Human wig-1, a p53 target gene that encodes a growth inhibitory zinc finger protein

ABSTRACT Specification and differentiation of the cardiac muscle lineage appear to require a combinatorial network of many factors. The cardiac muscle-restricted homeobox protein Csx/Nkx2.5 (Csx) is expressed in the precardiac mesoderm as well as the embryonic and adult heart. Targeted disruption of Csx causes embryonic lethality due to abnormal heart morphogenesis. The zinc finger transcription factor GATA4 is also expressed in the heart and has been shown to be essential for heart tube formation. GATA4 is known to activate many cardiac tissue-restricted genes. In this study, we tested whether Csx and GATA4 physically associate and cooperatively activate transcription of a target gene. Coimmunoprecipitation experiments demonstrate that Csx and GATA4 associate intracellularly. Interestingly, in vitro protein-protein interaction studies indicate that helix III of the homeodomain of Csx is required to interact with GATA4 and that the carboxy-terminal zinc finger of GATA4 is necessary to associate with Csx. Both regions are known to directly contact the cognate DNA sequences. The promoter-enhancer region of the atrial natriuretic factor (ANF) contains several putative Csx binding sites and consensus GATA4 binding sites. Transient-transfection assays indicate that Csx can activate ANF reporter gene expression to the same extent that GATA4 does in a DNA binding site-dependent manner. Coexpression of Csx and GATA4 synergistically activates ANF reporter gene expression. Mutational analyses suggest that this synergy requires both factors to fully retain their transcriptional activities, including the cofactor binding activity. These results demonstrate the first example of homeoprotein and zinc finger protein interaction in vertebrates to cooperatively regulate target gene expression. Such synergistic interaction among tissue-restricted transcription factors may be an important mechanism to reinforce tissue-specific developmental pathways.

Download Full-text

Isogenic Human Cell Lines for Drug Discovery: Regulation of Target Gene Expression by Engineered Zinc-Finger Protein Transcription Factors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104272663 ◽

2005 ◽

Vol 10 (4) ◽

pp. 304-313 ◽

Cited By ~ 11

Author(s):

Pei-Qi Liu ◽

Siyuan Tan ◽

Matthew C. Mendel ◽

Richard J. Murrills ◽

Bheem M. Bhat ◽

...

Keyword(s):

Cell Lines ◽

Zinc Finger ◽

Human Cell ◽

Target Gene ◽

Zinc Finger Protein ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Hek293 Cells ◽

Human Cell Lines ◽

Finger Protein

Isogenic cell lines differing only in the expression of the protein of interest provide the ideal platform for cell-based screening. However, related natural lines differentially expressing the therapeutic target of choice are rare. Here the authors report a strategy for drug screening employing isogenic human cell lines in which the expression of the target protein is regulated by a gene-specific engineered zinc-finger protein (ZFP) transcription factor (TF). To demonstrate this approach, a ZFP TF activator of the human parathyroid hormone receptor 1 (PTHR1) gene was identified and introduced into HEK293 cells (negative for PTHR1). Following induction of ZFP TF expression, this cell line produced functional PTHR1 protein, resulting in a robust and ligand-specific cyclic adenosine monophosphate (cAMP) response. Reciprocally, the natural expression of PTHR1 observed in SAOS2 cells was dramatically reduced by the introduction of the appropriate PTHR1-specific ZFP TF repressor. Moreover, this ZFP-driven PTHR1 repression selectively eliminated the functional cAMP response invoked by known ligands of PTHR1. These data establish ZFP TF–generated isogenic lines as a general approach for the identification of therapeutic agents specific for the target gene of interest.

Download Full-text

Specific Recognition of ZNF217 and Other Zinc Finger Proteins at a Surface Groove of C-Terminal Binding Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.00680-06 ◽

2006 ◽

Vol 26 (21) ◽

pp. 8159-8172 ◽

Cited By ~ 50

Author(s):

Kate G. R. Quinlan ◽

Marco Nardini ◽

Alexis Verger ◽

Pierangelo Francescato ◽

Paul Yaswen ◽

...

Keyword(s):

Crystal Structure ◽

Transcription Factors ◽

Zinc Finger ◽

Target Gene ◽

Zinc Finger Protein ◽

Zinc Finger Proteins ◽

Repressor Protein ◽

Finger Protein ◽

Surface Groove ◽

Binding Cleft

ABSTRACT Numerous transcription factors recruit C-terminal binding protein (CtBP) corepressors. We show that the large zinc finger protein ZNF217 contacts CtBP. ZNF217 is encoded by an oncogene frequently amplified in tumors. ZNF217 contains a typical Pro-X-Asp-Leu-Ser (PXDLS) motif that binds in CtBP's PXDLS-binding cleft. However, ZNF217 also contains a second motif, Arg-Arg-Thr (RRT), that binds a separate surface on CtBP. The crystal structure of CtBP bound to an RRTGAPPAL peptide shows that it contacts a surface crevice distinct from the PXDLS binding cleft. Interestingly, both PXDLS and RRT motifs are also found in other zinc finger proteins, such as RIZ. Finally, we show that ZNF217 represses several promoters, including one from a known CtBP target gene, and mutations preventing ZNF217's contact with CtBP reduce repression. These results identify a new CtBP interaction motif and establish ZNF217 as a transcriptional repressor protein that functions, at least in part, by associating with CtBP.

Download Full-text