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

2005 ◽  
Vol 10 (4) ◽  
pp. 304-313 ◽  
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.

scholarly journals The Cardiac Tissue-Restricted Homeobox Protein Csx/Nkx2.5 Physically Associates with the Zinc Finger Protein GATA4 and Cooperatively Activates Atrial Natriuretic Factor Gene Expression

1998 ◽  
Vol 18 (6) ◽  
pp. 3120-3129 ◽  
Author(s):  
Youngsook Lee ◽  
Tetsuo Shioi ◽  
Hideko Kasahara ◽  
Shawn M. Jobe ◽  
Russell J. Wiese ◽  
...  
Keyword(s):  
Gene Expression ◽  
Zinc Finger ◽  
Protein Interaction ◽  
Binding Sites ◽  
Atrial Natriuretic Factor ◽  
Target Gene ◽  
Cardiac Tissue ◽  
Zinc Finger Protein ◽  
Finger Protein ◽  
Homeobox 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.

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

Oncogene ◽  
2001 ◽  
Vol 20 (39) ◽  
pp. 5466-5474 ◽  
Author(s):  
Fredrik Hellborg ◽  
Wang Qian ◽  
Cristina Mendez-Vidal ◽  
Charlotte Asker ◽  
Maria Kost-Alimova ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Target Gene ◽  
Zinc Finger Protein ◽  
Finger Protein ◽  
Growth Inhibitory ◽  
P53 Target Gene

Proteomic Analysis of MCF-7 Cell Lines Expressing the Zinc-Finger or the Proline-Rich Domain of Retinoblastoma-Interacting-Zinc-Finger Protein

2006 ◽  
Vol 5 (5) ◽  
pp. 1176-1185 ◽  
Author(s):  
Angela Chambery ◽  
Annarita Farina ◽  
Antimo Di Maro ◽  
Mariangela Rossi ◽  
Ciro Abbondanza ◽  
...  
Keyword(s):  
Cell Lines ◽  
Zinc Finger ◽  
Proteomic Analysis ◽  
Zinc Finger Protein ◽  
Rich Domain ◽  
Finger Protein ◽  
Mcf 7

scholarly journals Progression of interleukin-2 (IL-2)-dependent rat T cell lymphoma lines to IL-2-independent growth following activation of a gene (Gfi-1) encoding a novel zinc finger protein.

1993 ◽  
Vol 13 (3) ◽  
pp. 1759-1768 ◽  
Author(s):  
C B Gilks ◽  
S E Bear ◽  
H L Grimes ◽  
P N Tsichlis
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Cell Lymphoma ◽  
Interleukin 2 ◽  
T Cell Lymphoma ◽  
Finger Protein ◽  
Cell Clones

During progression of Moloney murine leukemia virus (Mo-MuLV)-induced rat T cell lymphomas, growth selection results in the expansion of cell clones carrying increasing numbers of integrated proviruses. These new provirus insertions reproducibly contribute to enhanced growth, allowing the emergence of cell clones from the initially heterogeneous population of tumor cells. The Mo-MuLV-induced rat T cell lymphoma lines 2780d and 5675d, which are dependent on interleukin-2 (IL-2) for growth in culture (IL-2d), were placed in IL-2-free medium to select for IL-2-independent (IL-2i) mutants. Southern blot analysis of genomic DNA from these mutants, which was hybridized to a Mo-MuLV long terminal repeat probe, revealed that all mutants carried new provirus insertions (from one to four new proviruses per cell line). A locus of integration identified through cloning of the single new provirus detected in one of the IL-2i mutants, 2780i.5, was found to be the target of provirus insertion in 1 additional IL-2i cell line of 24 tested. A full-length cDNA of a gene (growth factor independence-1 [Gfi-1]) activated by promoter insertion in the 2780i.5 cells was cloned and shown to encode a novel zinc finger protein. Gfi-1 is expressed at low levels in IL-2d cell lines cultured in IL-2-containing medium and at high levels in most IL-2i cell lines, including the two harboring a provirus at this locus. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis. In mitogen-stimulated splenocytes, Gfi-1 expression begins to rise at 12 h after stimulation and reaches very high levels after 50 h, suggesting that it may be functionally involved in events occurring after the interaction of IL-2 with its receptor, perhaps during the transition from the G1 to the S phase of the cell cycle. In agreement with this, Gfi-1 does not induce the expression of IL-2. Expression of Gfi-1 in 2780d cells following transfer of a Gfi-1/LXSN retrovirus construct contributes to the emergence of the IL-2i phenotype.

scholarly journals Roles of Zinc Finger Protein 423 in Proliferation and Invasion of Cholangiocarcinoma through Oxidative Stress

Biomolecules ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 263
Author(s):  
Timpika Chaiprasert ◽  
Napat Armartmuntree ◽  
Anchalee Techasen ◽  
Chadamas Sakonsinsiri ◽  
Somchai Pinlaor ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Lines ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Expression Patterns ◽  
Immunohistochemical Analysis ◽  
Oxidative Stress Marker ◽  
Finger Protein ◽  
Hydrogen Peroxide Treatment ◽  
Proliferation And Invasion

Zinc finger protein 423 (ZNF423) is a transcriptional factor involved in the development and progression of cancers but has not yet been examined in cholangiocarcinoma (CCA), an oxidative stress-driven cancer of biliary epithelium. In this study, we hypothesized that oxidative stress mediated ZNF423 expression regulates its downstream genes resulting in CCA genesis. ZNF423 protein expression patterns and 8-oxodG (an oxidative stress marker) formation in CCA tissues were investigated using immunohistochemical analysis. The results showed that ZNF423 was overexpressed in CCA cells compared to normal bile duct cells adjacent of the tumor. Notably, ZNF423 expression was positively correlated with 8-oxodG formation. Moreover, ZNF423 expression in an immortalized cholangiocyte cell line (MMNK1) was increased by hydrogen peroxide-treatment, suggesting that oxidative stress induces ZNF423 expression. To investigate the roles of ZNF423 in CCA progression, ZNF423 mRNA was silenced using specific siRNA in CCA cell lines, KKU-100 and KKU-213. Silencing of ZNF423 significantly inhibits cell proliferation and invasion of both CCA cell lines. Taking all these results together, the present study denoted that ZNF423 is an oxidative stress-responsive gene with an oncogenic property contributing to the regulation of CCA genesis.

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