Metal-dependent binding of a nuclear factor to the rat metallothionein-I promoter.
Genomic footprinting studies in vivo and experiments using synthetic metal regulatory elements (MREs) in vitro suggest protein binding to the MREs of the mouse and rat metallothionein I (MT-I) genes. Using gel-retardation assays of promoter fragments, we observe a cadmium-dependent binding factor for the rat MT-I promoter in rat hepatoma cells. This factor is present in extracts from both uninduced and cadmium-induced cells, but requires the presence of cadmium to bind to the promoter. The formation of a cadmium-dependent complex is competed by an oligonucleotide containing two MREs. This competition is lost when when one of the MREs is mutated, indicating a requirement for at least two MREs for binding of this factor. The cadmium-dependent factor dissociates more rapidly from the MT-I promoter than does a factor that binds to a consensus Sp1 site present on the same DNA fragment. UV crosslinking analysis using nuclear extracts from cadmium induced cells, in the presence of an oligonucleotide probe containing both 5-bromodeoxyuridine and 32P-deoxycytidine, identifies a 39 kDalton protein associated with the metal inducible complex.
Andersen
R D
RD
Department of Biological Chemistry, University of California, Los Angeles 90024.
Taplitz
S J
SJ
Oberbauer
A M
AM
Calame
K L
KL
Herschman
H R
HR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002104:Cadmium / Q000494:pharmacology*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental; D008668:Metallothionein / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic / Q000187:drug effects; D051381:Rats; D015183:Restriction Mapping; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D014157:Transcription Factors; D002104:Cadmium; D008668:Metallothionein