Dynamics of the metal-dependent transcription factor complex in vivo at the mouse metallothionein-I promoter

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Abstract
The in vivo association of transcription factors with the metallothionein‐I promoter was examined using chromatin immunoprecipitation (ChIP) assays. The results demonstrated that c‐fos is rapidly recruited along with the metal response element‐binding transcription factor‐1 (MTF‐1) to this promoter in response to zinc or cadmium, and that this recruitment is reversed in the visceral yolk sac by a zinc‐deficient diet in vivo, and in cultured cells after lowering the zinc concentration in the medium or during prolonged zinc exposure. In contrast, the interactions of c‐jun, USF‐1, USF‐2 and Sp1 with this promoter are metal‐independent. Studies of knockout cells revealed that the recruitment of c‐fos to the MT‐I promoter requires MTF‐1, but that c‐fos is not essential for recruitment of MTF‐1 and metal‐induction of MT‐I gene expression. Studies of Hepa cells stably‐transfected with reporter genes driven by the MT‐I promoter suggested two in vivo binding sites for USF‐1 and ‐2. In contrast, Sp1 was apparently associated with a single binding site (upstream of –153 bp). In addition, maximal recruitment of c‐fos by metals required sequences and/or other proteins that interact upstream of –153 bp. In summary, these studies extend our understanding of the complexity and dynamics of the transcription factor complex that forms at the MT‐I promoter in vivo in response to metals.