Identification of mZnf8, a mouse Krüppel-like transcriptional repressor, as a novel nuclear interaction partner of Smad1.
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.
Jiao
Kai
K
Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University Medical School, Nashville, Tennessee 37232, USA.
Zhou
Yingna
Y
Hogan
Brigid L M
BL
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization; D051741:Kruppel-Like Transcription Factors; D008156:Luciferases / Q000378:metabolism; D008297:Male; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D051785:Smad Proteins; D051898:Smad1 Protein; D013091:Spermatogenesis; D013737:Testis / Q000196:embryology / Q000378:metabolism; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D019485:Bone Morphogenetic Proteins; D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C497399:SMAD1 protein, human; D051785:Smad Proteins; D051898:Smad1 Protein; C497401:Smad1 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; C497248:ZNF8 protein, human; C467908:Zfp128 protein, mouse; D012313:RNA; D008156:Luciferases; D005982:Glutathione Transferase