Identification of mZnf8, a Mouse Kruüppel-Like Transcriptional Repressor, as a Novel Nuclear Interaction Partner of Smad1

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Abstract
To identify novel genes that play critical roles in mediating bone morphogenetic protein (BMP) signal pathways, we performed a yeast two-hybrid screen using Smad1 as bait. A novel mouse Krüppel-type zinc finger protein, mZnf8, was isolated. Interactions between mZnf8 and Smad proteins were further analyzed with various in vitro and in vivo approaches, including mammalian two-hybrid, in vitro glutathione S-transferase pulldown, and copurification assays. Results from functional analysis indicate that mZnf8 is a nuclear transcriptional repressor. Overexpression of mZnf8 represses activity of BMP and transforming growth factor beta (TGF-β) reporters. Silencing the expression of endogenous mZnf8 with an RNA interference approach caused a significant increase in the expression of one BMP reporter. These results suggest that mZnf8 negatively regulates the TGF-β/BMP signaling pathway in vivo. Transcription of mZnf8 is ubiquitous in mouse embryos, but high levels are specifically observed in adult mouse testes, with the same cell- and stage-specific transcription pattern as Smad1. Our data support the hypothesis that mZnf8 plays critical roles in mediating BMP signaling during spermatogenesis.