Functional heterogeneity in the zinc fingers of metalloregulatory protein metal response element-binding transcription factor-1.
Metal response element-binding transcription factor-1 (MTF-1) is a unique, zinc-inducible transcription factor that binds to metal response elements in the metallothionein promoter and activates transcription in response to metals and oxidative stress. MTF-1 contains six zinc fingers of the Cys(2)-His(2) type. It was previously shown that MTF-1 is reversibly activated to bind DNA in response to changes in zinc status, unlike other zinc finger transcription factors, which do not appear to be reversibly activated by zinc in the cellular environment. Here we show that zinc fingers 2-4 constitute the core DNA-binding domain, whereas fingers 5 and 6 appear to be unnecessary for DNA binding in vitro. Deletion of finger 1 resulted in a protein that bound DNA constitutively in vitro. Furthermore, transfer of MTF-1 finger 1 to a position immediately preceding the three zinc fingers of Sp1 resulted in a chimeric protein that required exogenous zinc to activate DNA binding in vitro, unlike native Sp1, which binds DNA constitutively. Transient transfection experiments demonstrated that intact MTF-1 activated a reporter 2.5-4-fold above basal levels after metal treatment in mouse MTF-1 knockout cells, Drosophila SL2 cells, and yeast. However, the metal response was lost in all three systems when finger 1 was deleted, but was unaffected by deletion of fingers 5 and 6. These data suggest that finger 1 of MTF-1 constitutes a unique metal-sensing domain that, in cooperation with the transactivation domains, produces a zinc-sensing metalloregulatory transcription factor.
Bittel
D C
DC
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7421, USA.
Smirnova
I V
IV
Andrews
G K
GK
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004330:Drosophila; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry*; D015533:Transcriptional Activation; D016335:Zinc Fingers; D000097809:Transcription Factor MTF-1
D004247:DNA; D004268:DNA-Binding Proteins; D008668:Metallothionein; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D000097809:Transcription Factor MTF-1