Identification of mZnf8, a mouse Krüppel-like transcriptional repressor, as a novel nuclear interaction partner of Smad1

Mol Cell Biol. 2002 Nov;22(21):7633-44. doi: 10.1128/MCB.22.21.7633-7644.2002.

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

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

Substances

  • Bone Morphogenetic Proteins
  • DNA-Binding Proteins
  • Kruppel-Like Transcription Factors
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • SMAD1 protein, human
  • Smad Proteins
  • Smad1 Protein
  • Smad1 protein, mouse
  • Trans-Activators
  • Transcription Factors
  • Transforming Growth Factor beta
  • ZNF8 protein, human
  • Zfp128 protein, mouse
  • RNA
  • Luciferases
  • Glutathione Transferase