of the hsp70 promoter region (up to -300) were linked to the
luciferase gene and the constructs were transfected into mouse L cells to
examine their transcriptional activity. A strong enhancer activity was
defined in the HSP-MYCB element, but not in HSP-MYCA. Mutations introduced
within HSP- MYCB abolished the transcriptional activation. In synchronized
cells, pHB-Luc (a luciferase construct containing approximately 2.4 kb of
the hsp70 promoter region) as well as endogenous hsp70 showed two peaks of
expression; one in G1 and the other in the S phase. Site-directed
mutagenesis of HSP-MYCB in pHB-Luc abolished the expression peak in G1, but
not that in the S phase. To test promoter specificity, wild-type and mutant
HSP-MYCB elements were then linked to the luciferase gene in combination
with the hsp70 , the cyclin A or the PCNA promoter. Both in transient
experiments and established cell lines, a strong peak of expression in
mid-G1phase was observed with all the constructs containing wild-type
HSP-MYCB, but not with the constructs containing the mutant sequence. These
results suggest that the HSP-MYCB sequence is a G1-specific enhancer and is
responsible for cell cycle-dependent expression of hsp70.
ARTICLES
A novel G1-specific enhancer identified in the human heat shock protein 70 gene
Faculty of Pharmaceutical Sciences and 1 College of Medical Technology, Hokkaido University, Kita-ku, Sapporo 060, Japan.
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