Putative zinc-sensing zinc fingers of metal-response element-binding transcription factor-1 stabilize a metal-dependent chromatin complex on the endogenous metallothionein-I promoter.
The metalloregulatory functions of metal-response element-binding transcription factor-1 (MTF-1) have been mapped, in part, to its six highly conserved zinc fingers. Here we examined the ability of zinc finger deletion mutants of mouse MTF-1 to regulate the endogenous metallothionein-I (MT-I) gene in cells lacking endogenous MTF-1. MTF-1 knockout mouse embryo fibroblasts were transfected with expression vectors for FLAG-tagged MTF-1 (MTF-1flag) or finger deletion mutants of MTF-1flag and then assayed for metal induction of MT-I gene expression, nuclear translocation, and in vitro DNA-binding activity of MTF-1 and its stable association with the endogenous chromosomal MT-I promoter. Intact MTF-1flag restored metal responsiveness of the MT-I gene, underwent nuclear translocation, displayed increased in vitro DNA binding in response to zinc and less so to cadmium, and rapidly formed a stable complex with the MT-I promoter chromatin in response to both of these metals. In contrast, although deletion of finger 1, fingers 5 and 6, or finger 6 only had variable effects on the nuclear localization and in vitro DNA-binding activity of MTF-1, each of these finger-deletion mutants severely attenuated metal-induced MTF-1 binding to the MT-I promoter chromatin and activation of the endogenous MT-I gene. These results demonstrated that the metal-induced recruitment of MTF-1 to the MT-I promoter is a rate-limiting step in its metalloregulatory function and that an intact zinc finger domain is required for this recruitment. During the course of these studies, it was discovered that mouse MTF-1 is polymorphic. The impact of these polymorphisms on MTF-1 metalloregulatory functions is discussed.
Jiang
Huimin
H
Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas 66160-7421, USA.
Daniels
Patrick J
PJ
Andrews
Glen K
GK
D021581:Active Transport, Cell Nucleus; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002104:Cadmium / Q000737:chemistry; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D005822:Genetic Vectors; D006801:Humans; D008668:Metallothionein / Q000235:genetics*; D008670:Metals / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011110:Polymorphism, Genetic; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection; D015032:Zinc / Q000737:chemistry; D016335:Zinc Fingers; D000097809:Transcription Factor MTF-1
D002104:Cadmium; D002843:Chromatin; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D008668:Metallothionein; D008670:Metals; D012333:RNA, Messenger; D014157:Transcription Factors; D015032:Zinc; D000097809:Transcription Factor MTF-1