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-beta) 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-beta/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.
MeSH terms
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Amino Acid Sequence
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Animals
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Blotting, Northern
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Bone Morphogenetic Proteins / metabolism
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COS Cells
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Cell Nucleus / metabolism*
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Cloning, Molecular
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DNA-Binding Proteins / metabolism*
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Glutathione Transferase / metabolism
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Humans
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In Situ Hybridization
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Kruppel-Like Transcription Factors
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Luciferases / metabolism
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Male
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Mice
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Models, Biological
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Molecular Sequence Data
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Protein Binding
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RNA / metabolism
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Recombinant Fusion Proteins / metabolism
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Repressor Proteins / biosynthesis*
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Repressor Proteins / chemistry
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Repressor Proteins / physiology*
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Reverse Transcriptase Polymerase Chain Reaction
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Sequence Homology, Amino Acid
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Signal Transduction
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Smad Proteins
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Smad1 Protein
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Spermatogenesis
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Testis / embryology
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Testis / metabolism
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Time Factors
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Trans-Activators / metabolism*
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Transcription Factors / metabolism*
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Transcription, Genetic*
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Transfection
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Transforming Growth Factor beta / metabolism
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Two-Hybrid System Techniques
Substances
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Bone Morphogenetic Proteins
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DNA-Binding Proteins
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Kruppel-Like Transcription Factors
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Recombinant Fusion Proteins
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Repressor Proteins
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SMAD1 protein, human
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Smad Proteins
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Smad1 Protein
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Smad1 protein, mouse
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Trans-Activators
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Transcription Factors
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Transforming Growth Factor beta
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ZNF8 protein, human
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Zfp128 protein, mouse
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RNA
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Luciferases
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Glutathione Transferase