Interactions of nuclear proteins from uninduced, induced and superinduced HeLa cells with metal regulatory elements MRE3 and 4 of the human metallothionein IIa-encoding gene.
Transcriptional activation of metallothionein (MT)-encoding genes(MT) is regulated during heavy metal induction by short non-identical repeats, termed 'metal regulatory elements' (MRE), present in multiple imperfect copies in MT promoter regions of eukaryotes. Using mobility shift assays, we have studied the interaction between the human MRE 3 and 4 regions (hMRE3/4) of the MTIIa promoter and nuclear proteins from uninduced and Cd(2+)-induced HeLa cells, and from Cd(2+)-superinduced H454 cells, a HeLa-derived Cd(2+)-resistant cell isolate which overexpresses hMTIIa after exposure to metal. A specific complex with a similar electrophoretic mobility was formed in all three extracts. Dialysis of the extracts using EDTA inhibited the formation of the complexes, which could be reconstituted only after the addition of Zn2+. UV cross-linking analyses of the specific complexes formed by the three nuclear extracts interacting with the hMRE3/4 region revealed that in all of them polypeptides were present having similar electrophoretic mobilities and different molecular masses. Mobility shift assays showed no major differences in the binding of nuclear proteins from induced or uninduced cells. Proposed models of activation of metal-induced MT transcription are discussed.
Minichiello
L
L
Dipartimento di Biochimica e Biotecnologie Mediche, II Facoltà di Medicina e Chirurgia, Università degli Studi di Napoli, Italy.
Remondelli
P
P
Cigliano
S
S
Bonatti
S
S
Leone
A
A
D001483:Base Sequence; D002104:Cadmium / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008668:Metallothionein / Q000096:biosynthesis / Q000235:genetics*; D008670:Metals / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid
D004268:DNA-Binding Proteins; D008670:Metals; D009687:Nuclear Proteins; D002104:Cadmium; D008668:Metallothionein; D004492:Edetic Acid