Abstract
The Wilms tumor suppressor gene, wt1, encodes a zinc finger transcription factor that has been implicated in the regulation of a number of genes. Protein-protein interactions are known to modulate the transcription regulatory functions of Wilms tumor (WT1) and have also implicated WT1 in splicing. In this report, we identify a novel WT1-interacting protein, bone marrow zinc finger 2 (BMZF2), by affinity chromatography utilizing immobilized WT1 protein. BMZF2 is a potential transcription factor with 18 zinc fingers. The BMZF2 mRNA is mainly expressed in fetal tissues, and the protein is predominantly nuclear. Co-immunoprecipitation experiments are consistent with an in vivo association between WT1 and BMZF2. Glutathione S-transferase pulldown assays and far Western blots revealed that zinc fingers VI-X (amino acids 231-370) are required for interaction with the zinc finger region of WT1. Functionally, BMZF2 inhibits transcriptional activation by WT1. Moreover, a chimeric protein generated by fusion of BMZF2 to the GAL4 DNA-binding domain significantly decreases promoter activity of a reporter containing GAL4 DNA-binding sites, suggesting the presence of an active repressor domain within BMZF2. Our results suggest that BMZF2 interferes with the transactivation potential of WT1.
Highlights
Wilms tumor (WT)1 is a pediatric kidney cancer, occurring with a frequency of 1 in 10,000 children, usually before the age of 5 years [1]
Isolation of bone marrow zinc finger 2 (BMZF2) as a Novel WT1-interacting Protein— Genetic screens and chemical cross-linkers have identified a small number of proteins that interact with the WT1 zinc finger domain
We looked to employ a biochemical approach to validate the interaction of previously described WT1-interacting proteins, as well as identify potentially new protein(s) that could interact with the WT1 zinc finger domain
Summary
Wilms tumor (WT)1 is a pediatric kidney cancer, occurring with a frequency of 1 in 10,000 children, usually before the age of 5 years [1]. For the analysis of the interaction between endogenous WT1 and BMZF2, K562 cells were lysed in lysis buffer (20 mM Tris-HCl (pH 7.4), 10 mM KCl, 10 mM MgCl2, 2 mM EDTA, 10% glycerol, 1% Triton X-100, 2.5 mM -glycerol phosphate, 1 mM NaF, 1 mM DTT, 1 g/ml of aprotinin, 1 g/ml of leupeptin, 1 g/ml of Pefabloc, and 1 g/ml of pepstatin A) for 10 min on ice. Lysates were sonicated twice for 15 s and incubated with 420 mM NaCl in lysis buffer for 1 h on ice. Extracts were incubated with the indicated antisera or antibodies, and immune complexes were collected with protein A-Sepharose beads at 4 °C for 1 h.
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