scholarly journals Correlation between functional and binding activities of designer zinc-finger proteins

2007 ◽  
Vol 403 (1) ◽  
pp. 177-182 ◽  
Author(s):  
Jong Seok Kang
Keyword(s):  
Binding Affinity ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Zinc Finger Proteins ◽  
Sequence Specificity ◽  
Binding Assays ◽  
Finger Protein ◽  
Vitro Binding

Rapid progress in the ability to develop and utilize zinc-finger proteins with customized sequence specificity have led to their increasing use as tools for modulation of target gene transcription in the post-genomic era. In the present paper, a series of in vitro binding assays and in vivo reporter analyses were used to demonstrate that a zinc-finger protein can effectively specify a base at each position of the target site in vivo and that functional activity of the zinc-finger protein as either a transcriptional repressor or activator is positively correlated with its binding affinity. In addition, this correlation can be extended to artificial engineered zinc-finger proteins. These data suggest that the binding affinity of designer zinc-finger proteins with novel specificity might be a determinant for their ability to regulate transcription of a gene of interest.

scholarly journals A non-canonical monovalent zinc finger stabilizes the integration of Cfp1 into the H3K4 methyltransferase complex COMPASS

2019 ◽  
Author(s):  
Yidai Yang ◽  
Monika Joshi ◽  
Yoh-hei Takahashi ◽  
Zhibin Ning ◽  
Qianhui Qu ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Protein ◽  
H3k4 Methylation ◽  
Methyltransferase Activity ◽  
Vitro Binding ◽  
New Type ◽  
High Level ◽  
In Cells

Abstract COMPlex ASsociating with SET1 (COMPASS) is a histone H3 Lys-4 methyltransferase that typically marks the promoter region of actively transcribed genes. COMPASS is a multi-subunit complex in which the catalytic unit, SET1, is required for H3K4 methylation. An important subunit known to regulate SET1 methyltransferase activity is the CxxC zinc finger protein 1 (Cfp1). Cfp1 binds to COMPASS and is critical to maintain high level of H3K4me3 in cells but the mechanisms underlying its stimulatory activity is poorly understood. In this study, we show that Cfp1 only modestly activates COMPASS methyltransferase activity in vitro. Binding of Cfp1 to COMPASS is in part mediated by a new type of monovalent zinc finger (ZnF). This ZnF interacts with the COMPASS’s subunits RbBP5 and disruption of this interaction blunts its methyltransferase activity in cells and in vivo. Collectively, our studies reveal that a novel form of ZnF on Cfp1 enables its integration into COMPASS and contributes to epigenetic signaling.

scholarly journals Elevated Expression of Zinc Finger Protein 703 Promotes Cell Proliferation and Metastasis through PI3K/AKT/GSK-3β Signalling in Oral Squamous Cell Carcinoma

2017 ◽  
Vol 44 (3) ◽  
pp. 920-934 ◽  
Author(s):  
Hao Wang ◽  
Xubin Deng ◽  
Jinshan Zhang ◽  
Zhilin Ou ◽  
Jiajie Mai ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Specific Inhibitor ◽  
Finger Protein ◽  
Proliferation And Metastasis ◽  
Gsk 3Β

Background/Aims: Zinc finger protein 703 (ZNF703), initially identified as a novel oncogene in human breast cancer, is a member of the NET/NlZ family of zinc finger transcription factors. It is recognized that the overexpression of ZNF703 is associated with various types of human cancers, but the role and molecular mechanism of ZNF703 in oral squamous cell carcinoma (OSCC) are unknown. Methods: ZNF703 expression levels were examined in OSCC tissues and non-cancerous tissues by qRT-PCR and immunohistochemistry (IHC). The molecular mechanisms of ZNF703 and its effects on cell growth and metastasis were explored in vitro and in vivo using the CCK8 assay, colony formation assay, cell cycle analysis, migration and invasion assays, wound-healing assay, western blotting and xenograft experiments in nude mice. Results: In this study, ZNF703 was found to be upregulated in OSCC tissues compared to that in normal tissues at both mRNA and protein levels, and its expression level was closely correlated with the overall survival of patients with OSCC. Silencing of the ZNF703 gene in OSCC cells significantly inhibited cell growth and metastasis in vitro and in vivo. Conversely, the overexpression of ZNF703 in OSCC cells promoted cancer growth and metastasis in vitro. Mechanistically, ZNF703 activated the PI3K/AKT/GSK-3β signalling pathway and its downstream effectors, thus regulating the cell cycle and epithelial-mesenchymal transition (EMT). Furthermore, the promotive effects of ZNF703 on cellular proliferation and metastasis could be rescued by LY294002 (a PI3K-specific inhibitor) and MK2206 (an Akt-specific inhibitor). Conclusion: The results show that ZNF703 promotes cell growth and metastasis through PI3K/Akt/GSK-3β signalling in OSCC and that it may be a promising target in the treatment of patients with OSCC.

A feel for the template: zinc finger protein transcription factors and chromatin

2002 ◽  
Vol 80 (3) ◽  
pp. 321-333 ◽  
Author(s):  
Fyodor D Urnov
Keyword(s):  
Transcription Factors ◽  
Zinc Finger ◽  
Binding Sites ◽  
Transcriptional Control ◽  
Hormone Receptors ◽  
Zinc Finger Protein ◽  
Finger Protein ◽  
Cell Biol

Transcription factors and chromatin collaborate in bringing the eukaryotic genome to life. An important, and poorly understood, aspect of this collaboration involves targeting the regulators to correct binding sites in vivo. An implicit and insufficiently tested assumption in the field has been that chromatin simply obstructs most sites and leaves only a few functionally relevant ones accessible. The major class of transcription factors in all metazoa, zinc finger proteins (ZFPs), can bind to chromatin in vitro (as clearly shown for Sp1, GATA-1 and -4, and the nuclear hormone receptors, for example). Data on the accessibility of DNA within heterochromatin to nonhistone regulators (E.A. Sekinger and D.S. Gross. 2001. Mol. Cell 105: 403–414; C. Jolly et al. 2002. J. Cell. Biol. 156: 775–781) and the ability of the basal transcription machinery to reside within highly condensed chromatin (most recently, R. Christova and T. Oelgeschlaeger. 2002. Nat. Cell Biol. 4: 79–82) further weaken the argument that chromatin acts as an across-the-board deterrent to ZFP binding. These proteins, however, do not bind promiscuously in vivo, and recent data on human cells (C.E. Horak et al. 2002. Proc. Natl. Acad. Sci. U.S.A. 99: 2924–2929) confirm earlier data on budding yeast (B. Ren et al. 2000. Science (Washington, D.C.), 290: 2306–2309) that primary DNA sequence, i.e., density of binding sites per unit DNA length, is not the primary determinant of where a ZFP transcription factor will bind in vivo. This article reviews these data and uses ZFP transcription factors as a model system to compare in vitro binding to chromatin by transcription factors with their in vivo behavior in gene regulation. DNA binding domain structure, nonrandom nucleoprotein organization of chromatin at target promoters, and cooperativity of regulator action may all contribute to target site selection in vivo.Key words: zinc finger protein, chromatin, transcriptional control, nucleosome.

IL-7R–dependent survival and differentiation of early T-lineage progenitors is regulated by the BTB/POZ domain transcription factor Miz-1

Blood ◽  
2011 ◽  
Vol 117 (12) ◽  
pp. 3370-3381 ◽  
Author(s):  
Ingrid Saba ◽  
Christian Kosan ◽  
Lothar Vassen ◽  
Tarik Möröy
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Strong Reduction ◽  
Expression Levels ◽  
Cell Numbers ◽  
Finger Protein

Abstract T cells originate from early T lineage precursors that have entered the thymus and differentiate through well-defined steps. Mice deficient for the BTB/POZ domain of zinc finger protein-1 (Miz-1) almost entirely lack early T lineage precursors and have a CD4−CD8− to CD4+CD8+ block causing a strong reduction in thymic cellularity. Miz-1ΔPOZ pro-T cells cannot differentiate in vitro and are unable to relay signals from the interleukin-7R (IL-7R). Both STAT5 phosphorylation and Bcl-2 up-regulation are perturbed. The high expression levels of SOCS1 found in Miz-1ΔPOZ cells probably cause these alterations. Moreover, Miz-1 can bind to the SOCS1 promoter, suggesting that Miz-1 deficiency causes a deregulation of SOCS1. Transgenic overexpression of Bcl-2 or inhibition of SOCS1 restored pro-T cell numbers and their ability to differentiate, supporting the hypothesis that Miz-1 is required for the regulation of the IL-7/IL-7R/STAT5/Bcl-2 signaling pathway by monitoring the expression levels of SOCS1.

Repeated CT elements bound by zinc finger proteins control the absolute and relative activities of the two principal human c-myc promoters

1993 ◽  
Vol 13 (9) ◽  
pp. 5710-5724
Author(s):  
E DesJardins ◽  
N Hay
Keyword(s):  
Zinc Finger ◽  
Tandem Repeats ◽  
Zinc Finger Protein ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Maximal Activity ◽  
Single Copy ◽  
Promoter Usage

Transcription of the human proto-oncogene c-myc is governed by two tandem principal promoters, termed P1 and P2. In general, the downstream promoter, P2, is predominant, which is in contrast to the promoter occlusion phenomenon usually observed in genes containing tandem promoters. A shift in human c-myc promoter usage has been observed in some tumor cells and in certain physiological conditions. However, the mechanisms that regulate promoter usage are not well understood. The present studies identify regulators which are required to promote transcription from both human c-myc promoters, P1 and P2, and have a role in determining their relative activities in vivo. A novel regulatory region located 101 bp upstream of P1 was characterized and contains five tandem repeats of the consensus sequence CCCTCCCC (CT element). The integrity of the region containing all five elements is required to promote transcription from P1 and for maximal activity from P2 in vivo. A single copy of this same element, designated CT-I2, also appears in an inverted orientation 53 bp upstream of the P2 transcription start site. This element has an inhibitory effect on P1 transcription and is required for P2 transcription. The transcription factor Sp1 was identified as the factor that binds specifically to the tandem CT elements upstream of P1 and to the CT-I2 element upstream of P2. In addition, the recently cloned zinc finger protein ZF87, or MAZ, was also able to bind these same elements in vitro. The five tandem CT elements can be functionally replaced by a heterologous enhancer that only in the absence of CT-I2 reverses the promoter usage, similar to what is observed in the translocated c-myc allele of Burkitt's lymphoma cells.

Single zinc-finger extension: enhancing transcriptional activity and specificity of three-zinc-finger proteins

2005 ◽  
Vol 386 (2) ◽  
pp. 95-99 ◽  
Author(s):  
Alexander E.F. Smith ◽  
Farzin Farzaneh ◽  
Kevin G. Ford
Keyword(s):  
Transcription Factors ◽  
Fusion Protein ◽  
Zinc Finger ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Zinc Finger Protein ◽  
Zinc Finger Proteins ◽  
Finger Protein ◽  
Finger Extension ◽  
Vp16 Fusion

AbstractIn order to demonstrate that an existing zinc-finger protein can be simply modified to enhance DNA binding and sequence discrimination in both episomal and chromatin contexts using existing zinc-finger DNA recognition code data, and without recourse to phage display and selection strategies, we have examined the consequences of a single zinc-finger extension to a synthetic three-zinc-finger VP16 fusion protein, on transcriptional activation from model target promoters harbouring the zinc-finger binding sequences. We report a nearly 10-fold enhanced transcriptional activation by the four-zinc-finger VP16 fusion protein relative to the progenitor three-finger VP16 protein in transient assays and a greater than five-fold enhancement in stable reporter-gene expression assays. A marked decrease in transcriptional activation was evident for the four-zinc-finger derivative from mutated regulatory regions compared to the progenitor protein, as a result of recognition site-size extension. This discriminatory effect was shown to be protein concentration-dependent. These observations suggest that four-zinc-finger proteins are stable functional motifs that can be a significant improvement over the progenitor three-zinc-finger protein, both in terms of specificity and the ability to target transcriptional function to promoters, and that single zinc-finger extension can therefore have a significant impact on DNA zinc-finger protein interactions. This is a simple route for modifying or enhancing the binding properties of existing synthetic zinc-finger-based transcription factors and may be particularly suited for the modification of endogenous zinc-finger transcription factors for promoter biasing applications.

scholarly journals Two Isoforms of Npap60 (Nup50) Differentially Regulate Nuclear Protein Import

2010 ◽  
Vol 21 (4) ◽  
pp. 630-638 ◽  
Author(s):  
Yutaka Ogawa ◽  
Yoichi Miyamoto ◽  
Munehiro Asally ◽  
Masahiro Oka ◽  
Yoshinari Yasuda ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Time Lapse ◽  
Binding Assays ◽  
Vitro Binding ◽  
Importin Α ◽  
Nuclear Protein Import

Npap60 (Nup50) is a nucleoporin that binds directly to importin α. In humans, there are two Npap60 isoforms: the long (Npap60L) and short (Npap60S) forms. In this study, we provide both in vitro and in vivo evidence that Npap60L and Npap60S function differently in nuclear protein import. In vitro binding assays revealed that Npap60S stabilizes the binding of importin α to classical NLS-cargo, whereas Npap60L promotes the release of NLS-cargo from importin α. In vivo time-lapse experiments showed that when the Npap60 protein level is controlled, allowing CAS to efficiently promote the dissociation of the Npap60/importin α complex, Npap60S and Npap60L suppress and accelerate the nuclear import of NLS-cargo, respectively. These results demonstrate that Npap60L and Npap60S have opposing functions and suggest that Npap60L and Npap60S levels must be carefully controlled for efficient nuclear import of classical NLS-cargo in humans. This study provides novel evidence that nucleoporin expression levels regulate nuclear import efficiency.

The zinc finger protein GLI transforms primary cells in cooperation with adenovirus E1A

1991 ◽  
Vol 11 (3) ◽  
pp. 1724-1728 ◽  
Author(s):  
J M Ruppert ◽  
B Vogelstein ◽  
K W Kinzler
Keyword(s):  
Zinc Finger ◽  
Nude Mice ◽  
Zinc Finger Protein ◽  
Primary Cells ◽  
Adenovirus E1a ◽  
In Vitro Transformation ◽  
Finger Protein ◽  
Protein Functions

The GLI gene was previously isolated by virtue of its amplification in human glioblastomas. We have now found that GLI expression can result in the in vitro transformation of both primary and secondary rodent cells. When coexpressed with adenovirus E1A, the GLI protein functions analogously to RAS, resulting in the formation of dense foci of cells which are tumorigenic in nude mice.

scholarly journals Identification and purification of a protein that binds DNA cooperatively with the yeast SWI5 protein.

1993 ◽  
Vol 13 (9) ◽  
pp. 5524-5537 ◽  
Author(s):  
R M Brazas ◽  
D J Stillman
Keyword(s):  
Zinc Finger Protein ◽  
Cooperative Interaction ◽  
Protein Fusion ◽  
Glutathione S Transferase ◽  
Binding Studies ◽  
Eukaryotic Transcription ◽  
Low Concentrations ◽  
Vitro Binding

The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein required for the expression of the HO gene. A protein fusion between glutathione S-transferase and SWI5 was expressed in Escherichia coli and purified. The GST-SWI5 fusion protein formed only a low-affinity complex in vitro with the HO promoter, which was inhibited by low concentrations of nonspecific DNA. This result was surprising, since genetic evidence demonstrated that SWI5 functions at the HO promoter via this site in vivo. A yeast factor, GRF10 (also known as PHO2 and BAS2), that promoted high-affinity binding of SWI5 in the presence of a large excess of nonspecific carrier DNA was purified. Final purification of the 83-kDa GRF10 protein was achieved by cooperative interaction-based DNA affinity chromatography. In vitro binding studies demonstrated that SWI5 and GRF10 bind DNA cooperatively. Methylation interference and missing-nucleoside studies demonstrated that the two proteins bind at adjacent sites, with each protein making unique DNA contacts. SWI5 and GRF10 interactions were not detected in the absence of DNA. The role of cooperative DNA binding in determining promoter specificity of eukaryotic transcription factors is discussed.

Sign in / Sign up

Export Citation Format

Share Document