NUCLEAR PROTEINS BINDING TO THE HUMAN METALLOTHIONEIN-IIA GENE UPSTREAM SEQUENCES

Metallothionein genes are known to be transcriptionally regulated by a variety of factors such as heavy metals, glucocorticoids and cytokines, and have multiple regulatory elements in their 5'-flanking region. To study the interactions between these sequences and regulatory factors, HeLa cell nuclear proteins were analyzed by band-shift assay using a 95-base pair (bp) DNA probe containing a part of the human MT-II, gene upstream sequences. Consequently, two Zn-dependent DNA-binding proteins were detected. One of these showed properties almost identical with those of zinc regulatory factor (ZRF), which had been detected using an oligonucleotide probe containing the metal responsive element (MRE); namely, this protein is activated only by Zn, and requires not only MRE but also its flanking sequences for optimal DNA-binding. The other protein appears to be Spl, based on its recognition sequences specificity, In addition, by Southwestern blotting analysis of nuclear extracts using the 95-bp probe or MRE oligonucleotide probe, we detected a Zn-dependent DNA-binding protein with a molecular mass of 116 kDa, which is likely to be ZRF. Analysis of HeLa cell nuclear proteins fractionated by glycerol gradient centrifugation showed that ZRF is distinct from another MRE-binding protein, MREBP.