Nucleic Acids Research, Vol 24, Issue 16 3253-3260, Copyright © 1996 by Oxford University Press
T Hasegawa, X Zhou, LA Garrett, EC Ruteshouser, SN Maity and B de Crombrugghe
In vivo transient expression and in vitro transcription experiments
indicated that a segment between -170 and -40 bp upstream of the start of
transcription of the mouse proalpha2(I) collagen gene was essential to
activate transcription. DNase I protection experiments identified three
strong footprints in this segment. Experiments with deletion mutants
encompassing the sequences defined by these three footprints indicated that
each of the three elements contributed to the transcriptional activity of
the promoter. All three elements are GC- rich, redundant sites for a
complex set of DNA binding proteins that includes SP1, other proteins that
bind to an SP1 consensus site and proteins that bind to a Krox consensus
site. In addition, the segment corresponding to the most proximal footprint
also binds the multimeric CCAAT binding protein CBF. Addition of an excess
amount of oligo- nucleotides corresponding to either of the two distal
footprints significantly inhibited in vitro transcription of the -350 bp
proalpha2(I) collagen promoter. Anti-SP1 antibodies that completely
inhibited transcription of the early SV40 promoter had little effect on
transcription of the wild-type -350 bp promoter, suggesting that SP1 has
only a minor role in activity of this promoter. Our results show that the
segment between base pairs -170 and -40 of the proalpha2(I) collagen
promoter, which contains redundant binding sites for a complex set of
nuclear proteins, is essential in the transcriptional activity of this
promoter in fibroblasts.
ARTICLES
Evidence for three major transcription activation elements in the proximal mouse proalpha2(I) collagen promoter
Department of Molecular Genetics, The University of Texas M.D.Anderson Cancer Center, Houston 77030, USA.
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