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Sample records for gap zinc finger

  1. Identification of a novel zinc finger protein gene (ZNF298) in the GAP2 of human chromosome 21q

    SciTech Connect

    Shibuya, Kazunori; Kudoh, Jun; Okui, Michiyo; Shimizu, Nobuyoshi . E-mail: shimizu@dmb.med.keio.ac.jp

    2005-07-01

    We have isolated a novel zinc finger protein gene, designated ZNF298, as a candidate gene for a particular phenotype of Down syndrome or bipolar affective disorder (BPAD) which maps to human chromosome 21q22.3. ZNF298 gene consists of 25 exons spanning approximately 80 kb in a direction from the telomere to centromere. There are four kinds of transcripts that harbor three types of 3' UTR. These four transcripts (ZNF298a, ZNF298b, ZNF298c, and ZNF298d) contain putative open reading frames encoding 1178, 1198, 555, and 515 amino acids, respectively. ZNF298 gene was ubiquitously expressed in various tissues at very low level. The protein motif analysis revealed that ZNF298 proteins contain a SET [Su(var)3-9, Enhancer-of-zeste, Trithorax] domain, multiple C2H2-type zinc finger (ZnF{sub C}2H2) domains, several nuclear localization signals (NLSs), and PEST sequences. Nuclear localization of ZNF298 protein was confirmed by transfection of expression vector of GFP-tagged protein into two human cell lines. Interestingly, this gene crosses over a clone gap (GAP2) remaining in the band 21q22.3. We obtained the DNA fragments corresponding to GAP2 using ZNF298 cDNA sequence as anchor primers for PCR and determined its genomic DNA sequence.

  2. Prediction of zinc finger DNA binding protein.

    PubMed

    Nakata, K

    1995-04-01

    Using the neural network algorithm with back-propagation training procedure, we analysed the zinc finger DNA binding protein sequences. We incorporated the characteristic patterns around the zinc finger motifs TFIIIA type (Cys-X2-5-Cys-X12-13-His-X2-5-His) and the steroid hormone receptor type (Cys-X2-5-Cys-X12-15-Cys-X2-5-Cys-X15-16-Cys-X4-5-Cys-X8-10- Cys-X2-3-Cys) in the neural network algorithm. The patterns used in the neural network were the amino acid pattern, the electric charge and polarity pattern, the side-chain chemical property and subproperty patterns, the hydrophobicity and hydrophilicity patterns and the secondary structure propensity pattern. Two consecutive patterns were also considered. Each pattern was incorporated in the single layer perceptron algorithm and the combinations of patterns were considered in the two-layer perceptron algorithm. As for the TFIIIA type zinc finger DNA binding motifs, the prediction results of the two-layer perceptron algorithm reached up to 96.9% discrimination, and the prediction results of the discriminant analysis using the combination of several characters reached up to 97.0%. As for the steroid hormone receptor type zinc finger, the prediction results of neural network algorithm and the discriminant analyses reached up to 96.0%.

  3. [Effects of zinc-finger proteins and artificial zinc-finger proteins on microbial metabolisms--a review].

    PubMed

    Liu, Zhuo; Zhang, Fei; Zhao, Xinqing; Bai, Fengwu

    2014-03-01

    Zinc-finger proteins have been widely studied due to their highly conserved structures and DNA-binding specificity of zinc-finger domains. However, researches on the zinc-finger proteins from microorganisms, especially those from prokaryotes, are still very limited. This review focuses on the latest progress on microbial zinc-finger proteins, especially those from prokaryotes and the application of artificial zinc-finger proteins in the breeding of robust strains. Artificial zinc-finger proteins with transcriptional activation or repression domain can regulate the global gene transcription of microbial cells to acquire improved phenotypes, such as stress tolerance to heat, ethanol, butanol, and osmotic pressure. Using the zinc-finger domain as DNA scaffold in the construction of enzymatic system can enhance the catalytic efficiency and subsequently the production of specific metabolites. Currently, zinc-finger domains used in the construction of artificial transcription factor are usually isolated from mammalian cells. In the near future, novel transcription factors can be designed for strain development based on the natural zinc-finger domains from different microbes, which may be used to regulate the global gene expression of microbial cells more efficiently.

  4. Zinc finger proteins and the 3D organization of chromosomes.

    PubMed

    Feinauer, Christoph J; Hofmann, Andreas; Goldt, Sebastian; Liu, Lei; Máté, Gabriell; Heermann, Dieter W

    2013-01-01

    Zinc finger domains are one of the most common structural motifs in eukaryotic cells, which employ the motif in some of their most important proteins (including TFIIIA, CTCF, and ZiF268). These DNA binding proteins contain up to 37 zinc finger domains connected by flexible linker regions. They have been shown to be important organizers of the 3D structure of chromosomes and as such are called the master weaver of the genome. Using NMR and numerical simulations, much progress has been made during the past few decades in understanding their various functions and their ways of binding to the DNA, but a large knowledge gap remains to be filled. One problem of the hitherto existing theoretical models of zinc finger protein DNA binding in this context is that they are aimed at describing specific binding. Furthermore, they exclusively focus on the microscopic details or approach the problem without considering such details at all. We present the Flexible Linker Model, which aims explicitly at describing nonspecific binding. It takes into account the most important effects of flexible linkers and allows a qualitative investigation of the effects of these linkers on the nonspecific binding affinity of zinc finger proteins to DNA. Our results indicate that the binding affinity is increased by the flexible linkers by several orders of magnitude. Moreover, they show that the binding map for proteins with more than one domain presents interesting structures, which have been neither observed nor described before, and can be interpreted to fit very well with existing theories of facilitated target location. The effect of the increased binding affinity is also in agreement with recent experiments that until now have lacked an explanation. We further explore the class of proteins with flexible linkers, which are unstructured until they bind. We have developed a methodology to characterize these flexible proteins. Employing the concept of barcodes, we propose a measure to compare

  5. The generation of zinc finger proteins by modular assembly

    PubMed Central

    Bhakta, Mital; Segal, David J.

    2015-01-01

    The modular assembly (MA) method of generating engineered zinc finger proteins (ZFPs) was the first practical method for creating custom DNA-binding proteins. As such, MA has enabled a vast exploration of sequence-specific methods and reagents, ushering in the modern era of zinc finger-based applications that are described in this volume. The first zinc finger nuclease to cleave an endogenous site was created using MA, as was the first artificial transcription factor to enter phase II clinical trials. In recent years, other excellent methods have been developed that improved the affinity and specificity of the engineered ZFPs. However, MA is still used widely for many applications. This chapter will describe methods and give guidance for the creation of ZFPs using MA. Such ZFPs might be useful as starting materials to perform other methods described in this volume. Here, we also describe a single-strand annealing recombination assay for the initial testing of zinc finger nucleases. PMID:20680825

  6. Targeted mutagenesis of zebrafish: use of zinc finger nucleases.

    PubMed

    Leong, Ivone Un San; Lai, Daniel; Lan, Chuan-Ching; Johnson, Ross; Love, Donald R; Johnson, Ross; Love, Donald R

    2011-09-01

    The modeling of human disease in the zebrafish (Danio rerio) is moving away from chemical mutagensis and transient downregulation using morpholino oligomers to more targeted and stable transgenic methods. In this respect, zinc finger nucleases offer a means of introducing mutations at targeted sites at high efficiency. We describe here the development of zinc finger nucleases and their general use in model systems with a focus on the zebrafish.

  7. Targeted Mutagenesis in Zebrafish Using Customized Zinc Finger Nucleases

    PubMed Central

    Foley, Jonathan E.; Maeder, Morgan L.; Pearlberg, Joseph; Joung, J. Keith; Peterson, Randall T.; Yeh, Jing-Ruey J.

    2009-01-01

    Zebrafish mutants have traditionally been obtained using random mutagenesis or retroviral insertions, methods that cannot be targeted to a specific gene and require laborious gene mapping and sequencing. Recently, we and others have shown that customized zinc finger nucleases (ZFNs) can introduce targeted frame-shift mutations with high efficiency, thereby enabling directed creation of zebrafish gene mutations. Here we describe a detailed protocol for constructing ZFN expression vectors, for generating and introducing ZFN-encoding RNAs into zebrafish embryos, and for identifying ZFN-generated mutations in targeted genomic sites. All of our vectors and methods are compatible with previously described Zinc Finger Consortium reagents for constructing engineered zinc finger arrays. Using these methods, zebrafish founders carrying targeted mutations can be identified within four months. PMID:20010934

  8. Structure based design of protein linkers for zinc finger nuclease.

    PubMed

    Anand, Priya; Schug, Alexander; Wenzel, Wolfgang

    2013-10-01

    Zinc finger nucleases are a promising tool to edit DNA in many biological applications, in particular for gene knockout. Despite many efforts the number of genes that can be effectively targeted with ZFNs remains severely limited, as available constructs cannot address arbitrary gene sequences. Here, we develop a novel concept to significantly enhance the number of DNA sequences that can be targeted by ZFN. Using an efficient computational model, we provide an extensive library of possible linker molecules between individual zinc finger motifs in the construct that can skip up to 10 base pairs between adjacent zinc finger recognition sites in the DNA sequence, which increases the number of genes that can be efficiently targeted by more than an order of magnitude.

  9. Gamma Radiation-Induced Damage in the Zinc Finger of the Transcription Factor IIIA

    PubMed Central

    Miao, YuJi; Hu, XiaoDan; Min, Rui; Liu, PeiDang; Zhang, HaiQian

    2016-01-01

    A zinc finger motif is an element of proteins that can specifically recognize and bind to DNA. Because they contain multiple cysteine residues, zinc finger motifs possess redox properties. Ionizing radiation generates a variety of free radicals in organisms. Zinc finger motifs, therefore, may be a target of ionizing radiation. The effect of gamma radiation on the zinc finger motifs in transcription factor IIIA (TFIIIA), a zinc finger protein, was investigated. TFIIIA was exposed to different gamma doses from 60Co sources. The dose rates were 0.20 Gy/min and 800 Gy/h, respectively. The binding capacity of zinc finger motifs in TFIIIA was determined using an electrophoretic mobility shift assay. We found that 1000 Gy of gamma radiation impaired the function of the zinc finger motifs in TFIIIA. The sites of radiation-induced damage in the zinc finger were the thiol groups of cysteine residues and zinc (II) ions. The thiol groups were oxidized to form disulfide bonds and the zinc (II) ions were indicated to be reduced to zinc atoms. These results indicate that the zinc finger motif is a target domain for gamma radiation, which may decrease 5S rRNA expression via impairment of the zinc finger motifs in TFIIIA. PMID:27803644

  10. Emerging roles of zinc finger proteins in regulating adipogenesis

    PubMed Central

    Wei, Shengjuan; Zhang, Lifan; Zhou, Xiang; Du, Min; Jiang, Zhihua; Hausman, Gary J.; Bergen, Werner G.; Zan, Linsen; Dodson, Michael V.

    2014-01-01

    Proteins containing the zinc finger domain(s) are named zinc finger proteins (ZFPs), which are one of the largest classes of transcription factors in eukaryotic genomes. A large number of ZFPs have been studied and many of them were found to be involved regulating normal growth and development of cells and tissues through diverse signal transduction pathways. Recent studies revealed that a small but increasing number of ZFPs could function as key transcriptional regulators involved in adipogenesis. As the prevalence of obesity and metabolic disorders, the investigation of molecular regulatory mechanisms of adipocyte development must be more completely understood to develop novel and long term impact strategies for ameliorating obesity. In this review, we discuss recent work which has documented that ZFPs are important functional contributors to the regulation of adipogenesis. Taken altogether these data lead to the conclusion that ZFPs may become promising targets to combat human obesity. PMID:23760207

  11. Emerging roles of zinc finger proteins in regulating adipogenesis.

    PubMed

    Wei, Shengjuan; Zhang, Lifan; Zhou, Xiang; Du, Min; Jiang, Zhihua; Hausman, Gary J; Bergen, Werner G; Zan, Linsen; Dodson, Michael V

    2013-12-01

    Proteins containing the zinc finger domain(s) are named zinc finger proteins (ZFPs), one of the largest classes of transcription factors in eukaryotic genomes. A large number of ZFPs have been studied and many of them were found to be involved in regulating normal growth and development of cells and tissues through diverse signal transduction pathways. Recent studies revealed that a small but increasing number of ZFPs could function as key transcriptional regulators involved in adipogenesis. Due to the prevalence of obesity and metabolic disorders, the investigation of molecular regulatory mechanisms of adipocyte development must be more completely understood in order to develop novel and long-term impact strategies for ameliorating obesity. In this review, we discuss recent work that has documented that ZFPs are important functional contributors to the regulation of adipogenesis. Taken together, these data lead to the conclusion that ZFPs may become promising targets to combat human obesity.

  12. The creation of the artificial RING finger from the cross-brace zinc finger by {alpha}-helical region substitution

    SciTech Connect

    Miyamoto, Kazuhide; Togiya, Kayo

    2010-04-16

    The creation of the artificial RING finger as ubiquitin-ligating enzyme (E3) has been demonstrated. In this study, by the {alpha}-helical region substitution between the EL5 RING finger and the Williams-Beuren syndrome transcription factor (WSTF) PHD finger, the artificial E3 (WSTF PHD{sub R}ING finger) was newly created. The experiments of the chemical modification of residues Cys and the circular dichroism spectra revealed that the WSTF PHD{sub R}ING finger binds two zinc atoms and adopts the zinc-dependent ordered-structure. In the substrate-independent ubiquitination assay, the WSTF PHD{sub R}ING finger functions as E3 and was poly- or mono-ubiquitinated. The present strategy is very simple and convenient, and consequently it might be widely applicable to the creation of various artificial E3 RING fingers with the specific ubiquitin-conjugating enzyme (E2)-binding capability.

  13. Conformational Analysis on structural perturbations of the zinc finger NEMO

    NASA Astrophysics Data System (ADS)

    Godwin, Ryan; Salsbury, Freddie; Salsbury Group Team

    2014-03-01

    The NEMO (NF-kB Essential Modulator) Zinc Finger protein (2jvx) is a functional Ubiquitin-binding domain, and plays a role in signaling pathways for immune/inflammatory responses, apoptosis, and oncogenesis [Cordier et al., 2008]. Characterized by 3 cysteines and 1 histidine residue at the active site, the biologically occurring, bound zinc configuration is a stable structural motif. Perturbations of the zinc binding residues suggest conformational changes in the 423-atom protein characterized via analysis of all-atom molecular dynamics simulations. Structural perturbations include simulations with and without a zinc ion and with and without de-protonated cysteines, resulting in four distinct configurations. Simulations of various time scales show consistent results, yet the longest, GPU driven, microsecond runs show more drastic structural and dynamic fluctuations when compared to shorter duration time-scales. The last cysteine residue (26 of 28) and the helix on which it resides exhibit a secondary, locally unfolded conformation in addition to its normal bound conformation. Combined analytics elucidate how the presence of zinc and/or protonated cysteines impact the dynamics and energetic fluctuations of NEMO. Comprehensive Cancer Center of Wake Forest University Computational Biosciences shared resource supported by NCI CCSG P30CA012197.

  14. Systematic Characterization of the Zinc-Finger-Containing Proteins in the Mouse Transcriptome

    PubMed Central

    Ravasi, Timothy; Huber, Thomas; Zavolan, Mihaela; Forrest, Alistair; Gaasterland, Terry; Grimmond, Sean; Hume, David A.

    2003-01-01

    Zinc-finger-containing proteins can be classified into evolutionary and functionally divergent protein families that share one or more domains in which a zinc ion is tetrahedrally coordinated by cysteines and histidines. The zinc finger domain defines one of the largest protein superfamilies in mammalian genomes;46 different conserved zinc finger domains are listed in InterPro (http://www.ebi.ac.uk/InterPro). Zinc finger proteins can bind to DNA, RNA, other proteins, or lipids as a modular domain in combination with other conserved structures. Owing to this combinatorial diversity, different members of zinc finger superfamilies contribute to many distinct cellular processes, including transcriptional regulation, mRNA stability and processing, and protein turnover. Accordingly, mutations of zinc finger genes lead to aberrations in a broad spectrum of biological processes such as development, differentiation, apoptosis, and immunological responses. This study provides the first comprehensive classification of zinc finger proteins in a mammalian transcriptome. Specific detailed analysis of the SP/Krüppel-like factors and the E3 ubiquitin-ligase RING-H2 families illustrates the importance of such an analysis for a more comprehensive functional classification of large protein families. We describe the characterization of a new family of C2H2 zinc-finger-containing proteins and a new conserved domain characteristic of this family, the identification and characterization of Sp8, a new member of the Sp family of transcriptional regulators, and the identification of five new RING-H2 proteins. PMID:12819142

  15. A bacterial one-hybrid selection system for interrogating zinc finger-DNA interactions.

    PubMed

    Durai, Sundar; Bosley, Allen; Abulencia, Alice Bridgeman; Chandrasegaran, Srinivasan; Ostermeier, Marc

    2006-05-01

    We have developed two bacterial one-hybrid systems for interrogating and selecting zinc finger-DNA interactions. Our systems utilize two plasmids: a zinc finger-plasmid containing the gene for the zinc finger fused to a fragment of the alpha subunit of RNA polymerase and a reporter plasmid where the zinc finger-binding site is located upstream of a reporter gene-either the gene encoding the green fluorescent protein (GFP) or chloramphenicol acetyltransferase (CAT). Binding of the zinc finger domain to the target binding site results in a 10-fold increase in chloramphenicol resistance with the CAT reporter and an 8- to 22-fold increase in total cell fluorescence with the GFP reporter. The CAT reporter allows for sequence specific zinc fingers to be isolated in a single selection step whereas the GFP reporter enables quantitative evaluation of libraries using flow cytometry and theoretically allows for both negative and positive selection. Both systems have been used to select for zinc fingers that have affinity for the motif 5'-GGGGCAGAA-3' from a library of approximately 2 x 10(5) variants. The systems have been engineered to report on zinc finger-DNA binding with dissociation constants less than about 1 microM in order to be most applicable for evaluating binding specificity in an in vivo setting.

  16. Arsenite Interacts Selectively with Zinc Finger Proteins Containing C3H1 or C4 Motifs*

    PubMed Central

    Zhou, Xixi; Sun, Xi; Cooper, Karen L.; Wang, Feng; Liu, Ke Jian; Hudson, Laurie G.

    2011-01-01

    Arsenic inhibits DNA repair and enhances the genotoxicity of DNA-damaging agents such as benzo[a]pyrene and ultraviolet radiation. Arsenic interaction with DNA repair proteins containing functional zinc finger motifs is one proposed mechanism to account for these observations. Here, we report that arsenite binds to both CCHC DNA-binding zinc fingers of the DNA repair protein PARP-1 (poly(ADP-ribose) polymerase-1). Furthermore, trivalent arsenite coordinated with all three cysteine residues as demonstrated by MS/MS. MALDI-TOF-MS analysis of peptides harboring site-directed substitutions of cysteine with histidine residues within the PARP-1 zinc finger revealed that arsenite bound to peptides containing three or four cysteine residues, but not to peptides with two cysteines, demonstrating arsenite binding selectivity. This finding was not unique to PARP-1; arsenite did not bind to a peptide representing the CCHH zinc finger of the DNA repair protein aprataxin, but did bind to an aprataxin peptide mutated to a CCHC zinc finger. To investigate the impact of arsenite on PARP-1 zinc finger function, we measured the zinc content and DNA-binding capacity of PARP-1 immunoprecipitated from arsenite-exposed cells. PARP-1 zinc content and DNA binding were decreased by 76 and 80%, respectively, compared with protein isolated from untreated cells. We observed comparable decreases in zinc content for XPA (xeroderma pigmentosum group A) protein (CCCC zinc finger), but not SP-1 (specificity protein-1) or aprataxin (CCHH zinc finger). These findings demonstrate that PARP-1 is a direct molecular target of arsenite and that arsenite interacts selectively with zinc finger motifs containing three or more cysteine residues. PMID:21550982

  17. Role of zinc finger structure in nuclear localization of transcription factor Sp1

    SciTech Connect

    Ito, Tatsuo; Azumano, Makiko; Uwatoko, Chisana; Itoh, Kohji Kuwahara, Jun

    2009-02-27

    Transcription factor Sp1 is localized in the nucleus and regulates gene expression. Our previous study demonstrated that the carboxyl terminal region of Sp1 containing 3-zinc finger region as DNA binding domain can also serve as nuclear localization signal (NLS). However, the nuclear transport mechanism of Sp1 has not been well understood. In this study, we performed a gene expression study on mutant Sp1 genes causing a set of amino acid substitutions in zinc finger domains to elucidate nuclear import activity. Nuclear localization of the GFP-fused mutant Sp1 proteins bearing concomitant substitutions in the first and third zinc fingers was highly inhibited. These mutant Sp1 proteins had also lost the binding ability as to the GC box sequence. The results suggest that the overall tertiary structure formed by the three zinc fingers is essential for nuclear localization of Sp1 as well as dispersed basic amino acids within the zinc fingers region.

  18. New redesigned zinc-finger proteins: design strategy and its application.

    PubMed

    Negi, Shigeru; Imanishi, Miki; Matsumoto, Makoto; Sugiura, Yukio

    2008-01-01

    The design of DNA-binding proteins for the specific control of the gene expression is one of the big challenges for several research laboratories in the post-genomic era. An artificial transcription factor with the desired DNA binding specificity could work as a powerful tool and drug to regulate the target gene. The zinc-finger proteins, which typically contain many fingers linked in a tandem fashion, are some of the most intensively studied DNA-binding proteins. In particular, the Cys(2)His(2)-type zinc finger is one of the most common DNA-binding motifs in eukaryotes. A simple mode of DNA recognition by the Cys(2)His(2)-type zinc-finger domain provides an ideal framework for designing proteins with new functions. Our laboratory has utilized several design strategies to create new zinc-finger peptides/proteins by redesigning the Cys(2)His(2)-type zinc-finger motif. This review focuses on the aspects of design strategies, mainly from our recent results, for the creation of artificial zinc-finger proteins, and discusses the possible application of zinc-finger technology for gene regulation and gene therapy.

  19. Structural Analyses of Zinc Finger Domains for Specific Interactions with DNA.

    PubMed

    Eom, Ki Seong; Cheong, Jin Sung; Lee, Seung Jae

    2016-12-28

    Zinc finger proteins are among the most extensively applied metalloproteins in the field of biotechnology owing to their unique structural and functional aspects as transcriptional and translational regulators. The classical zinc fingers are the largest family of zinc proteins and they provide critical roles in physiological systems from prokaryotes to eukaryotes. Two cysteine and two histidine residues (Cys₂His₂) coordinate to the zinc ion for the structural functions to generate a ββα fold, and this secondary structure supports specific interactions with their binding partners, including DNA, RNA, lipids, proteins, and small molecules. In this account, the structural similarity and differences of well-known Cys₂His₂-type zinc fingers such as zinc interaction factor 268 (ZIF268), transcription factor IIIA (TFIIIA), GAGA, and Ros will be explained. These proteins perform their specific roles in species from archaea to eukaryotes and they show significant structural similarity; however, their aligned amino acids present low sequence homology. These zinc finger proteins have different numbers of domains for their structural roles to maintain biological progress through transcriptional regulations from exogenous stresses. The superimposed structures of these finger domains provide interesting details when these fingers are applied to specific gene binding and editing. The structural information in this study will aid in the selection of unique types of zinc finger applications in vivo and in vitro approaches, because biophysical backgrounds including complex structures and binding affinities aid in the protein design area.

  20. Editing the Trypanosoma cruzi genome with zinc finger nucleases.

    PubMed

    Burle-Caldas, Gabriela Assis; Grazielle-Silva, Viviane; Soares-Simões, Melissa; Schumann Burkard, Gabriela; Roditi, Isabel; DaRocha, Wanderson Duarte; Teixeira, Santuza M

    2017-03-01

    Gene function studies in Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, have been hindered by the lack of efficient genetic manipulation protocols. In most organisms, insertion and deletion of DNA fragments in the genome are dependent on the generation of double-stranded DNA break (DSB) and repair. By inducing a site-specific DSB, zinc finger nucleases (ZFNs) have proven to be useful to enhance gene editing in many cell types. Using a pair of ZFNs targeted to the T. cruzi gp72 gene, we were able to generate gp72 knockout parasites with improved efficiency compared to the conventional gene knockout protocol. We also provide evidence that, in T. cruzi, repair of DSBs generated by ZFNs occurs primarily by the homologous recombination pathway.

  1. Modular synthetic inverters from zinc finger proteins and small RNAs

    DOE PAGES

    Hsia, Justin; Holtz, William J.; Maharbiz, Michel M.; ...

    2016-02-17

    Synthetic zinc finger proteins (ZFPs) can be created to target promoter DNA sequences, repressing transcription. The binding of small RNA (sRNA) to ZFP mRNA creates an ultrasensitive response to generate higher effective Hill coefficients. Here we combined three “off the shelf” ZFPs and three sRNAs to create new modular inverters in E. coli and quantify their behavior using induction fold. We found a general ordering of the effects of the ZFPs and sRNAs on induction fold that mostly held true when combining these parts. We then attempted to construct a ring oscillator using our new inverters. In conclusion, our chosenmore » parts performed insufficiently to create oscillations, but we include future directions for improvement upon our work presented here.« less

  2. Modular synthetic inverters from zinc finger proteins and small RNAs

    SciTech Connect

    Hsia, Justin; Holtz, William J.; Maharbiz, Michel M.; Arcak, Murat; Keasling, Jay D.; Rao, Christopher V.

    2016-02-17

    Synthetic zinc finger proteins (ZFPs) can be created to target promoter DNA sequences, repressing transcription. The binding of small RNA (sRNA) to ZFP mRNA creates an ultrasensitive response to generate higher effective Hill coefficients. Here we combined three “off the shelf” ZFPs and three sRNAs to create new modular inverters in E. coli and quantify their behavior using induction fold. We found a general ordering of the effects of the ZFPs and sRNAs on induction fold that mostly held true when combining these parts. We then attempted to construct a ring oscillator using our new inverters. In conclusion, our chosen parts performed insufficiently to create oscillations, but we include future directions for improvement upon our work presented here.

  3. Enhanced protein production by engineered zinc finger proteins.

    PubMed

    Reik, Andreas; Zhou, Yuanyue; Collingwood, Trevor N; Warfe, Lyndon; Bartsevich, Victor; Kong, Yanhong; Henning, Karla A; Fallentine, Barrett K; Zhang, Lei; Zhong, Xiaohong; Jouvenot, Yann; Jamieson, Andrew C; Rebar, Edward J; Case, Casey C; Korman, Alan; Li, Xiao-Yong; Black, Amelia; King, David J; Gregory, Philip D

    2007-08-01

    Increasing the yield of therapeutic proteins from mammalian production cell lines reduces costs and decreases the time to market. To this end, we engineered a zinc finger protein transcription factor (ZFP TF) that binds a DNA sequence within the promoter driving transgene expression. This ZFP TF enabled >100% increase in protein yield from CHO cells in transient, stable, and fermentor production run settings. Expression vectors engineered to carry up to 10 ZFP binding sites further enhanced ZFP-mediated increases in protein production up to approximately 500%. The multimerized ZFP binding sites function independently of the promoter, and therefore across vector platforms. CHO cell lines stably expressing ZFP TFs demonstrated growth characteristics similar to parental cell lines. ZFP TF expression and gains in protein production were stable over >30 generations in the absence of antibiotic selection. Our results demonstrate that ZFP TFs can rapidly and stably increase protein production in mammalian cells.

  4. Generation of albino Xenopus tropicalis using zinc-finger nucleases.

    PubMed

    Nakajima, Keisuke; Nakajima, Taeko; Takase, Minoru; Yaoita, Yoshio

    2012-12-01

    To generate albino lines of Xenopus tropicalis, we injected fertilized eggs with mRNAs encoding zinc-finger nucleases (ZFNs) targeting the tyrosinase coding region. Surprisingly, vitiligo was observed on the skin of F0 frogs that had been injected with ZFN mRNAs, indicating that both tyrosinase genes in the genome were disrupted in all melanocytes within the vitiligo patches. Mutation analysis using genomic DNA from the skin revealed that two mosaic F0 frogs underwent spatially complex tyrosinase gene mutations. The data implies that the ZFN-induced tyrosinase gene ablations occurred randomly over space and time throughout the entire body, possibly until the young tadpole stage, and that melanocyte precursors lacking functional tyrosinase proliferated and formed vitiligo patches. Several albino X. tropicalis, which are compound heterozygotes for biallelic tyrosinase mutations, were obtained by mating the mosaic F0 frogs. To our knowledge, this is the first report of the albino vertebrates generated by the targeted gene knockout.

  5. Crystallographic and Biochemical Analysis of the Ran-Binding Zinc Finger Domain

    SciTech Connect

    Partridge, James R.; Schwartz, Thomas U.; MIT

    2009-08-13

    The nuclear pore complex (NPC) resides in circular openings within the nuclear envelope and serves as the sole conduit to facilitate nucleocytoplasmic transport in eukaryotes. The asymmetric distribution of the small G protein Ran across the nuclear envelope regulates directionality of protein transport. Ran interacts with the NPC of metazoa via two asymmetrically localized components, Nup153 at the nuclear face and Nup358 at the cytoplasmic face. Both nucleoporins contain a stretch of distinct, Ran-binding zinc finger domains. Here, we present six crystal structures of Nup153-zinc fingers in complex with Ran and a 1.48 {angstrom} crystal structure of RanGDP. Crystal engineering allowed us to obtain well diffracting crystals so that all ZnF-Ran complex structures are refined to high resolution. Each of the four zinc finger modules of Nup153 binds one Ran molecule in apparently non-allosteric fashion. The affinity is measurably higher for RanGDP than for RanGTP and varies modestly between the individual zinc fingers. By microcalorimetric and mutational analysis, we determined that one specific hydrogen bond accounts for most of the differences in the binding affinity of individual zinc fingers. Genomic analysis reveals that only in animals do NPCs contain Ran-binding zinc fingers. We speculate that these organisms evolved a mechanism to maintain a high local concentration of Ran at the vicinity of the NPC, using this zinc finger domain as a sink.

  6. Zinc finger peptide based optic sensor for detection of zinc ions.

    PubMed

    Verma, Neelam; Kaur, Gagandeep

    2016-12-15

    In the present work, polyacrylamide gel has been used as a matrix for the immobilization of zinc finger peptide and fluorescent dye acrydine orange on the micro well plate to fabricate the fluorescence based biosensor for the detection of zinc ions in milk samples. The fluorescent dye moves in the hydrophobic groove formed after folding of the peptide in the presence of zinc ions. Under optimized conditions, linear range was observed between 0.001µg/l to 10µg/l of Zinc ions, with a lowest detection limit of 0.001µg/l and response time of 5min. Presented biosensor has shown 20% decrease in fluorescent intensity values after 5 regenerations and stable for more than one month, stored at 4°C. Interference study with other metal ions like lead, cadmium and copper showed a negligible change in fluorescence intensity in comparison to zinc ions. Developed bio sensing system was found to be novel, quick, reliable, miniaturized, stable, reproducible and repeatable and specific for zinc ion, which has been applied to various milk samples.

  7. CD spectra show the relational style between Zic-, Gli-, Glis-zinc finger protein and DNA.

    PubMed

    Sakai-Kato, Kumiko; Ishiguro, Akira; Mikoshiba, Katsuhiko; Aruga, Jun; Utsunomiya-Tate, Naoko

    2008-01-01

    Zic family proteins have five C2H2-type zinc finger motifs. The Zic-zinc finger domains show high homology to the corresponding domains of the Gli and Glis families, which also contain five C2H2-type zinc finger motifs. The zinc finger motifs of the proteins of these three protein families form an alpha-helix conformation in solution. The addition of oligo DNA that included a Gli-binding sequence increased the alpha-helix content estimated by using circular dichroism spectroscopy. Comparison of the Zic-, Gli-, and Glis-zinc fingers indicated that the alpha-helix content after the addition of oligo DNA correlated well with the affinity of each zinc finger for the oligo DNA (correlation coefficient, 0.85). The importance of the zinc ion for protein folding was reflected in a reduction in the alpha-helix content upon removal of the zinc ion. Owing to the compact globular structure, the alpha-helix structure of the proteins of these three protein families is extremely thermally stable. These results suggest that the alpha-helix structure is important for DNA binding and profoundly related to functional and structural diversity among the three families.

  8. Inhibitors of HIV nucleocapsid protein zinc fingers as candidates for the treatment of AIDS.

    PubMed

    Rice, W G; Supko, J G; Malspeis, L; Buckheit, R W; Clanton, D; Bu, M; Graham, L; Schaeffer, C A; Turpin, J A; Domagala, J; Gogliotti, R; Bader, J P; Halliday, S M; Coren, L; Sowder, R C; Arthur, L O; Henderson, L E

    1995-11-17

    Strategies for the treatment of human immunodeficiency virus-type 1 (HIV-1) infection must contend with the obstacle of drug resistance. HIV-1 nucleocapsid protein zinc fingers are prime antiviral targets because they are mutationally intolerant and are required both for acute infection and virion assembly. Nontoxic disulfide-substituted benzamides were identified that attack the zinc fingers, inactivate cell-free virions, inhibit acute and chronic infections, and exhibit broad antiretroviral activity. The compounds were highly synergistic with other antiviral agents, and resistant mutants have not been detected. Zinc finger-reactive compounds may offer an anti-HIV strategy that restricts drug-resistance development.

  9. Evaluation of Novel Design Strategies for Developing Zinc Finger Nucleases Tools for Treating Human Diseases

    PubMed Central

    Bach, Christian; Sherman, William; Pallis, Jani; Bajwa, Hassan

    2014-01-01

    Zinc finger nucleases (ZFNs) are associated with cell death and apoptosis by binding at countless undesired locations. This cytotoxicity is associated with the binding ability of engineered zinc finger domains to bind dissimilar DNA sequences with high affinity. In general, binding preferences of transcription factors are associated with significant degenerated diversity and complexity which convolutes the design and engineering of precise DNA binding domains. Evolutionary success of natural zinc finger proteins, however, evinces that nature created specific evolutionary traits and strategies, such as modularity and rank-specific recognition to cope with binding complexity that are critical for creating clinical viable tools to precisely modify the human genome. Our findings indicate preservation of general modularity and significant alteration of the rank-specific binding preferences of the three-finger binding domain of transcription factor SP1 when exchanging amino acids in the 2nd finger. PMID:24808958

  10. Evaluation of Novel Design Strategies for Developing Zinc Finger Nucleases Tools for Treating Human Diseases

    DOE PAGES

    Bach, Christian; Sherman, William; Pallis, Jani; ...

    2014-01-01

    Zinc finger nucleases (ZFNs) are associated with cell death and apoptosis by binding at countless undesired locations. This cytotoxicity is associated with the binding ability of engineered zinc finger domains to bind dissimilar DNA sequences with high affinity. In general, binding preferences of transcription factors are associated with significant degenerated diversity and complexity which convolutes the design and engineering of precise DNA binding domains. Evolutionary success of natural zinc finger proteins, however, evinces that nature created specific evolutionary traits and strategies, such as modularity and rank-specific recognition to cope with binding complexity that are critical for creating clinical viable toolsmore » to precisely modify the human genome. Our findings indicate preservation of general modularity and significant alteration of the rank-specific binding preferences of the three-finger binding domain of transcription factor SP1 when exchanging amino acids in the 2nd finger.« less

  11. Solution structure of the zinc finger HIT domain in protein FON

    PubMed Central

    He, Fahu; Umehara, Takashi; Tsuda, Kengo; Inoue, Makoto; Kigawa, Takanori; Matsuda, Takayoshi; Yabuki, Takashi; Aoki, Masaaki; Seki, Eiko; Terada, Takaho; Shirouzu, Mikako; Tanaka, Akiko; Sugano, Sumio; Muto, Yutaka; Yokoyama, Shigeyuki

    2007-01-01

    The zinc finger HIT domain is a sequence motif found in many proteins, including thyroid hormone receptor interacting protein 3 (TRIP-3), which is possibly involved in maturity-onset diabetes of the young (MODY). Novel zinc finger motifs are suggested to play important roles in gene regulation and chromatin remodeling. Here, we determined the high-resolution solution structure of the zinc finger HIT domain in ZNHIT2 (protein FON) from Homo sapiens, by an NMR method based on 567 upper distance limits derived from NOE intensities measured in three-dimensional NOESY spectra. The structure yielded a backbone RMSD to the mean coordinates of 0.19 Å for the structured residues 12–48. The fold consists of two consecutive antiparallel β-sheets and two short C-terminal helices packed against the second β-sheet, and binds two zinc ions. Both zinc ions are coordinated tetrahedrally via a CCCC-CCHC motif to the ligand residues of the zf-HIT domain in an interleaved manner. The tertiary structure of the zinc finger HIT domain closely resembles the folds of the B-box, RING finger, and PHD domains with a cross-brace zinc coordination mode, but is distinct from them. The unique three-dimensional structure of the zinc finger HIT domain revealed a novel zinc-binding fold, as a new member of the treble clef domain family. On the basis of the structural data, we discuss the possible functional roles of the zinc finger HIT domain. PMID:17656577

  12. PRDM9 sticks its zinc fingers into recombination hotspots and between species.

    PubMed

    Sandovici, Ionel; Sapienza, Carmen

    2010-05-24

    Meiotic recombination events typically cluster within narrow regions of the genome termed hotspots. A series of recent papers reveals that PRDM9, a C2H2-type zinc-finger protein with histone H3 lysine 4 methyltransferase activity, plays a major role in the specification of hotspots. The zinc fingers that contact DNA in a sequence-dependent manner evolve rapidly and are under positive selection, leading to differences in the location of recombination hotspots as well as hybrid sterility.

  13. Spectroscopic characterization of copper(I) binding to apo and metal-reconstituted zinc finger peptides.

    PubMed

    Doku, Reginald T; Park, Grace; Wheeler, Korin E; Splan, Kathryn E

    2013-08-01

    Cu(I) exhibits high affinity for thiolate ligands, suggesting that thiol-rich zinc or iron binding sites may be subject to disruption during copper stress conditions. Zinc fingers constitute a large class of metalloproteins that use a combination of cysteine and histidine residues that bind Zn(II) as a structural element. Despite the shared preference of both copper and zinc for thiolate and amine coordination, the susceptibility of zinc finger domains toward copper substitution is not well studied. We report spectroscopic studies that characterize the Cu(I) binding properties of the zinc finger consensus peptides CP-CCHH, CP-CCHC, and CP-CCCC and the C-terminal zinc finger domain of HIV-1 nucleocapsid protein p7 (NCp7_C). Cu(I) binds to both the apopeptides and the Co(II)-substituted peptides, and the stoichiometry of Cu(I) binding is dependent on the number of cysteine thiols at the metal binding site. Fluorescence studies of the Zn(II)-NCp7_C complex indicate that Cu(I) also effectively competes with Zn(II) at the metal binding site, despite the high affinity of Zn(II) for the CCHC binding motif. Circular dichroism studies on both CP-CCHC and NCp7_C show that the conformations of the Cu(I)-bound complexes differ substantially from those of the Zn(II) species, implying that Cu(I) substitution is likely to impact zinc finger function. These results show that for the peptides studied here, Cu(I) is the thermodynamically favored metal despite the known high Zn(II) affinity of zinc finger domains, suggesting that Cu(I)-substituted zinc finger domains might be relevant in the context of both copper toxicity mechanisms and copper-responsive transcription factors.

  14. Targeted genome modification in mice using zinc-finger nucleases.

    PubMed

    Carbery, Iara D; Ji, Diana; Harrington, Anne; Brown, Victoria; Weinstein, Edward J; Liaw, Lucy; Cui, Xiaoxia

    2010-10-01

    Homologous recombination-based gene targeting using Mus musculus embryonic stem cells has greatly impacted biomedical research. This study presents a powerful new technology for more efficient and less time-consuming gene targeting in mice using embryonic injection of zinc-finger nucleases (ZFNs), which generate site-specific double strand breaks, leading to insertions or deletions via DNA repair by the nonhomologous end joining pathway. Three individual genes, multidrug resistant 1a (Mdr1a), jagged 1 (Jag1), and notch homolog 3 (Notch3), were targeted in FVB/N and C57BL/6 mice. Injection of ZFNs resulted in a range of specific gene deletions, from several nucleotides to >1000 bp in length, among 20-75% of live births. Modified alleles were efficiently transmitted through the germline, and animals homozygous for targeted modifications were obtained in as little as 4 months. In addition, the technology can be adapted to any genetic background, eliminating the need for generations of backcrossing to achieve congenic animals. We also validated the functional disruption of Mdr1a and demonstrated that the ZFN-mediated modifications lead to true knockouts. We conclude that ZFN technology is an efficient and convenient alternative to conventional gene targeting and will greatly facilitate the rapid creation of mouse models and functional genomics research.

  15. Targeted Genome Modification in Mice Using Zinc-Finger Nucleases

    PubMed Central

    Carbery, Iara D.; Ji, Diana; Harrington, Anne; Brown, Victoria; Weinstein, Edward J.; Liaw, Lucy; Cui, Xiaoxia

    2010-01-01

    Homologous recombination-based gene targeting using Mus musculus embryonic stem cells has greatly impacted biomedical research. This study presents a powerful new technology for more efficient and less time-consuming gene targeting in mice using embryonic injection of zinc-finger nucleases (ZFNs), which generate site-specific double strand breaks, leading to insertions or deletions via DNA repair by the nonhomologous end joining pathway. Three individual genes, multidrug resistant 1a (Mdr1a), jagged 1 (Jag1), and notch homolog 3 (Notch3), were targeted in FVB/N and C57BL/6 mice. Injection of ZFNs resulted in a range of specific gene deletions, from several nucleotides to >1000 bp in length, among 20–75% of live births. Modified alleles were efficiently transmitted through the germline, and animals homozygous for targeted modifications were obtained in as little as 4 months. In addition, the technology can be adapted to any genetic background, eliminating the need for generations of backcrossing to achieve congenic animals. We also validated the functional disruption of Mdr1a and demonstrated that the ZFN-mediated modifications lead to true knockouts. We conclude that ZFN technology is an efficient and convenient alternative to conventional gene targeting and will greatly facilitate the rapid creation of mouse models and functional genomics research. PMID:20628038

  16. [Breeding of robust industrial ethanol-tolerant Saccharomyces cerevisiae strain by artificial zinc finger protein library].

    PubMed

    Ma, Cui; Zhao, Xinqing; Li, Qian; Zhang, Mingming; Kim, Jin Soo; Bai, Fengwu

    2013-05-01

    Breeding of robust industrial Saccharomyces cerevisiae strains with high ethanol tolerance is of great significance for efficient fuel ethanol production. Zinc finger proteins play important roles in gene transcription and translation, and exerting control on the regulation of multiple genes. The sequence and localization of the zinc finger motif can be designed and engineered, and the artificial zinc finger protein can be used to regulate celluar metabolism. Stress tolerance of microbial strains is related to multiple genes. Therefore, it is possible to use artificially-designed zinc finger proteins to breed stress tolerant strains. In this study, a library containing artificial zinc finger protein encoding genes was transformed into the model yeast strain S288c. A recombinant strain named M01 with improved ethanol tolerance was obtained. The plasmid in M01 was isolated, and then transformed into the industrial yeast strain Sc4126. Ethanol tolerance of the recombinant strain of Sc4126 were significantly improved. When high gravity ethanol fermentation using 250 g/L glucose was performed, comparing with the wild-type strain, fermentation time of the recombinant strain was decreased by 24 h and the final ethanol concentration was enhanced by 6.3%. The results of this study demonstrate that artificial zinc finger proteins are able to exert control on stress tolerance of yeast strains, and these results provide basis to construct robust industrial yeast strains for efficient ethanol fermentation.

  17. A multiscale approach to simulating the conformational properties of unbound multi-C₂H₂ zinc finger proteins.

    PubMed

    Liu, Lei; Wade, Rebecca C; Heermann, Dieter W

    2015-09-01

    The conformational properties of unbound multi-Cys2 His2 (mC2H2) zinc finger proteins, in which zinc finger domains are connected by flexible linkers, are studied by a multiscale approach. Three methods on different length scales are utilized. First, atomic detail molecular dynamics simulations of one zinc finger and its adjacent flexible linker confirmed that the zinc finger is more rigid than the flexible linker. Second, the end-to-end distance distributions of mC2H2 zinc finger proteins are computed using an efficient atomistic pivoting algorithm, which only takes excluded volume interactions into consideration. The end-to-end distance distribution gradually changes its profile, from left-tailed to right-tailed, as the number of zinc fingers increases. This is explained by using a worm-like chain model. For proteins of a few zinc fingers, an effective bending constraint favors an extended conformation. Only for proteins containing more than nine zinc fingers, is a somewhat compacted conformation preferred. Third, a mesoscale model is modified to study both the local and the global conformational properties of multi-C2H2 zinc finger proteins. Simulations of the CCCTC-binding factor (CTCF), an important mC2H2 zinc finger protein for genome spatial organization, are presented.

  18. Transcriptional cofactors of the FOG family interact with GATA proteins by means of multiple zinc fingers.

    PubMed Central

    Fox, A H; Liew, C; Holmes, M; Kowalski, K; Mackay, J; Crossley, M

    1999-01-01

    Friend of GATA-1 (FOG-1) is a zinc finger protein that has been shown to interact physically with the erythroid DNA-binding protein GATA-1 and modulate its transcriptional activity. Recently, two new members of the FOG family have been identified: a mammalian protein, FOG-2, that also associates with GATA-1 and other mammalian GATA factors; and U-shaped, a Drosophila protein that interacts with the Drosophila GATA protein Pannier. FOG proteins contain multiple zinc fingers and it has been shown previously that the sixth finger of FOG-1 interacts specifically with the N-finger but not the C-finger of GATA-1. Here we show that fingers 1, 5 and 9 of FOG-1 also interact with the N-finger of GATA-1 and that FOG-2 and U-shaped also contain multiple GATA-interacting fingers. We define the key contact residues and show that these residues are highly conserved in GATA-interacting fingers. We examine the effect of selectively mutating the four interacting fingers of FOG-1 and show that each contributes to FOG-1's ability to modulate GATA-1 activity. Finally, we show that FOG-1 can repress GATA-1-mediated activation and present evidence that this ability involves the recently described CtBP co-repressor proteins that recognize all known FOG proteins. PMID:10329627

  19. Context-dependent DNA recognition code for C2H2 zinc-finger transcription factors

    PubMed Central

    Liu, Jiajian; Stormo, Gary D.

    2008-01-01

    Motivation: Modeling and identifying the DNA-protein recognition code is one of the most challenging problems in computational biology. Several quantitative methods have been developed to model DNA-protein interactions with specific focus on the C2H2 zinc-finger proteins, the largest transcription factor family in eukaryotic genomes. In many cases, they performed well. But the overall the predictive accuracy of these methods is still limited. One of the major reasons is all these methods used weight matrix models to represent DNA-protein interactions, assuming all base-amino acid contacts contribute independently to the total free energy of binding. Results: We present a context-dependent model for DNA–zinc-finger protein interactions that allows us to identify inter-positional dependencies in the DNA recognition code for C2H2 zinc-finger proteins. The degree of non-independence was detected by comparing the linear perceptron model with the non-linear neural net (NN) model for their predictions of DNA–zinc-finger protein interactions. This dependency is supported by the complex base-amino acid contacts observed in DNA–zinc-finger interactions from structural analyses. Using extensive published qualitative and quantitative experimental data, we demonstrated that the context-dependent model developed in this study can significantly improves predictions of DNA binding profiles and free energies of binding for both individual zinc fingers and proteins with multiple zinc fingers when comparing to previous positional-independent models. This approach can be extended to other protein families with complex base-amino acid residue interactions that would help to further understand the transcriptional regulation in eukaryotic genomes. Availability:The software implemented as c programs and are available by request. http://ural.wustl.edu/softwares.html Contact: stormo@ural.wustl.edu PMID:18586699

  20. A systematic survey of the Cys2His2 zinc finger DNA-binding landscape

    PubMed Central

    Persikov, Anton V.; Wetzel, Joshua L.; Rowland, Elizabeth F.; Oakes, Benjamin L.; Xu, Denise J.; Singh, Mona; Noyes, Marcus B.

    2015-01-01

    Cys2His2 zinc fingers (C2H2-ZFs) comprise the largest class of metazoan DNA-binding domains. Despite this domain's well-defined DNA-recognition interface, and its successful use in the design of chimeric proteins capable of targeting genomic regions of interest, much remains unknown about its DNA-binding landscape. To help bridge this gap in fundamental knowledge and to provide a resource for design-oriented applications, we screened large synthetic protein libraries to select binding C2H2-ZF domains for each possible three base pair target. The resulting data consist of >160 000 unique domain–DNA interactions and comprise the most comprehensive investigation of C2H2-ZF DNA-binding interactions to date. An integrated analysis of these independent screens yielded DNA-binding profiles for tens of thousands of domains and led to the successful design and prediction of C2H2-ZF DNA-binding specificities. Computational analyses uncovered important aspects of C2H2-ZF domain–DNA interactions, including the roles of within-finger context and domain position on base recognition. We observed the existence of numerous distinct binding strategies for each possible three base pair target and an apparent balance between affinity and specificity of binding. In sum, our comprehensive data help elucidate the complex binding landscape of C2H2-ZF domains and provide a foundation for efforts to determine, predict and engineer their DNA-binding specificities. PMID:25593323

  1. Solution structure of an archaeal DNA binding protein with an eukaryotic zinc finger fold.

    PubMed

    Guillière, Florence; Danioux, Chloé; Jaubert, Carole; Desnoues, Nicole; Delepierre, Muriel; Prangishvili, David; Sezonov, Guennadi; Guijarro, J Iñaki

    2013-01-01

    While the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1. The sequence of the protein consists of the classical eukaryotic C2H2 motif with the fourth histidine coordinating zinc missing, as well as of N- and C-terminal extensions. We showed that the protein AFV1p06 binds zinc and solved its solution structure by NMR. AFV1p06 displays a zinc finger fold with a novel structure extension and disordered N- and C-termini. Structure calculations show that a glutamic acid residue that coordinates zinc replaces the fourth histidine of the C2H2 motif. Electromobility gel shift assays indicate that the protein binds to DNA with different affinities depending on the DNA sequence. AFV1p06 is the first experimentally characterised archaeal zinc finger protein with a DNA binding activity. The AFV1p06 protein family has homologues in diverse viruses of hyperthermophilic archaea. A phylogenetic analysis points out a common origin of archaeal and eukaryotic C2H2 zinc fingers.

  2. Conservation, diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome

    PubMed Central

    Englbrecht, Claudia C; Schoof, Heiko; Böhm, Siegfried

    2004-01-01

    Background The classical C2H2 zinc finger domain is involved in a wide range of functions and can bind to DNA, RNA and proteins. The comparison of zinc finger proteins in several eukaryotes has shown that there is a lot of lineage specific diversification and expansion. Although the number of characterized plant proteins that carry the classical C2H2 zinc finger motifs is growing, a systematic classification and analysis of a plant genome zinc finger gene set is lacking. Results We found through in silico analysis 176 zinc finger proteins in Arabidopsis thaliana that hence constitute the most abundant family of putative transcriptional regulators in this plant. Only a minority of 33 A. thaliana zinc finger proteins are conserved in other eukaryotes. In contrast, the majority of these proteins (81%) are plant specific. They are derived from extensive duplication events and form expanded families. We assigned the proteins to different subgroups and families and focused specifically on the two largest and evolutionarily youngest families (A1 and C1) that are suggested to be primarily involved in transcriptional regulation. The newly defined family A1 (24 members) comprises proteins with tandemly arranged zinc finger domains. Family C1 (64 members), earlier described as the EPF-family in Petunia, comprises proteins with one isolated or two to five dispersed fingers and a mostly invariant QALGGH motif in the zinc finger helices. Based on the amino acid pattern in these helices we could describe five different signature sequences prevalent in C1 zinc finger domains. We also found a number of non-finger domains that are conserved in these families. Conclusions Our analysis of the few evolutionarily conserved zinc finger proteins of A. thaliana suggests that most of them could be involved in ancient biological processes like RNA metabolism and chromatin-remodeling. In contrast, the majority of the unique A. thaliana zinc finger proteins are known or suggested to be

  3. The artificial zinc finger coding gene 'Jazz' binds the utrophin promoter and activates transcription.

    PubMed

    Corbi, N; Libri, V; Fanciulli, M; Tinsley, J M; Davies, K E; Passananti, C

    2000-06-01

    Up-regulation of utrophin gene expression is recognized as a plausible therapeutic approach in the treatment of Duchenne muscular dystrophy (DMD). We have designed and engineered new zinc finger-based transcription factors capable of binding and activating transcription from the promoter of the dystrophin-related gene, utrophin. Using the recognition 'code' that proposes specific rules between zinc finger primary structure and potential DNA binding sites, we engineered a new gene named 'Jazz' that encodes for a three-zinc finger peptide. Jazz belongs to the Cys2-His2 zinc finger type and was engineered to target the nine base pair DNA sequence: 5'-GCT-GCT-GCG-3', present in the promoter region of both the human and mouse utrophin gene. The entire zinc finger alpha-helix region, containing the amino acid positions that are crucial for DNA binding, was specifically chosen on the basis of the contacts more frequently represented in the available list of the 'code'. Here we demonstrate that Jazz protein binds specifically to the double-stranded DNA target, with a dissociation constant of about 32 nM. Band shift and super-shift experiments confirmed the high affinity and specificity of Jazz protein for its DNA target. Moreover, we show that chimeric proteins, named Gal4-Jazz and Sp1-Jazz, are able to drive the transcription of a test gene from the human utrophin promoter.

  4. Zinc finger binding motifs do not explain recombination rate variation within or between species of Drosophila.

    PubMed

    Heil, Caiti S S; Noor, Mohamed A F

    2012-01-01

    In humans and mice, the Cys(2)His(2) zinc finger protein PRDM9 binds to a DNA sequence motif enriched in hotspots of recombination, possibly modifying nucleosomes, and recruiting recombination machinery to initiate Double Strand Breaks (DSBs). However, since its discovery, some researchers have suggested that the recombinational effect of PRDM9 is lineage or species specific. To test for a conserved role of PRDM9-like proteins across taxa, we use the Drosophila pseudoobscura species group in an attempt to identify recombination associated zinc finger proteins and motifs. We leveraged the conserved amino acid motifs in Cys(2)His(2) zinc fingers to predict nucleotide binding motifs for all Cys(2)His(2) zinc finger proteins in Drosophila pseudoobscura and identified associations with empirical measures of recombination rate. Additionally, we utilized recombination maps from D. pseudoobscura and D. miranda to explore whether changes in the binding motifs between species can account for changes in the recombination landscape, analogous to the effect observed in PRDM9 among human populations. We identified a handful of potential recombination-associated sequence motifs, but the associations are generally tenuous and their biological relevance remains uncertain. Furthermore, we found no evidence that changes in zinc finger DNA binding explains variation in recombination rate between species. We therefore conclude that there is no protein with a DNA sequence specific human-PRDM9-like function in Drosophila. We suggest these findings could be explained by the existence of a different recombination initiation system in Drosophila.

  5. Comparing zinc finger nucleases and transcription activator-like effector nucleases for gene targeting in Drosophila.

    PubMed

    Beumer, Kelly J; Trautman, Jonathan K; Christian, Michelle; Dahlem, Timothy J; Lake, Cathleen M; Hawley, R Scott; Grunwald, David J; Voytas, Daniel F; Carroll, Dana

    2013-10-03

    Zinc-finger nucleases have proven to be successful as reagents for targeted genome manipulation in Drosophila melanogaster and many other organisms. Their utility has been limited, however, by the significant failure rate of new designs, reflecting the complexity of DNA recognition by zinc fingers. Transcription activator-like effector (TALE) DNA-binding domains depend on a simple, one-module-to-one-base-pair recognition code, and they have been very productively incorporated into nucleases (TALENs) for genome engineering. In this report we describe the design of TALENs for a number of different genes in Drosophila, and we explore several parameters of TALEN design. The rate of success with TALENs was substantially greater than for zinc-finger nucleases , and the frequency of mutagenesis was comparable. Knockout mutations were isolated in several genes in which such alleles were not previously available. TALENs are an effective tool for targeted genome manipulation in Drosophila.

  6. Isolation of a zinc finger gene consistently deleted in DiGeorge syndrome.

    PubMed

    Aubry, M; Demczuk, S; Desmaze, C; Aikem, M; Aurias, A; Julien, J P; Rouleau, G A

    1993-10-01

    DiGeorge syndrome is a human developmental disorder resulting in hypoplasia of the thymus and parathyroids, and conotruncal heart defects. We recently isolated four genes with zinc finger DNA binding motifs mapping to chromosome 22q11.2 DiGeorge critical region. We now report that one of them, ZNF74 gene, is hemizygously deleted in 23 out of 24 DiGeorge syndrome patients tested. ZNF74 mRNA transcripts are detected in human and mouse embryos but not in adult tissues. Sequence analysis of a corresponding cDNA reveals an an open reading frame encoding 12 zinc finger motifs of the Kruppel/TFIIIA type as well as N-terminal and C-terminal non-zinc finger domains. These results suggest that changes in the dosage of a putative transcription factor through ZNF74 hemizygous deletion may be critical for DiGeorge developmental anomalies.

  7. Identification and localization of a novel zinc finger gene in developing chick skin and feather buds.

    PubMed

    Padanilam, B J; Solursh, M

    1996-03-07

    We have cloned and sequenced a cDNA encoding a novel zinc finger protein (Fzf-1) containing two tandem repeats of zinc finger motifs of the C2H2 type. The cDNA is 3.0 Kb long and has an open reading frame which codes for a protein of 789 amino acids. The expression pattern of the zinc finger gene was studied in chick embryonic skin and feathers by in situ hybridization. The expression of the gene is found to be temporally and spatially regulated. In stage 38 chick embryos, the transcripts are localized to the epidermis but in 10-day-old embryos, the signal is localized to the forming dermis. In 12-day-old chick, the transcripts are localized to the mesenchymal region of the elongated feather buds. Reverse transcription followed by Polymerase Chain Reaction (RT-PCR) did not detect the transcripts in any other tissues.

  8. The (unusual) aspartic acid in the metal coordination sphere of the prokaryotic zinc finger domain.

    PubMed

    D'Abrosca, Gianluca; Russo, Luigi; Palmieri, Maddalena; Baglivo, Ilaria; Netti, Fortuna; de Paola, Ivan; Zaccaro, Laura; Farina, Biancamaria; Iacovino, Rosa; Pedone, Paolo Vincenzo; Isernia, Carla; Fattorusso, Roberto; Malgieri, Gaetano

    2016-08-01

    The possibility of choices of protein ligands and coordination geometries leads to diverse Zn(II) binding sites in zinc-proteins, allowing a range of important biological roles. The prokaryotic Cys2His2 zinc finger domain (originally found in the Ros protein from Agrobacterium tumefaciens) tetrahedrally coordinates zinc through two cysteine and two histidine residues and it does not adopt a correct fold in the absence of the metal ion. Ros is the first structurally characterized member of a family of bacterial proteins that presents several amino acid changes in the positions occupied in Ros by the zinc coordinating residues. In particular, the second position is very often occupied by an aspartic acid although the coordination of structural zinc by an aspartate in eukaryotic zinc fingers is very unusual. Here, by appropriately mutating the protein Ros, we characterize the aspartate role within the coordination sphere of this family of proteins demonstrating how the presence of this residue only slightly perturbs the functional structure of the prokaryotic zinc finger domain while it greatly influences its thermodynamic properties.

  9. Exploring the DNA-binding specificities of zinc fingers with DNA microarrays

    PubMed Central

    Bulyk, Martha L.; Huang, Xiaohua; Choo, Yen; Church, George M.

    2001-01-01

    A key step in the regulation of networks that control gene expression is the sequence-specific binding of transcription factors to their DNA recognition sites. A more complete understanding of these DNA–protein interactions will permit a more comprehensive and quantitative mapping of the regulatory pathways within cells, as well as a deeper understanding of the potential functions of individual genes regulated by newly identified DNA-binding sites. Here we describe a DNA microarray-based method to characterize sequence-specific DNA recognition by zinc-finger proteins. A phage display library, prepared by randomizing critical amino acid residues in the second of three fingers of the mouse Zif268 domain, provided a rich source of zinc-finger proteins with variant DNA-binding specificities. Microarrays containing all possible 3-bp binding sites for the variable zinc fingers permitted the quantitation of the binding site preferences of the entire library, pools of zinc fingers corresponding to different rounds of selection from this library, as well as individual Zif268 variants that were isolated from the library by using specific DNA sequences. The results demonstrate the feasibility of using DNA microarrays for genome-wide identification of putative transcription factor-binding sites. PMID:11404456

  10. Solution NMR characterization of Sgf73(1-104) indicates that Zn ion is required to stabilize zinc finger motif

    SciTech Connect

    Lai, Chaohua; Wu, Minhao; Li, Pan; Shi, Chaowei; Tian, Changlin; Zang, Jianye

    2010-07-02

    Zinc finger motif contains a zinc ion coordinated by several conserved amino acid residues. Yeast Sgf73 protein was identified as a component of SAGA (Spt/Ada/Gcn5 acetyltransferase) multi-subunit complex and Sgf73 protein was known to contain two zinc finger motifs. Sgf73(1-104), containing the first zinc finger motif, was necessary to modulate the deubiquitinase activity of SAGA complex. Here, Sgf73(1-104) was over-expressed using bacterial expression system and purified for solution NMR (nuclear magnetic resonance) structural studies. Secondary structure and site-specific relaxation analysis of Sgf73(1-104) were achieved after solution NMR backbone assignment. Solution NMR and circular dichroism analysis of Sgf73(1-104) after zinc ion removal using chelation reagent EDTA (ethylene-diamine-tetraacetic acid) demonstrated that zinc ion was required to maintain stable conformation of the zinc finger motif.

  11. Characterization of Zinc finger protein 496 that interacts with Jumonji/Jarid2

    PubMed Central

    Mysliwiec, Matthew Robert; Kim, Tae-gyun; Lee, Youngsook

    2007-01-01

    Jumonij (JMJ)/Jarid2 plays important roles in embryonic development and functions as a transcriptional repressor. Using yeast two-hybrid screening, we have identified a cofactor of JMJ, the zinc finger protein 496 (Zfp496) that contains a SCAN, KRAB and zinc finger domain. Our molecular analyses indicate that Zfp496 functions as a transcriptional activator. Further, Zfp496 inhibits the transcriptional repression of JMJ and JMJ represses the transcriptional activation of Zfp496. This study demonstrates that JMJ physically and functionally interacts with Zfp496, which will provide important insights into endogenous target gene regulation by both factors. PMID:17521633

  12. Zinc finger nuclease technology: advances and obstacles in modelling and treating genetic disorders.

    PubMed

    Jabalameli, Hamid Reza; Zahednasab, Hamid; Karimi-Moghaddam, Amin; Jabalameli, Mohammad Reza

    2015-03-01

    Zinc finger nucleases (ZFNs) are engineered restriction enzymes designed to target specific DNA sequences within the genome. Assembly of zinc finger DNA-binding domain to a DNA-cleavage domain enables the enzyme machinery to target unique locus in the genome and invoke endogenous DNA repair mechanisms. This machinery offers a versatile approach in allele editing and gene therapy. Here we discuss the architecture of ZFNs and strategies for generating targeted modifications within the genome. We review advances in gene therapy and modelling of the disease using these enzymes and finally, discuss the practical obstacles in using this technology.

  13. Differential Binding of Monomethylarsonous Acid Compared to Arsenite and Arsenic Trioxide with Zinc Finger Peptides and Proteins

    PubMed Central

    2015-01-01

    Arsenic is an environmental toxin that enhances the carcinogenic effect of DNA-damaging agents, such as ultraviolet radiation and benzo[a]pyrene. Interaction with zinc finger proteins has been shown to be an important molecular mechanism for arsenic toxicity and cocarcinogenesis. Arsenicals such as arsenite, arsenic trioxide (ATO), and monomethylarsonous acid (MMA(III)) have been reported to interact with cysteine residues of zinc finger domains, but little is known about potential differences in their selectivity of interaction. Herein we analyzed the interaction of arsenite, MMA(III), and ATO with C2H2, C3H1, and C4 configurations of zinc fingers using UV–vis, cobalt, fluorescence, and mass spectrometry. We observed that arsenite and ATO both selectively bound to C3H1 and C4 zinc fingers, while MMA(III) interacted with all three configurations of zinc finger peptides. Structurally and functionally, arsenite and ATO caused conformational changes and zinc loss on C3H1 and C4 zinc finger peptide and protein, respectively, whereas MMA(III) changed conformation and displaced zinc on all three types of zinc fingers. The differential selectivity was also demonstrated in zinc finger proteins isolated from cells treated with these arsenicals. Our results show that trivalent inorganic arsenic compounds, arsenite and ATO, have the same selectivity and behavior when interacting with zinc finger proteins, while methylation removes the selectivity. These findings provide insights on the molecular mechanisms underlying the differential effects of inorganic versus methylated arsenicals, as well as the role of in vivo arsenic methylation in arsenic toxicity and carcinogenesis. PMID:24611629

  14. Differential binding of monomethylarsonous acid compared to arsenite and arsenic trioxide with zinc finger peptides and proteins.

    PubMed

    Zhou, Xixi; Sun, Xi; Mobarak, Charlotte; Gandolfi, A Jay; Burchiel, Scott W; Hudson, Laurie G; Liu, Ke Jian

    2014-04-21

    Arsenic is an environmental toxin that enhances the carcinogenic effect of DNA-damaging agents, such as ultraviolet radiation and benzo[a]pyrene. Interaction with zinc finger proteins has been shown to be an important molecular mechanism for arsenic toxicity and cocarcinogenesis. Arsenicals such as arsenite, arsenic trioxide (ATO), and monomethylarsonous acid (MMA(III)) have been reported to interact with cysteine residues of zinc finger domains, but little is known about potential differences in their selectivity of interaction. Herein we analyzed the interaction of arsenite, MMA(III), and ATO with C2H2, C3H1, and C4 configurations of zinc fingers using UV-vis, cobalt, fluorescence, and mass spectrometry. We observed that arsenite and ATO both selectively bound to C3H1 and C4 zinc fingers, while MMA(III) interacted with all three configurations of zinc finger peptides. Structurally and functionally, arsenite and ATO caused conformational changes and zinc loss on C3H1 and C4 zinc finger peptide and protein, respectively, whereas MMA(III) changed conformation and displaced zinc on all three types of zinc fingers. The differential selectivity was also demonstrated in zinc finger proteins isolated from cells treated with these arsenicals. Our results show that trivalent inorganic arsenic compounds, arsenite and ATO, have the same selectivity and behavior when interacting with zinc finger proteins, while methylation removes the selectivity. These findings provide insights on the molecular mechanisms underlying the differential effects of inorganic versus methylated arsenicals, as well as the role of in vivo arsenic methylation in arsenic toxicity and carcinogenesis.

  15. Transgenic mice expressing an artificial zinc finger regulator targeting an endogenous gene.

    PubMed

    Passananti, Claudio; Corbi, Nicoletta; Onori, Annalisa; Di Certo, Maria Grazia; Mattei, Elisabetta

    2010-01-01

    Zinc finger (ZF) proteins belonging to the Cys2-His2 class provide a simple and versatile framework to design novel artificial transcription factors (ATFs) targeted to the desired genes. Our work is based on ZF ATFs engineered to up-regulate the expression level of the dystrophin-related gene utrophin in Duchenne muscular dystrophy (DMD). In particular, on the basis of the "recognition code" that defines specific rules between zinc finger primary structure and potential DNA-binding sites we engineered and selected a new family of artificial transcription factors, whose DNA-binding domain consists in a three zinc finger peptide called "Jazz." Jazz protein binds specifically the 9 bp DNA sequence (5(')-GCT-GCT-GCG-3(')) present in the promoter region of both the human and mouse utrophin gene. We generated a transgenic mouse expressing Jazz protein fused to the strong transcriptional activation domain VP16 and under the control of the muscle specific promoter of the myosin light chain gene. Vp16-Jazz mice display a strong up-regulation of the utrophin at both mRNA and protein levels. To our knowledge, this represents the first example of a transgenic mouse expressing an artificial gene coding for a zinc finger-based transcription factor.

  16. DUF581 Is Plant Specific FCS-Like Zinc Finger Involved in Protein-Protein Interaction

    PubMed Central

    K, Muhammed Jamsheer; Laxmi, Ashverya

    2014-01-01

    Zinc fingers are a ubiquitous class of protein domain with considerable variation in structure and function. Zf-FCS is a highly diverged group of C2-C2 zinc finger which is present in animals, prokaryotes and viruses, but not in plants. In this study we identified that a plant specific domain of unknown function, DUF581 is a zf-FCS type zinc finger. Based on HMM-HMM comparison and signature motif similarity we named this domain as FCS-Like Zinc finger (FLZ) domain. A genome wide survey identified that FLZ domain containing genes are bryophytic in origin and this gene family is expanded in spermatophytes. Expression analysis of selected FLZ gene family members of A. thaliana identified an overlapping expression pattern suggesting a possible redundancy in their function. Unlike the zf-FCS domain, the FLZ domain found to be highly conserved in sequence and structure. Using a combination of bioinformatic and protein-protein interaction tools, we identified that FLZ domain is involved in protein-protein interaction. PMID:24901469

  17. High-frequency homologous recombination in plants mediated by zinc-finger nucleases.

    PubMed

    Wright, David A; Townsend, Jeffrey A; Winfrey, Ronnie Joe; Irwin, Phillip A; Rajagopal, Jyothi; Lonosky, Patricia M; Hall, Bradford D; Jondle, Michael D; Voytas, Daniel F

    2005-11-01

    Homologous recombination offers great promise for plant genome engineering. This promise has not been realized, however, because when DNA enters plant cells homologous recombination occurs infrequently and random integration predominates. Using a tobacco test system, we demonstrate that chromosome breaks created by zinc-finger nucleases greatly enhance the frequency of localized recombination. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene integrated at various chromosomal sites in 10 different transgenic tobacco lines. The reporter gene carried a recognition site for a zinc-finger nuclease, and protoplasts from each tobacco line were electroporated with both DNA encoding the nuclease and donor DNA to effect repair of the reporter. Homologous recombination occurred in more than 10% of the transformed protoplasts regardless of the reporter's chromosomal position. Approximately 20% of the GUS:NPTII reporter genes were repaired solely by homologous recombination, whereas the remainder had associated DNA insertions or deletions consistent with repair by both homologous recombination and non-homologous end joining. The DNA-binding domain encoded by zinc-finger nucleases can be engineered to recognize a variety of chromosomal target sequences. This flexibility, coupled with the enhancement in homologous recombination conferred by double-strand breaks, suggests that plant genome engineering through homologous recombination can now be reliably accomplished using zinc-finger nucleases.

  18. A zinc finger protein from Candida albicans is involved in sucrose utilization.

    PubMed Central

    Kelly, R; Kwon-Chung, K J

    1992-01-01

    A sucrose-inducible alpha-glucosidase activity that hydrolyzes sucrose in Candida albicans has been demonstrated previously. The enzyme is assayable in whole cells and was inhibited by both sucrose and maltose. A C. albicans gene (CASUC1) that affects sucrose utilization and alpha-glucosidase activity was cloned by expression in a Saccharomyces cerevisiae suc2 mutant (2102) devoid of invertase genes. CASUC1 enabled the S. cerevisiae mutant to utilize both sucrose and maltose. DNA sequence analysis revealed that CASUC1 encodes a putative zinc finger-containing protein with 28% identity to a maltose-regulatory gene (MAL63) of S. cerevisiae. The gene products of CASUC1 and MAL63 are approximately the same size (501 and 470 amino acids, respectively), and each contains a single zinc finger located at the N terminus. The zinc fingers of CASUC1 and MAL63 comprise six conserved cysteines (C6 zinc finger) and are of the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaavariable-Cys-Xaa2-Cys-+ ++Xaa6-Cys (where Xaan indicates a stretch of the indicated number of any amino acids). Both contain five amino acids in the variable region. CASUC1 also complemented the maltose utilization defect of an S. cerevisiae mutant (TCY-137) containing a defined mutation in a maltose-regulatory gene. The sucrose utilization defect of type II Candida stellatoidea, a sucrase-negative mutant of C. albicans, was corrected by CASUC1. Determinations of alpha-glucosidase activity in whole cells revealed that activity was restored in transformants cultivated on either sucrose or maltose. To our knowledge, this is the first zinc finger-encoding gene, as well as the first putative regulatory gene, to be identified in C. albicans. Images PMID:1729210

  19. The Dof domain, a zinc finger DNA-binding domain conserved only in higher plants, truly functions as a Cys2/Cys2 Zn finger domain.

    PubMed

    Umemura, Yoshimi; Ishiduka, Tomoko; Yamamoto, Rie; Esaka, Muneharu

    2004-03-01

    The Dof (DNA-binding with one finger) proteins are plant transcription factors that have a highly conserved DNA-binding domain, called the Dof domain. The Dof domain, which is composed of 52 amino acid residues, is similar to the Cys2/Cys2 zinc finger DNA-binding domain of GATA1 and steroid hormone receptors, but has a longer putative loop than that in the case of these zinc finger domains. The DNA-binding function of ascorbate oxidase gene binding protein (AOBP), a Dof protein, was investigated by gel retardation analysis. When Cys was replaced by His, the Dof domain could not function as a Cys3/His- or a Cys2/His2-type zinc finger. The characteristic longer loop was essential for DNA-binding activity. Furthermore, heavy metals such as Co(II), Ni(II), Cd(II), Cu(II), Hg(II), Fe(II), and Fe(III) inhibited the DNA-binding activity of the Dof domain. Manganese ion as well as zinc ion was coordinated by the Dof domain in vitro. On the other hand, the analysis using inductively coupled argon plasma mass spectrometry (ICP-MS) showed that the Dof domain contained zinc ion but not manganese ion. Thus, the Dof domain was proved to function as a Cys2/Cys2 zinc finger domain.

  20. EOR-2 is an obligate binding partner of the BTB-zinc finger protein EOR-1 in Caenorhabditis elegans.

    PubMed

    Howell, Kelly; Arur, Swathi; Schedl, Tim; Sundaram, Meera V

    2010-04-01

    BTB-zinc finger transcription factors play many important roles in metazoan development. In these proteins, the BTB domain is critical for dimerization and for recruiting cofactors to target genes. Identification of these cofactors is important for understanding how BTB-zinc finger proteins influence transcription. Here we show that the novel but conserved protein EOR-2 is an obligate binding partner of the BTB-zinc finger protein EOR-1 in Caenorhabditis elegans. EOR-1 and EOR-2 function together to promote multiple Ras/ERK-dependent cell fates during development, and we show that EOR-1 is a robust substrate of ERK in vitro. A point mutation (L81F) in the EOR-1 BTB domain reduces both ERK phosphorylation and EOR-2 binding and eliminates all detectable biological function without affecting EOR-1 expression levels, localization, or dimerization. This point mutation lies near the predicted charged pocket region of the EOR-1 BTB dimer, a region that, in other BTB-zinc finger proteins, has been proposed to interact with corepressors or coactivators. We also show that a conserved zinc finger-like motif in EOR-2 is required for binding to EOR-1, that the interaction between EOR-1 and EOR-2 is direct, and that EOR-2 can bind to the human BTB-zinc finger protein PLZF. We propose that EOR-2 defines a new family of cofactors for BTB-zinc finger transcription factors that may have conserved roles in other organisms.

  1. Mammalian spermatid specific protein, TP2, is a zinc metalloprotein with two finger motifs.

    PubMed

    Baskaran, R; Rao, M R

    1991-09-30

    An analysis of the recently reported cDNA derived amino acid sequences of mouse (Kleene and Flynn, J. Biol. Chem. 262, 17272-17277, 1987) and rat (Luersson et al., Nucl. Acids Res. 17, 3585, 1989). TP2 has revealed the presence of two potential zinc finger motifs involving cysteine and histidine residues. TP2, as purified from rat elongating spermatids, is shown here to contain 0.2 atoms of zinc bound per molecule of the protein by atomic absorption spectroscopy. On incubation with 10 microM ZnCl2, in vitro, and subsequent exhaustive dialysis, TP2 had 2 atoms of zinc bound per molecule. The involvement of cysteine residues of TP2 in coordination with zinc was also suggested by the observation that TP2 could be labeled, in situ, with iodoacetamidofluorescein only after preincubation of spermatid nuclei with EDTA. The zinc finger domains of TP2 may play an important role in initiation of chromatin condensation and/or cessation of transcriptional activity during mammalian spermiogenesis.

  2. Disruption of a novel imprinted zinc-finger gene, ZNF215, in Beckwith-Wiedemann syndrome.

    PubMed Central

    Alders, M; Ryan, A; Hodges, M; Bliek, J; Feinberg, A P; Privitera, O; Westerveld, A; Little, P F; Mannens, M

    2000-01-01

    The genetics of Beckwith-Wiedemann syndrome (BWS) is complex and is thought to involve multiple genes. It is known that three regions on chromosome 11p15 (BWSCR1, BWSCR2, and BWSCR3) may play a role in the development of BWS. BWSCR2 is defined by two BWS breakpoints. Here we describe the cloning and sequence analysis of 73 kb containing BWSCR2. Within this region, we detected a novel zinc-finger gene, ZNF215. We show that two of its five alternatively spliced transcripts are disrupted by both BWSCR2 breakpoints. Parts of the 3' end of these splice forms are transcribed from the antisense strand of a second zinc-finger gene, ZNF214. We show that ZNF215 is imprinted in a tissue-specific manner. PMID:10762538

  3. Zinc fingers as protein recognition motifs: structural basis for the GATA-1/friend of GATA interaction.

    PubMed

    Liew, Chu Kong; Simpson, Raina J Y; Kwan, Ann H Y; Crofts, Linda A; Loughlin, Fionna E; Matthews, Jacqueline M; Crossley, Merlin; Mackay, Joel P

    2005-01-18

    GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1/FOG interaction, we have determined the three-dimensional structure of a complex comprising the interaction domains of these proteins. The structure reveals how zinc fingers can act as protein recognition motifs. Details of the architecture of the contact domains and their physical properties provide a molecular explanation for how the GATA-1 mutations contribute to distinct but related genetic diseases.

  4. A zinc finger protein that regulates oligodendrocyte specification, migration and myelination in zebrafish

    PubMed Central

    Sidik, Harwin; Talbot, William S.

    2015-01-01

    Precise control of oligodendrocyte migration and development is crucial for myelination of axons in the central nervous system (CNS), but important questions remain unanswered about the mechanisms controlling these processes. In a zebrafish screen for myelination mutants, we identified a mutation in zinc finger protein 16-like (znf16l). znf16l mutant larvae have reduced myelin basic protein (mbp) expression and reduced CNS myelin. Marker, time-lapse and ultrastructural studies indicated that oligodendrocyte specification, migration and myelination are disrupted in znf16l mutants. Transgenic studies indicated that znf16l acts autonomously in oligodendrocytes. Expression of Zfp488 from mouse rescued mbp expression in znf16l mutants, indicating that these homologs have overlapping functions. Our results defined the function of a new zinc finger protein with specific function in oligodendrocyte specification, migration and myelination in the developing CNS. PMID:26459222

  5. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.

    PubMed Central

    Kim, Y G; Cha, J; Chandrasegaran, S

    1996-01-01

    A long-term goal in the field of restriction-modification enzymes has been to generate restriction endonucleases with novel sequence specificities by mutating or engineering existing enzymes. This will avoid the increasingly arduous task of extensive screening of bacteria and other microorganisms for new enzymes. Here, we report the deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of Fok I endonuclease. Both fusion proteins are active and under optimal conditions cleave DNA in a sequence-specific manner. Thus, the modular structure of Fok I endonuclease and the zinc finger motifs makes it possible to create "artificial" nucleases that will cut DNA near a predetermined site. This opens the way to generate many new enzymes with tailor-made sequence specificities desirable for various applications. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8577732

  6. Generalization through similarity: motif discourse in the discovery and elaboration of zinc finger proteins

    PubMed Central

    Condit, Celeste Michelle; Railsback, L Bruce

    2007-01-01

    Background Biological organisms and their components are better conceived within categories based on similarity rather than on identity. Biologists routinely operate with similarity-based concepts such as "model organism" and "motif." There has been little exploration of the characteristics of the similarity-based categories that exist in biology. This study uses the case of the discovery and classification of zinc finger proteins to explore how biological categories based in similarity are represented. Results The existence of a category of "zinc finger proteins" was based in 1) a lumpy gradient of similarity, 2) a link between function and structure, 3) establishment of a range of appearance across systems and organisms, and 4) an evolutionary locus as a historically based common-ground. Conclusion More systematic application of the idea of similarity-based categorization might eliminate the assumption that biological characteristics can only contribute to narrow categorization of humans. It also raises possibilities for refining data-driven exploration efforts. PMID:17915020

  7. Zinc Finger Protein5 Is Required for the Control of Trichome Initiation by Acting Upstream of Zinc Finger Protein8 in Arabidopsis1[C][W][OA

    PubMed Central

    Zhou, Zhongjing; An, Lijun; Sun, Lili; Zhu, Shuijin; Xi, Wanyan; Broun, Pierre; Yu, Hao; Gan, Yinbo

    2011-01-01

    Arabidopsis (Arabidopsis thaliana) trichome development is a model system for studying cell development, cell differentiation, and the cell cycle. Our previous studies have shown that the GLABROUS INFLORESCENCE STEMS (GIS) family genes, GIS, GIS2, and ZINC FINGER PROTEIN8 (ZFP8), control shoot maturation and epidermal cell fate by integrating gibberellins (GAs) and cytokinin signaling in Arabidopsis. Here, we show that a new C2H2 zinc finger protein, ZFP5, plays an important role in controlling trichome cell development through GA signaling. Overexpression of ZFP5 results in the formation of ectopic trichomes on carpels and other inflorescence organs. zfp5 loss-of-function mutants exhibit a reduced number of trichomes on sepals, cauline leaves, paraclades, and main inflorescence stems in comparison with wild-type plants. More importantly, it is found that ZFP5 mediates the regulation of trichome initiation by GAs. These results are consistent with ZFP5 expression patterns and the regional influence of GA on trichome initiation. The molecular analyses suggest that ZFP5 functions upstream of GIS, GIS2, ZFP8, and the key trichome initiation regulators GLABROUS1 (GL1) and GL3. Using a steroid-inducible activation of ZFP5 and chromatin immunoprecipitation experiments, we further demonstrate that ZFP8 is the direct target of ZFP5 in controlling epidermal cell differentiation. PMID:21803862

  8. Zinc Finger Transcription Factors as Novel Genetic Switches to Modulate Metastatic Progression of Breast Tumors

    DTIC Science & Technology

    2008-05-01

    Program, Biochemistry, Université de Montréal with Robert Cedergren 1999-2003 - Research Associate in Dr. Carlos Barbas, III laboratory...PCT/US03/03705 (2001). B. Peer Reviewed Publications (* most significant to proposed work) Blancafort, P., Ferbeyre, G., Sariol, C., and Cedergren , R...and Cedergren , R. The recognition of a non-canonical base pair by a zinc finger protein. Chem Biol. 1999; 6:585-97. Segal, D.J., Beerli R.R, Blancafort

  9. Role of the POZ zinc finger transcription factor FBI-1 in human and murine adipogenesis.

    PubMed

    Laudes, Matthias; Christodoulides, Constantinos; Sewter, Ciaran; Rochford, Justin J; Considine, Robert V; Sethi, Jaswinder K; Vidal-Puig, Antonio; O'Rahilly, Stephen

    2004-03-19

    Poxvirus zinc finger (POZ) zinc finger domain transcription factors have been shown to play a role in the control of growth arrest and differentiation in several types of mesenchymal cells but not, as yet, adipocytes. We found that a POZ domain protein, factor that binds to inducer of short transcripts-1 (FBI-1), was induced during both murine and human preadipocyte differentiation with maximal expression levels seen at days 2-4. FBI-1 mRNA was expressed in human adipose tissue with the highest levels found in samples from morbidly obese subjects. Murine cell lines constitutively expressing FBI-1 showed evidence for accelerated adipogenesis with earlier induction of markers of differentiation and enhanced lipid accumulation, suggesting that FBI-1 may be an active participant in the differentiation process. Consistent with the properties of this family of proteins in other cell systems, 3T3L1 cells stably overexpressing FBI-1 showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, FBI-1 reduced the transcriptional activity of the cyclin A promoter. Thus, FBI-1, a POZ zinc finger transcription factor, is induced during the early phases of human and murine preadipocyte differentiation where it may contribute to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation.

  10. Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase

    PubMed Central

    Minczuk, Michal; Papworth, Monika A.; Kolasinska, Paulina; Murphy, Michael P.; Klug, Aaron

    2006-01-01

    We used engineered zinc finger peptides (ZFPs) to bind selectively to predetermined sequences in human mtDNA. Surprisingly, we found that engineered ZFPs cannot be reliably routed to mitochondria by using only conventional mitochondrial targeting sequences. We here show that addition of a nuclear export signal allows zinc finger chimeric enzymes to be imported into human mitochondria. The selective binding of mitochondria-specific ZFPs to mtDNA was exemplified by targeting the T8993G mutation, which causes two mitochondrial diseases, neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) and also maternally inherited Leigh's syndrome. To develop a system that allows the monitoring of site-specific alteration of mtDNA we combined a ZFP with the easily assayed DNA-modifying activity of hDNMT3a methylase. Expression of the mutation-specific chimeric methylase resulted in the selective methylation of cytosines adjacent to the mutation site. This is a proof of principle that it is possible to target and alter mtDNA in a sequence-specific manner by using zinc finger technology. PMID:17170133

  11. A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter.

    PubMed

    Isalan, M; Klug, A; Choo, Y

    2001-07-01

    DNA-binding domains with predetermined sequence specificity are engineered by selection of zinc finger modules using phage display, allowing the construction of customized transcription factors. Despite remarkable progress in this field, the available protein-engineering methods are deficient in many respects, thus hampering the applicability of the technique. Here we present a rapid and convenient method that can be used to design zinc finger proteins against a variety of DNA-binding sites. This is based on a pair of pre-made zinc finger phage-display libraries, which are used in parallel to select two DNA-binding domains each of which recognizes given 5 base pair sequences, and whose products are recombined to produce a single protein that recognizes a composite (9 base pair) site of predefined sequence. Engineering using this system can be completed in less than two weeks and yields proteins that bind sequence-specifically to DNA with Kd values in the nanomolar range. To illustrate the technique, we have selected seven different proteins to bind various regions of the human immunodeficiency virus 1 (HIV-1) promoter.

  12. Interaction of Sp1 zinc finger with transport factor in the nuclear localization of transcription factor Sp1

    SciTech Connect

    Ito, Tatsuo; Kitamura, Haruka; Uwatoko, Chisana; Azumano, Makiko; Itoh, Kohji; Kuwahara, Jun

    2010-12-10

    Research highlights: {yields} Sp1 zinc fingers themselves interact with importin {alpha}. {yields} Sp1 zinc finger domains play an essential role as a nuclear localization signal. {yields} Sp1 can be transported into the nucleus in an importin-dependent manner. -- Abstract: Transcription factor Sp1 is localized in the nucleus and regulates the expression of many cellular genes, but the nuclear transport mechanism of Sp1 is not well understood. In this study, we revealed that GST-fused Sp1 protein bound to endogenous importin {alpha} in HeLa cells via the Sp1 zinc finger domains, which comprise the DNA binding domain of Sp1. It was found that the Sp1 zinc finger domains directly interacted with a wide range of importin {alpha} including the armadillo (arm) repeat domain and the C-terminal acidic domain. Furthermore, it turned out that all three zinc fingers of Sp1 are essential for binding to importin {alpha}. Taken together, these results suggest that the Sp1 zinc finger domains play an essential role as a NLS and Sp1 can be transported into the nucleus in an importin-dependent manner even though it possesses no classical NLSs.

  13. A theoretical investigation of DNA dynamics and desolvation kinetics for zinc finger proteinZif268

    PubMed Central

    2015-01-01

    Background Transcription factors, regulating the expression inventory of a cell, interact with its respective DNA subjugated by a specific recognition pattern, which if well exploited may ensure targeted genome engineering. The mostly widely studied transcription factors are zinc finger proteins that bind to its target DNA via direct and indirect recognition levels at the interaction interface. Exploiting the binding specificity and affinity of the interaction between the zinc fingers and the respective DNA can help in generating engineered zinc fingers for therapeutic applications. Experimental evidences lucidly substantiate the effect of indirect interaction like DNA deformation and desolvation kinetics, in empowering ZFPs to accomplish partial sequence specificity functioning around structural properties of DNA. Exploring the structure-function relationships of the existing zinc finger-DNA complexes at the indirect recognition level can aid in predicting the probable zinc fingers that could bind to any target DNA. Deformation energy, which defines the energy required to bend DNA from its native shape to its shape when bound to the ZFP, is an effect of indirect recognition mechanism. Water is treated as a co-reactant for unfurling the affinity studies in ZFP-DNA binding equilibria that takes into account the unavoidable change in hydration that occurs when these two solvated surfaces come into contact. Results Aspects like desolvation and DNA deformation have been theoretically investigated based on simulations and free energy perturbation data revealing a consensus in correlating affinity and specificity as well as stability for ZFP-DNA interactions. Greater loss of water at the interaction interface of the DNA calls for binding with higher affinity, eventually distorting the DNA to a greater extent accounted by the change in major groove width and DNA tilt, stretch and rise. Conclusion Most prediction algorithms for ZFPs do not account for water loss at the

  14. Synthetic zinc finger proteins: the advent of targeted gene regulation and genome modification technologies.

    PubMed

    Gersbach, Charles A; Gaj, Thomas; Barbas, Carlos F

    2014-08-19

    The understanding of gene regulation and the structure and function of the human genome increased dramatically at the end of the 20th century. Yet the technologies for manipulating the genome have been slower to develop. For instance, the field of gene therapy has been focused on correcting genetic diseases and augmenting tissue repair for more than 40 years. However, with the exception of a few very low efficiency approaches, conventional genetic engineering methods have only been able to add auxiliary genes to cells. This has been a substantial obstacle to the clinical success of gene therapies and has also led to severe unintended consequences in several cases. Therefore, technologies that facilitate the precise modification of cellular genomes have diverse and significant implications in many facets of research and are essential for translating the products of the Genomic Revolution into tangible benefits for medicine and biotechnology. To address this need, in the 1990s, we embarked on a mission to develop technologies for engineering protein-DNA interactions with the aim of creating custom tools capable of targeting any DNA sequence. Our goal has been to allow researchers to reach into genomes to specifically regulate, knock out, or replace any gene. To realize these goals, we initially focused on understanding and manipulating zinc finger proteins. In particular, we sought to create a simple and straightforward method that enables unspecialized laboratories to engineer custom DNA-modifying proteins using only defined modular components, a web-based utility, and standard recombinant DNA technology. Two significant challenges we faced were (i) the development of zinc finger domains that target sequences not recognized by naturally occurring zinc finger proteins and (ii) determining how individual zinc finger domains could be tethered together as polydactyl proteins to recognize unique locations within complex genomes. We and others have since used this modular

  15. Identifying key interactions stabilizing DOF zinc finger-DNA complexes using in silico approaches.

    PubMed

    Hamzeh-Mivehroud, Maryam; Moghaddas-Sani, Hakimeh; Rahbar-Shahrouziasl, Mahdieh; Dastmalchi, Siavoush

    2015-10-07

    DOF (DNA-binding with one finger) proteins, a family of DNA-binding transcription factors, are members of zinc fingers unique to plants. They are associated with different plant specific phenomena including germination, dormancy, light and defense responses. Until now, there is no report of experimentally solved structure for DOF proteins, making empirical investigation of DOF-DNA interaction more challenging. It has been shown that comparative modeling can be used to reliably predict the three-dimensional (3D) model of structurally unknown proteins whenever a suitable template is available. Furthermore, current molecular mechanics force fields allow prediction of interaction energies for macromolecular complexes. Therefore, the approaches considered in this work were to model the 3D structures of DOF zinc fingers (ZFs) from Arabidopsis thaliana complexed with DNA molecule, to calculate their binding energies, to identify key interactions established between ZFs and DNA, and to determine the impact of the different interactions on the binding energies. The results were used to predict the binding affinities for the novel designed ZFs and may be used in engineering DNA binding proteins.

  16. Molecular and functional characterization of two drought-induced zinc finger proteins, ZmZnF1 and ZmZnF2 from maize kernels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have isolated two cDNA clones encoding Zinc Finger proteins, designated as ZmZnF1 and ZmZnF2, from water-stressed maize kernels. Sequence analyses indicates that ZmZnF1 is homologous to the A20/AN1-type zinc finger protein and contains the zinc finger motif of Cx2–Cx10–CxCx4Cx2Hx5HxC. Whereas ZmZ...

  17. The KUP gene, located on human chromosome 14, encodes a protein with two distant zinc fingers.

    PubMed

    Chardin, P; Courtois, G; Mattei, M G; Gisselbrecht, S

    1991-04-11

    We have isolated a human cDNA (kup), encoding a new protein with two distantly spaced zinc fingers of the C2H2 type. This gene is highly conserved in mammals and is expressed mainly in hematopoietic cells and testis. Its expression was not higher in the various transformed cells tested than in the normal corresponding tissues. The kup gene is located in region q23-q24 of the long arm of human chromosome 14. The kup protein is 433 a.a. long, has a M.W. close to 50 kD and binds to DNA. Although the structure of the kup protein is unusual, the isolated fingers resemble closely those of the Krüppel family, suggesting that this protein is also a transcription factor. The precise function and DNA motif recognized by the kup protein remain to be determined.

  18. Characterization of How DNA Modifications Affect DNA Binding by C2H2 Zinc Finger Proteins

    PubMed Central

    Patel, A.; Hashimoto, H.; Zhang, X.; Cheng, X.

    2016-01-01

    Much is known about vertebrate DNA methylation and oxidation; however, much less is known about how modified cytosine residues within particular sequences are recognized. Among the known methylated DNA-binding domains, the Cys2-His2 zinc finger (ZnF) protein superfamily is the largest with hundreds of members, each containing tandem ZnFs ranging from 3 to >30 fingers. We have begun to biochemically and structurally characterize these ZnFs not only on their sequence specificity but also on their sensitivity to various DNA modifications. Rather than following published methods of refolding insoluble ZnF arrays, we have expressed and purified soluble forms of ZnFs, ranging in size from a tandem array of two to six ZnFs, from seven different proteins. We also describe a fluorescence polarization assay to measure ZnFs affinity with oligonucleotides containing various modifications and our approaches for cocrystallization of ZnFs with oligonucleotides. PMID:27372763

  19. A Zinc Finger Motif-Containing Protein Is Essential for Chloroplast RNA Editing

    PubMed Central

    Sun, Tao; Shi, Xiaowen; Friso, Giulia; Van Wijk, Klaas; Bentolila, Stephane; Hanson, Maureen R.

    2015-01-01

    C-to-U editing of transcripts in plant organelles is carried out by small (<400 kD) protein complexes called editosomes. Recognition of the proper C target for editing is mediated by pentatricopeptide repeat (PPR) containing proteins that recognize cis-elements. Members of two additional gene families, the RIP/MORF and ORRM families, have each been found to be required for editing of particular sets of Cs in mitochondria and/or chloroplasts. By co-immunoprecipitation of the chloroplast editing factor ORRM1, followed by mass spectrometry, we have now identified a member of the RanBP2 type zinc fingers (pFAM00641) protein family that is required for editing of 14 sites in chloroplasts and affects editing efficiency of another 16 chloroplast C targets. In yeast two-hybrid assays, OZ1 (Organelle Zinc finger 1) interacts with PPR site recognition factors whose cognate sites are affected when OZ1 is mutated. No interaction of OZ1 with the chloroplast editing factors RIP2 and RIP9 was detected; however, OZ1 interacts with ORRM1, which binds to RIP proteins, allowing us to build a model for the chloroplast RNA editosome. The RNA editosomes that act upon most chloroplast C targets are likely to contain a PPR protein recognition factor, either RIP2 or RIP9, ORRM1, and OZ1. The organelle zinc finger editing factor family (OZ) contains 4 members in Arabidopsis, three that are predicted to be targeted to chloroplasts and one to mitochondria. With the identification of OZ1, there are now 4 nuclear-encoded protein families known to be essential for plant organelle RNA editing. PMID:25768119

  20. The Zinc Finger of Prolyl Hydroxylase Domain Protein 2 Is Essential for Efficient Hydroxylation of Hypoxia-Inducible Factor α

    PubMed Central

    Arsenault, Patrick R.; Song, Daisheng; Chung, Yu Jin; Khurana, Tejvir S.

    2016-01-01

    Prolyl hydroxylase domain protein 2 (PHD2) (also known as EGLN1) is a key oxygen sensor in mammals that posttranslationally modifies hypoxia-inducible factor α (HIF-α) and targets it for degradation. In addition to its catalytic domain, PHD2 contains an evolutionarily conserved zinc finger domain, which we have previously proposed recruits PHD2 to the HSP90 pathway to promote HIF-α hydroxylation. Here, we provide evidence that this recruitment is critical both in vitro and in vivo. We show that in vitro, the zinc finger can function as an autonomous recruitment domain to facilitate interaction with HIF-α. In vivo, ablation of zinc finger function by a C36S/C42S Egln1 knock-in mutation results in upregulation of the erythropoietin gene, erythrocytosis, and augmented hypoxic ventilatory response, all hallmarks of Egln1 loss of function and HIF stabilization. Hence, the zinc finger ordinarily performs a critical positive regulatory function. Intriguingly, the function of this zinc finger is impaired in high-altitude-adapted Tibetans, suggesting that their adaptation to high altitude may, in part, be due to a loss-of-function EGLN1 allele. Thus, these findings have important implications for understanding both the molecular mechanism of the hypoxic response and human adaptation to high altitude. PMID:27325674

  1. The Electronic Behavior of Zinc-Finger Protein Binding Sites in the Context of the DNA Extended Ladder Model

    NASA Astrophysics Data System (ADS)

    Oiwa, Nestor; Cordeiro, Claudette; Heermann, Dieter

    2016-05-01

    Instead of ATCG letter alignments, typically used in bioinformatics, we propose a new alignment method using the probability distribution function of the bottom of the occupied molecular orbital (BOMO), highest occupied molecular orbital (HOMO) and lowest unoccupied orbital (LUMO). We apply the technique to transcription factors with Cys2His2 zinc fingers. These transcription factors search for binding sites, probing for the electronic patterns at the minor and major DNA groves. The eukaryotic Cys2His2 zinc finger proteins bind to DNA ubiquitously at highly conserved domains. They are responsible for gene regulation and the spatial organization of DNA. To study and understand these zinc finger DNA-protein interactions, we use the extended ladder in the DNA model proposed by Zhu, Rasmussen, Balatsky & Bishop (2007) te{Zhu-2007}. Considering one single spinless electron in each nucleotide π-orbital along a double DNA chain (dDNA), we find a typical pattern for the bottom of BOMO, HOMO and LUMO along the binding sites. We specifically looked at two members of zinc finger protein family: specificity protein 1 (SP1) and early grown response 1 transcription factors (EGR1). When the valence band is filled, we find electrons in the purines along the nucleotide sequence, compatible with the electric charges of the binding amino acids in SP1 and EGR1 zinc finger.

  2. The putative zinc finger of a caulimovirus is essential for infectivity but does not influence gene expression.

    PubMed

    Scholthof, H B; Wu, F C; Kiernan, J M; Shepherd, R J

    1993-04-01

    Plant pararetroviruses, such as caulimoviruses, and animal retroviruses have in common the presence of a highly conserved arrangement of cysteines and a histidine in the precursor of the capsid protein. The composition of these amino acids resembles a zinc finger element, a structure that is common to a class of eukaryotic proteins that regulate gene expression. The role of the putative zinc finger in the life-cycle of caulimoviruses was investigated by introducing specific mutations in the coat protein coding region of a cloned and infectious form of figwort mosaic virus, a caulimovirus. This mutated viral genome, which no longer encoded the conserved cysteine and histidine residues, was not infectious in plants. Transient expression assays in protoplasts showed that expression of a reporter gene inserted at different places in the genome was not detectably influenced by the coat protein or its putative zinc finger. It appears that the zinc finger-like element of caulimoviruses is not involved in the regulation of gene expression. These observations support a model which predicts a function of the zinc finger in specific recognition and packaging of viral RNA into virions prior to reverse transcription.

  3. Zinc finger nuclease technology: A stable tool for high efficiency transformation in bloodstream form T. brucei.

    PubMed

    Schumann, Gabriela; Kangussu-Marcolino, Monica M; Doiron, Nicholas; Käser, Sandro; de Assis Burle-Caldas, Gabriela; DaRocha, Wanderson D; Teixeira, Santuza M; Roditi, Isabel

    2017-04-01

    In Trypanosoma brucei, the generation of knockout mutants is relatively easy compared to other organisms as transfection methods are well established. These methods have their limitations, however, when it comes to the generation of genome-wide libraries that require a minimum of several hundred thousand transformants. Double-strand breaks with the meganuclease ISce-I dramatically increase transformation efficiency, but are not widely in use as cell lines need to be generated de novo before each transfection. Here we show that zinc finger nucleases are a robust and stable tool that can enhance transformation in bloodstream forms by more than an order of magnitude.

  4. The Cytoplasmic Zinc Finger Protein ZPR1 Accumulates in the Nucleolus of Proliferating Cells

    PubMed Central

    Galcheva-Gargova, Zoya; Gangwani, Laxman; Konstantinov, Konstantin N.; Mikrut, Monique; Theroux, Steven J.; Enoch, Tamar; Davis, Roger J.

    1998-01-01

    The zinc finger protein ZPR1 translocates from the cytoplasm to the nucleus after treatment of cells with mitogens. The function of nuclear ZPR1 has not been defined. Here we demonstrate that ZPR1 accumulates in the nucleolus of proliferating cells. The role of ZPR1 was examined using a gene disruption strategy. Cells lacking ZPR1 are not viable. Biochemical analysis demonstrated that the loss of ZPR1 caused disruption of nucleolar function, including preribosomal RNA expression. These data establish ZPR1 as an essential protein that is required for normal nucleolar function in proliferating cells. PMID:9763455

  5. ZFNGenome: A comprehensive resource for locating zinc finger nuclease target sites in model organisms

    PubMed Central

    2011-01-01

    Background Zinc Finger Nucleases (ZFNs) have tremendous potential as tools to facilitate genomic modifications, such as precise gene knockouts or gene replacements by homologous recombination. ZFNs can be used to advance both basic research and clinical applications, including gene therapy. Recently, the ability to engineer ZFNs that target any desired genomic DNA sequence with high fidelity has improved significantly with the introduction of rapid, robust, and publicly available techniques for ZFN design such as the Oligomerized Pool ENgineering (OPEN) method. The motivation for this study is to make resources for genome modifications using OPEN-generated ZFNs more accessible to researchers by creating a user-friendly interface that identifies and provides quality scores for all potential ZFN target sites in the complete genomes of several model organisms. Description ZFNGenome is a GBrowse-based tool for identifying and visualizing potential target sites for OPEN-generated ZFNs. ZFNGenome currently includes a total of more than 11.6 million potential ZFN target sites, mapped within the fully sequenced genomes of seven model organisms; S. cerevisiae, C. reinhardtii, A. thaliana, D. melanogaster, D. rerio, C. elegans, and H. sapiens and can be visualized within the flexible GBrowse environment. Additional model organisms will be included in future updates. ZFNGenome provides information about each potential ZFN target site, including its chromosomal location and position relative to transcription initiation site(s). Users can query ZFNGenome using several different criteria (e.g., gene ID, transcript ID, target site sequence). Tracks in ZFNGenome also provide "uniqueness" and ZiFOpT (Zinc Finger OPEN Targeter) "confidence" scores that estimate the likelihood that a chosen ZFN target site will function in vivo. ZFNGenome is dynamically linked to ZiFDB, allowing users access to all available information about zinc finger reagents, such as the effectiveness of a given

  6. Zinc Finger Transcription Factors as Novel Switches to Modulate Metastatic Progression of Breast Tumors

    DTIC Science & Technology

    2007-05-01

    Biochemistry, Université de Montréal (Canada) 1995-1999 - Ph.D. Program, Biochemistry, Université de Montréal with Robert Cedergren 1999-2003...work) Blancafort, P., Ferbeyre, G., Sariol, C., and Cedergren , R. Pol I-driven integrative expression vectors for yeast. Journal of Biotechnology...1997;56:41-7 Blancafort, P., Klinck, R., Steinberg, S., Scott, J.K. and Cedergren , R. The recognition of a non-canonical base pair by a zinc finger protein

  7. Identification and disruption of an Arabidopsis zinc finger gene controlling seed germination

    PubMed Central

    Papi, Maura; Sabatini, Sabrina; Bouchez, David; Camilleri, Christine; Costantino, Paolo; Vittorioso, Paola

    2000-01-01

    We describe here the Arabidopsis gene DAG1, encoding a zinc finger transcription factor of the Dof family, and show that it is involved in the control of seed germination. By a reverse genetics approach, we isolated an Arabidopsis mutant line with one T-DNA insertion in DAG1. Seeds from homozygous knockout dag1-1 plants do not develop dormancy and germinate also in the absence of light. Segregation analysis indicates that the effect of the mutation is maternal. Accordingly, in situ mRNA hybridizations reveal expression of DAG1 in the vascular tissue of the flower and maturing fruit but not in the seed. PMID:10640273

  8. The N-terminal zinc finger of the erythroid transcription factor GATA-1 binds GATC motifs in DNA.

    PubMed

    Newton, A; Mackay, J; Crossley, M

    2001-09-21

    The mammalian transcription factor GATA-1 is required for normal erythroid and megakaryocytic development. GATA-1 contains two zinc fingers, the C-terminal finger, which is known to bind (A/T)GATA(A/G) motifs in DNA and the N-finger, which is important for interacting with co-regulatory proteins such as Friend of GATA (FOG). We now show that, like the C-finger, the N-finger of GATA-1 is also capable of binding DNA but recognizes distinct sequences with the core GATC. We demonstrate that the GATA-1 N-finger can bind these sequences in vitro and that in cellular assays, GATA-1 can activate promoters containing GATC motifs. Experiments with mutant GATA-1 proteins confirm the importance of the N-finger, as the C-finger is not required for transactivation from GATC sites. Recently four naturally occurring mutations in GATA-1 have been shown to be associated with familial blood disorders. These mutations all map to the N-finger domain. We have investigated the effect of these mutations on the recognition of GATC sites by the N-finger and show that one mutation R216Q abolishes DNA binding, whereas the others have only minor effects.

  9. Members of the zinc finger protein gene family sharing a conserved N-terminal module.

    PubMed Central

    Rosati, M; Marino, M; Franzè, A; Tramontano, A; Grimaldi, G

    1991-01-01

    We report the isolation of human members of a sub-family of structurally related finger protein genes. These potentially encode polypeptides containing finger motifs of the Krüppel type at the C-terminus, and a conserved amino acid module at the N-terminus; because of its invariant location the latter is referred to as finger preceding box (FPB). The FPB, detected also in previously described finger proteins from human, mouse and Xenopus, extends over approximately 65 amino acids and appears to be composed of two contiguous modules: FPB-A (residues 1-42) and FPB-B (residues 43-65). The latter is absent in some of the members analyzed. Elements A and B and the zinc finger domain are encoded by separate exons in the ZNF2 gene, a human member of this sub-family. The positioning of introns within this gene is remarkable. One intron flanks and a second interrupts the first codon of the FPB-A and FPB-B modules, respectively. A third intron occurs a few nucleotides downstream of FPB-B marking its separation from the remainder of the coding sequences. This organization, together with the absence of FPB-B in some cDNAs, supports the hypothesis that mRNAs encoding polypeptides that include one, both or none of the FPB-A and FPB-B modules may be assembled through alternative splicing pathways. Northern analyses showed that members of this sub-family are expressed as multiple transcripts in several cell lines. The sequences of distinct cDNAs homologous to the ZNF2 gene indicate that alternative splicing events adjoin either coding or non coding exons to the FPB sequences. Images PMID:1945843

  10. Recent developments and clinical studies utilizing engineered zinc finger nuclease technology.

    PubMed

    Jo, Young-Il; Kim, Hyongbum; Ramakrishna, Suresh

    2015-10-01

    Efficient methods for creating targeted genetic modifications have long been sought for the investigation of gene function and the development of therapeutic modalities for various diseases, including genetic disorders. Although such modifications are possible using homologous recombination, the efficiency is extremely low. Zinc finger nucleases (ZFNs) are custom-designed artificial nucleases that make double-strand breaks at specific sequences, enabling efficient targeted genetic modifications such as corrections, additions, gene knockouts and structural variations. ZFNs are composed of two domains: (i) a DNA-binding domain comprised of zinc finger modules and (ii) the FokI nuclease domain that cleaves the DNA strand. Over 17 years after ZFNs were initially developed, a number of improvements have been made. Here, we will review the developments and future perspectives of ZFN technology. For example, ZFN activity and specificity have been significantly enhanced by modifying the DNA-binding domain and FokI cleavage domain. Advances in culture methods, such as the application of a cold shock and the use of small molecules that affect ZFN stability, have also increased ZFN activity. Furthermore, ZFN-induced mutant cells can be enriched using episomal surrogate reporters. Additionally, we discuss several ongoing clinical studies that are based on ZFN-mediated genome editing in humans. These breakthroughs have substantially facilitated the use of ZFNs in research, medicine and biotechnology.

  11. Deficiency of the zinc finger protein ZFP106 causes motor and sensory neurodegeneration

    PubMed Central

    Joyce, Peter I.; Fratta, Pietro; Landman, Allison S.; Mcgoldrick, Philip; Wackerhage, Henning; Groves, Michael; Busam, Bharani Shiva; Galino, Jorge; Corrochano, Silvia; Beskina, Olga A.; Esapa, Christopher; Ryder, Edward; Carter, Sarah; Stewart, Michelle; Codner, Gemma; Hilton, Helen; Teboul, Lydia; Tucker, Jennifer; Lionikas, Arimantas; Estabel, Jeanne; Ramirez-Solis, Ramiro; White, Jacqueline K.; Brandner, Sebastian; Plagnol, Vincent; Bennet, David L. H.; Abramov, Andrey Y.; Greensmith, Linda; Fisher, Elizabeth M. C.; Acevedo-Arozena, Abraham

    2016-01-01

    Zinc finger motifs are distributed amongst many eukaryotic protein families, directing nucleic acid–protein and protein–protein interactions. Zinc finger protein 106 (ZFP106) has previously been associated with roles in immune response, muscle differentiation, testes development and DNA damage, although little is known about its specific function. To further investigate the function of ZFP106, we performed an in-depth characterization of Zfp106 deficient mice (Zfp106−/−), and we report a novel role for ZFP106 in motor and sensory neuronal maintenance and survival. Zfp106−/− mice develop severe motor abnormalities, major deficits in muscle strength and histopathological changes in muscle. Intriguingly, despite being highly expressed throughout the central nervous system, Zfp106−/− mice undergo selective motor and sensory neuronal and axonal degeneration specific to the spinal cord and peripheral nervous system. Neurodegeneration does not occur during development of Zfp106−/− mice, suggesting that ZFP106 is likely required for the maintenance of mature peripheral motor and sensory neurons. Analysis of embryonic Zfp106−/− motor neurons revealed deficits in mitochondrial function, with an inhibition of Complex I within the mitochondrial electron transport chain. Our results highlight a vital role for ZFP106 in sensory and motor neuron maintenance and reveal a novel player in mitochondrial dysfunction and neurodegeneration. PMID:26604141

  12. piragua encodes a zinc finger protein required for development in Drosophila.

    PubMed

    Nazario-Yepiz, Nestor O; Riesgo-Escovar, Juan R

    2016-12-21

    We isolated and characterized embryonic lethal mutations in piragua (prg). The prg locus encodes a protein with an amino terminus Zinc Finger-Associated-Domain (ZAD) and nine C2H2 zinc fingers (ZF). prg mRNA and protein expression during embryogenesis is dynamic with widespread maternal contribution, and subsequent expression in epithelial precursors. About a quarter of prg mutant embryos do not develop cuticle, and from those that do a small fraction have cuticular defects. Roughly half of prg mutants die during embryogenesis. prg mutants have an extended phenocritical period encompassing embryogenesis and first instar larval stage, since the other half of prg mutants die as first or second instar larvae. During dorsal closure, time-lapse high-resolution imaging shows defects arising out of sluggishness in closure, resolving at times in failures of closure. prg is expressed in imaginal discs, and is required for imaginal development. prg was identified in imaginal tissue in a cell super competition screen, together with other genes, like flower. We find that flower mutations are also embryonic lethal with a similar phenocritical period and strong embryonic mutant phenotypes (head involution defects, primarily). The two loci interact genetically in the embryo, as they increase embryonic mortality to close to 90% with the same embryonic phenotypes (dorsal closure and head involution defects, plus lack of cuticle). Mutant prg clones generated in developing dorsal thorax and eye imaginal tissue have strong developmental defects (lack of bristles and ommatidial malformations). prg is required in several developmental morphogenetic processes.

  13. The zinc finger transcription factor 191 is required for early embryonic development and cell proliferation

    SciTech Connect

    Li Jianzhong; Chen Xia; Yang Hua; Wang Shuiliang; Guo Baoyu; Yu Long; Wang Zhugang; Fu Jiliang . E-mail: fu825@mail.tongji.edu.cn

    2006-12-10

    Human zinc finger protein 191 (ZNF191/ZNF24) was cloned and characterized as a SCAN family member, which shows 94% identity to its mouse homologue zinc finger protein 191 (Zfp191). ZNF191 can specifically interact with an intronic polymorphic TCAT repeat (HUMTH01) in the tyrosine hydroxylase (TH) gene. Allelic variations of HUMTH01 have been stated to have a quantitative silencing effect on TH gene expression and to correlate with quantitative and qualitative changes in the binding by ZNF191. Zfp191 is widely expressed during embryonic development and in multiple tissues and organs in adult. To investigate the functions of Zfp191 in vivo, we have used homologous recombination to generate mice that are deficient in Zfp191. Heterozygous Zfp191 {sup +/-} mice are normal and fertile. Homozygous Zfp191 {sup -/-} embryos are severely retarded in development and die at approximately 7.5 days post-fertilization. Unexpectedly, in Zfp191 {sup -/-} and Zfp191 {sup +/-} embryos, TH gene expression is not affected. Blastocyst outgrowth experiments and the RNA interference-mediated knockdown of ZNF191 in cultured cells revealed an essential role for Zfp191 in cell proliferation. In further agreement with this function, no viable Zfp191 {sup -/-} cell lines were obtained by derivation of embryonic stem (ES) cells from blastocysts of Zfp191 {sup +/-} intercrosses or by forced homogenotization of heterozygous ES cells at high concentrations of G418. These data show that Zfp191 is indispensable for early embryonic development and cell proliferation.

  14. Deficiency of the zinc finger protein ZFP106 causes motor and sensory neurodegeneration.

    PubMed

    Joyce, Peter I; Fratta, Pietro; Landman, Allison S; Mcgoldrick, Philip; Wackerhage, Henning; Groves, Michael; Busam, Bharani Shiva; Galino, Jorge; Corrochano, Silvia; Beskina, Olga A; Esapa, Christopher; Ryder, Edward; Carter, Sarah; Stewart, Michelle; Codner, Gemma; Hilton, Helen; Teboul, Lydia; Tucker, Jennifer; Lionikas, Arimantas; Estabel, Jeanne; Ramirez-Solis, Ramiro; White, Jacqueline K; Brandner, Sebastian; Plagnol, Vincent; Bennet, David L H; Abramov, Andrey Y; Greensmith, Linda; Fisher, Elizabeth M C; Acevedo-Arozena, Abraham

    2016-01-15

    Zinc finger motifs are distributed amongst many eukaryotic protein families, directing nucleic acid-protein and protein-protein interactions. Zinc finger protein 106 (ZFP106) has previously been associated with roles in immune response, muscle differentiation, testes development and DNA damage, although little is known about its specific function. To further investigate the function of ZFP106, we performed an in-depth characterization of Zfp106 deficient mice (Zfp106(-/-)), and we report a novel role for ZFP106 in motor and sensory neuronal maintenance and survival. Zfp106(-/-) mice develop severe motor abnormalities, major deficits in muscle strength and histopathological changes in muscle. Intriguingly, despite being highly expressed throughout the central nervous system, Zfp106(-/-) mice undergo selective motor and sensory neuronal and axonal degeneration specific to the spinal cord and peripheral nervous system. Neurodegeneration does not occur during development of Zfp106(-/-) mice, suggesting that ZFP106 is likely required for the maintenance of mature peripheral motor and sensory neurons. Analysis of embryonic Zfp106(-/-) motor neurons revealed deficits in mitochondrial function, with an inhibition of Complex I within the mitochondrial electron transport chain. Our results highlight a vital role for ZFP106 in sensory and motor neuron maintenance and reveal a novel player in mitochondrial dysfunction and neurodegeneration.

  15. Requiem: a novel zinc finger gene essential for apoptosis in myeloid cells.

    PubMed

    Gabig, T G; Mantel, P L; Rosli, R; Crean, C D

    1994-11-25

    To identify genes mediating programmed cell death triggered by interleukin 3 (IL-3)-deprivation of myeloid cells, the IL-3-dependent murine myeloid cell line FDCP-1 was used to screen a mammalian cell expression library for cDNAs that would promote survival following withdrawal of IL-3. A unique 892-base pair cDNA was cloned that prevented the programmed cell death response following IL-3 deprivation by causing antisense suppression of an endogenous 2.4-kilobase (kb) mRNA. A 2.3-kb cDNA containing the identical 892-base pair over-lapping sequence was cloned that encoded a deduced 371-amino acid protein containing a single Kruppel-type zinc finger and a cluster of 4 cysteine/histidine-rich repeats resembling atypical zinc fingers. The 2.4-kb mRNA was found to be ubiquitously expressed in murine tissues and its abundance in FDCP-1 cells was not altered in response to IL-3 deprivation. Since expression of this 2.4-kb mRNA was a prerequisite for the apoptosis response following IL-3 deprivation, the gene encoding it was named requiem. Requiem is likely to encode a transcription factor required for the apoptosis response following survival factor withdrawal from myeloid cells.

  16. KRAB-Zinc Finger Proteins: A Repressor Family Displaying Multiple Biological Functions

    PubMed Central

    Lupo, Angelo; Cesaro, Elena; Montano, Giorgia; Zurlo, Diana; Izzo, Paola; Costanzo, Paola

    2013-01-01

    Zinc finger proteins containing the Kruppel associated box (KRAB-ZFPs) constitute the largest individual family of transcriptional repressors encoded by the genomes of higher organisms. KRAB domain, positioned at the NH2 terminus of the KRAB-ZFPs, interacts with a scaffold protein, KAP-1, which is able to recruit various transcriptional factors causing repression of genes to which KRAB ZFPs bind. The relevance of such repression is reflected in the large number of the KRAB zinc finger protein genes in the human genome. However, in spite of their numerical abundance little is currently known about the gene targets and the physiological functions of KRAB- ZFPs. However, emerging evidence links the transcriptional repression mediated by the KRAB-ZFPs to cell proliferation, differentiation, apoptosis and cancer. Moreover, the fact that KRAB containing proteins are vertebrate-specific suggests that they have evolved recently, and that their key roles lie in some aspects of vertebrate development. In this review, we will briefly discuss some regulatory functions of the KRAB-ZFPs in different physiological and pathological states, thus contributing to better understand their biological roles. PMID:24294107

  17. Simultaneous Screening and Validation of Effective Zinc Finger Nucleases in Yeast

    PubMed Central

    Wang, Ling; Lin, Juan; Zhang, Tingting; Xu, Kun; Ren, Chonghua; Zhang, Zhiying

    2013-01-01

    Zinc finger nucleases (ZFNs) have been successfully used for genome modification in various cell types and species. However, construction of an effective ZFN remained challenging. Previous studies all focused on obtaining specific zinc finger proteins (ZFPs) first via bacterial 2-hybrid approach, and then fusing selected ZFPs to FokI nuclease domain. These assembled ZFNs have high rate of failing to cleave target sites in vivo. In this study, we developed a simultaneous screening and validation system to obtain effective ZFNs directly in yeast AH109. This system is based on Gal4 reporter system carrying a unique intermediate reporter plasmid with two 30-bp Gal4 homology arms and a ZFN target site. DNA double strand breaks introduced on target sequence by ZFNs were repaired by single strand annealing (SSA) mechanism, and the restored Gal4 drove reporter genes expression. Taking the advantage of OPEN (Oligomerized Pool ENgineering) selection, we constructed 3 randomized ZFNs libraries and 9 reporter strains for each target gene. We tested this system by taking goat α s1-casein as target gene following three-step selection. Consequently, 3 efficient pairs of ZFNs were obtained from positive colonies on selective medium. The ZFNs achieved a 15.9% disruption frequency in goat mammary epithelial cells. In conclusion, we created a novel system to obtain effective ZFNs directly with simultaneous screening and validation. PMID:23741369

  18. Upregulation of long noncoding RNA zinc finger antisense 1 enhances epithelial-mesenchymal transition in vitro and predicts poor prognosis in glioma.

    PubMed

    Lv, Qiao-Li; Chen, Shu-Hui; Zhang, Xue; Sun, Bao; Hu, Lei; Qu, Qiang; Huang, Yuan-Tao; Wang, Gui-Hua; Liu, Yan-Ling; Zhang, Ying-Ying; Zhou, Hong-Hao

    2017-03-01

    Increasing evidence indicates that long noncoding RNAs play important roles in development and progression of various cancers. Zinc finger antisense 1 is a novel long noncoding RNA whose clinical significance, biological function, and underlying mechanism are still undetermined in glioma. In this study, we reported that zinc finger antisense 1 expression was markedly upregulated in glioma and tightly correlated with clinical stage. Moreover, patients with high zinc finger antisense 1 expression had shorter survival. Multivariate Cox regression analysis provided a clue that, probably, zinc finger antisense 1 level could serve as an independent prognostic factor for glioma. Functionally, zinc finger antisense 1 acted as an oncogene in glioma because its knockdown could promote apoptosis and significantly inhibit cell proliferation, migration, and invasion. Furthermore, zinc finger antisense 1 silencing could result in cell cycle arrest at the G0/G1 phase and correspondingly decrease the percentage of S phase cells in both U87 and U251 cell lines. Moreover, it was found that silenced zinc finger antisense 1 could impair migration and invasion by inhibiting the epithelial-mesenchymal transition through reducing the expression of MMP2, MMP9, N-cadherin, Integrin β1, ZEB1, Twist, and Snail as well as increasing E-cadherin level in glioma. Taken together, our data identified that zinc finger antisense 1 might act as a valuable prognostic biomarker and potential therapeutic target for glioma.

  19. Classification of the treble clef zinc finger: noteworthy lessons for structure and function evolution

    PubMed Central

    Kaur, Gurmeet; Subramanian, Srikrishna

    2016-01-01

    Treble clef (TC) zinc fingers constitute a large fold-group of structural zinc-binding protein domains that mediate numerous cellular functions. We have analysed the sequence, structure, and function relationships among all TCs in the Protein Data Bank. This led to the identification of novel TCs, such as lsr2, YggX and TFIIIC τ 60 kDa subunit, and prediction of a nuclease-like function for the DUF1364 family. The structural malleability of TCs is evident from the many examples with variations to the core structural elements of the fold. We observe domains wherein the structural core of the TC fold is circularly permuted, and also some examples where the overall fold resembles both the TC motif and another unrelated fold. All extant TC families do not share a monophyletic origin, as several TC proteins are known to have been present in the last universal common ancestor and the last eukaryotic common ancestor. We identify several TCs where the zinc-chelating site and residues are not merely responsible for structure stabilization but also perform other functions, such as being redox active in C1B domain of protein kinase C, a nucleophilic acceptor in Ada and catalytic in organomercurial lyase, MerB. PMID:27562564

  20. Classification of the treble clef zinc finger: noteworthy lessons for structure and function evolution

    NASA Astrophysics Data System (ADS)

    Kaur, Gurmeet; Subramanian, Srikrishna

    2016-08-01

    Treble clef (TC) zinc fingers constitute a large fold-group of structural zinc-binding protein domains that mediate numerous cellular functions. We have analysed the sequence, structure, and function relationships among all TCs in the Protein Data Bank. This led to the identification of novel TCs, such as lsr2, YggX and TFIIIC τ 60 kDa subunit, and prediction of a nuclease-like function for the DUF1364 family. The structural malleability of TCs is evident from the many examples with variations to the core structural elements of the fold. We observe domains wherein the structural core of the TC fold is circularly permuted, and also some examples where the overall fold resembles both the TC motif and another unrelated fold. All extant TC families do not share a monophyletic origin, as several TC proteins are known to have been present in the last universal common ancestor and the last eukaryotic common ancestor. We identify several TCs where the zinc-chelating site and residues are not merely responsible for structure stabilization but also perform other functions, such as being redox active in C1B domain of protein kinase C, a nucleophilic acceptor in Ada and catalytic in organomercurial lyase, MerB.

  1. Protein kinase C epsilon is localized to the Golgi via its zinc-finger domain and modulates Golgi function.

    PubMed Central

    Lehel, C; Olah, Z; Jakab, G; Anderson, W B

    1995-01-01

    Protein kinase C (PKC) is a multigene family of serine/threonine kinases that are central to many signal transduction pathways. Among the PKC isozymes, only PKC epsilon has been reported to exhibit full oncogenic potential. PKC epsilon also displays unique substrate specificity and intracellular localization. To examine the interrelationship between the biological effects and domain structure of PKC epsilon, NIH 3T3 cells were stably transfected to overexpress different epitope-tagged fragments of PKC epsilon. The overexpressed proteins each contain the epsilon-tag peptide at the C terminus to allow ready detection with an antibody specific for the tag. The holo-PKC epsilon was found to localize with the Golgi network and other compartments, whereas the zinc-finger domain localized exclusively at the Golgi. Golgi-specific glycosaminoglycan sulfation was strongly inhibited in cells overexpressing either holo-PKC epsilon or its zinc-finger domain, while the secretion of sulfated glycosaminoglycans into the medium was impaired in cells expressing the PKC epsilon zinc-finger domain. Thus, these results suggest that PKC epsilon may be involved in specifically regulating Golgi-related processes. Further, the results indicate that PKC epsilon domains other than the kinase domain may also have biological activity and that the zinc-finger domain may function as a subcellular localization signal. Images Fig. 1 Fig. 2 Fig. 3 PMID:7877991

  2. Mining the Brassica oleracea genome for Q-type C2H2 zinc finger transcription factor proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Q-type zinc finger proteins have been studied in several plant species and have been associated with response to stress. A whole genome analysis of Arabidopsis identified 176 putative C2H2 transcription factors (TF). Q-type C2H2 TFs containing the QALGGH motif and are a subset of these. In Arabidops...

  3. Domain analysis of the Nematostella vectensis SNAIL ortholog reveals unique nucleolar localization that depends on the zinc-finger domains.

    PubMed

    Dattoli, Ada A; Hink, Mark A; DuBuc, Timothy Q; Teunisse, Bram J; Goedhart, Joachim; Röttinger, Eric; Postma, Marten

    2015-07-20

    SNAIL transcriptional factors are key regulators during development and disease. They arose early during evolution, and in cnidarians such as Nematostella vectensis, NvSNAILA/B are detected in invaginating tissues during gastrulation. The function of SNAIL proteins is well established in bilaterians but their roles in cnidarians remain unknown. The structure of NvSNAILA and B is similar to the human SNAIL1 and 2, including SNAG and zinc-finger domains. Here, we performed a molecular analysis on localization and mobility of NvSNAILA/B using mammalian cells and Nematostella embryos. NvSNAILA/B display nuclear localization and mobility similar to HsSNAIL1/2. Strikingly, NvSNAILA is highly enriched in the nucleoli and shuttles between the nucleoli and the nucleoplasm. Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility. Truncation of the C-terminal zinc-fingers, involved in DNA binding in higher organisms, significantly affects subcellular localization and mobility. Specifically, the zinc-finger domains are required for nucleolar enrichment of NvSNAILA. Differently from SNAIL transcriptional factors described before, NvSNAILA is specifically enriched in the nucleoli co-localizing with nucleolar markers even after nucleolar disruption. Our findings implicate additional roles for SNAG and zinc-finger domains, suggesting a role for NvSNAILA in the nucleolus.

  4. Mutations in the zinc fingers of ADR1 that change the specificity of DNA binding and transactivation.

    PubMed Central

    Thukral, S K; Morrison, M L; Young, E T

    1992-01-01

    ADR1 is a yeast transcription factor that contains two zinc fingers of the Cys-2-His-2 (C2H2) class. Mutations that change the specificity of DNA binding of ADR1 to its target site, upstream activation sequence 1 (UAS1), have been identified at three positions in the first zinc finger. Mutations Arg-115 to Gln, His-118 to Thr, and Arg-121 to Asn led to new specificities of DNA binding at adjacent positions 10, 9, and 8 (3'-GAG-5') in UAS1. Arg-115 is at the finger tip, and His-118 and Arg-121 are at positions 3 and 6, respectively, in the alpha helix of finger 1. One double mutant displayed the binding specificity expected from the properties of its constituent new-specificity mutations. Mutations in the second finger that allowed its binding site to be identified through loss-of-contact phenotypes were made. These mutations imply a tail-to-tail orientation of the two ADR1 monomers on their adjacent binding sites. Finger 1 is aligned on UAS1 in an amino-to-carboxyl-terminal orientation along the guanine-rich strand in a 3'-to-5' direction. One of the ADR1 mutants was functional in vivo with both its cognate binding site and wild-type UAS1, but the other two mutants were defective in transactivation despite their ability to bind with high affinity to their cognate binding sites. Images PMID:1588970

  5. An improved zinc-finger nuclease architecture for highly specific genome editing.

    PubMed

    Miller, Jeffrey C; Holmes, Michael C; Wang, Jianbin; Guschin, Dmitry Y; Lee, Ya-Li; Rupniewski, Igor; Beausejour, Christian M; Waite, Adam J; Wang, Nathaniel S; Kim, Kenneth A; Gregory, Philip D; Pabo, Carl O; Rebar, Edward J

    2007-07-01

    Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.

  6. The prokaryotic zinc-finger: structure, function and comparison with the eukaryotic counterpart.

    PubMed

    Malgieri, Gaetano; Palmieri, Maddalena; Russo, Luigi; Fattorusso, Roberto; Pedone, Paolo V; Isernia, Carla

    2015-12-01

    Classical zinc finger (ZF) domains were thought to be confined to the eukaryotic kingdom until the transcriptional regulator Ros protein was identified in Agrobacterium tumefaciens. The Ros Cys2 His2 ZF binds DNA in a peculiar mode and folds in a domain significantly larger than its eukaryotic counterpart consisting of 58 amino acids (the 9-66 region) arranged in a βββαα topology, and stabilized by a conserved, extensive, 15-residue hydrophobic core. The prokaryotic ZF domain, then, shows some intriguing new features that make it interestingly different from its eukaryotic counterpart. This review will focus on the prokaryotic ZFs, summarizing and discussing differences and analogies with the eukaryotic domains and providing important insights into their structure/function relationships.

  7. Tandem zinc-finger gene families in mammals: insights and unanswered questions.

    PubMed

    Shannon, M; Kim, J; Ashworth, L; Branscomb, E; Stubbs, L

    1998-01-01

    Evidence for the remarkable conservation of mammalian genomes, in both content and organization of resident genes, is rapidly emerging from comparative mapping studies. The frequent occurrence of familial gene clustering, presumably reflecting a history of tandem in situ duplications starting from a single ancestral gene, is also apparent from these analyses. Genes encoding Kruppel-type zinc-finger (ZNF) proteins, including those containing Kruppel-associated box (KRAB) motifs, are particularly prone to such clustered organization. Existing data suggest that genes in KRAB-ZNF gene clusters have diverged in sequence and expression patterns, possibly yielding families of proteins with distinct, yet related, functions. Comparative mapping studies indicate that at least some of the genes within these clusters in mammals were elaborated prior to the divergence of mammalian orders and, subsequently, have been conserved. These data suggest a possible role for these tandem KRAB-ZNF gene families in mammalian evolution.

  8. Targeted disruption of exogenous EGFP gene in medaka using zinc-finger nucleases.

    PubMed

    Ansai, Satoshi; Ochiai, Hiroshi; Kanie, Yuta; Kamei, Yasuhiro; Gou, Yuki; Kitano, Takeshi; Yamamoto, Takashi; Kinoshita, Masato

    2012-06-01

    Zinc-finger nucleases (ZFNs) are artificial enzymes that create site-specific double-strand breaks and thereby induce targeted genome editing. Here, we demonstrated successful gene disruption in somatic and germ cells of medaka (Oryzias latipes) using ZFN to target exogenous EGFP genes. Embryos that were injected with an RNA sequence pair coding for ZFNs showed mosaic loss of green fluorescent protein fluorescence in skeletal muscle. A number of mutations that included both deletions and insertions were identified within the ZFN target site in each embryo, whereas no mutations were found at the non-targeted sites. In addition, ZFN-induced mutations were introduced in germ cells and efficiently transmitted to the next generation. The mutation frequency varied (6-100%) in the germ cells from each founder, and a founder carried more than two types of mutation in germ cells. Our results have introduced the possibility of targeted gene disruption and reverse genetics in medaka.

  9. Disruption of the myostatin gene in porcine primary fibroblasts and embryos using zinc-finger nucleases.

    PubMed

    Huang, Xian-Ju; Zhang, Hong-Xiao; Wang, Huili; Xiong, Kai; Qin, Ling; Liu, Honglin

    2014-04-01

    Myostatin represses muscle growth by negatively regulating the number and size of muscle fibers. Myostatin lossof- function can result in the double-muscling phenotype and increased muscle mass. Thus, knockout of myostatin gene could improve the quality of meat from mammals. In the present study, zinc finger nucleases, a useful tool for generating gene knockout animals, were designed to target exon 1 of the myostatin gene. The designed ZFNs were introduced into porcine primary fibroblasts and early implantation embryos via electroporation and microinjection, respectively. Mutations around the ZFNs target site were detected in both primary fibroblasts and blastocysts. The proportion of mutant fibroblast cells and blastocyst was 4.81% and 5.31%, respectively. Thus, ZFNs can be used to knockout myostatin in porcine primary fibroblasts and early implantation embryos.

  10. Identification and characterization of a zinc finger gene (ZNF213) from 16p13.3.

    PubMed

    Chen, X; Hamon, M; Deng, Z; Centola, M; Sood, R; Taylor, K; Kastner, D L; Fischel-Ghodsian, N

    1999-02-16

    During our search for the familial Mediterranean fever (FMF) gene, we identified by cDNA selection a 1.2 kb cDNA fragment representing a novel human gene that is expressed in a wide variety of tissues. This gene spans approx. 8.0 kb genomic DNA and has seven exons. Its 3' untranslated region contains a long tandem repeat that gives rise to a polymorphism with two alleles of approx. 1.1 kb and 1.0 kb, with the 1.1 kb allele in strong linkage disequilibrium with FMF in patients of different ethnic backgrounds. However, both genetic and mutational analyses have excluded this gene as the one responsible for FMF. The predicted 424 amino acid protein, designated ZNF213, contains three C2H2 zinc fingers, a Kruppel associated A box and a leucine rich motif (LeR domain/SCAN box), strongly suggestive of a transcription factor.

  11. Importance of long-time simulations for rare event sampling in zinc finger proteins.

    PubMed

    Godwin, Ryan; Gmeiner, William; Salsbury, Freddie R

    2016-01-01

    Molecular dynamics (MD) simulation methods have seen significant improvement since their inception in the late 1950s. Constraints of simulation size and duration that once impeded the field have lessened with the advent of better algorithms, faster processors, and parallel computing. With newer techniques and hardware available, MD simulations of more biologically relevant timescales can now sample a broader range of conformational and dynamical changes including rare events. One concern in the literature has been under which circumstances it is sufficient to perform many shorter timescale simulations and under which circumstances fewer longer simulations are necessary. Herein, our simulations of the zinc finger NEMO (2JVX) using multiple simulations of length 15, 30, 1000, and 3000 ns are analyzed to provide clarity on this point.

  12. Disruption of the Myostatin Gene in Porcine Primary Fibroblasts and Embryos Using Zinc-Finger Nucleases

    PubMed Central

    Huang, Xian-Ju; Zhang, Hong-Xiao; Wang, Huili; Xiong, Kai; Qin, Ling; Liu, Honglin

    2014-01-01

    Myostatin represses muscle growth by negatively regulating the number and size of muscle fibers. Myostatin loss-of-function can result in the double-muscling phenotype and increased muscle mass. Thus, knockout of myostatin gene could improve the quality of meat from mammals. In the present study, zinc finger nucleases, a useful tool for generating gene knockout animals, were designed to target exon 1 of the myostatin gene. The designed ZFNs were introduced into porcine primary fibroblasts and early implantation embryos via electroporation and microinjection, respectively. Mutations around the ZFNs target site were detected in both primary fibroblasts and blastocysts. The proportion of mutant fibroblast cells and blastocyst was 4.81% and 5.31%, respectively. Thus, ZFNs can be used to knockout myostatin in porcine primary fibroblasts and early implantation embryos. PMID:24802055

  13. Knockout of Myostatin by Zinc-finger Nuclease in Sheep Fibroblasts and Embryos

    PubMed Central

    Zhang, Xuemei; Wang, Liqin; Wu, Yangsheng; Li, Wenrong; An, Jing; Zhang, Fuchun; Liu, Mingjun

    2016-01-01

    Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause “double-muscling” trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN) which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos. PMID:27189642

  14. Nuclear translocation of {alpha}N-catenin by the novel zinc finger transcriptional repressor ZASC1

    SciTech Connect

    Bogaerts, Sven; Vanlandschoot, Ann; Hengel, Jolanda van; Roy, Frans van . E-mail: F.Vanroy@dmbr.UGent.be

    2005-11-15

    Alpha-catenins anchor the transmembrane cell-cell adhesion molecule E-cadherin indirectly to the actin cytoskeleton through interaction with {beta}-catenin or plakoglobin. Three different {alpha}-catenins are known at present: {alpha}E-, {alpha}T-, and {alpha}N-catenin. Despite their different expression patterns, no functional differences between the {alpha}-catenins are known. In a yeast two-hybrid screening with {alpha}N-catenin as bait, we identified the Cys{sub 2}-His{sub 2} zinc finger protein ZASC1. The mRNA and protein of ZASC1 were ubiquitously expressed in various cell lines and human tissues. Our results suggest an association of the ZASC1 protein with DNA, and luciferase reporter assays revealed that ZASC1 is a transcriptional repressor. Upon transient overexpression, the ZASC1 protein localized in the nucleus, to where it was able to recruit cytoplasmic {alpha}N-catenin. Neither the highly related {alpha}E-catenin nor {alpha}T-catenin interacted with ZASC1. By interchanging parts of {alpha}N-catenin and {alpha}E-catenin cDNAs, we were able to narrow down the interaction region of {alpha}N-catenin to two limited amino-terminal regions. On the other hand, the interaction of ZASC1 with {alpha}N-catenin can be mediated by the domain comprising zinc fingers six to eight of ZASC1. The interaction and nuclear cotranslocation of a neural {alpha}-catenin with a putative proto-oncogene product as reported here provides novel insights into the signaling functions of {alpha}-catenins.

  15. Predicting DNA recognition by Cys2His2 zinc finger proteins

    PubMed Central

    Persikov, Anton V.; Osada, Robert; Singh, Mona

    2009-01-01

    Motivation: Cys2His2 zinc finger (ZF) proteins represent the largest class of eukaryotic transcription factors. Their modular structure and well-conserved protein-DNA interface allow the development of computational approaches for predicting their DNA-binding preferences even when no binding sites are known for a particular protein. The ‘canonical model’ for ZF protein-DNA interaction consists of only four amino acid nucleotide contacts per zinc finger domain. Results: We present an approach for predicting ZF binding based on support vector machines (SVMs). While most previous computational approaches have been based solely on examples of known ZF protein–DNA interactions, ours additionally incorporates information about protein–DNA pairs known to bind weakly or not at all. Moreover, SVMs with a linear kernel can naturally incorporate constraints about the relative binding affinities of protein-DNA pairs; this type of information has not been used previously in predicting ZF protein-DNA binding. Here, we build a high-quality literature-derived experimental database of ZF–DNA binding examples and utilize it to test both linear and polynomial kernels for predicting ZF protein–DNA binding on the basis of the canonical binding model. The polynomial SVM outperforms previously published prediction procedures as well as the linear SVM. This may indicate the presence of dependencies between contacts in the canonical binding model and suggests that modification of the underlying structural model may result in further improved performance in predicting ZF protein–DNA binding. Overall, this work demonstrates that methods incorporating information about non-binding and relative binding of protein–DNA pairs have great potential for effective prediction of protein–DNA interactions. Availability: An online tool for predicting ZF DNA binding is available at http://compbio.cs.princeton.edu/zf/. Contact: mona@cs.princeton.edu Supplementary information

  16. Asymmetrical roles of zinc fingers in dynamic DNA-scanning process by the inducible transcription factor Egr-1.

    PubMed

    Zandarashvili, Levani; Vuzman, Dana; Esadze, Alexandre; Takayama, Yuki; Sahu, Debashish; Levy, Yaakov; Iwahara, Junji

    2012-06-26

    Egr-1 is an inducible transcription factor that recognizes 9-bp target DNA sites via three zinc finger domains and activates genes in response to cellular stimuli such as synaptic signals and vascular stresses. Using spectroscopic and computational approaches, we have studied structural, dynamic, and kinetic aspects of the DNA-scanning process in which Egr-1 is nonspecifically bound to DNA and perpetually changes its location on DNA. Our NMR data indicate that Egr-1 undergoes highly dynamic domain motions when scanning DNA. In particular, the zinc finger 1 (ZF1) of Egr-1 in the nonspecific complex is mainly dissociated from DNA and undergoes collective motions on a nanosecond timescale, whereas zinc fingers 2 and 3 (ZF2 and ZF3, respectively) are bound to DNA. This was totally unexpected because the previous crystallographic studies of the specific complex indicated that all of Egr-1's three zinc fingers are equally involved in binding to a target DNA site. Mutations that are expected to enhance ZF1's interactions with DNA and with ZF2 were found to reduce ZF1's domain motions in the nonspecific complex suggesting that these interactions dictate the dynamic behavior of ZF1. By experiment and computation, we have also investigated kinetics of Egr-1's translocation between two nonspecific DNA duplexes. Our data on the wild type and mutant proteins suggest that the domain dynamics facilitate Egr-1's intersegment transfer that involves transient bridging of two DNA sites. These results shed light on asymmetrical roles of the zinc finger domains for Egr-1 to scan DNA efficiently in the nucleus.

  17. The calculation of band gap energy in zinc oxide films

    NASA Astrophysics Data System (ADS)

    Arif, Ali; Belahssen, Okba; Gareh, Salim; Benramache, Said

    2015-01-01

    We investigated the optical properties of undoped zinc oxide thin films as the n-type semiconductor; the thin films were deposited at different precursor molarities by ultrasonic spray and spray pyrolysis techniques. The thin films were deposited at different substrate temperatures ranging between 200 and 500 °C. In this paper, we present a new approach to control the optical gap energy of ZnO thin films by concentration of the ZnO solution and substrate temperatures from experimental data, which were published in international journals. The model proposed to calculate the band gap energy with the Urbach energy was investigated. The relation between the experimental data and theoretical calculation suggests that the band gap energies are predominantly estimated by the Urbach energies, film transparency, and concentration of the ZnO solution and substrate temperatures. The measurements by these proposal models are in qualitative agreements with the experimental data; the correlation coefficient values were varied in the range 0.96-0.99999, indicating high quality representation of data based on Equation (2), so that the relative errors of all calculation are smaller than 4%. Thus, one can suppose that the undoped ZnO thin films are chemically purer and have many fewer defects and less disorder owing to an almost complete chemical decomposition and contained higher optical band gap energy.

  18. Revisiting and re-engineering the classical zinc finger peptide: consensus peptide-1 (CP-1).

    PubMed

    Besold, Angelique N; Widger, Leland R; Namuswe, Frances; Michalek, Jamie L; Michel, Sarah L J; Goldberg, David P

    2016-04-01

    Zinc plays key structural and catalytic roles in biology. Structural zinc sites are often referred to as zinc finger (ZF) sites, and the classical ZF contains a Cys2His2 motif that is involved in coordinating Zn(II). An optimized Cys2His2 ZF, named consensus peptide 1 (CP-1), was identified more than 20 years ago using a limited set of sequenced proteins. We have reexamined the CP-1 sequence, using our current, much larger database of sequenced proteins that have been identified from high-throughput sequencing methods, and found the sequence to be largely unchanged. The CCHH ligand set of CP-1 was then altered to a CAHH motif to impart hydrolytic activity. This ligand set mimics the His2Cys ligand set of peptide deformylase (PDF), a hydrolytically active M(II)-centered (M = Zn or Fe) protein. The resultant peptide [CP-1(CAHH)] was evaluated for its ability to coordinate Zn(II) and Co(II) ions, adopt secondary structure, and promote hydrolysis. CP-1(CAHH) was found to coordinate Co(II) and Zn(II) and a pentacoordinate geometry for Co(II)-CP-1(CAHH) was implicated from UV-vis data. This suggests a His2Cys(H2O)2 environment at the metal center. The Zn(II)-bound CP-1(CAHH) was shown to adopt partial secondary structure by 1-D (1)H NMR spectroscopy. Both Zn(II)-CP-1(CAHH) and Co(II)-CP-1(CAHH) show good hydrolytic activity toward the test substrate 4-nitrophenyl acetate, exhibiting faster rates than most active synthetic Zn(II) complexes.

  19. SCAN domain-containing 2 gene (SCAND2) is a novel nuclear protein derived from the zinc finger family by exon shuffling.

    PubMed

    Dupuy, Denis; Dupérat, Véronique Guyonnet; Arveiler, Benoît

    2002-05-01

    The SCAN domain is a recently recognized protein domain that characterizes a subfamily of the Krüppel-like zinc finger proteins. We have previously described a novel SCAN domain-containing 2 gene (SCAND2) that does not belong to the zinc finger family. We report structural and sequence analyzes of all known members of the SCAN family and use these data to illustrate a model of gene family evolution. Most of the SCAN containing genes share common gene organization features that support the proposed origin for SCAND2 by disruption of an ancestral SCAN-zinc finger gene by a retroposition event and subsequent exon shuffling.

  20. A novel Zinc finger protein, ZCCHC11, interacts with TIFA and modulates TLR signaling.

    PubMed

    Minoda, Yasumasa; Saeki, Kazuko; Aki, Daisuke; Takaki, Hiromi; Sanada, Takahito; Koga, Keiko; Kobayashi, Takashi; Takaesu, Giichi; Yoshimura, Akihiko

    2006-06-09

    Toll-like receptors (TLRs) play an important role as a sensor of microbial pathogens in the innate immune response. TLRs transmit signals through the recruitment of adaptor proteins including tumor necrosis factor-associated factor 6 (TRAF6), which mediates the activation of IkappaB kinase (IKK). TIFA (TRAF-interacting protein with a forkhead-associated (FHA) domain) has been shown to bind to TRAF6 and activate IKK by promoting the oligomerization and ubiquitin-ligase activity of TRAF6. FHA domains preferentially bind to phospho-threonine residues in their targets. Here, we identified a novel zinc finger protein, ZCCHC11, that interacts with TIFA from phosphoproteins of a macrophage cell line, RAW 264.7, by using affinity purification with GST-TIFA and mass spectrometric analysis. By a search of the EST database, we found a 200kDa full-length form (ZCCHC11L). ZCCHC11L was mostly located to the nucleus, but translocated into the cytoplasm in response to LPS and bound to TIFA. Overexpression and knockdown by siRNA indicated that ZCCHC11 functions as a negative regulator of TLR-mediated NF-kappaB activation. The N-terminal region (ZCCHC11S) including C2H2-type [corrected] Zn-finger motif was sufficient for suppression of NF-kappaB. We propose that ZCCHC11 is a unique TLR signal regulator, which interacts with TIFA after LPS treatment and suppresses the TRAF6-dependent activation of NF-kappaB.

  1. Functional and structural basis of the nuclear localization signal in the ZIC3 zinc finger domain

    PubMed Central

    Hatayama, Minoru; Tomizawa, Tadashi; Sakai-Kato, Kumiko; Bouvagnet, Patrice; Kose, Shingo; Imamoto, Naoko; Yokoyama, Shigeyuki; Utsunomiya-Tate, Naoko; Mikoshiba, Katsuhiko; Kigawa, Takanori; Aruga, Jun

    2008-01-01

    Disruptions in ZIC3 cause heterotaxy, a congenital anomaly of the left–right axis. ZIC3 encodes a nuclear protein with a zinc finger (ZF) domain that contains five tandem C2H2 ZF motifs. Missense mutations in the first ZF motif (ZF1) result in defective nuclear localization, which may underlie the pathogenesis of heterotaxy. Here we revealed the structural and functional basis of the nuclear localization signal (NLS) of ZIC3 and investigated its relationship to the defect caused by ZF1 mutation. The ZIC3 NLS was located in the ZF2 and ZF3 regions, rather than ZF1. Several basic residues interspersed throughout these regions were responsible for the nuclear localization, but R320, K337 and R350 were particularly important. NMR structure analysis revealed that ZF1–4 had a similar structure to GLI ZF, and the basic side chains of the NLS clustered together in two regions on the protein surface, similar to classical bipartite NLSs. Among the residues for the ZF1 mutations, C253 and H286 were positioned for the metal chelation, whereas W255 was positioned in the hydrophobic core formed by ZF1 and ZF2. Tryptophan 255 was a highly conserved inter-finger connector and formed part of a structural motif (tandem CXW-C-H-H) that is shared with GLI, Glis and some fungal ZF proteins. Furthermore, we found that knockdown of Karyopherin α1/α6 impaired ZIC3 nuclear localization, and physical interactions between the NLS and the nuclear import adapter proteins were disturbed by mutations in the NLS but not by W255G. These results indicate that ZIC3 is imported into the cell nucleus by the Karyopherin (Importin) system and that the impaired nuclear localization by the ZF1 mutation is not due to a direct influence on the NLS. PMID:18716025

  2. Embryonic neural inducing factor churchill is not a DNA-binding zinc finger protein: solution structure reveals a solvent-exposed beta-sheet and zinc binuclear cluster.

    PubMed

    Lee, Brian M; Buck-Koehntop, Bethany A; Martinez-Yamout, Maria A; Dyson, H Jane; Wright, Peter E

    2007-08-31

    Churchill is a zinc-containing protein that is involved in neural induction during embryogenesis. At the time of its discovery, it was thought on the basis of sequence alignment to contain two zinc fingers of the C4 type. Further, binding of an N-terminal GST-Churchill fusion protein to a particular DNA sequence was demonstrated by immunoprecipitation selection assay, suggesting that Churchill may function as a transcriptional regulator by sequence-specific DNA binding. We show by NMR solution structure determination that, far from containing canonical C4 zinc fingers, the protein contains three bound zinc ions in novel coordination sites, including an unusual binuclear zinc cluster. The secondary structure of Churchill is also unusual, consisting of a highly solvent-exposed single-layer beta-sheet. Hydrogen-deuterium exchange and backbone relaxation measurements reveal that Churchill is unusually dynamic on a number of time scales, with the exception of regions surrounding the zinc coordinating sites, which serve to stabilize the otherwise unstructured N terminus and the single-layer beta-sheet. No binding of Churchill to the previously identified DNA sequence could be detected, and extensive searches using DNA sequence selection techniques could find no other DNA sequence that was bound by Churchill. Since the N-terminal amino acids of Churchill form part of the zinc-binding motif, the addition of a fusion protein at the N terminus causes loss of zinc and unfolding of Churchill. This observation most likely explains the published DNA-binding results, which would arise due to non-specific interaction of the unfolded protein in the immunoprecipitation selection assay. Since Churchill does not appear to bind DNA, we suggest that it may function in embryogenesis as a protein-interaction factor.

  3. Zinc finger protein 804A (ZNF804A) and verbal deficits in individuals with autism

    PubMed Central

    Anitha, Ayyappan; Thanseem, Ismail; Nakamura, Kazuhiko; Vasu, Mahesh M.; Yamada, Kazuo; Ueki, Takatoshi; Iwayama, Yoshimi; Toyota, Tomoko; Tsuchiya, Kenji J.; Iwata, Yasuhide; Suzuki, Katsuaki; Sugiyama, Toshiro; Tsujii, Masatsugu; Yoshikawa, Takeo; Mori, Norio

    2014-01-01

    Background In a genome-wide association study of autism, zinc finger protein 804A (ZNF804A) single nucleotide polymorphisms (SNPs) were found to be nominally associated in verbally deficient individuals with autism. Zinc finger protein 804A copy number variations (CNVs) have also been observed in individuals with autism. In addition, ZNF804A is known to be involved in theory of mind (ToM) tasks, and ToM deficits are deemed responsible for the communication and social challenges faced by individuals with autism. We hypothesized that ZNF804A could be a risk gene for autism. Methods We examined the genetic association and CNVs of ZNF804A in 841 families in which 1 or more members had autism. We compared the expression of ZNF804A in the postmortem brains of individuals with autism (n = 8) and controls (n = 13). We also assessed in vitro the effect of ZNF804A silencing on the expression of several genes known to be involved in verbal efficiency and social cognition. Results We found that rs7603001 was nominally associated with autism (p = 0.018). The association was stronger (p = 0.008) in the families of individuals with autism who were verbally deficient (n = 761 families). We observed ZNF804A CNVs in 7 verbally deficient boys with autism. In ZNF804A knockdown cells, the expression of synaptosomal-associated protein, 25kDa (SNAP25) was reduced compared with controls (p = 0.009). The expression of ZNF804A (p = 0.009) and SNAP25 (p = 0.009) were reduced in the anterior cingulate gyrus (ACG) of individuals with autism. There was a strong positive correlation between the expression of ZNF804A and SNAP25 in the ACG (p < 0.001). Limitations Study limitations include our small sample size of postmortem brains. Conclusion Our results suggest that ZNF804A could be a potential candidate gene mediating the intermediate phenotypes associated with verbal traits in individuals with autism. PMID:24866414

  4. Rmt1 catalyzes zinc-finger independent arginine methylation of the small ribosomal protein Rps2 in Saccharomyces cerevisiae

    PubMed Central

    Lipson, Rebecca S.; Webb, Kristofor J.; Clarke, Steven G.

    2010-01-01

    Rps2/rpS2 is a well conserved protein of the eukaryotic ribosomal small subunit. Rps2 has previously been shown to contain asymmetric dimethylarginine residues, the addition of which is catalyzed by zinc-finger-containing arginine methyltransferase 3 (Rmt3) in the fission yeast Schizosaccharomyces pombe and protein arginine methyltransferase 3 (PRMT3) in mammalian cells. Here we demonstrate that despite the lack of a zinc-finger-containing homolog of Rmt3/PRMT3 in the budding yeast Saccharomyces cerevisiae, Rps2 is partially modified to generate asymmetric dimethylarginine and monomethylarginine residues. We find that this modification of Rps2 is dependent upon the major arginine methyltransferase 1 (Rmt1) in S. cerevisiae. These results are suggestive of a role for Rmt1 in modifying the function of Rps2 in a manner distinct from that occurring in S. pombe and mammalian cells. PMID:20035717

  5. Transcriptional regulation of the MHC class I HLA-A11 promoter by the zinc finger protein ZFX.

    PubMed Central

    L'Haridon, M; Paul, P; Xerri, J G; Dastot, H; Dolliger, C; Schmid, M; de Angelis, N; Grollet, L; Sigaux, F; Degos, L; Gazin, C

    1996-01-01

    Regulation of the human MHC class I HLA-A11 promoter is governed by a complex array of regulatory elements. One of these elements, shown here to be critical for the transcriptional activity of the promoter, was used to screen a lambda gt11 library and allowed the identification of a cDNA which coded for the zinc finger protein ZFX. ZFX was shown to bind the sequences AGGGCCCCA and AGGCCCCGA, located respectively at positions -271 to -263 and -242 to -234 of the HLA-A11 promoter, with similar affinities through its three C-terminal zinc fingers. ZFX575, a short isoform of ZFX, activates transcription from the HLA-All promoter in a Leydig cell line. PMID:8657576

  6. A DNA-binding protein containing two widely separated zinc finger motifs that recognize the same DNA sequence.

    PubMed

    Fan, C M; Maniatis, T

    1990-01-01

    We have isolated a full-length cDNA clone encoding a protein (PRDII-BF1) that binds specifically to a positive regulatory domain (PRDII) of the human IFN-beta gene promoter, and to a similar sequence present in a number of other promoters and enhancers. The sequence of this protein reveals two novel structural features. First, it is the largest sequence-specific DNA-binding protein reported to date (298 kD). Second, it contains two widely separated sets of C2-H2-type zinc fingers. Remarkably, each set of zinc fingers binds to the same DNA sequence motif with similar affinities and methylation interference patterns. Thus, this protein may act by binding simultaneously to reiterated copies of the same recognition sequence. Although the function of PRDII-BF1 is not known, the level of its mRNA is inducible by serum and virus, albeit with different kinetics.

  7. Rmt1 catalyzes zinc-finger independent arginine methylation of ribosomal protein Rps2 in Saccharomyces cerevisiae

    SciTech Connect

    Lipson, Rebecca S.; Webb, Kristofor J.; Clarke, Steven G.

    2010-01-22

    Rps2/rpS2 is a well conserved protein of the eukaryotic ribosomal small subunit. Rps2 has previously been shown to contain asymmetric dimethylarginine residues, the addition of which is catalyzed by zinc-finger-containing arginine methyltransferase 3 (Rmt3) in the fission yeast Schizosaccharomyces pombe and protein arginine methyltransferase 3 (PRMT3) in mammalian cells. Here, we demonstrate that despite the lack of a zinc-finger-containing homolog of Rmt3/PRMT3 in the budding yeast Saccharomyces cerevisiae, Rps2 is partially modified to generate asymmetric dimethylarginine and monomethylarginine residues. We find that this modification of Rps2 is dependent upon the major arginine methyltransferase 1 (Rmt1) in S. cerevisiae. These results are suggestive of a role for Rmt1 in modifying the function of Rps2 in a manner distinct from that occurring in S. pombe and mammalian cells.

  8. Diagnostic Potential of Zinc Finger Protein-Specific Autoantibodies and Associated Linear B-Cell Epitopes in Colorectal Cancer

    PubMed Central

    O’Reilly, Julie-Ann; Fitzgerald, Jenny; Fitzgerald, Seán; Kenny, Dermot; Kay, Elaine W.; O’Kennedy, Richard; Kijanka, Gregor S.

    2015-01-01

    Colorectal cancer is one of the most common cancers worldwide with almost 700,000 deaths every year. Detection of colorectal cancer at an early stage significantly improves patient survival. Cancer-specific autoantibodies found in sera of cancer patients can be used for pre-symptomatic detection of the disease. In this study we assess the zinc finger proteins ZNF346, ZNF638, ZNF700 and ZNF768 as capture antigens for the detection of autoantibodies in colorectal cancer. Sera from 96 patients with colorectal cancer and 35 control patients with no evidence of cancer on colonoscopy were analysed for the presence of ZNF-specific autoantibodies using an indirect ELISA. Autoantibodies to individual ZNF proteins were detected in 10–20% of colorectal cancer patients and in 0–5.7% of controls. A panel of all four ZNF proteins resulted in an assay specificity of 91.4% and sensitivity of 41.7% for the detection of cancer patients in a cohort of non-cancer controls and colorectal cancer patients. Clinicopathological and survival analysis revealed that ZNF autoantibodies were independent of disease stage and did not correlate with disease outcome. Since ZNF autoantibodies were shared between patients and corresponding ZNF proteins showed similarities in their zinc finger motifs, we performed an in silico epitope sequence analysis. Zinc finger proteins ZNF700 and ZNF768 showed the highest sequence similarity with a bl2seq score of 262 (E-value 1E-81) and their classical C2H2 ZNF motifs were identified as potential epitopes contributing to their elevated immunogenic potential. Our findings show an enhanced and specific immunogenicity to zinc finger proteins, thereby providing a multiplexed autoantibody assay for minimally invasive detection of colorectal cancer. PMID:25875936

  9. Dynamics of linker residues modulate the nucleic acid binding properties of the HIV-1 nucleocapsid protein zinc fingers.

    PubMed

    Zargarian, Loussiné; Tisné, Carine; Barraud, Pierre; Xu, Xiaoqian; Morellet, Nelly; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

    2014-01-01

    The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity.

  10. p53 Gene Repair with Zinc Finger Nucleases Optimised by Yeast 1-Hybrid and Validated by Solexa Sequencing

    PubMed Central

    Herrmann, Frank; Garriga-Canut, Mireia; Baumstark, Rebecca; Fajardo-Sanchez, Emmanuel; Cotterell, James; Minoche, André; Himmelbauer, Heinz; Isalan, Mark

    2011-01-01

    The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wild-type (wt) p53 function have been investigated. In this study, we applied an approach using gene repair with zinc finger nucleases (ZFNs). We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries. We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation ‘hotspots’. The ZFNs were first validated using in vitro cleavage assays and in vivo episomal gene repair assays in HEK293T cells. Subsequently, the ZFNs were used to restore wt-p53 status in the SF268 human cancer cell line, via ZFN-induced homologous recombination. The frequency of gene repair and mutation by non-homologous end-joining was then ascertained in several cancer cell lines, using a deep sequencing strategy. Our Y1H system facilitates the generation and optimisation of novel, sequence-specific four- to six-finger peptides, and the p53-specific ZFN described here can be used to mutate or repair p53 in genomic loci. PMID:21695267

  11. Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases.

    PubMed

    Watanabe, Takahito; Ochiai, Hiroshi; Sakuma, Tetsushi; Horch, Hadley W; Hamaguchi, Naoya; Nakamura, Taro; Bando, Tetsuya; Ohuchi, Hideyo; Yamamoto, Takashi; Noji, Sumihare; Mito, Taro

    2012-01-01

    Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy.

  12. MZF6D, a novel KRAB zinc-finger gene expressed exclusively in meiotic male germ cells.

    PubMed

    Looman, Camilla; Mark, Charlotta; Abrink, Magnus; Hellman, Lars

    2003-08-01

    Spermatogenesis takes place in the seminiferous tubule in the testes and culminates in the production of spermatozoa (male gametes). Here we report the identification of a novel mouse zinc-finger gene, MZF6D, which is selectively expressed in meiotic spermatocytes. The MZF6D protein contains an N-terminally located repressor domain, a KRAB domain, followed by at least seven successive Krüppel zinc-finger motifs. The KRAB domain of MZF6D, which consists of a KRAB A box and the newly identified KRAB C box, has previously been shown to interact with TIF1beta, which is the common corepressor of all KRAB zinc-finger proteins. Northern blot analysis shows that the expression of MZF6D is restricted to testes. This was confirmed by RT-PCR analysis of a panel of mouse tissues. In situ hybridization of sections from adult mouse testes localizes the expression to meiotic spermatocytes, suggesting a specific role for MZF6D in the regulation of spermatogenesis.

  13. Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases

    PubMed Central

    Watanabe, Takahito; Ochiai, Hiroshi; Sakuma, Tetsushi; Horch, Hadley W.; Hamaguchi, Naoya; Nakamura, Taro; Bando, Tetsuya; Ohuchi, Hideyo; Yamamoto, Takashi; Noji, Sumihare; Mito, Taro

    2012-01-01

    Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy. PMID:22910363

  14. Zinc-finger nickase-mediated insertion of the lysostaphin gene into the beta-casein locus in cloned cows.

    PubMed

    Liu, Xu; Wang, Yongsheng; Guo, Wenjiang; Chang, Bohao; Liu, Jun; Guo, Zekun; Quan, Fusheng; Zhang, Yong

    2013-01-01

    Zinc-finger nickases (ZFNickases) are a type of programmable nuclease that can be engineered from zinc-finger nucleases to induce site-specific single-strand breaks or nicks in genomic DNA, which result in homology-directed repair. Although zinc-finger nuclease-mediated gene disruption has been demonstrated in pigs and cattle, they have not been used to target gene addition into an endogenous gene locus in any large domestic species. Here we show in bovine fetal fibroblasts that targeting ZFNickases to the endogenous β-casein (CSN2) locus stimulates lysostaphin gene addition by homology-directed repair. We find that ZFNickase-treated cells can be successfully used in somatic cell nuclear transfer, resulting in live-born gene-targeted cows. Furthermore, the gene-targeted cows secrete lysostaphin in their milk and in vitro assays demonstrate the milk's ability to kill Staphylococcus aureus. Our success with this strategy will facilitate new transgenic technologies beneficial to both agriculture and biomedicine.

  15. Activation of transcriptional activities of AP-1 and SRE by a new zinc-finger protein ZNF641

    SciTech Connect

    Qi Xingzhu; Li Yongqing; Xiao Jing; Yuan Wuzhou; Yan Yan; Wang Yuequn; Liang Shuyuan; Zhu Chuanbing; Chen Yingduan; Liu Mingyao . E-mail: mliu@ibt.tamhsc.edu; Wu Xiushan

    2006-01-27

    Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes in cell signal transduction connecting cell-surface receptors to critical regulatory targets within cells and control cell survival, adaptation, and proliferation. Previous studies revealed that zinc-finger proteins are involved in the regulation of the MAPK signaling pathways. Here, we report the identification and characterization of a novel human zinc-finger protein, ZNF641. The cDNA of ZNF641 is 4.9 kb, encoding 438 amino acids in the nucleus. The protein is highly conserved in evolution across different vertebrate species from mouse to human. Northern blot analysis indicates that ZNF641 is expressed in most of the examined human tissues, with a high level in skeletal muscle. Overexpression of pCMV-Tag2B-ZNF641 in the COS-7 cells activates the transcriptional activities of AP-1 and SRE. Deletion analysis indicates that the linker between KRAB box and C{sub 2}H{sub 2}-type zinc-fingers represents the basal activation domain. These results suggest that ZNF641 may be a positive regulator in MAPK-mediated signaling pathways that lead to the activation of AP-1 and SRE.

  16. The C2H2 zinc finger genes of Strongylocentrotus purpuratus and their expression in embryonic development.

    PubMed

    Materna, Stefan C; Howard-Ashby, Meredith; Gray, Rachel F; Davidson, Eric H

    2006-12-01

    The C2H2 zinc finger is one of the most abundant protein domains and is thought to have been extensively replicated in diverse animal clades. Some well-studied proteins that contain this domain are transcriptional regulators. As part of an attempt to delineate all transcription factors encoded in the Strongylocentrotus purpuratus genome, we identified the C2H2 zinc finger genes indicated in the sequence, and examined their involvement in embryonic development. We found 377 zinc finger genes in the sea urchin genome, about half the number found in mice or humans. Their expression was measured by quantitative PCR. Up to the end of gastrulation less than a third of these genes is expressed, and about 75% of the expressed genes are maternal; both parameters distinguish these from all other classes of regulatory genes as measured in other studies. Spatial expression pattern was determined by whole mount in situ hybridization for 43 genes transcribed at a sufficient level, and localized expression was observed in diverse embryonic tissues. These genes may execute important regulatory functions in development. However, the functional meaning of the majority of this large gene family remains undefined.

  17. The DNA-Binding Domain of Human PARP-1 Interacts with DNA Single-Strand Breaks as a Monomer through Its Second Zinc Finger

    PubMed Central

    Eustermann, Sebastian; Videler, Hortense; Yang, Ji-Chun; Cole, Paul T.; Gruszka, Dominika; Veprintsev, Dmitry; Neuhaus, David

    2011-01-01

    Poly(ADP-ribose)polymerase-1 (PARP-1) is a highly abundant chromatin-associated enzyme present in all higher eukaryotic cell nuclei, where it plays key roles in the maintenance of genomic integrity, chromatin remodeling and transcriptional control. It binds to DNA single- and double-strand breaks through an N-terminal region containing two zinc fingers, F1 and F2, following which its C-terminal catalytic domain becomes activated via an unknown mechanism, causing formation and addition of polyadenosine-ribose (PAR) to acceptor proteins including PARP-1 itself. Here, we report a biophysical and structural characterization of the F1 and F2 fingers of human PARP-1, both as independent fragments and in the context of the 24-kDa DNA-binding domain (F1 + F2). We show that the fingers are structurally independent in the absence of DNA and share a highly similar structural fold and dynamics. The F1 + F2 fragment recognizes DNA single-strand breaks as a monomer and in a single orientation. Using a combination of NMR spectroscopy and other biophysical techniques, we show that recognition is primarily achieved by F2, which binds the DNA in an essentially identical manner whether present in isolation or in the two-finger fragment. F2 interacts much more strongly with nicked or gapped DNA ligands than does F1, and we present a mutational study that suggests origins of this difference. Our data suggest that different DNA lesions are recognized by the DNA-binding domain of PARP-1 in a highly similar conformation, helping to rationalize how the full-length protein participates in multiple steps of DNA single-strand breakage and base excision repair. PMID:21262234

  18. Heritable Targeted Gene Disruption in Zebrafish Using Designed Zinc Finger Nucleases

    PubMed Central

    Doyon, Yannick; McCammon, Jasmine M; Miller, Jeffrey C; Faraji, Farhoud; Ngo, Catherine; Katibah, George E; Amora, Rainier; Hocking, Toby D; Zhang, Lei; Rebar, Edward J; Gregory, Philip D; Urnov, Fyodor D; Amacher, Sharon L

    2009-01-01

    We describe here the use of zinc finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), where targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury genes. In both cases, injection of ZFN-encoding mRNA into 1-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Disrupted ntl alleles were transmitted from ZFN mRNA-injected founder animals in over half the adults tested at frequencies averaging 20%. The frequency and precision of gene disruption events observed, in combination with the ability to design ZFNs against any locus, open fundamentally novel avenues of experimentation, and suggest that ZFN technology may be widely applied to many organisms that allow mRNA delivery into the fertilized egg. PMID:18500334

  19. Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases.

    PubMed

    Cai, Yujia; Bak, Rasmus O; Mikkelsen, Jacob Giehm

    2014-04-24

    Future therapeutic use of engineered site-directed nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), relies on safe and effective means of delivering nucleases to cells. In this study, we adapt lentiviral vectors as carriers of designer nuclease proteins, providing efficient targeted gene disruption in vector-treated cell lines and primary cells. By co-packaging pairs of ZFN proteins with donor RNA in 'all-in-one' lentiviral particles, we co-deliver ZFN proteins and the donor template for homology-directed repair leading to targeted DNA insertion and gene correction. Comparative studies of ZFN activity in a predetermined target locus and a known nearby off-target locus demonstrate reduced off-target activity after ZFN protein transduction relative to conventional delivery approaches. Additionally, TALEN proteins are added to the repertoire of custom-designed nucleases that can be delivered by protein transduction. Altogether, our findings generate a new platform for genome engineering based on efficient and potentially safer delivery of programmable nucleases.DOI: http://dx.doi.org/10.7554/eLife.01911.001.

  20. Multiparameter functional diversity of human C2H2 zinc finger proteins

    PubMed Central

    Schmitges, Frank W.; Radovani, Ernest; Najafabadi, Hamed S.; Barazandeh, Marjan; Campitelli, Laura F.; Yin, Yimeng; Jolma, Arttu; Zhong, Guoqing; Guo, Hongbo; Kanagalingam, Tharsan; Dai, Wei F.; Taipale, Jussi; Emili, Andrew; Greenblatt, Jack F.; Hughes, Timothy R.

    2016-01-01

    C2H2 zinc finger proteins represent the largest and most enigmatic class of human transcription factors. Their C2H2-ZF arrays are highly variable, indicating that most will have unique DNA binding motifs. However, most of the binding motifs have not been directly determined. In addition, little is known about whether or how these proteins regulate transcription. Most of the ∼700 human C2H2-ZF proteins also contain at least one KRAB, SCAN, BTB, or SET domain, suggesting that they may have common interacting partners and/or effector functions. Here, we report a multifaceted functional analysis of 131 human C2H2-ZF proteins, encompassing DNA binding sites, interacting proteins, and transcriptional response to genetic perturbation. We confirm the expected diversity in DNA binding motifs and genomic binding sites, and provide motif models for 78 previously uncharacterized C2H2-ZF proteins, most of which are unique. Surprisingly, the diversity in protein–protein interactions is nearly as high as diversity in DNA binding motifs: Most C2H2-ZF proteins interact with a unique spectrum of co-activators and co-repressors. Thus, multiparameter diversification likely underlies the evolutionary success of this large class of human proteins. PMID:27852650

  1. Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases

    PubMed Central

    Merlin, Christine; Beaver, Lauren E.; Taylor, Orley R.; Wolfe, Scot A.; Reppert, Steven M.

    2013-01-01

    The development of reverse-genetic tools in “nonmodel” insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases that generate mutations at targeted genomic sequences. We focused our ZFN approach on targeting the type 2 vertebrate-like cryptochrome gene of the monarch (designated cry2), which encodes a putative transcriptional repressor of the monarch circadian clockwork. Co-injections of mRNAs encoding ZFNs targeting the second exon of monarch cry2 into “one nucleus” stage embryos led to high-frequency nonhomologous end-joining-mediated, mutagenic lesions in the germline (up to 50%). Heritable ZFN-induced lesions in two independent lines produced truncated, nonfunctional CRY2 proteins, resulting in the in vivo disruption of circadian behavior and the molecular clock mechanism. Our work genetically defines CRY2 as an essential transcriptional repressor of the monarch circadian clock and provides a proof of concept for the use of ZFNs for manipulating genes in the monarch butterfly genome. Importantly, this approach could be used in other lepidopterans and “nonmodel” insects, thus opening new avenues to decipher the molecular underpinnings of a variety of biological processes. PMID:23009861

  2. Zinc-finger Nucleases as a Novel Therapeutic Strategy for Targeting Hepatitis B Virus DNAs

    PubMed Central

    Cradick, Thomas J; Keck, Kathy; Bradshaw, Shannon; Jamieson, Andrew C; McCaffrey, Anton P

    2010-01-01

    Hepatitis B virus (HBV) chronically infects 350–400 million people worldwide and causes >1 million deaths yearly. Current therapies prevent new viral genome formation, but do not target pre-existing viral genomic DNA, thus curing only ~1/2 of patients. We targeted HBV DNA for cleavage using zinc-finger nucleases (ZFNs), which cleave as dimers. Co-transfection of our ZFN pair with a target plasmid containing the HBV genome resulted in specific cleavage. After 3 days in culture, 26% of the target remained linear, whereas ~10% was cleaved and misjoined tail-to-tail. Notably, ZFN treatment decreased levels of the hepatitis C virus pregenomic RNA by 29%. A portion of cleaved plasmids are repaired in cells, often with deletions and insertions. To track misrepair, we introduced an XbaI restriction site in the spacer between the ZFN sites. Targeted cleavage and misrepair destroys the XbaI site. After 3 days in culture, ~6% of plasmids were XbaI-resistant. Thirteen of 16 clones sequenced contained frameshift mutations that would lead to truncations of the viral core protein. These results demonstrate, for the first time, the possibility of targeting episomal viral DNA genomes using ZFNs. PMID:20160705

  3. Epigenetic regulation of puberty via Zinc finger protein-mediated transcriptional repression

    PubMed Central

    Lomniczi, Alejandro; Wright, Hollis; Castellano, Juan Manuel; Matagne, Valerie; Toro, Carlos A.; Ramaswamy, Suresh; Plant, Tony M.; Ojeda, Sergio R.

    2015-01-01

    In primates, puberty is unleashed by increased GnRH release from the hypothalamus following an interval of juvenile quiescence. GWAS implicates Zinc finger (ZNF) genes in timing human puberty. Here we show that hypothalamic expression of several ZNFs decreased in agonadal male monkeys in association with the pubertal reactivation of gonadotropin secretion. Expression of two of these ZNFs, GATAD1 and ZNF573, also decreases in peripubertal female monkeys. However, only GATAD1 abundance increases when gonadotropin secretion is suppressed during late infancy. Targeted delivery of GATAD1 or ZNF573 to the rat hypothalamus delays puberty by impairing the transition of a transcriptional network from an immature repressive epigenetic configuration to one of activation. GATAD1 represses transcription of two key puberty-related genes, KISS1 and TAC3, directly, and reduces the activating histone mark H3K4me2 at each promoter via recruitment of histone demethylase KDM1A. We conclude that GATAD1 epitomizes a subset of ZNFs involved in epigenetic repression of primate puberty. PMID:26671628

  4. Improved Cell-Penetrating Zinc-Finger Nuclease Proteins for Precision Genome Engineering

    PubMed Central

    Liu, Jia; Gaj, Thomas; Wallen, Mark C; Barbas, Carlos F

    2015-01-01

    Safe, efficient, and broadly applicable methods for delivering site-specific nucleases into cells are needed in order for targeted genome editing to reach its full potential for basic research and medicine. We previously reported that zinc-finger nuclease (ZFN) proteins have the innate capacity to cross cell membranes and induce genome modification via their direct application to human cells. Here, we show that incorporation of tandem nuclear localization signal (NLS) repeats into the ZFN protein backbone enhances cell permeability nearly 13-fold and that single administration of multi-NLS ZFN proteins leads to genome modification rates of up to 26% in CD4+ T cells and 17% in CD34+ hematopoietic stem/progenitor cells. In addition, we show that multi-NLS ZFN proteins attenuate off-target effects and that codelivery of ZFN protein pairs facilitates dual gene modification frequencies of 20–30% in CD4+ T cells. These results illustrate the applicability of ZFN protein delivery for precision genome engineering. PMID:25756962

  5. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

    PubMed Central

    Santiago, Yolanda; Chan, Edmond; Liu, Pei-Qi; Orlando, Salvatore; Zhang, Lin; Urnov, Fyodor D.; Holmes, Michael C.; Guschin, Dmitry; Waite, Adam; Miller, Jeffrey C.; Rebar, Edward J.; Gregory, Philip D.; Klug, Aaron; Collingwood, Trevor N.

    2008-01-01

    Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural—but imperfect—DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR−/− cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2–3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR−/− cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production. PMID:18359850

  6. Precise genome modification in the crop species Zea mays using zinc-finger nucleases.

    PubMed

    Shukla, Vipula K; Doyon, Yannick; Miller, Jeffrey C; DeKelver, Russell C; Moehle, Erica A; Worden, Sarah E; Mitchell, Jon C; Arnold, Nicole L; Gopalan, Sunita; Meng, Xiangdong; Choi, Vivian M; Rock, Jeremy M; Wu, Ying-Ying; Katibah, George E; Zhifang, Gao; McCaskill, David; Simpson, Matthew A; Blakeslee, Beth; Greenwalt, Scott A; Butler, Holly J; Hinkley, Sarah J; Zhang, Lei; Rebar, Edward J; Gregory, Philip D; Urnov, Fyodor D

    2009-05-21

    Agricultural biotechnology is limited by the inefficiencies of conventional random mutagenesis and transgenesis. Because targeted genome modification in plants has been intractable, plant trait engineering remains a laborious, time-consuming and unpredictable undertaking. Here we report a broadly applicable, versatile solution to this problem: the use of designed zinc-finger nucleases (ZFNs) that induce a double-stranded break at their target locus. We describe the use of ZFNs to modify endogenous loci in plants of the crop species Zea mays. We show that simultaneous expression of ZFNs and delivery of a simple heterologous donor molecule leads to precise targeted addition of an herbicide-tolerance gene at the intended locus in a significant number of isolated events. ZFN-modified maize plants faithfully transmit these genetic changes to the next generation. Insertional disruption of one target locus, IPK1, results in both herbicide tolerance and the expected alteration of the inositol phosphate profile in developing seeds. ZFNs can be used in any plant species amenable to DNA delivery; our results therefore establish a new strategy for plant genetic manipulation in basic science and agricultural applications.

  7. Zinc finger antiviral protein inhibits coxsackievirus B3 virus replication and protects against viral myocarditis.

    PubMed

    Li, Min; Yan, Kepeng; Wei, Lin; Yang, Jie; Lu, Chenyu; Xiong, Fei; Zheng, Chunfu; Xu, Wei

    2015-11-01

    The host Zinc finger antiviral protein (ZAP) has been reported exhibiting antiviral activity against positive-stranded RNA viruses (Togaviridae), negative-stranded RNA viruses (Filoviridae) and retroviruses (Retroviridae). However, whether ZAP restricts the infection of enterovirus and the development of enterovirus mediated disease remains unknown. Here, we reported the antiviral properties of ZAP against coxsackievirus B3 (CVB3), a single-stranded RNA virus of the Enterovirus genus within the Picornaviridae as a major causative agent of viral myocarditis (VMC). We found that the expression of ZAP was significantly induced after CVB3 infection in heart tissues of VMC mice. ZAP potently inhibited CVB3 replication in cells after infection, while overexpression of ZAP in mice significantly increased the resistance to CVB3 replication and viral myocarditis by significantly reducing cardiac inflammatory cytokine production. The ZAP-responsive elements (ZREs) were mapped to the 3'UTR and 5'UTR of viral RNA. Taken together, ZAP confers resistance to CVB3 infection via directly targeting viral RNA and protects mice from acute myocarditis by suppressing viral replication and cardiac inflammatory cytokine production. Our finding further expands ZAP's range of viral targets, and suggests ZAP as a potential therapeutic target for viral myocarditis caused by CVB3.

  8. The zinc finger protein ZPR1 is a potential modifier of spinal muscular atrophy

    PubMed Central

    Ahmad, Saif; Wang, Yi; Shaik, Gouse M.; Burghes, Arthur H.; Gangwani, Laxman

    2012-01-01

    Spinal muscular atrophy (SMA) is caused by mutation of the Survival Motor Neurons 1 (SMN1) gene and is characterized by degeneration of spinal motor neurons. The severity of SMA is primarily influenced by the copy number of the SMN2 gene. Additional modifier genes that lie outside the SMA locus exist and one gene that could modify SMA is the Zinc Finger Protein (ZPR1) gene. To test the significance of ZPR1 downregulation in SMA, we examined the effect of reduced ZPR1 expression in mice with mild and severe SMA. We report that the reduced ZPR1 expression causes increase in the loss of motor neurons, hypermyelination in phrenic nerves, increase in respiratory distress and disease severity and reduces the lifespan of SMA mice. The deficiency of SMN-containing sub-nuclear bodies correlates with the severity of SMA. ZPR1 is required for the accumulation of SMN in sub-nuclear bodies. Further, we report that ZPR1 overexpression increases levels of SMN and promotes accumulation of SMN in sub-nuclear bodies in SMA patient fibroblasts. ZPR1 stimulates neurite growth and rescues axonal growth defects in SMN-deficient spinal cord neurons from SMA mice. These data suggest that the severity of disease correlates negatively with ZPR1 levels and ZPR1 may be a protective modifier of SMA. PMID:22422766

  9. ZAS: C2H2 zinc finger proteins involved in growth and development.

    PubMed

    Wu, Lai-Chu

    2002-01-01

    A ZAS gene encodes a large protein with two separate C2H2 zinc finger pairs that independently bind to specific DNA sequences, including the kappaB motif. Three paralogous mammalian genes, ZAS1, ZAS2, and ZAS3, and a related Drosophila gene, Schnurri, have been cloned and characterized. The ZAS genes encode transcriptional proteins that activate or repress the transcription of a variety of genes involved in growth, development, and metastasis. In addition, ZAS3 associates with a TNF receptor-associated factor to inhibit NF-kappaB- and JNK/ SAPK-mediated signaling of TNF-alpha. Genetic experiments show that ZAS3 deficiency leads to proliferation of cells and tumor formation in mice. The data suggest that ZAS3 is important in controlling cell growth, apoptosis, and inflammation. The potent vasoactive hormone endothelin and transcription factor AP2 gene families also each consist of three members. The ZAS, endothelin, and transcription factor AP2 genes form several linkage groups. Knowledge of the chromosomal locations of these genes provides valuable clues to the evolution of the vertebrate genome.

  10. The zinc fingers of YY1 bind single-stranded RNA with low sequence specificity.

    PubMed

    Wai, Dorothy C C; Shihab, Manar; Low, Jason K K; Mackay, Joel P

    2016-11-02

    Classical zinc fingers (ZFs) are traditionally considered to act as sequence-specific DNA-binding domains. More recently, classical ZFs have been recognised as potential RNA-binding modules, raising the intriguing possibility that classical-ZF transcription factors are involved in post-transcriptional gene regulation via direct RNA binding. To date, however, only one classical ZF-RNA complex, that involving TFIIIA, has been structurally characterised. Yin Yang-1 (YY1) is a multi-functional transcription factor involved in many regulatory processes, and binds DNA via four classical ZFs. Recent evidence suggests that YY1 also interacts with RNA, but the molecular nature of the interaction remains unknown. In the present work, we directly assess the ability of YY1 to bind RNA using in vitro assays. Systematic Evolution of Ligands by EXponential enrichment (SELEX) was used to identify preferred RNA sequences bound by the YY1 ZFs from a randomised library over multiple rounds of selection. However, a strong motif was not consistently recovered, suggesting that the RNA sequence selectivity of these domains is modest. YY1 ZF residues involved in binding to single-stranded RNA were identified by NMR spectroscopy and found to be largely distinct from the set of residues involved in DNA binding, suggesting that interactions between YY1 and ssRNA constitute a separate mode of nucleic acid binding. Our data are consistent with recent reports that YY1 can bind to RNA in a low-specificity, yet physiologically relevant manner.

  11. Efficient generation of myostatin (MSTN) biallelic mutations in cattle using zinc finger nucleases.

    PubMed

    Luo, Junjie; Song, Zhiyuan; Yu, Shengli; Cui, Dan; Wang, Benli; Ding, Fangrong; Li, Song; Dai, Yunping; Li, Ning

    2014-01-01

    Genetically engineered zinc-finger nucleases (ZFNs) are useful for marker-free gene targeting using a one-step approach. We used ZFNs to efficiently disrupt bovine myostatin (MSTN), which was identified previously as the gene responsible for double muscling in cattle. The mutation efficiency of bovine somatic cells was approximately 20%, and the biallelic mutation efficiency was 8.3%. To evaluate the function of the mutated MSTN locus before somatic cell nuclear transfer, MSTN mRNA and protein expression was examined in four mutant cell colonies. We generated marker-gene-free cloned cattle, in which the MSTN biallelic mutations consisted of a 6-bp deletion in one of the alleles and a 117-bp deletion and 9-bp insertion in the other allele, resulting in at least four distinct mRNA splice variants. In the MSTN mutant cattle, the total amount of MSTN protein with the C-terminal domain was reduced by approximately 50%, and hypertrophied muscle fibers of the quadriceps and the double-muscled phenotype appeared at one month of age. Our proof-of-concept study is the first to produce MSTN mutations in cattle, and may allow the development of genetically modified strains of double-muscled cattle.

  12. The zinc fingers of YY1 bind single-stranded RNA with low sequence specificity

    PubMed Central

    Wai, Dorothy C.C.; Shihab, Manar; Low, Jason K.K.; Mackay, Joel P.

    2016-01-01

    Classical zinc fingers (ZFs) are traditionally considered to act as sequence-specific DNA-binding domains. More recently, classical ZFs have been recognised as potential RNA-binding modules, raising the intriguing possibility that classical-ZF transcription factors are involved in post-transcriptional gene regulation via direct RNA binding. To date, however, only one classical ZF-RNA complex, that involving TFIIIA, has been structurally characterised. Yin Yang-1 (YY1) is a multi-functional transcription factor involved in many regulatory processes, and binds DNA via four classical ZFs. Recent evidence suggests that YY1 also interacts with RNA, but the molecular nature of the interaction remains unknown. In the present work, we directly assess the ability of YY1 to bind RNA using in vitro assays. Systematic Evolution of Ligands by EXponential enrichment (SELEX) was used to identify preferred RNA sequences bound by the YY1 ZFs from a randomised library over multiple rounds of selection. However, a strong motif was not consistently recovered, suggesting that the RNA sequence selectivity of these domains is modest. YY1 ZF residues involved in binding to single-stranded RNA were identified by NMR spectroscopy and found to be largely distinct from the set of residues involved in DNA binding, suggesting that interactions between YY1 and ssRNA constitute a separate mode of nucleic acid binding. Our data are consistent with recent reports that YY1 can bind to RNA in a low-specificity, yet physiologically relevant manner. PMID:27369384

  13. Overexpression and clinical significance of MYC-associated zinc finger protein in pancreatic carcinoma

    PubMed Central

    Zhu, Xiaonian; Luo, Wei; Liang, Wenjin; Tang, Fen; Bei, Chunhua; Ren, Yuan; Qin, Linyuan; Tan, Chao; Zhang, Ying; Tan, Shengkui

    2016-01-01

    This study aimed to investigate the expression and clinical significance of MYC-associated zinc finger protein (MAZ) in pancreatic carcinoma (PC), and the biological functions of MAZ in PC cells. MAZ expression was detected in 57 PC tissues and 41 paired adjacent nontumor tissues by immunohistochemistry. Compared to the expression in adjacent nontumor tissues, MAZ was significantly higher expressed in PC tissues (P<0.0001). In addition, MAZ expression had a significant correlation with certain clinical characteristics of PC patients, such as age, tumor diameter, tumor number, and the serum level of CA199 (P<0.05). The survival analysis showed that the survival time of PC patients with high expression of MAZ was significantly lower than patients with low expression of MAZ (P=0.0365). After MAZ was knocked down in PANC-1 cells by RNA interference, the cells’ ability to proliferate, invade, and migrate was decreased significantly (P<0.01). Moreover, MAZ expression was found to be associated with Ki-67, a cell proliferation marker, in PC tissues, further supporting the idea that MAZ promotes PC cell proliferation. Our study clarifies an oncogenic role of MAZ in pathogenesis of PC and provides MAZ as a biomarker in the treatment and prognosis of PC. PMID:28008270

  14. Expression and prognostic significance of zinc fingers and homeoboxes family members in renal cell carcinoma

    PubMed Central

    Jeong, Dae Cheon; Han, Myoung-Eun; Kim, Ji-Young; Liu, Liangwen; Jung, Jin-Sup; Oh, Sae-Ock

    2017-01-01

    Zinc fingers and homeoboxes (ZHX) is a transcription repressor family that contains three members; ZHX1, ZHX2, and ZHX3. Although ZHX family members have been associated with the progression of cancer, their values as prognostic factors in cancer patients have been poorly examined. Renal cell carcinoma (RCC) is a highly heterogeneous, aggressive cancer that responds variably to treatment. Thus, prognostic molecular markers are required to evaluate disease progression and to improve the survival. In clear cell RCC (ccRCC), ZHX1 and ZHX3 expression were found to be down-regulated but ZHX2 was up-regulated, and the expressions of ZHX1 and ZHX3 were significantly associated with pathological stage. Furthermore, Kaplan-Meier and multivariate regression analysis showed that reduction in the mRNA expression of ZHX1 was associated with poorer survival. Taken together, the present study shows loss of ZHX1 is correlated with ccRCC progression and suggests it is an independent prognostic marker in ccRCC. PMID:28152006

  15. Repression of vascular endothelial growth factor A in glioblastoma cells using engineered zinc finger transcription factors.

    PubMed

    Snowden, Andrew W; Zhang, Lei; Urnov, Fyodor; Dent, Carolyn; Jouvenot, Yann; Zhong, Xiaohong; Rebar, Edward J; Jamieson, Andrew C; Zhang, H Steven; Tan, Siyuan; Case, Casey C; Pabo, Carl O; Wolffe, Alan P; Gregory, Philip D

    2003-12-15

    Angiogenic factors are necessary for tumor proliferation and thus are attractive therapeutic targets. In this study, we have used engineered zinc finger protein (ZFP) transcription factors (TFs) to repress expression of vascular endothelial growth factor (VEGF)-A in human cancer cell lines. We create potent transcriptional repressors by fusing a designed ZFP targeted to the VEGF-A promoter with either the ligand-binding domain of thyroid hormone receptor alpha or its viral relative, vErbA. Moreover, this ZFP-vErbA repressor binds its intended target site in vivo and mediates the specific deacetylation of histones H3 and H4 at the targeted promoter, a result that emulates the natural repression mechanism of these domains. The potential therapeutic relevance of ZFP-mediated VEGF-A repression was addressed using the highly tumorigenic glioblastoma cell line U87MG. Despite the aberrant overexpression of VEGF-A in this cell line, engineered ZFP TFs were able to repress the expression of VEGF-A by >20-fold. The VEGF-A levels observed after ZFP TF-mediated repression were comparable to those of a nonangiogenic cancer line (U251MG), suggesting that the degree of repression obtained with the ZFP TF would be sufficient to suppress tumor angiogenesis. Thus, engineered ZFP TFs are shown to be potent regulators of gene expression with therapeutic promise in the treatment of disease.

  16. Increased expression of Zinc finger protein 267 in non-alcoholic fatty liver disease

    PubMed Central

    Schnabl, Bernd; Czech, Barbara; Valletta, Daniela; Weiss, Thomas S; Kirovski, Georgi; Hellerbrand, Claus

    2011-01-01

    Hepatocellular lipid accumulation is a hallmark of non-alcoholic fatty liver disease (NAFLD), which encompasses a spectrum ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) and ultimately cirrhosis. Zinc finger protein 267 (ZNF267) belongs to the family of Kruppel-like transcription factors, which regulate diverse biological processes that include development, proliferation, and differentiation. We have previously demonstrated that ZNF267 expression is up-regulated in liver cirrhosis and is further increased in hepatocellular carcinoma (HCC). Here, we analyzed the expression of ZNF267 in tissue specimens of NAFLD patients and found a significant up-regulation compared to normal liver tissue. Noteworthy, ZNF267 mRNA was already significantly increased in steatotic liver tissue without inflammation. In line with this, incubation of primary human hepatocytes with palmitic acid induced a dose-dependent lipid accumulation and corresponding dose-dependent ZNF267 induction in vitro. Furthermore, hepatocellular lipid accumulation induced formation of reactive oxygen species (ROS), and also chemically induced ROS formation increased ZNF267 mRNA expression. In summary with previous findings, which revealed ZNF267 as pro-fibrogenic and pro-cancerogenic factor in chronic liver disease, the present study further suggests ZNF267 as promising therapeutic target particularly for NAFLD patients. In addition, it further indicates that hepatic steatosis per se has pathophysiological relevance and should not be considered as benign. PMID:22076166

  17. Zinc finger transcription factor Slug is a novel target gene of aryl hydrocarbon receptor

    SciTech Connect

    Ikuta, Togo; Kawajiri, Kaname . E-mail: kawajiri@cancer-c.pref.saitama.jp

    2006-11-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. We previously showed that AhR localizes predominantly in the cytoplasm under high cell densities of a keratinocytes cell line, HaCaT, but accumulates in the nucleus at low cell densities. In the current report, we show that the Slug, which is a member of the snail/slug family of zinc finger transcriptional repressors critical for induction of epithelial-mesenchymal transitions (EMT), is activated transcriptionally in accordance with nuclear accumulation of AhR. By reporter assay of the promoter of the Slug gene, gel shift and chromatin immunoprecipitation analyses showed AhR directly binds to xenobiotic responsive element 5 at - 0.7 kb of the gene. AhR-targeted gene silencing by small interfering RNA duplexes led to the abolishment of not only CYP1A1 but also Slug induction by 3-methycholanthrene. The Slug was co-localized to the AhR at the wound margins of HaCaT cells, where apparent nuclear distribution of AhR and Slug was observed. The induced Slug was associated with reduction of an epithelial marker of cytokeratin-18 and with an increase in the mesenchymal marker, fibronectin. Taken together, these findings suggest that AhR participated in Slug induction, which, in turn, regulates cellular physiology including cell adhesion and migration.

  18. The role of the DNA-binding One Zinc Finger (DOF) transcription factor family in plants.

    PubMed

    Noguero, Mélanie; Atif, Rana Muhammad; Ochatt, Sergio; Thompson, Richard D

    2013-08-01

    The DOF (DNA-binding One Zinc Finger) family of transcription factors is involved in many fundamental processes in higher plants, including responses to light and phytohormones as well as roles in seed maturation and germination. DOF transcription factor genes are restricted in their distribution to plants, where they are in many copies in both gymnosperms and angiosperms and also present in lower plants such as the moss Physcomitrella patens and in the alga Chlamydomonas reinhardtii which possesses a single DOF gene. DOF transcription factors bind to their promoter targets at the consensus sequence AAAG. This binding depends upon the presence of the highly conserved DOF domain in the protein. Depending on the target gene, DOF factor binding may activate or repress transcription. DOF factors are expressed in most if not all tissues of higher plants, but frequently appear to be functionally redundant. Recent next-generation sequencing data provide a more comprehensive survey of the distribution of DOF sequence classes among plant species and within tissue types, and clues as to the evolution of functions assumed by this transcription factor family. DOFs do not appear to be implicated in the initial differentiation of the plant body plan into organs via the resolution of meristematic zones, in contrast to MADS-box and homeobox transcription factors, which are found in other non-plant eukaryotes, and this may reflect a more recent evolutionary origin.

  19. Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases.

    PubMed

    Merlin, Christine; Beaver, Lauren E; Taylor, Orley R; Wolfe, Scot A; Reppert, Steven M

    2013-01-01

    The development of reverse-genetic tools in "nonmodel" insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases that generate mutations at targeted genomic sequences. We focused our ZFN approach on targeting the type 2 vertebrate-like cryptochrome gene of the monarch (designated cry2), which encodes a putative transcriptional repressor of the monarch circadian clockwork. Co-injections of mRNAs encoding ZFNs targeting the second exon of monarch cry2 into "one nucleus" stage embryos led to high-frequency nonhomologous end-joining-mediated, mutagenic lesions in the germline (up to 50%). Heritable ZFN-induced lesions in two independent lines produced truncated, nonfunctional CRY2 proteins, resulting in the in vivo disruption of circadian behavior and the molecular clock mechanism. Our work genetically defines CRY2 as an essential transcriptional repressor of the monarch circadian clock and provides a proof of concept for the use of ZFNs for manipulating genes in the monarch butterfly genome. Importantly, this approach could be used in other lepidopterans and "nonmodel" insects, thus opening new avenues to decipher the molecular underpinnings of a variety of biological processes.

  20. Abnormal spermatogenesis and male infertility in testicular zinc finger protein Zfp318-knockout mice.

    PubMed

    Ishizuka, Masamichi; Ohtsuka, Eri; Inoue, Atsuto; Odaka, Mirei; Ohshima, Hirotaka; Tamura, Norihisa; Yoshida, Kaoru; Sako, Norihisa; Baba, Tadashi; Kashiwabara, Shin-Ichi; Okabe, Masaru; Noguchi, Junko; Hagiwara, Hiromi

    2016-09-01

    Zfp318, a mouse gene with a Cys2/His2 zinc finger motif, is mainly expressed in germ cells in the testis. It encodes two alternative transcripts, which regulate androgen receptor-mediated transcriptional activation or repression by overexpression of them. However, the role of Zfp318 is still obscure in vivo, especially in spermatogenesis. To elucidate the role of Zfp318 during gamete production, we established a knockout mouse line. Zfp318-null male mice exhibited infertility, whereas Zfp318-null female mice displayed normal fertility. ZFP318 was expressed during multiple stages of spermatogenesis, from spermatocytes to round spermatids. The nuclei of secondary spermatocytes showed high levels of expression. Histological analysis and quantitative analysis of DNA content showed decreased numbers of both spermatids in the seminiferous tubules and mature spermatozoa in the epididymides of Zfp318-null mice. These results suggest that Zfp318 is expressed as a functional protein in testicular germ cells and plays an important role in meiosis during spermatogenesis.

  1. A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking.

    PubMed

    Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

    2015-05-18

    An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes.

  2. midlife crisis encodes a conserved zinc-finger protein required to maintain neuronal differentiation in Drosophila.

    PubMed

    Carney, Travis D; Struck, Adam J; Doe, Chris Q

    2013-10-01

    Stem cells generate progeny that undergo terminal differentiation. The initiation and maintenance of the differentiated status is crucial for tissue development, function and homeostasis. Drosophila neural stem cells (neuroblasts) are a model for stem cell self-renewal and differentiation; they divide asymmetrically to self-renew and generate the neurons and glia of the CNS. Here we report the identification of midlife crisis (mdlc; CG4973) as a gene required for the maintenance of neuronal differentiation and for neuroblast proliferation in Drosophila. mdlc encodes a ubiquitously expressed zinc-finger-containing protein with conserved orthologs from yeast to humans that are reported to have a role in RNA splicing. Using clonal analysis, we demonstrate that mdlc mutant neurons initiate but fail to complete differentiation, as judged by the loss of the pro-differentiation transcription factor Prospero, followed by derepression of the neuroblast factors Deadpan, Asense and Cyclin E. RNA-seq shows that loss of Mdlc decreases pros transcript levels and results in aberrant pros splicing. Importantly, misexpression of the full-length human ortholog, RNF113A, completely rescues all CNS defects in mdlc mutants. We conclude that Mdlc plays an essential role in maintaining neuronal differentiation, raising the possibility that RNF113A regulates neuronal differentiation in the human CNS.

  3. midlife crisis encodes a conserved zinc-finger protein required to maintain neuronal differentiation in Drosophila

    PubMed Central

    Carney, Travis D.; Struck, Adam J.; Doe, Chris Q.

    2013-01-01

    Stem cells generate progeny that undergo terminal differentiation. The initiation and maintenance of the differentiated status is crucial for tissue development, function and homeostasis. Drosophila neural stem cells (neuroblasts) are a model for stem cell self-renewal and differentiation; they divide asymmetrically to self-renew and generate the neurons and glia of the CNS. Here we report the identification of midlife crisis (mdlc; CG4973) as a gene required for the maintenance of neuronal differentiation and for neuroblast proliferation in Drosophila. mdlc encodes a ubiquitously expressed zinc-finger-containing protein with conserved orthologs from yeast to humans that are reported to have a role in RNA splicing. Using clonal analysis, we demonstrate that mdlc mutant neurons initiate but fail to complete differentiation, as judged by the loss of the pro-differentiation transcription factor Prospero, followed by derepression of the neuroblast factors Deadpan, Asense and Cyclin E. RNA-seq shows that loss of Mdlc decreases pros transcript levels and results in aberrant pros splicing. Importantly, misexpression of the full-length human ortholog, RNF113A, completely rescues all CNS defects in mdlc mutants. We conclude that Mdlc plays an essential role in maintaining neuronal differentiation, raising the possibility that RNF113A regulates neuronal differentiation in the human CNS. PMID:24026126

  4. Regulation of hepatic lipogenesis by the zinc finger protein Zbtb20

    PubMed Central

    Liu, Gan; Zhou, Luting; Zhang, Hai; Chen, Rong; Zhang, Ye; Li, Ling; Lu, Jun-Yu; Jiang, Hui; Liu, Dong; Qi, Shasha; Jiang, Ying-Ming; Yin, Kai; Xie, Zhifang; Shi, Yuguang; Liu, Yong; Cao, Xuetao; Chen, Yu-Xia; Zou, Dajin; Zhang, Weiping J.

    2017-01-01

    Hepatic de novo lipogenesis (DNL) converts carbohydrates into triglycerides and is known to influence systemic lipid homoeostasis. Here, we demonstrate that the zinc finger protein Zbtb20 is required for DNL. Mice lacking Zbtb20 in the liver exhibit hypolipidemia and reduced levels of liver triglycerides, along with impaired hepatic lipogenesis. The expression of genes involved in glycolysis and DNL, including that of two ChREBP isoforms, is decreased in livers of knockout mice. Zbtb20 binds to and enhances the activity of the ChREBP-α promoter, suggesting that altered metabolic gene expression is mainly driven by ChREBP. In addition, ChREBP-β overexpression largely restores hepatic expression of genes involved in glucose and lipid metabolism, and increases plasma and liver triglyceride levels in knockout mice. Finally, we show that Zbtb20 ablation protects from diet-induced liver steatosis and improves hepatic insulin resistance. We suggest ZBTB20 is an essential regulator of hepatic lipogenesis and may be a therapeutic target for the treatment of fatty liver disease. PMID:28327662

  5. Selection for a zinc-finger protein contributes to seed oil increase during soybean domestication.

    PubMed

    Li, Qing-Tian; Lu, Xiang; Song, Qingxin; Chen, Hao-Wei; Wei, Wei; Tao, Jian-Jun; Bian, Xiao-Hua; Shen, Ming; Ma, Biao; Zhang, Wan-Ke; Bi, Ying-Dong; Li, Wei; Lai, Yong-Cai; Lam, Sin Man; Shui, Guanghou; Chen, Shou-Yi; Zhang, Jin-Song

    2017-02-09

    Seed oil is a momentous agronomical trait of soybean targeted by domestication in breeding. Although multiple oil-related genes have been uncovered, the knowledge of regulatory mechanism of seed oil biosynthesis is currently limited. We demonstrate that seed-preferred gene GmZF351 encoding tandem CCCH zinc finger protein is selected during domestication. Further analysis shows that GmZF351 facilitates oil accumulation by directly activating WRI1, BCCP2, KASIII, TAG1 and OLEO2 in transgenic Arabidopsis seeds. Overexpression of GmZF351 in transgenic soybean also activates lipid biosynthesis genes, thereby accelerating seed oil accumulation. ZF351 haplotype from Glycine max group and Glycine soja subgroup III correlates well with high gene expression level, seed oil contents and promoter activity, suggesting that selection of GmZF351 expression leads to increased seed oil content in cultivated soybean. Our study provides novel insights into the regulatory mechanism for seed oil accumulation and manipulation of GmZF351 may have great potential in improvement of oil production in soybean and other related crops.

  6. Artificial zinc finger DNA binding domains: versatile tools for genome engineering and modulation of gene expression.

    PubMed

    Hossain, Mir A; Barrow, Joeva J; Shen, Yong; Haq, Md Imdadul; Bungert, Jörg

    2015-11-01

    Genome editing and alteration of gene expression by synthetic DNA binding activities gained a lot of momentum over the last decade. This is due to the development of new DNA binding molecules with enhanced binding specificity. The most commonly used DNA binding modules are zinc fingers (ZFs), TALE-domains, and the RNA component of the CRISPR/Cas9 system. These binding modules are fused or linked to either nucleases that cut the DNA and induce DNA repair processes, or to protein domains that activate or repress transcription of genes close to the targeted site in the genome. This review focuses on the structure, design, and applications of ZF DNA binding domains (ZFDBDs). ZFDBDs are relatively small and have been shown to penetrate the cell membrane without additional tags suggesting that they could be delivered to cells without a DNA or RNA intermediate. Advanced algorithms that are based on extensive knowledge of the mode of ZF/DNA interactions are used to design the amino acid composition of ZFDBDs so that they bind to unique sites in the genome. Off-target binding has been a concern for all synthetic DNA binding molecules. Thus, increasing the specificity and affinity of ZFDBDs will have a significant impact on their use in analytical or therapeutic settings.

  7. Deleterious Mutations in the Zinc-Finger 469 Gene Cause Brittle Cornea Syndrome

    PubMed Central

    Abu, Almogit; Frydman, Moshe; Marek, Dina; Pras, Eran; Nir, Uri; Reznik-Wolf, Haike; Pras, Elon

    2008-01-01

    Brittle cornea syndrome (BCS) is an autosomal-recessive disorder characterized by a thin cornea that tends to perforate, causing progressive visual loss and blindness. Additional systemic symptoms such as joint hypermotility, hyperlaxity of the skin, and kyphoscoliosis place BCS among the connective-tissue disorders. Previously, we assigned the disease gene to a 4.7 Mb interval on chromosome 16q24. In order to clone the BCS gene, we first narrowed the disease locus to a 2.8 Mb interval and systematically sequenced genes expressed in connective tissue in this chromosomal segment. We have identified two frameshift mutations in the Zinc-Finger 469 gene (ZNF469). In five unrelated patients of Tunisian Jewish ancestry, we found a 1 bp deletion at position 5943 (5943 delA), and in an inbred Palestinian family we detected a single-nucleotide deletion at position 9527 (9527 delG). The function of ZNF469 is unknown. However, a 30% homology to a number of collagens suggests that it could act as a transcription factor involved in the synthesis and/or organization of collagen fibers. PMID:18452888

  8. The Drosophila Zinc Finger Transcription Factor Ouija Board Controls Ecdysteroid Biosynthesis through Specific Regulation of spookier

    PubMed Central

    Komura-Kawa, Tatsuya; Hirota, Keiko; Shimada-Niwa, Yuko; Yamauchi, Rieko; Shimell, MaryJane; Shinoda, Tetsuro; Fukamizu, Akiyoshi; O’Connor, Michael B.; Niwa, Ryusuke

    2015-01-01

    Steroid hormones are crucial for many biological events in multicellular organisms. In insects, the principal steroid hormones are ecdysteroids, which play essential roles in regulating molting and metamorphosis. During larval and pupal development, ecdysteroids are synthesized in the prothoracic gland (PG) from dietary cholesterol via a series of hydroxylation and oxidation steps. The expression of all but one of the known ecdysteroid biosynthetic enzymes is restricted to the PG, but the transcriptional regulatory networks responsible for generating such exquisite tissue-specific regulation is only beginning to be elucidated. Here, we report identification and characterization of the C2H2-type zinc finger transcription factor Ouija board (Ouib) necessary for ecdysteroid production in the PG in the fruit fly Drosophila melanogaster. Expression of ouib is predominantly limited to the PG, and genetic null mutants of ouib result in larval developmental arrest that can be rescued by administrating an active ecdysteroid. Interestingly, ouib mutant animals exhibit a strong reduction in the expression of one ecdysteroid biosynthetic enzyme, spookier. Using a cell culture-based luciferase reporter assay, Ouib protein stimulates transcription of spok by binding to a specific ~15 bp response element in the spok PG enhancer element. Most remarkable, the developmental arrest phenotype of ouib mutants is rescued by over-expression of a functionally-equivalent paralog of spookier. These observations imply that the main biological function of Ouib is to specifically regulate spookier transcription during Drosophila development. PMID:26658797

  9. Expression of the zinc-finger antiviral protein inhibits alphavirus replication.

    PubMed

    Bick, Matthew J; Carroll, John-William N; Gao, Guangxia; Goff, Stephen P; Rice, Charles M; MacDonald, Margaret R

    2003-11-01

    The rat zinc-finger antiviral protein (ZAP) was recently identified as a host protein conferring resistance to retroviral infection. We analyzed ZAP's ability to inhibit viruses from other families and found that ZAP potently inhibits the replication of multiple members of the Alphavirus genus within the Togaviridae, including Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. However, expression of ZAP did not induce a broad-spectrum antiviral state as some viruses, including vesicular stomatitis virus, poliovirus, yellow fever virus, and herpes simplex virus type 1, replicated to normal levels in ZAP-expressing cells. We determined that ZAP expression inhibits Sindbis virus replication after virus penetration and entry, but before the amplification of newly synthesized plus strand genomic RNA. Using a temperature-sensitive Sindbis virus mutant expressing luciferase, we further showed that translation of incoming viral RNA is blocked by ZAP expression. Elucidation of the antiviral mechanism by which ZAP inhibits Sindbis virus translation may lead to the development of agents with broad activity against alphaviruses.

  10. TRIM25 Enhances the Antiviral Action of Zinc-Finger Antiviral Protein (ZAP)

    PubMed Central

    Lau, Zerlina; Cheung, Pamela; Schneider, William M.; Bozzacco, Leonia; Buehler, Eugen; Takaoka, Akinori; Rice, Charles M.; Felsenfeld, Dan P.; MacDonald, Margaret R.

    2017-01-01

    The host factor and interferon (IFN)-stimulated gene (ISG) product, zinc-finger antiviral protein (ZAP), inhibits a number of diverse viruses by usurping and intersecting with multiple cellular pathways. To elucidate its antiviral mechanism, we perform a loss-of-function genome-wide RNAi screen to identify cellular cofactors required for ZAP antiviral activity against the prototype alphavirus, Sindbis virus (SINV). In order to exclude off-target effects, we carry out stringent confirmatory assays to verify the top hits. Important ZAP-liaising partners identified include proteins involved in membrane ion permeability, type I IFN signaling, and post-translational protein modification. The factor contributing most to the antiviral function of ZAP is TRIM25, an E3 ubiquitin and ISG15 ligase. We demonstrate here that TRIM25 interacts with ZAP through the SPRY domain, and TRIM25 mutants lacking the RING or coiled coil domain fail to stimulate ZAP’s antiviral activity, suggesting that both TRIM25 ligase activity and its ability to form oligomers are critical for its cofactor function. TRIM25 increases the modification of both the short and long ZAP isoforms by K48- and K63-linked polyubiquitin, although ubiquitination of ZAP does not directly affect its antiviral activity. However, TRIM25 is critical for ZAP’s ability to inhibit translation of the incoming SINV genome. Taken together, these data uncover TRIM25 as a bona fide ZAP cofactor that leads to increased ZAP modification enhancing its translational inhibition activity. PMID:28060952

  11. Donor plasmid design for codon and single base genome editing using zinc finger nucleases.

    PubMed

    Pruett-Miller, Shondra M; Davis, Gregory D

    2015-01-01

    In recent years, CompoZr zinc finger nuclease (ZFN) technology has matured to the point that a user-defined double strand break (DSB) can be placed at virtually any location in the human genome within 50 bp of a desired site. Such high resolution ZFN engineering is well within the conversion tract limitations demarcated by the mammalian DNA repair machinery, resulting in a nearly universal ability to create point mutations throughout the human genome. Additionally, new architectures for targeted nuclease engineering have been rapidly developed, namely transcription activator like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems, further expanding options for placement of DSBs. This new capability has created a need to explore the practical limitations of delivering plasmid-based information to the sites of chromosomal double strand breaks so that nuclease-donor methods can be widely deployed in fundamental and therapeutic research. In this chapter, we explore a ZFN-compatible donor design in the context of codon changes at an endogenous locus encoding the human RSK2 kinase.

  12. Heritable Targeted Inactivation of Myostatin Gene in Yellow Catfish (Pelteobagrus fulvidraco) Using Engineered Zinc Finger Nucleases

    PubMed Central

    Li, Kui; Xu, Zhiqiang; Liang, Dong; Li, Jingyun; Li, Junbo; Jia, Wenshuang; Li, Yuehua; Dong, Xiaohua; Cao, Shasha; Wang, Xiaoxiao; Pan, Jianlin; Zhao, Qingshun

    2011-01-01

    Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater aquaculture species in China. However, its small size and lower meat yield limit its edible value. Myostatin (MSTN) is a negative regulator of mammalian muscle growth. But, the function of Mstn in fish remains elusive. To explore roles of mstn gene in fish growth and create a strain of yellow catfish with high amount of muscle mass, we performed targeted disruption of mstn in yellow catfish using engineered zinc-finger nucleases (ZFNs). Employing zebrafish embryos as a screening system to identify ZFN activity, we obtained one pair of ZFNs that can edit mstn in yellow catfish genome. Using the ZFNs, we successfully obtained two founders (Founder July29-7 and Founder July29-8) carrying mutated mstn gene in their germ cells. The mutated mstn allele inherited from Founder July29-7 was a null allele (mstnnju6) containing a 4 bp insertion, predicted to encode function null Mstn. The mutated mstn inherited from Founder July29-8 was a complex type of mutation (mstnnju7), predicted to encode a protein lacking two amino acids in the N-terminal secretory signal of Mstn. Totally, we obtained 6 mstnnju6/+ and 14 mstnnju7/+ yellow catfish. To our best knowledge, this is the first endogenous gene knockout in aquaculture fish. Our result will help in understanding the roles of mstn gene in fish. PMID:22194943

  13. Efficient Generation of Myostatin (MSTN) Biallelic Mutations in Cattle Using Zinc Finger Nucleases

    PubMed Central

    Yu, Shengli; Cui, Dan; Wang, Benli; Ding, Fangrong; Li, Song; Dai, Yunping; Li, Ning

    2014-01-01

    Genetically engineered zinc-finger nucleases (ZFNs) are useful for marker-free gene targeting using a one-step approach. We used ZFNs to efficiently disrupt bovine myostatin (MSTN), which was identified previously as the gene responsible for double muscling in cattle. The mutation efficiency of bovine somatic cells was approximately 20%, and the biallelic mutation efficiency was 8.3%. To evaluate the function of the mutated MSTN locus before somatic cell nuclear transfer, MSTN mRNA and protein expression was examined in four mutant cell colonies. We generated marker-gene-free cloned cattle, in which the MSTN biallelic mutations consisted of a 6-bp deletion in one of the alleles and a 117-bp deletion and 9-bp insertion in the other allele, resulting in at least four distinct mRNA splice variants. In the MSTN mutant cattle, the total amount of MSTN protein with the C-terminal domain was reduced by approximately 50%, and hypertrophied muscle fibers of the quadriceps and the double-muscled phenotype appeared at one month of age. Our proof-of-concept study is the first to produce MSTN mutations in cattle, and may allow the development of genetically modified strains of double-muscled cattle. PMID:24743319

  14. A conserved CCCH-type zinc finger protein regulates mRNA nuclear adenylation and export.

    PubMed

    Hurt, Jessica A; Obar, Robert A; Zhai, Bo; Farny, Natalie G; Gygi, Steven P; Silver, Pamela A

    2009-04-20

    Coupling of messenger RNA (mRNA) nuclear export with prior processing steps aids in the fidelity and efficiency of mRNA transport to the cytoplasm. In this study, we show that the processes of export and polyadenylation are coupled via the Drosophila melanogaster CCCH-type zinc finger protein CG6694/dZC3H3 through both physical and functional interactions. We show that depletion of dZC3H3 from S2R+ cells results in transcript hyperadenylation. Using targeted coimmunoprecipitation and liquid chromatography mass spectrometry (MS)/MS techniques, we characterize interactions of known components of the mRNA nuclear export and polyadenylation machineries with dZC3H3. Furthermore, we demonstrate the functional conservation of this factor, as depletion of its human homologue ZC3H3 by small interfering RNA results in an mRNA export defect in human cells as well. Nuclear polyadenylated (poly(A)) RNA in ZC3H3-depleted cells is sequestered in foci removed from SC35-containing speckles, indicating a shift from the normal subnuclear distribution of poly(A) RNA. Our data suggest a model wherein ZC3H3 interfaces between the polyadenylation machinery, newly poly(A) mRNAs, and factors for transcript export.

  15. NMR chemical shift perturbation mapping of DNA binding by a zinc-finger domain from the yeast transcription factor ADR1.

    PubMed Central

    Schmiedeskamp, M.; Rajagopal, P.; Klevit, R. E.

    1997-01-01

    Mutagenesis studies have revealed that the minimal DNA-binding domain of the yeast transcription factor ADR1 consists of two Cys2-His2 zinc fingers plus an additional 20 residues proximal and N-terminal to the fingers. We have assigned NMR 1H, 15N, and 13C chemical shifts for the entire minimal DNA-binding domain of ADR1 both free and bound to specific DNA. 1H chemical shift values suggest little structural difference between the zinc fingers in this construct and in single-finger constructs, and 13C alpha chemical shift index analysis indicates little change in finger structure upon DNA binding. 1H chemical shift perturbations upon DNA binding are observed, however, and these are mapped to define the protein-DNA interface. The two zinc fingers appear to bind DNA with different orientations, as the entire helix of finger 1 is perturbed, while only the extreme N-terminus of the finger 2 helix is affected. Furthermore, residues N-terminal to the first finger undergo large chemical shift changes upon DNA binding suggesting a role at the protein-DNA interface. A striking correspondence is observed between the protein-DNA interface mapped by chemical shift changes and that previously mapped by mutagenesis. PMID:9300483

  16. NMR Scalar Couplings across Intermolecular Hydrogen Bonds between Zinc-Finger Histidine Side Chains and DNA Phosphate Groups.

    PubMed

    Chattopadhyay, Abhijnan; Esadze, Alexandre; Roy, Sourav; Iwahara, Junji

    2016-10-10

    NMR scalar couplings across hydrogen bonds represent direct evidence for the partial covalent nature of hydrogen bonds and provide structural and dynamic information on hydrogen bonding. In this article, we report heteronuclear (15)N-(31)P and (1)H-(31)P scalar couplings across the intermolecular hydrogen bonds between protein histidine (His) imidazole and DNA phosphate groups. These hydrogen-bond scalar couplings were observed for the Egr-1 zinc-finger-DNA complex. Although His side-chain NH protons are typically undetectable in heteronuclear (1)H-(15)N correlation spectra due to rapid hydrogen exchange, this complex exhibited two His side-chain NH signals around (1)H 14.3 ppm and (15)N 178 ppm at 35 °C. Through various heteronuclear multidimensional NMR experiments, these signals were assigned to two zinc-coordinating His side chains in contact with DNA phosphate groups. The data show that the Nδ1 atoms of these His side chains are protonated and exhibit the (1)H-(15)N cross-peaks. Using heteronuclear (1)H, (15)N, and (31)P NMR experiments, we observed the hydrogen-bond scalar couplings between the His (15)Nδ1/(1)Hδ1 and DNA phosphate (31)P nuclei. These results demonstrate the direct involvement of the zinc-coordinating His side chains in the recognition of DNA by the Cys2His2-class zinc fingers in solution.

  17. Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases.

    PubMed

    Kawai, Narudo; Ochiai, Hiroshi; Sakuma, Tetsushi; Yamada, Lixy; Sawada, Hitoshi; Yamamoto, Takashi; Sasakura, Yasunori

    2012-06-01

    Zinc-finger nucleases (ZFNs) are engineered nucleases that induce DNA double-strand breaks (DSBs) at target sequences. They have been used as tools for generating targeted mutations in the genomes of multiple organisms in both animals and plants. The DSB induced by ZFNs is repaired by non-homologous end joining (NHEJ) or by homologous recombination (HR) mechanisms. Non-homologous end joining induces some errors because it is independent of a reference DNA sequence. Through the NHEJ mechanism, ZFNs generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of ZFNs in the basal chordate Ciona intestinalis. As the target of ZFNs, we chose an enhanced green fluorescent protein (EGFP) gene artificially inserted in the C. intestinalis genome because this locus is neutral for the development and growth of C. intestinalis, and the efficiency of mutagenesis with ZFNs can thus be determined without any bias. We introduced EGFP -ZFN mRNAs into the embryos of an EGFP -transgenic line and observed the mutation frequency in the target site of EGFP . We also examined the effects of the EGFP -ZFNs at off-target sites resembling the EGFP target sequence in the C. intestinalis genome in order to examine the specificity of ZFNs. We further investigated the influence of ZFNs on embryogenesis, and showed that adequate amounts of ZFNs, which do not disrupt embryogenesis, can efficiently induce mutations on the on-target site with less effect on the off-target sites. This suggests that target mutagenesis with ZFNs will be a powerful technique in C. intestinalis.

  18. The B2 flowering time locus of beet encodes a zinc finger transcription factor

    PubMed Central

    Dally, Nadine; Xiao, Ke; Holtgräwe, Daniela; Jung, Christian

    2014-01-01

    Sugar beet (Beta vulgaris) is a biennial root crop that grows vegetatively in the first year and starts shoot elongation (bolting) and flowering after exposure to cold temperatures over winter. Early bolting before winter is controlled by the dominant allele of the B locus. Recently, the BOLTING TIME CONTROL 1 (BTC1) gene has been cloned from this locus. BTC1 promotes early bolting through repression of the downstream bolting repressor B. vulgaris FLOWERING LOCUS T1 (BvFT1) and activation of the downstream floral activator BvFT2. We have identified a new bolting locus B2 acting epistatically to B. B2 houses a transcription factor which is diurnally regulated and acts like BTC1 upstream of BvFT1 and BvFT2. It was termed BvBBX19 according to its closest homolog from Arabidopsis thaliana. The encoded protein has two conserved domains with homology to zinc finger B-boxes. Ethyl methanesulfonate-induced mutations within the second B-box caused up-regulation of BvFT1 and complete down-regulation of BvFT2. In Arabidopsis, the expression of FT is promoted by the B-box containing protein CONSTANS (CO). We performed a phylogenetic analysis with B-box genes from beet and A. thaliana but only BvCOL1 clustered with CO. However, BvCOL1 had been excluded as a CO ortholog by previous studies. Therefore, a new model for flowering induction in beet is proposed in which BTC1 and BvBBX19 complement each other and thus acquire a CO function to regulate their downstream targets BvFT1 and BvFT2. PMID:24965366

  19. A pair of mouse KRAB zinc finger proteins modulates multiple indicators of female reproduction.

    PubMed

    Krebs, Christopher J; Robins, Diane M

    2010-04-01

    Krüppel-associated box-zinc finger proteins (KRAB-ZFPs) are the largest class of transcriptional regulators in mammals, yet few have been assigned biological roles. Cloning the genes underlying the regulator of sex-limitation (rsl) phenotype, in which the normally male-specific sex-limited protein (SLP) is expressed in female mice, identified two KRAB-ZFPs, Rsl1 and Rsl2, as influencing sexually dimorphic liver gene expression. Combined absence of both repressors in rsl mice leads to increased expression in female liver of major urinary proteins (MUPs) and certain enzymes of steroid metabolism, as well as SLP. We hypothesized that this altered gene expression might affect reproductive physiology in rsl females. Urinary MUP (uMUP) concentration varied with the estrous cycle in both wt and rsl females but was consistently higher in rsl urine. A behavioral odor test revealed that wild-type (wt) males preferred rsl to wt females, possibly due to elevated uMUPs providing greater pheromone presentation. To ascribe activity to Rsl1, Rsl2, or both, the genes were individually expressed as liver-specific transgenes. RSL2 overexpression accentuated uMUP fluctuations across the estrous cycle, whereas RSL1 overexpression did not. In addition, puberty onset, as indicated by vaginal opening (VO), occurred 2 days earlier in rsl females, and excess RSL2, but not RSL1, restored VO timing to wt. Hence, transcriptional repression by RSL in liver modifies female mouse reproduction via targets that likely impact both hormonal and pheromonal cues. The large and rapidly diversifying KRAB-ZFP family may modulate biological processes, including reproduction, to confer individual differences that may isolate populations and ultimately lead to speciation.

  20. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases

    PubMed Central

    Moehle, Erica A.; Rock, Jeremy M.; Lee, Ya-Li; Jouvenot, Yann; DeKelver, Russell C.; Gregory, Philip D.; Urnov, Fyodor D.; Holmes, Michael C.

    2007-01-01

    Efficient incorporation of novel DNA sequences into a specific site in the genome of living human cells remains a challenge despite its potential utility to genetic medicine, biotechnology, and basic research. We find that a precisely placed double-strand break induced by engineered zinc finger nucleases (ZFNs) can stimulate integration of long DNA stretches into a predetermined genomic location, resulting in high-efficiency site-specific gene addition. Using an extrachromosomal DNA donor carrying a 12-bp tag, a 900-bp ORF, or a 1.5-kb promoter-transcription unit flanked by locus-specific homology arms, we find targeted integration frequencies of 15%, 6%, and 5%, respectively, within 72 h of treatment, and with no selection for the desired event. Importantly, we find that the integration event occurs in a homology-directed manner and leads to the accurate reconstruction of the donor-specified genotype at the endogenous chromosomal locus, and hence presumably results from synthesis-dependent strand annealing repair of the break using the donor DNA as a template. This site-specific gene addition occurs with no measurable increase in the rate of random integration. Remarkably, we also find that ZFNs can drive the addition of an 8-kb sequence carrying three distinct promoter-transcription units into an endogenous locus at a frequency of 6%, also in the absence of any selection. These data reveal the surprising versatility of the specialized polymerase machinery involved in double-strand break repair, illuminate a powerful approach to mammalian cell engineering, and open the possibility of ZFN-driven gene addition therapy for human genetic disease. PMID:17360608

  1. Generation and genetic engineering of human induced pluripotent stem cells using designed zinc finger nucleases.

    PubMed

    Ramalingam, Sivaprakash; London, Viktoriya; Kandavelou, Karthikeyan; Cebotaru, Liudmila; Guggino, William; Civin, Curt; Chandrasegaran, Srinivasan

    2013-02-15

    Zinc finger nucleases (ZFNs) have become powerful tools to deliver a targeted double-strand break at a pre-determined chromosomal locus in order to insert an exogenous transgene by homology-directed repair. ZFN-mediated gene targeting was used to generate both single-allele chemokine (C-C motif) receptor 5 (CCR5)-modified human induced pluripotent stem cells (hiPSCs) and biallele CCR5-modified hiPSCs from human lung fibroblasts (IMR90 cells) and human primary cord blood mononuclear cells (CBMNCs) by site-specific insertion of stem cell transcription factor genes flanked by LoxP sites into the endogenous CCR5 locus. The Oct4 and Sox2 reprogramming factors, in combination with valproic acid, induced reprogramming of human lung fibroblasts to form CCR5-modified hiPSCs, while 5 factors, Oct4/Sox2/Klf4/Lin28/Nanog, induced reprogramming of CBMNCs. Subsequent Cre recombinase treatment of the CCR5-modified IMR90 hiPSCs resulted in the removal of the Oct4 and Sox2 transgenes. Further genetic engineering of the single-allele CCR5-modified IMR90 hiPSCs was achieved by site-specific addition of the large CFTR transcription unit to the remaining CCR5 wild-type allele, using CCR5-specific ZFNs and a donor construct containing tdTomato and CFTR transgenes flanked by CCR5 homology arms. CFTR was expressed efficiently from the endogenous CCR5 locus of the CCR5-modified tdTomato/CFTR hiPSCs. These results suggest that it might be feasible to use ZFN-evoked strategies to (1) generate precisely targeted genetically well-defined patient-specific hiPSCs, and (2) then to reshape their function by targeted addition and expression of therapeutic genes from the CCR5 chromosomal locus for autologous cell-based transgene-correction therapy to treat various recessive monogenic human diseases in the future.

  2. Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases.

    PubMed

    Osiak, Anna; Radecke, Frank; Guhl, Eva; Radecke, Sarah; Dannemann, Nadine; Lütge, Fabienne; Glage, Silke; Rudolph, Cornelia; Cantz, Tobias; Schwarz, Klaus; Heilbronn, Regine; Cathomen, Toni

    2011-01-01

    Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.

  3. Treatment of traumatic brain injury using zinc-finger protein gene therapy targeting VEGF-A.

    PubMed

    Siddiq, Ishita; Park, Eugene; Liu, Elaine; Spratt, S Kaye; Surosky, Richard; Lee, Gary; Ando, Dale; Giedlin, Marty; Hare, Gregory M T; Fehlings, Michael G; Baker, Andrew J

    2012-11-20

    Vascular endothelial growth factor (VEGF) plays a role in angiogenesis and has been shown to be neuroprotective following central nervous system trauma. In the present study we evaluated the pro-angiogenic and neuroprotective effects of an engineered zinc-finger protein transcription factor transactivator targeting the vascular endothelial growth factor A (VEGF-ZFP). We used two virus delivery systems, adeno-virus and adeno-associated virus, to examine the effects of early and delayed VEGF-A upregulation after brain trauma, respectively. Male Sprague-Dawley rats were subject to a unilateral fluid percussion injury (FPI) of moderate severity (2.2-2.5 atm) followed by intracerebral microinjection of either adenovirus vector (Adv) or an adeno-associated vector (AAV) carrying the VEGF-ZFP construct. Adv-VEGF-ZFP-treated animals had significantly fewer TUNEL positive cells in the injured penumbra of the cortex (p<0.001) and hippocampus (p=0.001) relative to untreated rats at 72 h post-injury. Adv-VEGF-ZFP treatment significantly improved fEPSP values (p=0.007) in the CA1 region relative to injury alone. Treatment with AAV2-VEGF-ZFP resulted in improved post-injury microvascular diameter and improved functional recovery on the balance beam and rotarod task at 30 days post-injury. Collectively, the results provide supportive evidence for the concept of acute and delayed treatment following TBI using VEGF-ZFP to induce angiogenesis, reduce cell death, and enhance functional recovery.

  4. Targeting Serous Epithelial Ovarian Cancer with Designer Zinc Finger Transcription Factors*

    PubMed Central

    Lara, Haydee; Wang, Yuhua; Beltran, Adriana S.; Juárez-Moreno, Karla; Yuan, Xinni; Kato, Sumie; Leisewitz, Andrea V.; Cuello Fredes, Mauricio; Licea, Alexei F.; Connolly, Denise C.; Huang, Leaf; Blancafort, Pilar

    2012-01-01

    Ovarian cancer is the leading cause of death among gynecological malignancies. It is detected at late stages when the disease is spread through the abdominal cavity in a condition known as peritoneal carcinomatosis. Thus, there is an urgent need to develop novel therapeutic interventions to target advanced stages of ovarian cancer. Mammary serine protease inhibitor (Maspin) represents an important metastasis suppressor initially identified in breast cancer. Herein we have generated a sequence-specific zinc finger artificial transcription factor (ATF) to up-regulate the Maspin promoter in aggressive ovarian cancer cell lines and to interrogate the therapeutic potential of Maspin in ovarian cancer. We found that although Maspin was expressed in some primary ovarian tumors, the promoter was epigenetically silenced in cell lines derived from ascites. Transduction of the ATF in MOVCAR 5009 cells derived from ascitic cultures of a TgMISIIR-TAg mouse model of ovarian cancer resulted in tumor cell growth inhibition, impaired cell invasion, and severe disruption of actin cytoskeleton. Systemic delivery of lipid-protamine-RNA nanoparticles encapsulating a chemically modified ATF mRNA resulted in inhibition of ovarian cancer cell growth in nude mice accompanied with Maspin re-expression in the treated tumors. Gene expression microarrays of ATF-transduced cells revealed an exceptional specificity for the Maspin promoter. These analyses identified novel targets co-regulated with Maspin in human short-term cultures derived from ascites, such as TSPAN12, that could mediate the anti-metastatic phenotype of the ATF. Our work outlined the first targeted, non-viral delivery of ATFs into tumors with potential clinical applications for metastatic ovarian cancers. PMID:22782891

  5. Zinc finger homeobox is required for the differentiation of serotonergic neurons in the sea urchin embryo

    PubMed Central

    Yaguchi, Junko; Angerer, Lynne M.; Inaba, Kazuo; Yaguchi, Shunsuke

    2012-01-01

    Serotonergic neurons differentiate in the neurogenic animal plate ectoderm of the sea urchin embryo. The regulatory mechanisms that control the specification or differentiation of these neurons in the sea urchin embryo are not yet understood, although, after the genome was sequenced, many genes encoding transcription factors expressed in this region were identified. Here, we report that zinc finger homeobox (zfhx1/z81) is expressed in serotonergic neural precursor cells, using double in situ hybridization screening with a serotonergic neural marker, tryptophan 5-hydroxylase (tph) encoding a serotonin synthase that is required for the differentiation of serotonergic neurons. zfhx1/z81 begins to be expressed at gastrula stage in individual cells in the anterior neuroectoderm, some of which also express delta. zfhx1/z81 expression gradually disappears as neural differentiation begins with tph expression. When the translation of Zfhx1/Z81 is blocked by morpholino injection, embryos express neither tph nor the neural marker synaptotagminB in cells of the animal plate, and serotonergic neurons do not differentiate. In contrast, Zfhx1/Z81 morphants do express fez, another neural precursor marker, which appears to function in the initial phase of specification/differentiation of serotonergic neurons. In addition, zfhx1/z81 is one of the targets suppressed in the animal plate by anti-neural signals such as Nodal as well as Delta-Notch. We conclude that Zfhx1/Z81 functions during the specification of individual anterior neural precursors and promotes the expression of tph and synaptotagminB, required for the differentiation of serotonergic neurons. PMID:22210002

  6. Selection-Independent Generation of Gene Knockout Mouse Embryonic Stem Cells Using Zinc-Finger Nucleases

    PubMed Central

    Osiak, Anna; Radecke, Frank; Guhl, Eva; Radecke, Sarah; Dannemann, Nadine; Lütge, Fabienne; Glage, Silke; Rudolph, Cornelia; Cantz, Tobias; Schwarz, Klaus; Heilbronn, Regine; Cathomen, Toni

    2011-01-01

    Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10−6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells. PMID:22194948

  7. Dorsal root ganglion myeloid zinc finger protein 1 contributes to neuropathic pain after peripheral nerve trauma.

    PubMed

    Li, Zhisong; Gu, Xiyao; Sun, Linlin; Wu, Shaogen; Liang, Lingli; Cao, Jing; Lutz, Brianna Marie; Bekker, Alex; Zhang, Wei; Tao, Yuan-Xiang

    2015-04-01

    Peripheral nerve injury-induced changes in gene transcription and translation in primary sensory neurons of the dorsal root ganglion (DRG) are considered to contribute to neuropathic pain genesis. Transcription factors control gene expression. Peripheral nerve injury increases the expression of myeloid zinc finger protein 1 (MZF1), a transcription factor, and promotes its binding to the voltage-gated potassium 1.2 (Kv1.2) antisense (AS) RNA gene in the injured DRG. However, whether DRG MZF1 participates in neuropathic pain is still unknown. Here, we report that blocking the nerve injury-induced increase of DRG MZF1 through microinjection of MZF1 siRNA into the injured DRG attenuated the initiation and maintenance of mechanical, cold, and thermal pain hypersensitivities in rats with chronic constriction injury (CCI) of the sciatic nerve, without affecting locomotor functions and basal responses to acute mechanical, heat, and cold stimuli. Mimicking the nerve injury-induced increase of DRG MZF1 through microinjection of recombinant adeno-associated virus 5 expressing full-length MZF1 into the DRG produced significant mechanical, cold, and thermal pain hypersensitivities in naive rats. Mechanistically, MZF1 participated in CCI-induced reductions in Kv1.2 mRNA and protein and total Kv current and the CCI-induced increase in neuronal excitability through MZF1-triggered Kv1.2 AS RNA expression in the injured DRG neurons. MZF1 is likely an endogenous trigger of neuropathic pain and might serve as a potential target for preventing and treating this disorder.

  8. Nuclear gene targeting in Chlamydomonas using engineered zinc-finger nucleases.

    PubMed

    Sizova, Irina; Greiner, Andre; Awasthi, Mayanka; Kateriya, Suneel; Hegemann, Peter

    2013-03-01

    The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide range of tools for genetic manipulation have been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here, we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc-finger nucleases (ZFNs). Our approach includes (i) design of gene-specific ZFNs using available online tools, (ii) evaluation of the designed ZFNs in a Chlamydomonas in situ model system, (iii) optimization of ZFN activity by modification of the nuclease domain, and (iv) application of the most suitable enzymes for mutagenesis of an endogenous gene. Initially, we designed a set of ZFNs to target the COP3 gene that encodes the light-activated ion channel channelrhodopsin-1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non-functional aminoglycoside 3'-phosphotransferase VIII (aphVIII) selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co-transformation of this recipient strain with the engineered ZFNs and an aphVIII DNA template, we were able to restore marker activity and select paromomycin-resistant (Pm-R) clones with expressing nucleases. Of these Pm-R clones, 1% also contained a modified COP3 locus. In cases where cells were co-transformed with a modified COP3 template, the COP3 locus was specifically modified by homologous recombination between COP3 and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes in Chlamydomonas.

  9. Molecular cloning and characterization of a gene encoding RING zinc finger ankyrin protein from drought-tolerant Artemisia desertorum.

    PubMed

    Yang, Xiuhong; Sun, Chao; Hu, Yuanlei; Lin, Zhongping

    2008-03-01

    A RING zinc finger ankyrin protein gene,designated AdZFP1, was isolated from drought-tolerant Artemisia desertorum Spreng by mRNA differential display and RACE. Its cDNA was 1723 bp and encoded a putative protein of 445 amino acids with a predicted molecular mass of 47.9 kDa and an isoelectric point (pI) of 7.49. A typical C3HC4- type RING finger domain was found at the C-terminal region of the AdZFP1 protein,and several groups of ankyrin repeats were found at the N-terminal region. Alignments of amino acid sequence showed that AdZFP1 was 66% identical to the Arabidopsis thaliana putative RING zinc finger ankyrin protein AAN31869. Transcriptional analysis showed that AdZFP1 was inducible under drought stress in root,stem and leaf of the plant.Semi-quantitative reverse- transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the transcript of AdZFP1 was strongly induced by exogenous abscisic acid (ABA) and also by salinity,cold and heat to some extent. Overexpression of the AdZFP1 gene in transgenic tobacco enhanced their tolerance to drought stress.

  10. Solution Structure of the Cuz1 AN1 Zinc Finger Domain: An Exposed LDFLP Motif Defines a Subfamily of AN1 Proteins

    PubMed Central

    Sun, Zhen-Yu J.; Bhanu, Meera K.; Allan, Martin G.; Arthanari, Haribabu; Wagner, Gerhard; Hanna, John

    2016-01-01

    Zinc binding domains are common and versatile protein structural motifs that mediate diverse cellular functions. Among the many structurally distinct families of zinc finger (ZnF) proteins, the AN1 domain remains poorly characterized. Cuz1 is one of two AN1 ZnF proteins in the yeast S. cerevisiae, and is a stress-inducible protein that functions in protein degradation through direct interaction with the proteasome and Cdc48. Here we report the solution structure of the Cuz1 AN1 ZnF which reveals a compact C6H2 zinc-coordinating domain that resembles a two-finger hand holding a tri-helical clamp. A central phenylalanine residue sits between the two zinc-coordinating centers. The position of this phenylalanine, just before the penultimate zinc-chelating cysteine, is strongly conserved from yeast to man. This phenylalanine shows an exceptionally slow ring-flipping rate which likely contributes to the high rigidity and stability of the AN1 domain. In addition to the zinc-chelating residues, sequence analysis of Cuz1 indicates a second highly evolutionarily conserved motif. This LDFLP motif is shared with three human proteins—Zfand1, AIRAP, and AIRAP-L—the latter two of which share similar cellular functions with Cuz1. The LDFLP motif, while embedded within the zinc finger domain, is surface exposed, largely uninvolved in zinc chelation, and not required for the overall fold of the domain. The LDFLP motif was dispensable for Cuz1's major known functions, proteasome- and Cdc48-binding. These results provide the first structural characterization of the AN1 zinc finger domain, and suggest that the LDFLP motif may define a sub-family of evolutionarily conserved AN1 zinc finger proteins. PMID:27662200

  11. A survey of well conserved families of C2H2 zinc-finger genes in Daphnia

    PubMed Central

    2010-01-01

    Background A recent comparative genomic analysis tentatively identified roughly 40 orthologous groups of C2H2 Zinc-finger proteins that are well conserved in "bilaterians" (i.e. worms, flies, and humans). Here we extend that analysis to include a second arthropod genome from the crustacean, Daphnia pulex. Results Most of the 40 orthologous groups of C2H2 zinc-finger proteins are represented by just one or two proteins within each of the previously surveyed species. Likewise, Daphnia were found to possess a similar number of orthologs for all of these small orthology groups. In contrast, the number of Sp/KLF homologs tends to be greater and to vary between species. Like the corresponding mammalian Sp/KLF proteins, most of the Drosophila and Daphnia homologs can be placed into one of three sub-groups: Class I-III. Daphnia were found to have three Class I proteins that roughly correspond to their Drosophila counterparts, dSP1, btd, CG5669, and three Class II proteins that roughly correspond to Luna, CG12029, CG9895. However, Daphnia have four additional KLF-Class II proteins that are most similar to the vertebrate KLF1/2/4 proteins, a subset not found in Drosophila. Two of these four proteins are encoded by genes linked in tandem. Daphnia also have three KLF-Class III members, one more than Drosophila. One of these is a likely Bteb2 homolog, while the other two correspond to Cabot and KLF13, a vertebrate homolog of Cabot. Conclusion Consistent with their likely roles as fundamental determinants of bilaterian form and function, most of the 40 groups of C2H2 zinc-finger proteins are conserved in kind and number in Daphnia. However, the KLF family includes several additional genes that are most similar to genes present in vertebrates but missing in Drosophila. PMID:20433734

  12. The N-terminal zinc finger domain of Tgf2 transposase contributes to DNA binding and to transposition activity

    PubMed Central

    Jiang, Xia-Yun; Hou, Fei; Shen, Xiao-Dan; Du, Xue-Di; Xu, Hai-Li; Zou, Shu-Ming

    2016-01-01

    Active Hobo/Activator/Tam3 (hAT) transposable elements are rarely found in vertebrates. Previously, goldfish Tgf2 was found to be an autonomously active vertebrate transposon that is efficient at gene-transfer in teleost fish. However, little is known about Tgf2 functional domains required for transposition. To explore this, we first predicted in silico a zinc finger domain in the N-terminus of full length Tgf2 transposase (L-Tgf2TPase). Two truncated recombinant Tgf2 transposases with deletions in the N-terminal zinc finger domain, S1- and S2-Tgf2TPase, were expressed in bacteria from goldfish cDNAs. Both truncated Tgf2TPases lost their DNA-binding ability in vitro, specifically at the ends of Tgf2 transposon than native L-Tgf2TPase. Consequently, S1- and S2-Tgf2TPases mediated gene transfer in the zebrafish genome in vivo at a significantly (p < 0.01) lower efficiency (21%–25%), in comparison with L-Tgf2TPase (56% efficiency). Compared to L-Tgf2TPase, truncated Tgf2TPases catalyzed imprecise excisions with partial deletion of TE ends and/or plasmid backbone insertion/deletion. The gene integration into the zebrafish genome mediated by truncated Tgf2TPases was imperfect, creating incomplete 8-bp target site duplications at the insertion sites. These results indicate that the zinc finger domain in Tgf2 transposase is involved in binding to Tgf2 terminal sequences, and loss of those domains has effects on TE transposition. PMID:27251101

  13. Transcriptome wide identification, phylogenetic analysis, and expression profiling of zinc-finger transcription factors from Crocus sativus L.

    PubMed

    Malik, Aubid Hussain; Ashraf, Nasheeman

    2017-02-28

    Crocus sativus belongs to Iridaceae family and is the only plant species which produces apocarotenoids like crocin, picrocrocin, and safranal in significant quantities. Besides their organoleptic properties, Crocus apocarotenoids have been found to possess remarkable pharmacological potential. Although apocarotenoid biosynthetic pathway has been worked out to a great degree, but the mechanism that regulates the tissue and developmental stage-specific production of Crocus apocarotenoids is not known. To identify the genes regulating apocarotenoid biosynthesis in Crocus, transcriptome wide identification of zinc-finger transcription factors was undertaken. 81 zinc-finger transcription factors were identified which grouped into eight subfamilies. C2H2, C3H, and AN20/AN1 were the major subfamilies with 29, 20, and 14 members, respectively. Expression profiling revealed CsSAP09 as a potential candidate for regulation of apocarotenoid biosynthesis. CsSAP09 was found to be highly expressed in stigma at anthesis stage corroborating with the accumulation pattern of apocarotenoids. CsSAP09 was nuclear localized and activated reporter gene transcription in yeast. It was highly induced in response to oxidative, salt and dehydration stresses, ABA and methyl jasmonate. Furthermore, upstream region of CsSAP09 was found to contain stress and light responsive elements. To our knowledge, this is the first report on the study of a gene family in C. sativus and may provide basic insights into the putative role of zinc finger genes. It may also serve as a valuable resource for functional characterization of these genes aimed towards unraveling their role in regulation of apocarotenoid biosynthesis.

  14. A Soybean C2H2-Type Zinc Finger Gene GmZF1 Enhanced Cold Tolerance in Transgenic Arabidopsis

    PubMed Central

    Ma, Xue-Feng; Xu, Zhao-Shi; Liu, Meng-Meng; Shan, Shu-Guang; Cheng, Xian-Guo

    2014-01-01

    Zinc finger proteins were involved in response to different environmental stresses in plant species. A typical Cys2/His2-type (C2H2-type) zinc finger gene GmZF1 from soybean was isolated and was composed of 172 amino acids containing two conserved C2H2-type zinc finger domains. Phylogenetic analysis showed that GmZF1 was clustered on the same branch with six C2H2-type ZFPs from dicotyledonous plants excepting for GsZFP1, and distinguished those from monocotyledon species. The GmZF1 protein was localized at the nucleus, and has specific binding activity with EP1S core sequence, and nucleotide mutation in the core sequence of EPSPS promoter changed the binding ability between GmZF1 protein and core DNA element, implying that two amino acid residues, G and C boxed in core sequence TGACAGTGTCA possibly play positive regulation role in recognizing DNA-binding sites in GmZF1 proteins. High accumulation of GmZF1 mRNA induced by exogenous ABA suggested that GmZF1 was involved in an ABA-dependent signal transduction pathway. Over-expression of GmZF1 significantly improved the contents of proline and soluble sugar and decreased the MDA contents in the transgenic lines exposed to cold stress, indicating that transgenic Arabidopsis carrying GmZF1 gene have adaptive mechanisms to cold stress. Over-expression of GmZF1 also increased the expression of cold-regulated cor6.6 gene by probably recognizing protein-DNA binding sites, suggesting that GmZF1 from soybean could enhance the tolerance of Arabidopsis to cold stress by regulating expression of cold-regulation gene in the transgenic Arabidopsis. PMID:25286048

  15. Arsenite Targets the Zinc Finger Domains of Tet Proteins and Inhibits Tet-Mediated Oxidation of 5-Methylcytosine.

    PubMed

    Liu, Shuo; Jiang, Ji; Li, Lin; Amato, Nicholas J; Wang, Zi; Wang, Yinsheng

    2015-10-06

    Arsenic toxicity is a serious public health problem worldwide that brings more than 100 million people into the risk of arsenic exposure from groundwater and food contamination. Although there is accumulating evidence linking arsenic exposure with aberrant cytosine methylation in the global genome or at specific genomic loci, very few have investigated the impact of arsenic on the oxidation of 5-methylcytosine (5-mC) mediated by the Ten-eleven translocation (Tet) family of proteins. Owing to the high binding affinity of As(III) toward cysteine residues, we reasoned that the highly conserved C3H-type zinc fingers situated in Tet proteins may constitute potential targets for arsenic binding. Herein, we found that arsenite could bind directly to the zinc fingers of Tet proteins in vitro and in cells, and this interaction substantially impaired the catalytic efficiency of Tet proteins in oxidizing 5-mC to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC). Treatments with arsenite also led to a dose-dependent decrease in the level of 5-hmC, but not 5-mC, in DNA isolated from HEK293T cells overexpressing the catalytic domain of any of the three Tet proteins and from mouse embryonic stem cells. Together, our study unveiled, for the first time, that arsenite could alter epigenetic signaling by targeting the zinc fingers of Tet proteins and perturbing the Tet-mediated oxidation of 5-mC in vitro and in cells. Our results offer important mechanistic understanding of arsenic epigenotoxicity and carcinogenesis in mammalian systems and may lead to novel approaches for the chemoprevention of arsenic toxicity.

  16. The single Cys2-His2 zinc finger domain of the GAGA protein flanked by basic residues is sufficient for high-affinity specific DNA binding.

    PubMed Central

    Pedone, P V; Ghirlando, R; Clore, G M; Gronenborn, A M; Felsenfeld, G; Omichinski, J G

    1996-01-01

    Specific DNA binding to the core consensus site GAGAGAG has been shown with an 82-residue peptide (residues 310-391) taken from the Drosophila transcription factor GAGA. Using a series of deletion mutants, it was demonstrated that the minimal domain required for specific binding (residues 310-372) includes a single zinc finger of the Cys2-His2 family and a stretch of basic amino acids located on the N-terminal end of the zinc finger. In gel retardation assays, the specific binding seen with either the peptide or the whole protein is zinc dependent and corresponds to a dissociation constant of approximately 5 x 10(-9) M for the purified peptide. It has previously been thought that a single zinc finger of the Cys2-His2 family is incapable of specific, high-affinity binding to DNA. The combination of an N-terminal basic region with a single Cys2-His2 zinc finger in the GAGA protein can thus be viewed as a novel DNA binding domain. This raises the possibility that other proteins carrying only one Cys2-His2 finger are also capable of high-affinity specific binding to DNA. Images Fig. 2 Fig. 3 Fig. 4 PMID:8610125

  17. Structural analysis of MED-1 reveals unexpected diversity in the mechanism of DNA recognition by GATA-type zinc finger domains.

    PubMed

    Lowry, Jason A; Gamsjaeger, Roland; Thong, Sock Yue; Hung, Wendy; Kwan, Ann H; Broitman-Maduro, Gina; Matthews, Jacqueline M; Maduro, Morris; Mackay, Joel P

    2009-02-27

    MED-1 is a member of a group of divergent GATA-type zinc finger proteins recently identified in several species of Caenorhabditis. The med genes are transcriptional regulators that are involved in the specification of the mesoderm and endoderm precursor cells in nematodes. Unlike other GATA-type zinc fingers that recognize the consensus sequence (A/C/T)GATA(A/G), the MED-1 zinc finger (MED1zf) binds the larger and atypical site GTATACT(T/C)(3). We have examined the basis for this unusual DNA specificity using a range of biochemical and biophysical approaches. Most strikingly, we show that although the core of the MED1zf structure is similar to that of GATA-1, the basic tail C-terminal to the zinc finger unexpectedly adopts an alpha-helical structure upon binding DNA. This additional helix appears to contact the major groove of the DNA, making contacts that explain the extended DNA consensus sequence observed for MED1zf. Our data expand the versatility of DNA recognition by GATA-type zinc fingers and perhaps shed new light on the DNA-binding properties of mammalian GATA factors.

  18. A novel zinc-finger HIT protein with an additional PAPA-1-like region from Suaeda liaotungensis K. enhanced transgenic Arabidopsis drought and salt stresses tolerance.

    PubMed

    Li, Xiao-Lan; Hu, Yu-Xin; Yang, Xing; Yu, Xiao-Dong; Li, Qiu-Li

    2014-12-01

    Zinc-finger HIT belongs to the cross-brace zinc finger protein family and is involved in the regulation of plant defense and stress responses. In this study, we cloned a full-length zinc-finger HIT gene (1,377 bp) named SlPAPA1 using polymerase chain reaction from Suaeda liaotungensis K. and investigated its function by overexpression in transgenic Arabidopsis. SlPAPA1 contains a zinc-finger HIT domain and a Pim-1-associated protein-1 (PAP-1)-associated protein-1-like (PAPA-1-like) conserved region. Its expression in S. liaotungensis was induced by drought, high-salt, and cold (4 °C) stresses and by abscisic acid (ABA). Subcellular localization experiments in onion epidermal cells indicated that SlPAPA1 is localized in the nucleus. Yeast-one hybrid assays showed that SlPAPA1 functions as a transcriptional activator. SlPAPA1 transgenic Arabidopsis displayed a higher survival ratio and lower rate of water loss under drought stress; a higher germination ratio, higher survival ratio, and lower root inhibition rate under salt stress; and a lower germination ratio and root inhibition rate under ABA treatment, compared with wild-type Arabidopsis. These results suggested that SlPAPA1 functions as a stress-responsive zinc-finger HIT protein involved in the ABA-dependent signaling pathway and may have potential applications in transgenic breeding to enhance crops abiotic stress tolerances.

  19. Identification of off-target cleavage sites of zinc finger nucleases and TAL effector nucleases using predictive models.

    PubMed

    Fine, Eli J; Cradick, Thomas J; Bao, Gang

    2014-01-01

    Using engineered nucleases, such as Zinc Finger Nucleases (ZFNs) or Transcription Activator-Like Effector Nucleases (TALENs), to make targeted genomic modifications has become a common technique to create new model organisms and custom cell lines, and has shown great promise for disease treatment. However, these nucleases have the potential for off-target cleavage that could confound interpretation of experimental results and be detrimental for therapeutic use. Here, we describe a method to test for nuclease cleavage at potential off-target sites predicted by bioinformatics models.

  20. Double-finger-gate controlled spin-resolved resonant quantum transport in the presence of a Rashba-Zeeman gap.

    PubMed

    Tang, Chi-Shung; Tseng, Shu-Ting; Gudmundsson, Vidar; Cheng, Shun-Jen

    2015-03-04

    We investigate double finger gate (DFG) controlled spin-resolved resonant transport properties in an n-type quantum channel with a Rashba-Zeeman (RZ) subband energy gap. By appropriately tuning the DFG in the strong Rashba coupling regime, resonant state structures in conductance can be found that are sensitive to the length of the DFG system. Furthermore, a hole-like bound state feature below the RZ gap and an electron-like quasi-bound state feature at the threshold of the upper spin branch can be found that is insensitive to the length of the DFG system.

  1. Regulation of Nitrogen Metabolism by GATA Zinc Finger Transcription Factors in Yarrowia lipolytica

    PubMed Central

    2017-01-01

    ABSTRACT Fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeast Yarrowia lipolytica, growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors on lipid metabolism in Y. lipolytica. Deletion of the GATA transcription factor genes gzf3 and gzf2 resulted in nitrogen source-specific growth defects and greater accumulation of lipids when the cells were growing on a simple nitrogen source. Deletion of gzf1, which is most similar to activators of genes repressed by nitrogen catabolite repression in filamentous ascomycetes, did not affect growth on the nitrogen sources tested. We examined gene expression of wild-type and GATA transcription factor mutants on simple and complex nitrogen sources and found that expression of enzymes involved in malate metabolism, beta-oxidation, and ammonia utilization are strongly upregulated on a simple nitrogen source. Deletion of gzf3 results in overexpression of genes with GATAA sites in their promoters, suggesting that it acts as a repressor, while gzf2 is required for expression of ammonia utilization genes but does not grossly affect the transcription level of genes predicted to be controlled by nitrogen catabolite repression. Both GATA transcription factor mutants exhibit decreased expression of genes controlled by carbon catabolite repression via the repressor mig1, including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism. IMPORTANCE Nitrogen source is commonly used to control lipid production in industrial fungi. Here we identified regulators of nitrogen catabolite repression in the oleaginous yeast Y

  2. Genome editing with CompoZr custom zinc finger nucleases (ZFNs).

    PubMed

    Hansen, Keith; Coussens, Matthew J; Sago, Jack; Subramanian, Shilpi; Gjoka, Monika; Briner, Dave

    2012-06-14

    Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes. Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA. Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency. While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site. The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the

  3. Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance

    PubMed Central

    Shahbazi Dastjerdeh, Mansoureh; Kouhpayeh, Shirin; Sabzehei, Faezeh; Khanahmad, Hossein; Salehi, Mansour; Mohammadi, Zahra; Shariati, Laleh; Hejazi, Zahra; Rabiei, Parisa; Manian, Mostafa

    2016-01-01

    Background: The evolution of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) has been accelerated recently by the indiscriminate application of antibiotics. Antibiotic resistance has challenged the success of medical interventions and therefore is considered a hazardous threat to human health. Objectives: The present study aimed to describe the use of zinc finger nuclease (ZFN) technology to target and disrupt a plasmid-encoded β-lactamase, which prevents horizontal gene transfer-mediated evolution of ARBs. Materials and Methods: An engineered ZFN was designed to target a specific sequence in the ampicillin resistance gene (ampR) of the pTZ57R plasmid. The Escherichia coli bacteria already contained the pZFN kanamycin-resistant (kanaR) plasmid as the case or the pP15A, kanaR empty vector as the control, were transformed with the pTZ57R; the ability of the designed ZFN to disrupt the β-lactamase gene was evaluated with the subsequent disturbed ability of the bacteria to grow on ampicillin (amp) and ampicillin-kanamycin (amp-kana)-containing media. The effect of mild hypothermia on the ZFN gene targeting efficiency was also evaluated. Results: The growth of bacteria in the case group on the amp and amp-kana-containing media was significantly lower compared with the control group at 37°C (P < 0.001). Despite being more efficient in hypothermic conditions at 30°C (P < 0.001), there were no significant associations between the incubation temperature and the ZFN gene targeting efficiency. Conclusions: Our findings revealed that the ZFN technology could be employed to overcome ampicillin resistance by the targeted disruption of the ampicillin resistance gene, which leads to inactivation of β-lactam synthesis. Therefore, ZFN technology could be engaged to decrease the antibiotic resistance issue with the construction of a ZFN archive against different ARGs. To tackle the resistance issue at the environmental level, recombinant phages

  4. ExpandplusCrystal Structures of Poly(ADP-ribose) Polymerase-1 (PARP-1) Zinc Fingers Bound to DNA

    SciTech Connect

    M Langelier; J Planck; S Roy; J Pascal

    2011-12-31

    Poly(ADP-ribose) polymerase-1 (PARP-1) has two homologous zinc finger domains, Zn1 and Zn2, that bind to a variety of DNA structures to stimulate poly(ADP-ribose) synthesis activity and to mediate PARP-1 interaction with chromatin. The structural basis for interaction with DNA is unknown, which limits our understanding of PARP-1 regulation and involvement in DNA repair and transcription. Here, we have determined crystal structures for the individual Zn1 and Zn2 domains in complex with a DNA double strand break, providing the first views of PARP-1 zinc fingers bound to DNA. The Zn1-DNA and Zn2-DNA structures establish a novel, bipartite mode of sequence-independent DNA interaction that engages a continuous region of the phosphodiester backbone and the hydrophobic faces of exposed nucleotide bases. Biochemical and cell biological analysis indicate that the Zn1 and Zn2 domains perform distinct functions. The Zn2 domain exhibits high binding affinity to DNA compared with the Zn1 domain. However, the Zn1 domain is essential for DNA-dependent PARP-1 activity in vitro and in vivo, whereas the Zn2 domain is not strictly required. Structural differences between the Zn1-DNA and Zn2-DNA complexes, combined with mutational and structural analysis, indicate that a specialized region of the Zn1 domain is re-configured through the hydrophobic interaction with exposed nucleotide bases to initiate PARP-1 activation.

  5. The mouse genome displays highly dynamic populations of KRAB-zinc finger protein genes and related genetic units

    PubMed Central

    Kauzlaric, Annamaria; Ecco, Gabriela; Cassano, Marco; Duc, Julien; Imbeault, Michael; Trono, Didier

    2017-01-01

    KRAB-containing poly-zinc finger proteins (KZFPs) constitute the largest family of transcription factors encoded by mammalian genomes, and growing evidence indicates that they fulfill functions critical to both embryonic development and maintenance of adult homeostasis. KZFP genes underwent broad and independent waves of expansion in many higher vertebrates lineages, yet comprehensive studies of members harbored by a given species are scarce. Here we present a thorough analysis of KZFP genes and related units in the murine genome. We first identified about twice as many elements than previously annotated as either KZFP genes or pseudogenes, notably by assigning to this family an entity formerly considered as a large group of Satellite repeats. We then could delineate an organization in clusters distributed throughout the genome, with signs of recombination, translocation, duplication and seeding of new sites by retrotransposition of KZFP genes and related genetic units (KZFP/rGUs). Moreover, we harvested evidence indicating that closely related paralogs had evolved through both drifting and shifting of sequences encoding for zinc finger arrays. Finally, we could demonstrate that the KAP1-SETDB1 repressor complex tames the expression of KZFP/rGUs within clusters, yet that the primary targets of this regulation are not the KZFP/rGUs themselves but enhancers contained in neighboring endogenous retroelements and that, underneath, KZFPs conserve highly individualized patterns of expression. PMID:28334004

  6. Expression of myeloid zinc finger 1 and the correlation to clinical aspects of oral squamous cell carcinoma.

    PubMed

    Ko, Chung-Po; Yang, Li-Chiu; Chen, Chih-Jung; Yeh, Kun-Tu; Lin, Shu-Hui; Yang, Shun-Fa; Chen, Mu-Kuan; Lin, Chiao-Wen

    2015-09-01

    The myeloid zinc finger 1 (MZF1) is a zinc finger transcription factor which regulates myeloid differentiation and oncogenesis. However, little information is available concerning the MZF1 expression in oral squamous cell carcinoma (OSCC) and its correlation with patients' prognosis. We detected the expression of MZF1 in 274 patients with OSCC using tissue microarrays (TMAs) and evaluated the associations between nuclear MZF1 expression and the clinical parameters of OSCC patients. We found that nuclear MZF1 expression was present in 190/274 (69.3 %) cases, and loss of nuclear expression of MZF1 was associated with more advanced clinical stages (p = 0.011) and larger tumor size (p = 0.002), but not associated with positive lymph node metastasis and distal metastasis. Importantly, tongue squamous cell carcinomas (SCC) patients with negative nuclear MZF1 expression had significantly worse overall survival rates (log-rank test, p = 0.028). In conclusion, our results revealed that the loss of nuclear expression of MZF1 in OSCC samples can predict the progression of OSCC and the survival of OSCC patients in Taiwan.

  7. Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase.

    PubMed

    Yoshida, Wataru; Kezuka, Aki; Murakami, Yoshiyuki; Lee, Jinhee; Abe, Koichi; Motoki, Hiroaki; Matsuo, Takafumi; Shimura, Nobuaki; Noda, Mamoru; Igimi, Shizunobu; Ikebukuro, Kazunori

    2013-11-01

    An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268-luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF-luciferase fusion protein. By means of the automatic analyzer with ZF-luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0×10 to 1.0×10(6) copies.

  8. Repeatable Construction Method for Engineered Zinc Finger Nuclease Based on Overlap Extension PCR and TA-Cloning

    PubMed Central

    Fujii, Wataru; Kano, Kiyoshi; Sugiura, Koji; Naito, Kunihiko

    2013-01-01

    Zinc finger nuclease (ZFN) is a useful tool for endogenous site-directed genome modification. The development of an easier, less expensive and repeatedly usable construction method for various sequences of ZFNs should contribute to the further widespread use of this technology. Here, we establish a novel construction method for ZFNs. Zinc finger (ZF) fragments were synthesized by PCR using short primers coding DNA recognition helices of the ZF domain. DNA-binding domains composed of 4 to 6 ZFs were synthesized by overlap extension PCR of these PCR products, and the DNA-binding domains were joined with a nuclease vector by TA cloning. The short primers coding unique DNA recognition helices can be used repeatedly for other ZFN constructions. By using this novel OLTA (OverLap extension PCR and TA-cloning) method, arbitrary ZFN vectors were synthesized within 3 days, from the designing to the sequencing of the vector. Four different ZFN sets synthesized by OLTA showed nuclease activities at endogenous target loci. Genetically modified mice were successfully generated using ZFN vectors constructed by OLTA. This method, which enables the construction of intended ZFNs repeatedly and inexpensively in a short period of time, should contribute to the advancement of ZFN technology. PMID:23536890

  9. MYC associated zinc finger protein promotes the invasion and metastasis of hepatocellular carcinoma by inducing epithelial mesenchymal transition

    PubMed Central

    Liu, Wei; Ren, Yuan; Bei, Chunhua; Qin, Linyuan; Miao, Xueyan; Tang, Fen; Tang, Guifang; Tan, Shengkui

    2016-01-01

    MYC associated zinc finger protein (MAZ) plays a key role in regulation of gene expression and tumor development. Studies have shown that deregulated expression of MAZ is closely related to the progression of tumors such as glioblastoma, breast cancer, prostate cancer and liposarcoma. However, the role of MAZ in hepatocellular carcinoma (HCC) has not been fully elucidated. Here, we found that expression of MAZ was increased in HCC and correlated to the distant metastasis of HCC. Moreover, we found that MAZ had a relationship with zinc finger E-box binding homeobox 1 and 2 (ZEB1 and ZEB2), two important mesenchymal markers in epithelial-mesenchymal transition (EMT) that were over-expressed in HCC. After knocking-down MAZ expression in HCC cell lines using RNA interruption, HCC cell proliferation, tumorigenesis, invasion and migration were significantly inhibited. In addition, we found that expression of other EMT markers was also changed besides ZEB1 and ZEB2 by decreasing MAZ expression, both detected in vivo and in vitro assays. Therefore, we conclude that MAZ can promote the invasion and metastasis of HCC by inducing EMT. PMID:27861158

  10. Rearrangement of side-chains in a Zif268 mutant highlights the complexities of zinc finger-DNA recognition.

    PubMed

    Miller, J C; Pabo, C O

    2001-10-19

    Structural and biochemical studies of Cys(2)His(2) zinc finger proteins initially led several groups to propose a "recognition code" involving a simple set of rules relating key amino acid residues in the zinc finger protein to bases in its DNA site. One recent study from our group, involving geometric analysis of protein-DNA interactions, has discussed limitations of this idea and has shown how the spatial relationship between the polypeptide backbone and the DNA helps to determine what contacts are possible at any given position in a protein-DNA complex. Here we report a study of a zinc finger variant that highlights yet another source of complexity inherent in protein-DNA recognition. In particular, we find that mutations can cause key side-chains to rearrange at the protein-DNA interface without fundamental changes in the spatial relationship between the polypeptide backbone and the DNA. This is clear from a simple analysis of the binding site preferences and co-crystal structures for the Asp20-->Ala point mutant of Zif268. This point mutation in finger one changes the specificity of the protein from GCG TGG GCG to GCG TGG GC(G/T), and we have solved crystal structures of the D20A mutant bound to both types of sites. The structure of the D20A mutant bound to the GCG site reveals that contacts from key residues in the recognition helix are coupled in complex ways. The structure of the complex with the GCT site also shows an important new water molecule at the protein-DNA interface. These side-chain/side-chain interactions, and resultant changes in hydration at the interface, affect binding specificity in ways that cannot be predicted either from a simple recognition code or from analysis of spatial relationships at the protein-DNA interface. Accurate computer modeling of protein-DNA interfaces remains a challenging problem and will require systematic strategies for modeling side-chain rearrangements and change in hydration.

  11. Zinc Finger Takes on a Whole New Meaning: Reducing and Monitoring Zinc Blanks in the Isotope Lab

    NASA Astrophysics Data System (ADS)

    Wilkes, E. B.; Wasylenki, L. E.; Anbar, A. D.

    2010-12-01

    In terms of avoiding contamination, zinc is one of the most difficult elements to study isotopically. The reason for this is that zinc stearate is a very common mold release agent in the production of plastics, including those most often used in isotope geochemistry clean labs. While polyethylene bottles, polypropylene centrifuge tubes, pipette tips, and Kimwipes are all potential sources of contaminant zinc, by far the largest amount of zinc is introduced to the laboratory by gloves. Most items can be effectively rid of zinc by soaking in dilute hydrochloric acid, but gloves cannot be cleaned easily, and use of gloves can quickly lead to contamination on many surfaces throughout the lab. We recently conducted several experiments in which dissolved zinc was partly adsorbed onto synthetic Mn oxyhydroxide particles. The dissolved and adsorbed pools were separated by filtration, purified with ion exchange chemistry, and analyzed for isotope composition by MC-ICP-MS. We used a commercially purchased ICP standard solution both as our standard (delta66/64Zn = 0) and as the source of the zinc in the experiments. Whenever gloves were worn during purification, process blanks contained as much as 150 ng Zn, and both the dissolved and adsorbed pools of zinc came out enriched in heavy isotopes relative to the starting pool, contrary to our expectation of mass balance. When gloves were not worn, blanks were <10 ng, and, as expected, one pool of Zn was lighter and one heavier than the standard. Zinc leached from two different brands of vinyl gloves, including one brand recommended to us for being “low” in zinc, measured +10‰ relative to our standard. We therefore concluded that glove zinc contaminated most of our experimental samples. We were only able to see such clear evidence of contamination because (1) we were doing an experiment in which we expected one light and one heavy pool of zinc compared to our standard, and (2) we happened to use an ICP standard solution for

  12. STP1, a gene involved in pre-tRNA processing, encodes a nuclear protein containing zinc finger motifs.

    PubMed Central

    Wang, S S; Stanford, D R; Silvers, C D; Hopper, A K

    1992-01-01

    STP1 is an unessential yeast gene involved in the removal of intervening sequences from some, but not all, families of intervening sequence-containing pre-tRNAs. Previously, we proposed that STP1 might encode a product that generates pre-tRNA conformations efficiently recognized by tRNA-splicing endonuclease. To test the predictions of this model, we have undertaken a molecular analysis of the STP1 gene and its products. The STP1 locus is located on chromosome IV close to at least two other genes involved in RNA splicing: PRP3 and SPP41. The STP1 open reading frame (ORF) could encode a peptide of 64,827 Da; however, inspection of putative transcriptional and translational regulatory signals and mapping of the 5' ends of mRNA provide evidence that translation of the STP1 ORF usually initiates at a second AUG to generate a protein of 58,081 Da. The STP1 ORF contains three putative zinc fingers. The first of these closely resembles both the DNA transcription factor consensus and the Xenopus laevis p43 RNA-binding protein consensus. The third motif more closely resembles the fingers found in spliceosomal proteins. Employing antisera to the endogenous STP1 protein and to STP1-LacZ fusion proteins, we show that the STP1 protein is localized to nuclei. The presence of zinc finger motifs and the nuclear location of the STP1 protein support the model that this gene product is involved directly in pre-tRNA splicing. Images PMID:1588961

  13. Tailor-Made Zinc-Finger Transcription Factors Activate FLO11 Gene Expression with Phenotypic Consequences in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Shieh, Jia-Ching; Cheng, Yu-Che; Su, Mao-Chang; Moore, Michael; Choo, Yen; Klug, Aaron

    2007-01-01

    Cys2His2 zinc fingers are eukaryotic DNA-binding motifs, capable of distinguishing different DNA sequences, and are suitable for engineering artificial transcription factors. In this work, we used the budding yeast Saccharomyces cerevisiae to study the ability of tailor-made zinc finger proteins to activate the expression of the FLO11 gene, with phenotypic consequences. Two three-finger peptides were identified, recognizing sites from the 5′ UTR of the FLO11 gene with nanomolar DNA-binding affinity. The three-finger domains and their combined six-finger motif, recognizing an 18-bp site, were fused to the activation domain of VP16 or VP64. These transcription factor constructs retained their DNA-binding ability, with the six-finger ones being the highest in affinity. However, when expressed in haploid yeast cells, only one three-finger recombinant transcription factor was able to activate the expression of FLO11 efficiently. Unlike in the wild-type, cells with such transcriptional activation displayed invasive growth and biofilm formation, without any requirement for glucose depletion. The VP16 and VP64 domains appeared to act equally well in the activation of FLO11 expression, with comparable effects in phenotypic alteration. We conclude that the functional activity of tailor-made transcription factors in cells is not easily predicted by the in vitro DNA-binding activity. PMID:17710146

  14. Structural and dynamical characterization of the Miz-1 zinc fingers 5-8 by solution-state NMR.

    PubMed

    Bernard, David; Bédard, Mikaël; Bilodeau, Josée; Lavigne, Pierre

    2013-10-01

    Myc-interacting zinc finger protein-1 (Miz-1) is a BTB/POZ transcription factor that activates the transcription of cytostatic genes, such as p15(INK4B) or p21(CIP1). The C-terminus of Miz-1 contains 13 consensus C2H2 zinc finger domains (ZF). ZFs 1-4 have been shown to interact with SMAD3/4, while the remaining ZFs are expected to bind the promoters of target genes. We have noted unusual features in ZF 5 and the linker between ZFs 5 and 6. Indeed, a glutamate is found instead of the conserved basic residue two positions before the second zinc-coordinating histidine on the ZF 5 helix, and the linker sequence is DTDKE in place of the classical TGEKP sequence. In a canonical ββα fold, such unusual primary structure elements should cause severe electrostatic repulsions. In this context, we have characterized the structure and the dynamics of a Miz-1 construct comprising ZFs 5-8 (Miz 5-8) by solution-state NMR. Whilst ZFs 5, 7 and 8 were shown to adopt the classical ββα fold for C2H2 ZFs, the number of long-range NOEs was insufficient to define a classical fold for ZF 6. We show by using (15)N-relaxation dispersion experiments that this lack of NOEs is due to the presence of extensive motions on the μs-ms timescale. Since this negatively charged region would have to be located near the phosphodiester backbone in a DNA complex, we propose that in addition to promoting conformational searches, it could serve as a hinge region to keep ZFs 1-4 away from DNA.

  15. Zinc finger proteins as templates for metal ion exchange: Substitution effects on the C-finger of HIV nucleocapsid NCp7 using M(chelate) species (M=Pt, Pd, Au).

    PubMed

    de Paula, Queite A; Mangrum, John B; Farrell, Nicholas P

    2009-10-01

    The interactions of monofunctional [MCl(chelate)] compounds (M=Pt(II), Pd(II) or Au(III) and chelate=diethylenetriamine, dien or 2,2',2''-terpyridine, terpy) with the C-terminal finger of the HIV nucleocapsid NCp7 zinc finger (ZF) were studied by mass spectrometry and circular dichroism spectroscopy. In the case of [M(dien)] species, Pt(II) and Pd(II) behaved in a similar fashion with evidence of adducts caused by displacement of Pt-Cl or Pd-Cl by zinc-bound thiolate. Labilization, presumably under the influence of the strong trans influence of thiolate, resulted in loss of ligand (dien) as well as zinc ejection and formation of species with only Pd(II) or Pt(II) bound to the finger. For both Au(III) compounds the reactions were very fast and only "gold fingers" with no ancillary ligands were observed. For all terpyridine compounds ligand scrambling and metal exchange occurred with formation of [Zn(terpy)](2+). The results conform well to those proposed from the study of model Zn compounds such as N,N'-bis(2-mercapto-ethyl)-1,4-diazacycloheptanezinc(II), [Zn(bme-dach)](2). The possible structures of the adducts formed are discussed and, for Pt(II) and Pd(II), the evidence for possible expansion of the zinc coordination sphere from four- to five-coordinate is discussed. This observation reinforces the possibility of change in geometry for zinc in biology, even in common "structural" sites in metalloenzymes. The results further show that the extent and rate of zinc displacement by inorganic compounds can be modulated by the nature (metal, ligands) of the reacting compound.

  16. Concise review: putting a finger on stem cell biology: zinc finger nuclease-driven targeted genetic editing in human pluripotent stem cells.

    PubMed

    Collin, Joseph; Lako, Majlinda

    2011-07-01

    Human pluripotent stem cells (hPSCs) encompassing human embryonic stem cells and human induced pluripotent stem cells (hiPSCs) have a wide appeal for numerous basic biology studies and for therapeutic applications because of their potential to give rise to almost any cell type in the human body and immense ability to self-renew. Much attention in the stem cell field is focused toward the study of gene-based anomalies relating to the causative affects of human disease and their correction with the potential for patient-specific therapies using gene corrected hiPSCs. Therefore, the genetic manipulation of stem cells is clearly important for the development of future medicine. Although successful targeted genetic engineering in hPSCs has been reported, these cases are surprisingly few because of inherent technical limitations with the methods used. The development of more robust and efficient means by which to achieve specific genomic modifications in hPSCs has far reaching implications for stem cell research and its applications. Recent proof-of-principle reports have shown that genetic alterations with minimal toxicity are now possible through the use of zinc finger nucleases (ZFNs) and the inherent DNA repair mechanisms within the cell. In light of recent comprehensive reviews that highlight the applications, methodologies, and prospects of ZFNs, this article focuses on the application of ZFNs to stem cell biology, discussing the published work to date, potential problems, and future uses for this technology both experimentally and therapeutically.

  17. Identification of genes encoding zinc finger motifs in the cardiovascular system.

    PubMed

    Wang, R; Hwang, D M; Cukerman, E; Liew, C C

    1997-01-01

    The Zn2+-finger DNA-binding domain has been identified in several developmental control proteins, transcription factors and gene products associated with diseases, as well as in several RNA-binding proteins. We applied library screening, expressed sequence tagging (EST sequencing), Zn2+-binding assays and Northern blot hybridization, in order to characterize novel cDNA clones of the human cardiovascular system which contain Zn2+-finger motifs. An embryonic (8-10 weeks gestation) heart lambda ZAP Express cDNA library was screened with an oligonucleotide probe deduced from a consensus amino acid sequence which is highly conserved for Zn2+-finger proteins, and approximately 350 positive clones were isolated from 1 x 10(4) plaque-forming units (pfu) initially plated. The isolated clones were classified as known and novel following single pass automated DNA sequencing. Analysis of Northern blot hybridization delineated the tissue specificity of these clones, as well as their association with cardiac growth and development. Existence of Zn2+-finger motifs in the novel clones was confirmed by Zn2+-binding assay. In this report, we present the characterization of eight novel clones, including the complete cDNA sequences of one of these clones (HHZ-123).

  18. Two zinc finger proteins, OMA-1 and OMA-2, are redundantly required for oocyte maturation in C. elegans.

    PubMed

    Detwiler, M R; Reuben, M; Li, X; Rogers, E; Lin, R

    2001-08-01

    Oocytes are released from meiotic prophase I arrest through a process termed oocyte maturation. We present here a genetic characterization of oocyte maturation, using C. elegans as a model system. We show that two TIS11 zinc finger-containing proteins, OMA-1 and OMA-2, express specifically in maturing oocytes and function redundantly in oocyte maturation. Oocytes in oma-1;oma-2 mutants initiate but do not complete maturation and arrest at a defined point in prophase I. Two maturation signal-induced molecular events, including the maintenance of activated MAP kinase, do not occur in Oma oocytes. The Oma prophase arrest is released by inactivation of a MYT-1-like kinase, suggesting that OMA-1 and OMA-2 function upstream of MYT-1 as positive regulators of prophase progression during meiotic maturation.

  19. A novel zinc-finger-like gene from Tamarix hispida is involved in salt and osmotic tolerance.

    PubMed

    An, Yan; Wang, Yucheng; Lou, Lingling; Zheng, Tangchun; Qu, Guan-Zheng

    2011-11-01

    In the present study, a zinc-finger-like cDNA (ThZFL) was cloned from the Tamarix hispida. Northern blot analysis showed that the expression of ThZFL can be induced by salt, osmotic stress and ABA treatment. Overexpression of the ThZFL confers salt and osmotic stress tolerance in both yeast Saccharomyces cerevisiae and tobacco. Furthermore, MDA levels in ThZFL transformed tobacco were significantly decreased compared with control plants under salt and osmotic stress, suggesting ThZFL may confer stress tolerance by decreasing membrane lipid peroxidation. Subcellular localization analysis showed the ThZFL protein is localized in the cell wall. Our results indicated the ThZFL gene is an excellent candidate for genetic engineering to improve salt and osmotic tolerance in agricultural plants.

  20. Functional dichotomy and distinct nanoscale assemblies of a cell cycle-controlled bipolar zinc-finger regulator

    PubMed Central

    Mignolet, Johann; Holden, Seamus; Bergé, Matthieu; Panis, Gaël; Eroglu, Ezgi; Théraulaz, Laurence; Manley, Suliana; Viollier, Patrick H

    2016-01-01

    Protein polarization underlies differentiation in metazoans and in bacteria. How symmetric polarization can instate functional asymmetry remains elusive. Here, we show by super-resolution photo-activated localization microscopy and edgetic mutations that the bitopic zinc-finger protein ZitP implements specialized developmental functions – pilus biogenesis and multifactorial swarming motility – while shaping distinct nanoscale (bi)polar architectures in the asymmetric model bacterium Caulobacter crescentus. Polar assemblage and accumulation of ZitP and its effector protein CpaM are orchestrated in time and space by conserved components of the cell cycle circuitry that coordinate polar morphogenesis with cell cycle progression, and also act on the master cell cycle regulator CtrA. Thus, this novel class of potentially widespread multifunctional polarity regulators is deeply embedded in the cell cycle circuitry. DOI: http://dx.doi.org/10.7554/eLife.18647.001 PMID:28008851

  1. Evolutionary expansion and divergence in a large family of primate-specific zinc finger transcription factor genes

    SciTech Connect

    Hamilton, A T; Huntley, S; Tran-Gyamfi, M; Baggott, D; Gordon, L; Stubbs, L

    2005-09-28

    Although most genes are conserved as one-to-one orthologs in different mammalian orders, certain gene families have evolved to comprise different numbers and types of protein-coding genes through independent series of gene duplications, divergence and gene loss in each evolutionary lineage. One such family encodes KRAB-zinc finger (KRAB-ZNF) genes, which are likely to function as transcriptional repressors. One KRAB-ZNF subfamily, the ZNF91 clade, has expanded specifically in primates to comprise more than 110 loci in the human genome, yielding large gene clusters in human chromosomes 19 and 7 and smaller clusters or isolated copies at other chromosomal locations. Although phylogenetic analysis indicates that many of these genes arose before the split between old world monkeys and new world monkeys, the ZNF91 subfamily has continued to expand and diversify throughout the evolution of apes and humans. The paralogous loci are distinguished by sequence divergence within their zinc finger arrays indicating a selection for proteins with different DNA binding specificities. RT-PCR and in situ hybridization data show that some of these ZNF genes can have tissue-specific expression patterns, however many KRAB-ZNFs that are near-ubiquitous could also be playing very specific roles in halting target pathways in all tissues except for a few, where the target is released by the absence of its repressor. The number of variant KRAB-ZNF proteins is increased not only because of the large number of loci, but also because many loci can produce multiple splice variants, which because of the modular structure of these genes may have separate and perhaps even conflicting regulatory roles. The lineage-specific duplication and rapid divergence of this family of transcription factor genes suggests a role in determining species-specific biological differences and the evolution of novel primate traits.

  2. An over expression APP model for anti-Alzheimer disease drug screening created by zinc finger nuclease technology.

    PubMed

    Zhang, Xiaojing; Li, Hui; Mao, Yiqing; Li, Zhixin; Wang, Rong; Guo, Tingting; Jin, Ling; Song, Rongjing; Xu, Wei; Zhou, Na; Zhang, Yizhuang; Hu, Ruobi; Wang, Xi; Huang, Huakang; Lei, Zhen; Niu, Gang; Irwin, David M; Tan, Huanran

    2013-01-01

    Zinc Finger Nucleases (ZFNs), famous for their ability to precisely and efficiently modify specific genomic loci, have been employed in numerous transgenic model organism and cell constructions. Here we employ the ZFNs technology, with homologous recombination (HR), to construct sequence-specific Amyloid Precursor Protein (APP) knock-in cells. With the use of ZFNs, we established APP knock in cell lines with gene-modification efficiencies of about 7%. We electroporated DNA fragment containing the promoter and the protein coding regions of the zinc finger nucleases into cells, instead of the plasmids, to avoid problems associated with off target homologous recombination, and adopted a pair of mutated FokI cleavage domains to reduce the toxic effects of the ZFNs on cell growth. Since over-expression of APP, or a subdomain of it, might lead to an immediately lethal effect, we used the Cre-LoxP System to regulate APP expression. Our genetically transformed cell lines, w5c1 and s12c8, showed detectable APP and Amyloid β (Aβ) production. The Swedish double mutation in the APP coding sequence enhanced APP and Aβ abundance. What is more, the activity of the three key secretases in Aβ formation could be modulated, indicating that these transgenic cells have potential for drug screening to modify amyloid metabolism in cells. Our transformed cells could readily be propagated in culture and should provide an excellent experimental medium for elucidating aspects of the molecular pathogenesis of Alzheimer's disease, especially those concerning the amyloidogenic pathways involving mutations in the APP coding sequence. The cellular models may also serve as a tool for deriving potentially useful therapeutic agents.

  3. Evidence of positive selection and concerted evolution in the rapidly evolving PRDM9 zinc finger domain in goats and sheep.

    PubMed

    Ahlawat, S; Sharma, P; Sharma, R; Arora, R; Verma, N K; Brahma, B; Mishra, P; De, S

    2016-12-01

    Meiotic recombination contributes to augmentation of genetic diversity, exclusion of deleterious alleles and proper segregation of chromatids. PRDM9 has been identified as the gene responsible for specifying the location of recombination hotspots during meiosis and is also the only known vertebrate gene associated with reproductive isolation between species. PRDM9 encodes a protein with a highly variable zinc finger (ZF) domain that varies between as well as within species. In the present study, the ZF domain of PRDM9 on chromosome 1 was characterized for the first time in 15 goat breeds and 25 sheep breeds of India. A remarkable variation in the number and sequence of ZF domains was observed. The number of ZF repeats in the ZF array varied from eight to 12 yielding five homozygous and 10 heterozygous genotypes. The number of different ZF domains was 84 and 52 producing 36 and 26 unique alleles in goats and sheep respectively. The posterior mean of dN/dS or omega values were calculated using the codeml tool of pamlx to identify amino acids that are evolving positively in goats and sheep, as positions -1, +3 and +6 in the ZF domain have been reported to experience strong positive selection across different lineages. Our study identified sites -5, -1, +3, +4 and +6 to be experiencing positive selection. Small ruminant zinc fingers were also found to be evolving under concerted evolution. Our results demonstrate the existence of a vast diversity of PRDM9 in goats and sheep, which is in concert with reports in many metazoans.

  4. Engineering zinc finger protein transcription factors to downregulate the epithelial glycoprotein-2 promoter as a novel anti-cancer treatment.

    PubMed

    Gommans, Willemijn M; McLaughlin, Pamela M J; Lindhout, Beatrice I; Segal, David J; Wiegman, D J; Haisma, Hidde J; van der Zaal, Bert J; Rots, Marianne G

    2007-05-01

    Zinc finger protein transcription factors (ZFP-TFs) are emerging as powerful novel tools for the treatment of many different diseases. ZFPs are DNA-binding motifs and consist of modular zinc finger domains. Each domain can be engineered to recognize a specific DNA triplet, and stitching six domains together results in the recognition of a gene-specific sequence. Inhibition of gene expression can be achieved by fusing a repressor domain to these DNA-binding motifs. In this study, we engineered ZFP-TFs to downregulate the activity of the epithelial glycoprotein-2 (EGP-2) promoter. The protein EGP-2 is overexpressed in a wide variety of cancer types and EGP-2 downregulation has been shown to result in a decreased oncogenic potential of tumor cells. Therefore, downregulation of EGP-2 expression by ZFP-TFs provides a novel anti-cancer therapeutic. Using a straightforward strategy, we engineered a 3-ZFP that could bind a 9 bp sequence within the EGP-2 promoter. After the addition of a repressor domain, this 3-ZFP-TF could efficiently downregulate EGP-2 promoter activity by 60%. To demonstrate the flexibility of this technology, we coupled an activation domain to the engineered ZFP, resulting in a nearly 200% increase in EGP-2 promoter activity. To inhibit the endogenous EGP-2 promoter, we engineered 6-ZFP-TFs. Although none of the constructed ZFP-TFs could convincingly modulate the endogenous promoter, efficient and specific inhibition of the exogenous promoter was observed. Overall, ZFP-TFs are versatile bi-directional modulators of gene expression and downregulation of EGP-2 promoter activity using ZFP-TFs can ultimately result in a novel anti-cancer treatment.

  5. Inhibition of binding of tomato yellow leaf curl virus rep to its replication origin by artificial zinc-finger protein.

    PubMed

    Mori, Tomoaki; Takenaka, Kosuke; Domoto, Fumiya; Aoyama, Yasuhiro; Sera, Takashi

    2013-06-01

    Previously we demonstrated that inhibition of replication-associated protein (Rep) binding to its replication origin by artificial zinc-finger proteins (AZPs) is a powerful method to prevent plant virus infection in vivo. In the present study, we applied the AZP technology to Tomato yellow leaf curl virus (TYLCV), which is a limiting factor in tomato cultivation worldwide. First, we determined 5'-ATCGGTGT ATCGGTGT-3' in the 195-bp intergenic region of the TYLCV-Israel strain, a strain reported first among TYLCV strains, as the Rep-binding site by gel shift assays. We then constructed a 6-finger AZP that bound to a 19-bp DNA including the Rep-binding site. We demonstrated that the binding affinity of the AZP was >1,000-fold greater than that of Rep and that the AZP inhibited Rep binding completely in vitro. Because the binding capability of the AZP was same as that of the AZP previously designed for geminivirus-resistant Arabidopsis thaliana, we predict that the present AZP will prevent TYLCV infection in vivo.

  6. Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays.

    PubMed

    Lam, Kathy N; van Bakel, Harm; Cote, Atina G; van der Ven, Anton; Hughes, Timothy R

    2011-06-01

    C2H2 zinc fingers (C2H2-ZFs) are the most prevalent type of vertebrate DNA-binding domain, and typically appear in tandem arrays (ZFAs), with sequential C2H2-ZFs each contacting three (or more) sequential bases. C2H2-ZFs can be assembled in a modular fashion, providing one explanation for their remarkable evolutionary success. Given a set of modules with defined three-base specificities, modular assembly also presents a way to construct artificial proteins with specific DNA-binding preferences. However, a recent survey of a large number of three-finger ZFAs engineered by modular assembly reported high failure rates (∼70%), casting doubt on the generality of modular assembly. Here, we used protein-binding microarrays to analyze 28 ZFAs that failed in the aforementioned study. Most (17) preferred specific sequences, which in all but one case resembled the intended target sequence. Like natural ZFAs, the engineered ZFAs typically yielded degenerate motifs, binding dozens to hundreds of related individual sequences. Thus, the failure of these proteins in previous assays is not due to lack of sequence-specific DNA-binding activity. Our findings underscore the relevance of individual C2H2-ZF sequence specificities within tandem arrays, and support the general ability of modular assembly to produce ZFAs with sequence-specific DNA-binding activity.

  7. Evi9 Encodes a Novel Zinc Finger Protein That Physically Interacts with BCL6, a Known Human B-Cell Proto-Oncogene Product

    PubMed Central

    Nakamura, Takuro; Yamazaki, Yukari; Saiki, Yuriko; Moriyama, Masatsugu; Largaespada, David A.; Jenkins, Nancy A.; Copeland, Neal G.

    2000-01-01

    Evi9 is a common site of retroviral integration in BXH2 murine myeloid leukemias. Here we show that Evi9 encodes a novel zinc finger protein with three tissue-specific isoforms: Evi9a (773 amino acids [aa]) contains two C2H2-type zinc finger motifs, a proline-rich region, and an acidic domain; Evi9b (486 aa) lacks the first zinc finger motif and part of the proline-rich region; Evi9c (239 aa) lacks all but the first zinc finger motif. Proviral integration sites are located in the first intron of the gene and lead to increased gene expression. Evi9a and Evi9c, but not Evi9b, show transforming activity for NIH 3T3 cells, suggesting that Evi9 is a dominantly acting proto-oncogene. Immunolocalization studies show that Evi9c is restricted to the cytoplasm whereas Evi9a and Evi9b are located in the nucleus, where they form a speckled localization pattern identical to that observed for BCL6, a human B-cell proto-oncogene product. Coimmunoprecipitation and glutathione S-transferase pull-down experiments show that Evi9a and Evi9b, but not Evi9c, physically interact with BCL6, while deletion mutagenesis localized the interaction domains in or near the second zinc finger and POZ domains of Evi9 and BCL6, respectively. These results suggest that Evi9 is a leukemia disease gene that functions, in part, through its interaction with BCL6. PMID:10757802

  8. Zinc finger oxidation of Fpg/Nei DNA glycosylases by 2-thioxanthine: biochemical and X-ray structural characterization

    PubMed Central

    Biela, Artur; Coste, Franck; Culard, Françoise; Guerin, Martine; Goffinont, Stéphane; Gasteiger, Karola; Cieśla, Jarosław; Winczura, Alicja; Kazimierczuk, Zygmunt; Gasparutto, Didier; Carell, Thomas; Tudek, Barbara; Castaing, Bertrand

    2014-01-01

    DNA glycosylases from the Fpg/Nei structural superfamily are base excision repair enzymes involved in the removal of a wide variety of mutagen and potentially lethal oxidized purines and pyrimidines. Although involved in genome stability, the recent discovery of synthetic lethal relationships between DNA glycosylases and other pathways highlights the potential of DNA glycosylase inhibitors for future medicinal chemistry development in cancer therapy. By combining biochemical and structural approaches, the physical target of 2-thioxanthine (2TX), an uncompetitive inhibitor of Fpg, was identified. 2TX interacts with the zinc finger (ZnF) DNA binding domain of the enzyme. This explains why the zincless hNEIL1 enzyme is resistant to 2TX. Crystal structures of the enzyme bound to DNA in the presence of 2TX demonstrate that the inhibitor chemically reacts with cysteine thiolates of ZnF and induces the loss of zinc. The molecular mechanism by which 2TX inhibits Fpg may be generalized to all prokaryote and eukaryote ZnF-containing Fpg/Nei-DNA glycosylases. Cell experiments show that 2TX can operate in cellulo on the human Fpg/Nei DNA glycosylases. The atomic elucidation of the determinants for the interaction of 2TX to Fpg provides the foundation for the future design and synthesis of new inhibitors with high efficiency and selectivity. PMID:25143530

  9. Binding studies with mutants of Zif268. Contribution of individual side chains to binding affinity and specificity in the Zif268 zinc finger-DNA complex.

    PubMed

    Elrod-Erickson, M; Pabo, C O

    1999-07-02

    The Zif268 zinc finger-DNA complex has served as a model system for understanding how Cys2His2 type zinc fingers recognize DNA. Structural studies of the Zif268-DNA complex revealed that residues at four positions in the alpha helix of each zinc finger play key roles in recognition, but there has been no information about the precise contributions of individual residues. Here we report the results of binding studies involving five mutants of Zif268 that have changes in the base-contacting residues of finger one. These studies let us evaluate the contributions that Arg18 (position -1 of the alpha helix), Asp20 (position 2), Glu21 (position 3), and Arg24 (position 6) make to the overall energy of DNA binding. Our results confirm the important role played by these arginines. By comparing the affinities of the wild type and mutant peptides for various sites, we also prove that Asp20 and Glu21 play important roles in determining binding site specificity.

  10. Cleavage and polyadenylation specificity factor 30: An RNA-binding zinc-finger protein with an unexpected 2Fe–2S cluster

    PubMed Central

    Shimberg, Geoffrey D.; Michalek, Jamie L.; Oluyadi, Abdulafeez A.; Rodrigues, Andria V.; Zucconi, Beth E.; Neu, Heather M.; Ghosh, Shanchari; Sureschandra, Kanisha; Wilson, Gerald M.; Stemmler, Timothy L.; Michel, Sarah L. J.

    2016-01-01

    Cleavage and polyadenylation specificity factor 30 (CPSF30) is a key protein involved in pre-mRNA processing. CPSF30 contains five Cys3His domains (annotated as “zinc-finger” domains). Using inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy, and UV-visible spectroscopy, we report that CPSF30 is isolated with iron, in addition to zinc. Iron is present in CPSF30 as a 2Fe–2S cluster and uses one of the Cys3His domains; 2Fe–2S clusters with a Cys3His ligand set are rare and notably have also been identified in MitoNEET, a protein that was also annotated as a zinc finger. These findings support a role for iron in some zinc-finger proteins. Using electrophoretic mobility shift assays and fluorescence anisotropy, we report that CPSF30 selectively recognizes the AU-rich hexamer (AAUAAA) sequence present in pre-mRNA, providing the first molecular-based evidence to our knowledge for CPSF30/RNA binding. Removal of zinc, or both zinc and iron, abrogates binding, whereas removal of just iron significantly lessens binding. From these data we propose a model for RNA recognition that involves a metal-dependent cooperative binding mechanism. PMID:27071088

  11. Alternating zinc fingers in the human male associated protein ZFY: 2D NMR structure of an even finger and implications for jumping-linker DNA recognition

    SciTech Connect

    Kochoyan, M.; Havel, T.F.; Dahl, C.E. ); Nguyen, D.T.; Keutmann, H.T. ); Weiss, M.A. Massachusetts General Hospital, Boston )

    1991-04-09

    ZFY, a sex-related Zn-finger protein encoded by the human Y chromosome, is distinguished from the general class of Zn-finger proteins by the presence of a two-finger repeat. Whereas odd-numbered domains and linkers fit a general consensus, even-numbered domains and linkers exhibit systematic differences. Because this alternation may have fundamental implications for the mechanism of protein-DNA recognition, the authors have undertaken biochemical and structural studies of fragments of ZFY. They describe here the solution structure of a representative nonconsensus (even-numbered) Zn finger based on 2D NMR studies of a 30-residue peptide. Structural modeling by distance geometry and simulated annealing (DG/SA) demonstrates that this peptide folds as a miniglobular domain containing a C-terminal {beta}-hairpin and N-terminal {alpha}-helix ({beta}{beta}{alpha} motif). These features are similar to (but not identical with) those previously described in consensus-type Zn fingers (derived from ADR1 and Xfin); the similarities suggest that even and odd ZFY domains bind DNA by a common mechanism. A model of the protein-DNA complex (designated the jumping-linker model) is presented and discussed in terms of the ZFY two-finger repeat. In this model every other linker is proposed to cross the minor groove by means of a putative finger/linker submotif HX{sub 4}HX{sub 3}-hydrophobic residue-X{sub 3}.

  12. Plant architecture and grain yield are regulated by the novel DHHC-type zinc finger protein genes in rice (Oryza sativa L.).

    PubMed

    Zhou, Bo; Lin, Jian Zhong; Peng, Dan; Yang, Yuan Zhu; Guo, Ming; Tang, Dong Ying; Tan, Xiaofeng; Liu, Xuan Ming

    2017-01-01

    In many plants, architecture and grain yield are affected by both the environment and genetics. In rice, the tiller is a vital factor impacting plant architecture and regulated by many genes. In this study, we cloned a novel DHHC-type zinc finger protein gene Os02g0819100 and its alternative splice variant OsDHHC1 from the cDNA of rice (Oryza sativa L.), which regulate plant architecture by altering the tiller in rice. The tillers increased by about 40% when this type of DHHC-type zinc finger protein gene was over-expressed in Zhong Hua 11 (ZH11) rice plants. Moreover, the grain yield of transgenic rice increased approximately by 10% compared with wild-type ZH11. These findings provide an important genetic engineering approach for increasing rice yields.

  13. Characteristics of the interaction of a synthetic human tristetraprolin tandem zinc finger peptide with AU-rich element-containing RNA substrates.

    PubMed

    Blackshear, Perry J; Lai, Wi S; Kennington, Elizabeth A; Brewer, Gary; Wilson, Gerald M; Guan, Xiaoju; Zhou, Pei

    2003-05-30

    Tristetraprolin (TTP) and its two known mammalian family members are tandem CCCH zinc finger proteins that can bind to AU-rich elements (AREs) in cellular mRNAs and destabilize those transcripts, apparently by initiating their deadenylation. Previous studies have shown that the approximately 70-amino acid tandem zinc finger domain of TTP is required and sufficient for RNA binding, and that the integrity of both zinc fingers is also required. However, little is known about the kinetics or structure of the peptide-RNA interaction, in part because of difficulties in obtaining soluble recombinant protein or peptides. We characterized the binding of a synthetic 73-amino acid peptide from human TTP to the tumor necrosis factor (TNF) ARE by gel mobility shift analyses and fluorescence anisotropy experiments. Both types of studies yielded a peptide-RNA dissociation constant of approximately 10 nM. Surprisingly, we found that the "footprint" from the TNF ARE required for peptide binding was only approximately 9 bases and that two molecules of peptide could bind to probes containing as little as 19 bases. An identical recombinant peptide exhibited gel shift characteristics similar to those of the synthetic peptide. NMR analysis of the 15N-labeled recombinant peptide suggested that its first zinc finger was structured in solution but that the second was not. The titration of oligonucleotides representing 17, 13, and even 9 bases of the TNF ARE caused an essentially identical, dramatic shift of existing resonances, and the appearance of new resonances in the peptide spectra, so that all amino acids could be assigned. These data suggest that this TTP peptide-RNA complex is structured in solution and might be amenable to NMR structure determination.

  14. The Solanum lycopersicum Zinc Finger2 Cysteine-2/Histidine-2 Repressor-Like Transcription Factor Regulates Development and Tolerance to Salinity in Tomato and Arabidopsis1[W

    PubMed Central

    Hichri, Imène; Muhovski, Yordan; Žižková, Eva; Dobrev, Petre I.; Franco-Zorrilla, Jose Manuel; Solano, Roberto; Lopez-Vidriero, Irene; Motyka, Vaclav; Lutts, Stanley

    2014-01-01

    The zinc finger superfamily includes transcription factors that regulate multiple aspects of plant development and were recently shown to regulate abiotic stress tolerance. Cultivated tomato (Solanum lycopersicum Zinc Finger2 [SIZF2]) is a cysteine-2/histidine-2-type zinc finger transcription factor bearing an ERF-associated amphiphilic repression domain and binding to the ACGTCAGTG sequence containing two AGT core motifs. SlZF2 is ubiquitously expressed during plant development, and is rapidly induced by sodium chloride, drought, and potassium chloride treatments. Its ectopic expression in Arabidopsis (Arabidopsis thaliana) and tomato impaired development and influenced leaf and flower shape, while causing a general stress visible by anthocyanin and malonyldialdehyde accumulation. SlZF2 enhanced salt sensitivity in Arabidopsis, whereas SlZF2 delayed senescence and improved tomato salt tolerance, particularly by maintaining photosynthesis and increasing polyamine biosynthesis, in salt-treated hydroponic cultures (125 mm sodium chloride, 20 d). SlZF2 may be involved in abscisic acid (ABA) biosynthesis/signaling, because SlZF2 is rapidly induced by ABA treatment and 35S::SlZF2 tomatoes accumulate more ABA than wild-type plants. Transcriptome analysis of 35S::SlZF2 revealed that SlZF2 both increased and reduced expression of a comparable number of genes involved in various physiological processes such as photosynthesis, polyamine biosynthesis, and hormone (notably ABA) biosynthesis/signaling. Involvement of these different metabolic pathways in salt stress tolerance is discussed. PMID:24567191

  15. BrRZFP1 a Brassica rapa C3HC4-type RING zinc finger protein involved in cold, salt and dehydration stress.

    PubMed

    Jung, Y J; Lee, I H; Nou, I S; Lee, K D; Rashotte, A M; Kang, K K

    2013-03-01

    C3HC4-type RING zinc finger proteins are known to be essential in the regulation of plant processes, including responses to abiotic stress. Here, we identify, clone and examine the first C3HC4-type RING zinc finger protein (BrRZFP1) from Brassica rapa under stress conditions. Phylogenetic analysis of BrRZFP1 revealed strong sequence similarity to C3HC4-type zinc finger proteins from Arabidopsis that are induced by abiotic stresses. Diverse environmental stresses, including salt and cold, were found to induce BrRZFP1 transcripts greater than eightfold in B. rapa. Additional strong induction was shown of the stress hormone abscisic acid, together suggesting that BrRZFP1 could play a role as a general stress modulator. Similar profiles of induction for each of these stresses was found in both root and shoot tissues, although at much higher levels in roots. Constitutive expression of BrRZFP1 in Nicotiana tabacum was conducted to further analyse how changes in gene expression levels would affect plant stress responses. BrRZFP1 overexpression conferred increased tolerance to cold, salt and dehydration stresses. This was observed in several assays examining growth status throughout development, including increased germination, fresh weight and length of shoots and roots, as well as enhanced chlorophyll retention. These results suggest that the transcription factor BrRZFP1 is an important determinant of stress response in plants and that changes in its expression level in plants could increase stress tolerance.

  16. The PR/SET domain zinc finger protein Prdm4 regulates gene expression in embryonic stem cells but plays a nonessential role in the developing mouse embryo.

    PubMed

    Bogani, Debora; Morgan, Marc A J; Nelson, Andrew C; Costello, Ita; McGouran, Joanna F; Kessler, Benedikt M; Robertson, Elizabeth J; Bikoff, Elizabeth K

    2013-10-01

    Prdm4 is a highly conserved member of the Prdm family of PR/SET domain zinc finger proteins. Many well-studied Prdm family members play critical roles in development and display striking loss-of-function phenotypes. Prdm4 functional contributions have yet to be characterized. Here, we describe its widespread expression in the early embryo and adult tissues. We demonstrate that DNA binding is exclusively mediated by the Prdm4 zinc finger domain, and we characterize its tripartite consensus sequence via SELEX (systematic evolution of ligands by exponential enrichment) and ChIP-seq (chromatin immunoprecipitation-sequencing) experiments. In embryonic stem cells (ESCs), Prdm4 regulates key pluripotency and differentiation pathways. Two independent strategies, namely, targeted deletion of the zinc finger domain and generation of a EUCOMM LacZ reporter allele, resulted in functional null alleles. However, homozygous mutant embryos develop normally and adults are healthy and fertile. Collectively, these results strongly suggest that Prdm4 functions redundantly with other transcriptional partners to cooperatively regulate gene expression in the embryo and adult animal.

  17. Sequences homologous to the human x- and y-borne zinc finger protein genes (ZFX/Y) are autosomal in monotreme mannals

    SciTech Connect

    Watson, J.M.; Frost, C.; Graves, M.J.A. ); Spencer, J.A. )

    1993-02-01

    The human zinc finger protein genes (ZFX/Y) were identified as a result of a systematic search for the testis-determining factor gene on the human Y chromosome. Although they play no direct role in sex determination, they are of particular interest because they are highly conserved among mammals, birds, and amphibians and because, in eutherian mammals at least, they have active alleles on both the X and the Y chromosomes outside the pseudoautosomal region. We used in situ hybridization to localize the homologues of the zinc finger protein gene to chromosome 1 of the Australian echidna and to an equivalent position on chromosomes 1 and 2 of the playtpus. The localization to platypus chromosome 1 was confirmed by Southern analysis of a Chinese hamster [times] platypus cell hybrid retaining most of platypus chromosome 1. This localization is consistent with the cytological homology of chromosome 1 between the two species. The zinc finger protein gene homologues were localized to regions of platypus chromosomes 1 and 2 that included a number of other genes situated near ZFX on the short arm of the human X chromosome. These results support the hypothesis that many of the genes located on the short arm of the human X were originally autosomal and have been translocated to the X chromosome since the eutherian-metatherian divergence. 34 refs., 3 figs., 2 tabs.

  18. The Solanum lycopersicum Zinc Finger2 cysteine-2/histidine-2 repressor-like transcription factor regulates development and tolerance to salinity in tomato and Arabidopsis.

    PubMed

    Hichri, Imène; Muhovski, Yordan; Žižkova, Eva; Dobrev, Petre I; Franco-Zorrilla, Jose Manuel; Solano, Roberto; Lopez-Vidriero, Irene; Motyka, Vaclav; Lutts, Stanley

    2014-04-01

    The zinc finger superfamily includes transcription factors that regulate multiple aspects of plant development and were recently shown to regulate abiotic stress tolerance. Cultivated tomato (Solanum lycopersicum Zinc Finger2 [SIZF2]) is a cysteine-2/histidine-2-type zinc finger transcription factor bearing an ERF-associated amphiphilic repression domain and binding to the ACGTCAGTG sequence containing two AGT core motifs. SlZF2 is ubiquitously expressed during plant development, and is rapidly induced by sodium chloride, drought, and potassium chloride treatments. Its ectopic expression in Arabidopsis (Arabidopsis thaliana) and tomato impaired development and influenced leaf and flower shape, while causing a general stress visible by anthocyanin and malonyldialdehyde accumulation. SlZF2 enhanced salt sensitivity in Arabidopsis, whereas SlZF2 delayed senescence and improved tomato salt tolerance, particularly by maintaining photosynthesis and increasing polyamine biosynthesis, in salt-treated hydroponic cultures (125 mm sodium chloride, 20 d). SlZF2 may be involved in abscisic acid (ABA) biosynthesis/signaling, because SlZF2 is rapidly induced by ABA treatment and 35S::SlZF2 tomatoes accumulate more ABA than wild-type plants. Transcriptome analysis of 35S::SlZF2 revealed that SlZF2 both increased and reduced expression of a comparable number of genes involved in various physiological processes such as photosynthesis, polyamine biosynthesis, and hormone (notably ABA) biosynthesis/signaling. Involvement of these different metabolic pathways in salt stress tolerance is discussed.

  19. Zinc Finger Domain of the PRDM9 Gene on Chromosome 1 Exhibits High Diversity in Ruminants but Its Paralog PRDM7 Contains Multiple Disruptive Mutations

    PubMed Central

    Ahlawat, Sonika; Sharma, Priyanka; Sharma, Rekha; Arora, Reena; De, Sachinandan

    2016-01-01

    PRDM9 is the sole hybrid sterility gene identified so far in vertebrates. PRDM9 gene encodes a protein with an immensely variable zinc-finger (ZF) domain that determines the site of meiotic recombination hotspots genome-wide. In this study, the terminal ZF domain of PRDM9 on bovine chromosome 1 and its paralog on chromosome 22 were characterized in 225 samples from five ruminant species (cattle, yak, mithun, sheep and goat). We found extraordinary variation in the number of PRDM9 zinc fingers (6 to 12). We sequenced PRDM9 ZF encoding region from 15 individuals (carrying the same ZF number in both copies) and found 43 different ZF domain sequences. Ruminant zinc fingers of PRDM9 were found to be diversifying under positive selection and concerted evolution, specifically at positions involved in defining their DNA-binding specificity, consistent with the reports from other vertebrates such as mice, humans, equids and chimpanzees. ZF-encoding regions of the PRDM7, a paralog of PRDM9 on bovine chromosome 22 and on unknown chromosomes in other studied species were found to contain 84 base repeat units as in PRDM9, but there were multiple disruptive mutations after the first repeat unit. The diversity of the ZFs suggests that PRDM9 may activate recombination hotspots that are largely unique to each ruminant species. PMID:27203728

  20. Zinc Finger Domain of the PRDM9 Gene on Chromosome 1 Exhibits High Diversity in Ruminants but Its Paralog PRDM7 Contains Multiple Disruptive Mutations.

    PubMed

    Ahlawat, Sonika; Sharma, Priyanka; Sharma, Rekha; Arora, Reena; De, Sachinandan

    2016-01-01

    PRDM9 is the sole hybrid sterility gene identified so far in vertebrates. PRDM9 gene encodes a protein with an immensely variable zinc-finger (ZF) domain that determines the site of meiotic recombination hotspots genome-wide. In this study, the terminal ZF domain of PRDM9 on bovine chromosome 1 and its paralog on chromosome 22 were characterized in 225 samples from five ruminant species (cattle, yak, mithun, sheep and goat). We found extraordinary variation in the number of PRDM9 zinc fingers (6 to 12). We sequenced PRDM9 ZF encoding region from 15 individuals (carrying the same ZF number in both copies) and found 43 different ZF domain sequences. Ruminant zinc fingers of PRDM9 were found to be diversifying under positive selection and concerted evolution, specifically at positions involved in defining their DNA-binding specificity, consistent with the reports from other vertebrates such as mice, humans, equids and chimpanzees. ZF-encoding regions of the PRDM7, a paralog of PRDM9 on bovine chromosome 22 and on unknown chromosomes in other studied species were found to contain 84 base repeat units as in PRDM9, but there were multiple disruptive mutations after the first repeat unit. The diversity of the ZFs suggests that PRDM9 may activate recombination hotspots that are largely unique to each ruminant species.

  1. Design of a Zinc-Finger Hydrolase with a Synthetic αββ Protein

    PubMed Central

    Srivastava, Kinshuk Raj; Durani, Susheel

    2014-01-01

    Recent advances in protein design have opened avenues for the creation of artificial enzymes needed for biotechnological and pharmaceutical applications. However, designing efficient enzymes remains an unrealized ambition, as the design must incorporate a catalytic apparatus specific for the desired reaction. Here we present a de novo design approach to evolve a minimal carbonic anhydrase mimic. We followed a step-by-step design of first folding the main chain followed by sequence variation for substrate binding and catalysis. To optimize the fold, we designed an αββ protein based on a Zn-finger. We then inverse-designed the sequences to provide stability to the fold along with flexibility of linker regions to optimize Zn binding and substrate hydrolysis. The resultant peptides were synthesized and assessed for Zn and substrate binding affinity by fluorescence and ITC followed by evaluation of catalytic efficiency with UV-based enzyme kinetic assays. We were successful in mimicking carbonic anhydrase activity in a peptide of twenty two residues, using p-nitrophenyl acetate as a CO2 surrogate. Although our design had modest activity, being a simple structure is an advantage for further improvement in efficiency. Our approach opens a way forward to evolving an efficient biocatalyst for any industrial reaction of interest. PMID:24816915

  2. The Zinc-Finger Protein SOP1 Is Required for a Subset of the Nuclear Exosome Functions in Arabidopsis

    PubMed Central

    Hématy, Kian; Bellec, Yannick; Podicheti, Ram; Bouteiller, Nathalie; Anne, Pauline; Morineau, Céline; Haslam, Richard P.; Beaudoin, Frederic; Napier, Johnathan A.; Mockaitis, Keithanne; Gagliardi, Dominique; Vaucheret, Hervé; Lange, Heike; Faure, Jean-Denis

    2016-01-01

    Correct gene expression requires tight RNA quality control both at transcriptional and post-transcriptional levels. Using a splicing-defective allele of PASTICCINO2 (PAS2), a gene essential for plant development, we isolated suppressor mutations modifying pas2-1 mRNA profiles and restoring wild-type growth. Three suppressor of pas2 (sop) mutations modified the degradation of mis-spliced pas2-1 mRNA species, allowing the synthesis of a functional protein. Cloning of the suppressor mutations identified the core subunit of the exosome SOP2/RRP4, the exosome nucleoplasmic cofactor SOP3/HEN2 and a novel zinc-finger protein SOP1 that colocalizes with HEN2 in nucleoplasmic foci. The three SOP proteins counteract post-transcriptional (trans)gene silencing (PTGS), which suggests that they all act in RNA quality control. In addition, sop1 mutants accumulate some, but not all of the misprocessed mRNAs and other types of RNAs that are observed in exosome mutants. Taken together, our data show that SOP1 is a new component of nuclear RNA surveillance that is required for the degradation of a specific subset of nuclear exosome targets. PMID:26828932

  3. The zinc finger proteins ZNF644 and WIZ regulate the G9a/GLP complex for gene repression

    PubMed Central

    Bian, Chunjing; Chen, Qiang; Yu, Xiaochun

    2015-01-01

    The G9a/GLP complex mediates mono- and dimethylation of Lys9 of histone H3 at specific gene loci, which is associated with transcriptional repression. However, the molecular mechanism by which the G9a/GLP complex is targeted to the specific gene loci for H3K9 methylation is unclear. In this study, with unbiased protein affinity purification, we found ZNF644 and WIZ as two core subunits in the G9a/GLP complex. ZNF644 and WIZ interact with the transcription activation domain of G9a and GLP, respectively. Moreover, both ZNF644 and WIZ contain multiple zinc finger motifs that recognize consensus DNA sequences. ZNF644 and WIZ target G9a and GLP to the chromatin and mediate the G9a/GLP complex-dependent H3K9 methylation as well as gene repression. Thus, our studies reveal two key subunits in the G9a/GLP complex that regulate the function of this histone methyltransferase complex. DOI: http://dx.doi.org/10.7554/eLife.05606.001 PMID:25789554

  4. A homozygous mutation in a novel zinc-finger protein, ERIS, is responsible for Wolfram syndrome 2.

    PubMed

    Amr, Sami; Heisey, Cindy; Zhang, Min; Xia, Xia-Juan; Shows, Kathryn H; Ajlouni, Kamel; Pandya, Arti; Satin, Leslie S; El-Shanti, Hatem; Shiang, Rita

    2007-10-01

    A single missense mutation was identified in a novel, highly conserved zinc-finger gene, ZCD2, in three consanguineous families of Jordanian descent with Wolfram syndrome (WFS). It had been shown that these families did not have mutations in the WFS1 gene (WFS1) but were mapped to the WFS2 locus at 4q22-25. A G-->C transversion at nucleotide 109 predicts an amino acid change from glutamic acid to glutamine (E37Q). Although the amino acid is conserved and the mutation is nonsynonymous, the pathogenesis for the disorder is because the mutation also causes aberrant splicing. The mutation was found to disrupt messenger RNA splicing by eliminating exon 2, and it results in the introduction of a premature stop codon. Mutations in WFS1 have also been found to cause low-frequency nonsyndromic hearing loss, progressive hearing loss, and isolated optic atrophy associated with hearing loss. Screening of 377 probands with hearing loss did not identify mutations in the WFS2 gene. The WFS1-encoded protein, Wolframin, is known to localize to the endoplasmic reticulum and plays a role in calcium homeostasis. The ZCD2-encoded protein, ERIS (endoplasmic reticulum intermembrane small protein), is also shown to localize to the endoplasmic reticulum but does not interact directly with Wolframin. Lymphoblastoid cells from affected individuals show a significantly greater rise in intracellular calcium when stimulated with thapsigargin, compared with controls, although no difference was observed in resting concentrations of intracellular calcium.

  5. Generation of Knockout Rats with X-Linked Severe Combined Immunodeficiency (X-SCID) Using Zinc-Finger Nucleases

    PubMed Central

    Mashimo, Tomoji; Takizawa, Akiko; Voigt, Birger; Yoshimi, Kazuto; Hiai, Hiroshi; Kuramoto, Takashi; Serikawa, Tadao

    2010-01-01

    Background Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES) cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs) were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc. Methodology/Principal Findings We report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg) locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID). Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype. Conclusions and Significance The efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies. PMID:20111598

  6. Comparative analysis of a conserved zinc finger gene cluster on human chromosome 19q and mouse chromosome 7.

    PubMed

    Shannon, M; Ashworth, L K; Mucenski, M L; Lamerdin, J E; Branscomb, E; Stubbs, L

    1996-04-01

    Several lines of evidence now suggest that many of the zinc-finger-containing (ZNF) genes in the human genome are arranged in clusters. However, little is known about the structure or function of the clusters or about their conservation throughout evolution. Here, we report the analysis of a conserved ZNF gene cluster located in human chromosome 19q13.2 and mouse chromosome 7. Our results indicate that the human cluster consists of at least 10 related Kruppel-associated box (KRAB)-containing ZNF genes organized in tandem over a distance of 350-450 kb. Two cDNA clones representing genes in the murine cluster have been studied in detail. The KRAB A domains of these genes are nearly identical and are highly similar to human 19q13.2-derived KRAB sequences, but DNA-binding ZNF domains and other portions of the genes differ considerably. The two murine genes display distinct expression patterns, but are coexpressed in some adult tissues. These studies pave the way for a systematic analysis of the evolution of structure and function of genes within the numerous clustered ZNF families located on human chromosome 19 and elsewhere in the human and mouse genomes.

  7. PEI1, an embryo-specific zinc finger protein gene required for heart-stage embryo formation in Arabidopsis.

    PubMed Central

    Li, Z; Thomas, T L

    1998-01-01

    We used virtual subtraction, a new gene isolation strategy, to isolate several genes of interest that are expressed in Arabidopsis embryos. These genes have demonstrated biological properties or have the potential to be involved in important biological processes. One gene isolated by virtual subtraction is PEI. It encodes a protein containing a Cys3His zinc finger domain associated with a number of animal and fungal transcription factors. In situ hybridization results showed that PEI1 is expressed throughout the embryo from globular to late cotyledon stage. Transgenic Arabidopsis plants expressing a PEI1 antisense gene produced white seeds in which embryo development did not progress through heart stage. Aberrant embryos failed to form cotyledons, but the embryonic root appeared to be normal. Aberrant embryos did not turn green, and the expression of genes involved in photomorphogenesis was drastically attenuated. In culture, aberrant embryos did not form true leaves, but root formation was apparently normal. These results suggest that PEI1 is an embryo-specific transcription factor that plays an important role during Arabidopsis embryogenesis, functioning primarily in the apical domain of the embryo. PMID:9501112

  8. Dissection of splicing regulation at an endogenous locus by zinc-finger nuclease-mediated gene editing.

    PubMed

    Cristea, Sandra; Gregory, Philip D; Urnov, Fyodor D; Cost, Gregory J

    2011-02-08

    Sequences governing RNA splicing are difficult to study in situ due to the great difficulty of traditional targeted mutagenesis. Zinc-finger nuclease (ZFN) technology allows for the rapid and efficient introduction of site-specific mutations into mammalian chromosomes. Using a ZFN pair along with a donor plasmid to manipulate the outcomes of DNA repair, we introduced several discrete, targeted mutations into the fourth intron of the endogenous BAX gene in Chinese hamster ovary cells. Putative lariat branch points, the polypyrimidine tract, and the splice acceptor site were targeted. We recovered numerous otherwise isogenic clones carrying the intended mutations and analyzed the effect of each on BAX pre-mRNA splicing. Mutation of one of three possible branch points, the polypyrimidine tract, and the splice acceptor site all caused exclusion of exon five from BAX mRNA. Interestingly, these exon-skipping mutations allowed usage of cryptic splice acceptor sites within intron four. These data demonstrate that ZFN-mediated gene editing is a highly effective tool for dissection of pre-mRNA splicing regulatory sequences in their endogenous context.

  9. Non-integrating gamma-retroviral vectors as a versatile tool for transient zinc-finger nuclease delivery.

    PubMed

    Bobis-Wozowicz, Sylwia; Galla, Melanie; Alzubi, Jamal; Kuehle, Johannes; Baum, Christopher; Schambach, Axel; Cathomen, Toni

    2014-04-11

    Designer nucleases, like zinc-finger nucleases (ZFNs), represent valuable tools for targeted genome editing. Here, we took advantage of the gamma-retroviral life cycle and produced vectors to transfer ZFNs in the form of protein, mRNA and episomal DNA. Transfer efficacy and ZFN activity were assessed in quantitative proof-of-concept experiments in a human cell line and in mouse embryonic stem cells. We demonstrate that retrovirus-mediated protein transfer (RPT), retrovirus-mediated mRNA transfer (RMT), and retrovirus-mediated episome transfer (RET) represent powerful methodologies for transient protein delivery or protein expression. Furthermore, we describe complementary strategies to augment ZFN activity after gamma-retroviral transduction, including serial transduction, proteasome inhibition, and hypothermia. Depending on vector dose and target cell type, gene disruption frequencies of up to 15% were achieved with RPT and RMT, and >50% gene knockout after RET. In summary, non-integrating gamma-retroviral vectors represent a versatile tool to transiently deliver ZFNs to human and mouse cells.

  10. Enhanced cellulase production from Trichoderma reesei Rut-C30 by engineering with an artificial zinc finger protein library.

    PubMed

    Zhang, Fei; Bai, Fengwu; Zhao, Xinqing

    2016-10-01

    Trichoderma reesei Rut-C30 is a well-known cellulase producer, and improvement of its cellulase production is of great interest. An artificial zinc finger protein (AZFP) library is constructed for expression in T. reesei Rut-C30, and a mutant strain T. reesei U3 is selected based on its enhanced cellulase production. The U3 mutant shows a 55% rise in filter paper activity and 8.1-fold increased β-glucosidase activity, when compared to the native strain T. reesei Rut-C30. It is demonstrated that enhanced β-glucosidase activity was due to elevated transcription level of β-glucosidase gene in the U3 mutant. Moreover, significant elevation in transcription levels of several putative Azfp-U3 target genes is detected in the U3 mutant, including genes encoding hypothetical transcription factors and a putative glycoside hydrolase. Furthermore, U3 cellulase shows 115% higher glucose yield from pretreated corn stover, when compared to the cellulase of T. reesei Rut-C30. These results demonstrate that AZFP can be used to improve cellulase production in T. reesei Rut-C30. Our current work offers the establishment of an alternative strategy to develop fungal cell factories for improved production of high value industrial products.

  11. A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control

    PubMed Central

    Huang, Xin-Yuan; Chao, Dai-Yin; Gao, Ji-Ping; Zhu, Mei-Zhen; Shi, Min; Lin, Hong-Xuan

    2009-01-01

    Abiotic stresses, such as drought and salinity, lead to crop growth damage and a decrease in crop yields. Stomata control CO2 uptake and optimize water use efficiency, thereby playing crucial roles in abiotic stress tolerance. Hydrogen peroxide (H2O2) is an important signal molecule that induces stomatal closure. However, the molecular pathway that regulates the H2O2 level in guard cells remains largely unknown. Here, we clone and characterize DST (drought and salt tolerance)—a previously unknown zinc finger transcription factor that negatively regulates stomatal closure by direct modulation of genes related to H2O2 homeostasis—and identify a novel pathway for the signal transduction of DST-mediated H2O2-induced stomatal closure. Loss of DST function increases stomatal closure and reduces stomatal density, consequently resulting in enhanced drought and salt tolerance in rice. These findings provide an interesting insight into the mechanism of stomata-regulated abiotic stress tolerance, and an important genetic engineering approach for improving abiotic stress tolerance in crops. PMID:19651988

  12. Zinc Finger and X-Linked Factor (ZFX) Binds to Human SET Transcript 2 Promoter and Transactivates SET Expression.

    PubMed

    Xu, Siliang; Duan, Ping; Li, Jinping; Senkowski, Tristan; Guo, Fengbiao; Chen, Haibin; Romero, Alberto; Cui, Yugui; Liu, Jiayin; Jiang, Shi-Wen

    2016-10-20

    SET (SE Translocation) protein carries out multiple functions including those for protein phosphatase 2A (PP2A) inhibition, histone modification, DNA repair, and gene regulation. SET overexpression has been detected in brain neurons of patients suffering Alzheimer's disease, follicle theca cells of Polycystic Ovary Syndrome (PCOS) patients, and ovarian cancer cells, indicating that SET may play a pathological role for these disorders. SET transcript 2, produced by a specific promoter, represents a major transcript variant in different cell types. In this study, we characterized the transcriptional activation of human SET transcript 2 promoter in HeLa cells. Promoter deletion experiments and co-transfection assays indicated that ZFX, the Zinc finger and X-linked transcription factor, was able to transactivate the SET promoter. A proximal promoter region containing four ZFX-binding sites was found to be critical for the ZFX-mediated transactivation. Mutagenesis study indicated that the ZFX-binding site located the closest to the transcription start site accounted for most of the ZFX-mediated transactivity. Manipulation of ZFX levels by overexpression or siRNA knockdown confirmed the significance and specificity of the ZFX-mediated SET promoter activation. Chromatin immunoprecipitation results verified the binding of ZFX to its cognate sites in the SET promoter. These findings have led to identification of ZFX as an upstream factor regulating SET gene expression. More studies are required to define the in vivo significance of this mechanism, and specifically, its implication for several benign and malignant diseases related to SET dysregulation.

  13. Kruppel-like zinc finger protein Glis2 is essential for the maintenance of normal renal functions.

    PubMed

    Kim, Yong-Sik; Kang, Hong Soon; Herbert, Ronald; Beak, Ju Youn; Collins, Jennifer B; Grissom, Sherry F; Jetten, Anton M

    2008-04-01

    To obtain insight into the physiological functions of the Krüppel-like zinc finger protein Gli-similar 2 (Glis2), mice deficient in Glis2 expression were generated. Glis2 mutant (Glis2(mut)) mice exhibit significantly shorter life spans than do littermate wild-type (WT) mice due to the development of progressive chronic kidney disease with features resembling nephronophthisis. Glis2(mut) mice develop severe renal atrophy involving increased cell death and basement membrane thickening in the proximal convoluted tubules. This development is accompanied by infiltration of lymphocytic inflammatory cells and interstitial/glomerular fibrosis. The severity of the fibrosis, inflammatory infiltrates, and glomerular and tubular changes progresses with age. Blood urea nitrogen and creatinine increase, and Glis2(mut) mice develop proteinuria and ultimately die prematurely of renal failure. A comparison of the gene expression profiles of kidneys from 25-day-old/60-day-old WT and Glis2(mut) mice by microarray analysis showed increased expressions of many genes involved in immune responses/inflammation and fibrosis/tissue remodeling in kidneys of Glis2(mut) mice, including several cytokines and adhesion and extracellular matrix proteins. Our data demonstrate that a deficiency in Glis2 expression leads to tubular atrophy and progressive fibrosis, similar to nephronophthisis, that ultimately results in renal failure. Our study indicates that Glis2 plays a critical role in the maintenance of normal kidney architecture and functions.

  14. Conversion of Human Fibroblasts to Stably Self-Renewing Neural Stem Cells with a Single Zinc-Finger Transcription Factor

    PubMed Central

    Shahbazi, Ebrahim; Moradi, Sharif; Nemati, Shiva; Satarian, Leila; Basiri, Mohsen; Gourabi, Hamid; Zare Mehrjardi, Narges; Günther, Patrick; Lampert, Angelika; Händler, Kristian; Hatay, Firuze Fulya; Schmidt, Diana; Molcanyi, Marek; Hescheler, Jürgen; Schultze, Joachim L.; Saric, Tomo; Baharvand, Hossein

    2016-01-01

    Summary Direct conversion of somatic cells into neural stem cells (NSCs) by defined factors holds great promise for mechanistic studies, drug screening, and potential cell therapies for different neurodegenerative diseases. Here, we report that a single zinc-finger transcription factor, Zfp521, is sufficient for direct conversion of human fibroblasts into long-term self-renewable and multipotent NSCs. In vitro, Zfp521-induced NSCs maintained their characteristics in the absence of exogenous factor expression and exhibited morphological, molecular, developmental, and functional properties that were similar to control NSCs. In addition, the single-seeded induced NSCs were able to form NSC colonies with efficiency comparable with control NSCs and expressed NSC markers. The converted cells were capable of surviving, migrating, and attaining neural phenotypes after transplantation into neonatal mouse and adult rat brains, without forming tumors. Moreover, the Zfp521-induced NSCs predominantly expressed rostral genes. Our results suggest a facilitated approach for establishing human NSCs through Zfp521-driven conversion of fibroblasts. PMID:27052315

  15. Waking up dormant tumor suppressor genes with zinc fingers, TALEs and the CRISPR/dCas9 system

    PubMed Central

    Garcia-Bloj, Benjamin; Moses, Colette; Sgro, Agustin; Plani-Lam, Janice; Arooj, Mahira; Duffy, Ciara; Thiruvengadam, Shreyas; Sorolla, Anabel; Rashwan, Rabab; Mancera, Ricardo L.; Leisewitz, Andrea; Swift-Scanlan, Theresa; Corvalan, Alejandro H.; Blancafort, Pilar

    2016-01-01

    The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO, in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases. PMID:27528034

  16. Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs.

    PubMed

    Qian, Lili; Tang, Maoxue; Yang, Jinzeng; Wang, Qingqing; Cai, Chunbo; Jiang, Shengwang; Li, Hegang; Jiang, Ke; Gao, Pengfei; Ma, Dezun; Chen, Yaoxing; An, Xiaorong; Li, Kui; Cui, Wentao

    2015-09-24

    Myostatin (MSTN) is a dominant inhibitor of skeletal muscle development and growth. Mutations in MSTN gene can lead to muscle hypertrophy or double-muscled (DM) phenotype in cattle, sheep, dog and human. However, there has not been reported significant muscle phenotypes in pigs in association with MSTN mutations. Pigs are an important source of meat production, as well as serve as a preferred animal model for the studies of human disease. To study the impacts of MSTN mutations on skeletal muscle growth in pigs, we generated MSTN-mutant Meishan pigs with no marker gene via zinc finger nucleases (ZFN) technology. The MSTN-mutant pigs developed and grew normally, had increased muscle mass with decreased fat accumulation compared with wild type pigs, and homozygote MSTN mutant (MSTN(-/-)) pigs had apparent DM phenotype, and individual muscle mass increased by 100% over their wild-type controls (MSTN(+/+)) at eight months of age as a result of myofiber hyperplasia. Interestingly, 20% MSTN-mutant pigs had one extra thoracic vertebra. The MSTN-mutant pigs will not only offer a way of fast genetic improvement of lean meat for local fat-type indigenous pig breeds, but also serve as an important large animal model for biomedical studies of musculoskeletal formation, development and diseases.

  17. Genome-wide identification, evolution and expression analysis of the grape (Vitis vinifera L.) zinc finger-homeodomain gene family.

    PubMed

    Wang, Hao; Yin, Xiangjing; Li, Xiaoqin; Wang, Li; Zheng, Yi; Xu, Xiaozhao; Zhang, Yucheng; Wang, Xiping

    2014-04-03

    Plant zinc finger-homeodomain (ZHD) genes encode a family of transcription factors that have been demonstrated to play an important role in the regulation of plant growth and development. In this study, we identified a total of 13 ZHD genes (VvZHD) in the grape genome that were further classified into at least seven groups. Genome synteny analysis revealed that a number of VvZHD genes were present in the corresponding syntenic blocks of Arabidopsis, indicating that they arose before the divergence of these two species. Gene expression analysis showed that the identified VvZHD genes displayed distinct spatiotemporal expression patterns, and were differentially regulated under various stress conditions and hormone treatments, suggesting that the grape VvZHDs might be also involved in plant response to a variety of biotic and abiotic insults. Our work provides insightful information and knowledge about the ZHD genes in grape, which provides a framework for further characterization of their roles in regulation of stress tolerance as well as other aspects of grape productivity.

  18. The Arabidopsis Gene zinc finger protein 3(ZFP3) Is Involved in Salt Stress and Osmotic Stress Response

    PubMed Central

    Zhang, Aidong; Liu, Dongdong; Hua, Changmei; Yan, An; Liu, Bohan; Wu, Minjie; Liu, Yihua; Huang, Linli; Ali, Imran; Gan, Yinbo

    2016-01-01

    Plants are continuously challenged by various abiotic and biotic stresses. To tide over these adversities, plants evolved intricate regulatory networks to adapt these unfavorable environments. So far, many researchers have clarified the molecular and genetic pathways involved in regulation of stress responses. However, the mechanism through which these regulatory networks operate is largely unknown. In this study, we cloned a C2H2-type zinc finger protein gene ZFP3 from Arabidopsis thaliana and investigated its function in salt and osmotic stress response. Our results showed that the expression level of ZFP3 was highly suppressed by NaCl, mannitol and sucrose. Constitutive expression of ZFP3 enhanced tolerance of plants to salt and osmotic stress while the zfp3 mutant plants displays reduced tolerance in Arabidopsis. Gain- and Loss-of-function studies of ZFP3 showed that ZFP3 significantly changes proline accumulation and chlorophyll content. Furthermore, over-expression of ZFP3 induced the expressions of stress-related gene KIN1, RD22, RD29B and AtP5CS1. These results suggest that ZFP3 is involved in salt and osmotic stress response. PMID:27977750

  19. [Cloning and expression pattern of a zinc finger protein gene ShSAP1 in Saccharum officinarum].

    PubMed

    Li, Xiaojun; Cai, Wenwei; Zhang, Shuzhen; Xu, Liping; Chen, Ping; Wang, Jungang

    2011-06-01

    In plants, proteins with A20/AN1 zinc finger domain are involved in stress responses, named as "Stress Associated Protein" (SAP) gene family. Based on Expressed Sequence Tag (EST) sequences information in Badila Saccharum officinarum mature related cDNA library, we cloned an SAP gene from sugarcane full length cDNA library, named ShSAP1 (GenBank: Accession No. HM991960). To characterize ShSAP1, we analyzed its genome structure and expression pattern. Southern blot analysis showed ShSAP1 was present as one or two copy in the genome of Badila. Comparison of ShSAP1 1 008 bp full length cDNA with a genomic frangment (2 241 bp) generated by PCR amplification and sequencing, revealed the presence of two introns (202 bp and 1 052 bp) located in the 5'UTR region. Semiquantitative RT-PCR analysis found ShSAP1 expressed in leaves, roots and stalk in mature sugarcane. Compared with immature stems, ShSAP1 expressed higher in mature stalk. ShSAP1 was induced by different types of treatments, such as salt (200 mmol/L NaCl), drought (10% PEG 6 000), GA3 (200 mg/L), ABA (100 micromol/L) and ET (1 mmol/L) during sugarcane seedling stage. These results indicated that ShSAP1 may function in sugarcane maturation and abiotic stress response processes.

  20. Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer.

    PubMed

    Bao, Lei; Chen, HaiDe; Jong, UiMyong; Rim, CholHo; Li, WenLing; Lin, XiJuan; Zhang, Dan; Luo, Qiong; Cui, Chun; Huang, HeFeng; Zhang, Yan; Xiao, Lei; Fu, ZhiXin

    2014-02-01

    Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs. Conventional gene targeting in pig somatic cells is extremely inefficient. Zinc-finger nuclease (ZFN) technology has been shown to be a powerful tool for efficiently inducing mutations in the genome. However, ZFN-mediated targeting in pigs has rarely been achieved. Here, we used ZFNs to knock out the porcine α-1, 3-galactosyl-transferase (GGTA1) gene, which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation. Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1. Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4% and 5.2%, respectively. The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer (SCNT). Three GGTA1 null piglets were born, and one knockout primary fibroblast cell line was established from a cloned fetus. Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane. Functionally, GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum. This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs. GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.

  1. Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs

    PubMed Central

    Qian, Lili; Tang, Maoxue; Yang, Jinzeng; Wang, Qingqing; Cai, Chunbo; Jiang, Shengwang; Li, Hegang; Jiang, Ke; Gao, Pengfei; Ma, Dezun; Chen, Yaoxing; An, Xiaorong; Li, Kui; Cui, Wentao

    2015-01-01

    Myostatin (MSTN) is a dominant inhibitor of skeletal muscle development and growth. Mutations in MSTN gene can lead to muscle hypertrophy or double-muscled (DM) phenotype in cattle, sheep, dog and human. However, there has not been reported significant muscle phenotypes in pigs in association with MSTN mutations. Pigs are an important source of meat production, as well as serve as a preferred animal model for the studies of human disease. To study the impacts of MSTN mutations on skeletal muscle growth in pigs, we generated MSTN-mutant Meishan pigs with no marker gene via zinc finger nucleases (ZFN) technology. The MSTN-mutant pigs developed and grew normally, had increased muscle mass with decreased fat accumulation compared with wild type pigs, and homozygote MSTN mutant (MSTN−/−) pigs had apparent DM phenotype, and individual muscle mass increased by 100% over their wild-type controls (MSTN+/+) at eight months of age as a result of myofiber hyperplasia. Interestingly, 20% MSTN-mutant pigs had one extra thoracic vertebra. The MSTN-mutant pigs will not only offer a way of fast genetic improvement of lean meat for local fat-type indigenous pig breeds, but also serve as an important large animal model for biomedical studies of musculoskeletal formation, development and diseases. PMID:26400270

  2. scratch, a pan-neural gene encoding a zinc finger protein related to snail, promotes neuronal development.

    PubMed

    Roark, M; Sturtevant, M A; Emery, J; Vaessin, H; Grell, E; Bier, E

    1995-10-01

    The Drosophila scratch (scrt) gene is expressed in most or all neuronal precursor cells and encodes a predicted zinc finger transcription factor closely related to the product of the mesoderm determination gene snail (sna). Adult flies homozygous for scrt null alleles have a reduced number of photoreceptors in the eye, and embryos lacking the function of both scrt and the pan-neural gene deadpan (dpn), which encodes a basic helix-loop-helix (bHLH) protein, exhibit a significant loss of neurons. Conversely, ectopic expression of a scrt transgene during embryonic and adult development leads to the production of supernumerary neurons. Consistent with scrt functioning as a transcription factor, various genes are more broadly expressed than normal in scrt null mutants. Reciprocally, these same genes are expressed at reduced levels in response to ectopic scrt expression. We propose that scrt promotes neuronal cell fates by suppressing expression of genes promoting non-neuronal cell fates. We discuss the similarities between the roles of the ancestrally related scrt, sna, and escargot (esc) genes in regulating cell fate choices.

  3. The multi zinc-finger protein Trps1 acts as a regulator of histone deacetylation during mitosis.

    PubMed

    Wuelling, Manuela; Pasdziernik, Markus; Moll, Carina N; Thiesen, Andrea M; Schneider, Sabine; Johannes, Christian; Vortkamp, Andrea

    2013-07-15

    TRPS1, the gene mutated in human "Tricho-Rhino-Phalangeal syndrome," encodes a multi zinc-finger nuclear regulator of chondrocyte proliferation and differentiation. Here, we have identified a new function of Trps1 in controlling mitotic progression in chondrocytes. Loss of Trps1 in mice leads to an increased proportion of cells arrested in mitosis and, subsequently, to chromosome segregation defects. Searching for the molecular basis of the defect, we found that Trps1 acts as regulator of histone deacetylation. Trps1 interacts with two histone deacetylases, Hdac1 and Hdac4, thereby increasing their activity. Loss of Trps1 results in histone H3 hyperacetylation, which is maintained during mitosis. Consequently, chromatin condensation and binding of HP1 is impaired, and Trps1-deficient chondrocytes accumulate in prometaphase. Overexpression of Hdac4 rescues the mitotic defect of Trps1-deficient chondrocytes, identifying Trps1 as an important regulator of chromatin deacetylation during mitosis in chondrocytes. Our data provide the first evidence that the control of mitosis can be linked to the regulation of chondrocyte differentiation by epigenetic consequences of altered Hdac activity.

  4. Site-specific host gene modification by zinc finger nucleases: pointing the way to drug free control of HIV-1?

    PubMed Central

    Sasson, Sarah C; Kelleher, Anthony D

    2014-01-01

    Anti-retroviral therapy (ART) for human immunodeficiency virus-1 (HIV-1) infection has transformed its clinical course with spectacular reductions in morbidity and mortality, turning this once fatal diagnosis into a manageable chronic infection. However, ART has its limitations. Current ART does not eliminate the virus. Interruption of therapy results in rapid rebound of the virus, and such rebounds are associated with excess morbidity and mortality. This means that therapy once started is for life. This raises the issues of drug resistance due to suboptimal compliance, cumulative toxicities and mounting costs. Efforts to control the virus through novel interventions, particularly through cell or gene therapy have had a resurgence of interest as a single patient was apparently cured by an allogeneic stem cell transplantation from a donor who carried homozygous mutations that disable expression of the HIV-1 co-receptor CCR5. This paper reviews the state of play of gene therapy for HIV infection in the context of a recent paper showing the safety and feasibility of an approach that involves the ex vivo disruption of the ccr5 gene in autologous CD4 T cells using a virally delivered zinc finger nuclease, before their expansion and reinfusion. Although there are still considerable challenges, this approach may point towards a future drug free therapy for HIV-1 infection. PMID:25505967

  5. Minimum length of direct repeat sequences required for efficient homologous recombination induced by zinc finger nuclease in yeast.

    PubMed

    Ren, ChongHua; Yan, Qiang; Zhang, ZhiYing

    2014-10-01

    Zinc finger nuclease (ZFN) technology is a powerful molecular tool for targeted genome modifications and genetic engineering. However, screening for specific ZFs and validation of ZFN activity are labor intensive and time consuming. We previously designed a yeast-based ZFN screening and validation system by inserting a ZFN binding site flanked by a 164 bp direct repeat sequence into the middle of a Gal4 transcription factor, disrupting the open reading frame of the yeast Gal4 gene. Expression of the ZFN causes a double stranded break at its binding site, which promotes the cellular DNA repair system to restore expression of a functional Gal transcriptional factor via homologous recombination. Expression of Gal4 transcription factor leads to activation of three reporter genes in an AH109 yeast two-hybrid strain. However, the 164 bp direct repeat appears to generate spontaneous homologous recombination frequently, resulting in many false positive ZFNs. To overcome this, a series of DNA fragments of various lengths from 10 to 150 bp with 10 bp increase each and 164 bp direct repeats flanking the ZFN binding site were designed and constructed. The results demonstrated that the minimum length required for ZFN-induced homologous recombination was 30 bp, which almost eliminated spontaneous recombination. Using the 30 bp direct repeat sequence, ZFN could efficiently induce homologous recombination, while false positive ZFNs resulting from spontaneous homologous recombination were minimized. Thus, this study provided a simple, fast and sensitive ZFN screening and activity validation system in yeast.

  6. The Fruitless gene in Nasonia displays complex sex-specific splicing and contains new zinc finger domains.

    PubMed

    Bertossa, Rinaldo C; van de Zande, Louis; Beukeboom, Leo W

    2009-07-01

    The transcription factor Fruitless exerts a broad range of functions during Drosophila development, the most apparent of which is the determination of sexual behavior in males. Although fruitless sequences are found in other insect orders, little is known about fruitless structure and function outside Diptera. We have performed a thorough analysis of fruitless transcripts in the haplo-diploid wasp Nasonia vitripennis and found both sex-specific and non-sex-specific transcripts similar to those found in Drosophila. In Nasonia, however, a novel, large fruitless transcript is present in females only. Putative binding sites for sex-specific splicing factors found in Nasonia fruitless and doublesex as well as Apis mellifera doublesex transcripts were sufficient to identify a corresponding female-specific fruitless exon in A. mellifera, suggesting that similar factors in both hymenopteran species could be responsible for sex-specific splicing of both genes. Furthermore, new C(2)H(2) zinc finger domains found in Nasonia fruitless transcripts were also identified in the fruitless locus of major holometabolous insect species but not in drosophilids. Conservation of important domains and sex-specific splicing in Diptera and Hymenoptera support the hypothesis that fruitless is an ancient gene and has conserved functions in insects. Considerable divergences in other parts of the gene are expected to underlie species-specific differences and may help to explain diversity observed in insect sexual behaviors.

  7. A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control.

    PubMed

    Huang, Xin-Yuan; Chao, Dai-Yin; Gao, Ji-Ping; Zhu, Mei-Zhen; Shi, Min; Lin, Hong-Xuan

    2009-08-01

    Abiotic stresses, such as drought and salinity, lead to crop growth damage and a decrease in crop yields. Stomata control CO(2) uptake and optimize water use efficiency, thereby playing crucial roles in abiotic stress tolerance. Hydrogen peroxide (H(2)O(2)) is an important signal molecule that induces stomatal closure. However, the molecular pathway that regulates the H(2)O(2) level in guard cells remains largely unknown. Here, we clone and characterize DST (drought and salt tolerance)-a previously unknown zinc finger transcription factor that negatively regulates stomatal closure by direct modulation of genes related to H(2)O(2) homeostasis-and identify a novel pathway for the signal transduction of DST-mediated H(2)O(2)-induced stomatal closure. Loss of DST function increases stomatal closure and reduces stomatal density, consequently resulting in enhanced drought and salt tolerance in rice. These findings provide an interesting insight into the mechanism of stomata-regulated abiotic stress tolerance, and an important genetic engineering approach for improving abiotic stress tolerance in crops.

  8. Rice zinc finger protein DST enhances grain production through controlling Gn1a/OsCKX2 expression.

    PubMed

    Li, Shuyu; Zhao, Bingran; Yuan, Dingyang; Duan, Meijuan; Qian, Qian; Tang, Li; Wang, Bao; Liu, Xiaoqiang; Zhang, Jie; Wang, Jun; Sun, Jiaqiang; Liu, Zhao; Feng, Yu-Qi; Yuan, Longping; Li, Chuanyou

    2013-02-19

    The phytohormone cytokinin (CK) positively regulates the activity and function of the shoot apical meristem (SAM), which is a major parameter determining seed production. The rice (Oryza sativa L.) Gn1a/OsCKX2 (Grain number 1a/Cytokinin oxidase 2) gene, which encodes a cytokinin oxidase, has been identified as a major quantitative trait locus contributing to grain number improvement in rice breeding practice. However, the molecular mechanism of how the expression of OsCKX2 is regulated in planta remains elusive. Here, we report that the zinc finger transcription factor DROUGHT AND SALT TOLERANCE (DST) directly regulates OsCKX2 expression in the reproductive meristem. DST-directed expression of OsCKX2 regulates CK accumulation in the SAM and, therefore, controls the number of the reproductive organs. We identify that DST(reg1), a semidominant allele of the DST gene, perturbs DST-directed regulation of OsCKX2 expression and elevates CK levels in the reproductive SAM, leading to increased meristem activity, enhanced panicle branching, and a consequent increase of grain number. Importantly, the DST(reg1) allele provides an approach to pyramid the Gn1a-dependent and Gn1a-independent effects on grain production. Our study reveals that, as a unique regulator of reproductive meristem activity, DST may be explored to facilitate the genetic enhancement of grain production in rice and other small grain cereals.

  9. Multi-reporter selection for the design of active and more specific zinc-finger nucleases for genome editing

    PubMed Central

    Oakes, Benjamin L.; Xia, Danny F.; Rowland, Elizabeth F.; Xu, Denise J.; Ankoudinova, Irina; Borchardt, Jennifer S.; Zhang, Lei; Li, Patrick; Miller, Jeffrey C.; Rebar, Edward J.; Noyes, Marcus B.

    2016-01-01

    Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences. While many nuclease platforms have proven functional, all can produce unanticipated off-target lesions and have difficulty discriminating between homologous sequences, limiting their therapeutic application. Here we describe a multi-reporter selection system that allows the screening of large protein libraries to uncover variants able to discriminate between sequences with substantial homology. We have used this system to identify zinc-finger nucleases that exhibit high cleavage activity (up to 60% indels) at their targets within the CCR5 and HBB genes and strong discrimination against homologous sequences within CCR2 and HBD. An unbiased screen for off-target lesions using a novel set of CCR5-targeting nucleases confirms negligible CCR2 activity and demonstrates minimal off-target activity genome wide. This system offers a straightforward approach to generate nucleases that discriminate between similar targets and provide exceptional genome-wide specificity. PMID:26738816

  10. ZNF307 (Zinc Finger Protein 307) Acts as a Negative Regulator of Pressure Overload-Induced Cardiac Hypertrophy.

    PubMed

    Yu, Chang-Jiang; Liang, Chen; Li, Yu-Xia; Hu, Qing-Qing; Zheng, Wei-Wan; Niu, Na; Yang, Xu; Wang, Zi-Rui; Yu, Xiao-Di; Zhang, Bao-Long; Song, Bin-Lin; Zhang, Zhi-Ren

    2017-04-01

    Pathological cardiac hypertrophy is a key risk factor for heart failure. We found that the protein expression levels of the ZNF307 (zinc finger protein 307) were significantly increased in heart samples from both human patients with dilated cardiomyopathy and mice subjected to aortic banding. Therefore, we aimed to elucidate the role of ZNF307 in the development of cardiac hypertrophy and to explore the signal transduction events that mediate the effect of ZNF307 on cardiac hypertrophy, using cardiac-specific ZNF307 transgenic (ZNF307-TG) mice and ZNF307 global knockout (ZNF307-KO) mice. The results showed that the deletion of ZNF307 potentiated aortic banding-induced pathological cardiac hypertrophy, fibrosis, and cardiac dysfunction; however, the aortic banding-induced cardiac hypertrophic phenotype was dramatically diminished by ZNF307 overexpression in mouse heart. Mechanistically, the antihypertrophic effects mediated by ZNF307 in response to pathological stimuli were associated with the direct inactivation of NF-κB (nuclear factor-κB) signaling and blockade of the nuclear translocation of NF-κB subunit p65. Furthermore, the overexpression of a degradation-resistant mutant of IκBα (IκBα(S32A/S36A)) reversed the exacerbation of cardiac hypertrophy, fibrosis, and dysfunction shown in aortic banding-treated ZNF307-KO mice. In conclusion, our findings demonstrate that ZNF307 ameliorates pressure overload-induced cardiac hypertrophy by inhibiting the activity of NF-κB-signaling pathway.

  11. The phytochrome-interacting vascular plant one-zinc finger1 and VOZ2 redundantly regulate flowering in Arabidopsis.

    PubMed

    Yasui, Yukiko; Mukougawa, Keiko; Uemoto, Mitsuhiro; Yokofuji, Akira; Suzuri, Ryota; Nishitani, Aiko; Kohchi, Takayuki

    2012-08-01

    The timing of the transition to flowering in plants is regulated by various environmental factors, including daylength and light quality. Although the red/far-red photoreceptor phytochrome B (phyB) represses flowering by indirectly regulating the expression of a key flowering regulator, FLOWERING LOCUS T (FT), the mechanism of phyB signaling for flowering is largely unknown. Here, we identified two Arabidopsis thaliana genes, VASCULAR PLANT ONE-ZINC FINGER1 (VOZ1) and VOZ2, which are highly conserved throughout land plant evolution, as phyB-interacting factors. voz1 voz2 double mutants, but neither single mutant, showed a late-flowering phenotype under long-day conditions, which indicated that VOZ1 and VOZ2 redundantly promote flowering. voz1 voz2 mutations suppressed the early-flowering phenotype of the phyB mutant, and FT expression was repressed in the voz1 voz2 mutant. Green fluorescent protein-VOZ2 signal was observed in the cytoplasm, and interaction of VOZ proteins with phyB was indicated to occur in the cytoplasm under far-red light. However, VOZ2 protein modified to localize constitutively in the nucleus promoted flowering. In addition, the stability of VOZ2 proteins in the nucleus was modulated by light quality in a phytochrome-dependent manner. We propose that partial translocation of VOZ proteins from the cytoplasm to the nucleus mediates the initial step of the phyB signal transduction pathway that regulates flowering.

  12. Multiple interferon stimulated genes synergize with the zinc finger antiviral protein to mediate anti-alphavirus activity.

    PubMed

    Karki, Sophiya; Li, Melody M H; Schoggins, John W; Tian, Suyan; Rice, Charles M; MacDonald, Margaret R

    2012-01-01

    The zinc finger antiviral protein (ZAP) is a host factor that mediates inhibition of viruses in the Filoviridae, Retroviridae and Togaviridae families. We previously demonstrated that ZAP blocks replication of Sindbis virus (SINV), the prototype Alphavirus in the Togaviridae family at an early step prior to translation of the incoming genome and that synergy between ZAP and one or more interferon stimulated genes (ISGs) resulted in maximal inhibitory activity. The present study aimed to identify those ISGs that synergize with ZAP to mediate Alphavirus inhibition. Using a library of lentiviruses individually expressing more than 350 ISGs, we screened for inhibitory activity in interferon defective cells with or without ZAP overexpression. Confirmatory tests of the 23 ISGs demonstrating the largest infection reduction in combination with ZAP revealed that 16 were synergistic. Confirmatory tests of all potentially synergistic ISGs revealed 15 additional ISGs with a statistically significant synergistic effect in combination with ZAP. These 31 ISGs are candidates for further mechanistic studies. The number and diversity of the identified ZAP-synergistic ISGs lead us to speculate that ZAP may play an important role in priming the cell for optimal ISG function.

  13. Zinc finger nuclease: a new approach for excising HIV-1 proviral DNA from infected human T cells.

    PubMed

    Qu, Xiying; Wang, Pengfei; Ding, Donglin; Wang, Xiaohui; Zhang, Gongmin; Zhou, Xin; Liu, Lin; Zhu, Xiaoli; Zeng, Hanxian; Zhu, Huanzhang

    2014-09-01

    A major reason that Acquired Immune Deficiency Syndrome (AIDS) cannot be completely cured is the human immunodeficiency virus 1 (HIV-1) provirus integrated into the human genome. Though existing therapies can inhibit replication of HIV-1, they cannot eradicate it. A molecular therapy gains popularity due to its specifically targeting to HIV-1 infected cells and effectively removing the HIV-1, regardless of viral genes being active or dormant. Now, we propose a new method which can excellently delete the HIV provirus from the infected human T cell genome. First, we designed zinc-finger nucleases (ZFNs) that target a sequence within the long terminal repeat (LTR) U3 region that is highly conserved in whole clade. Then, we screened out one pair of ZFN and named it as ZFN-U3. We discovered that ZFN-U3 can exactly target and eliminate the full-length HIV-1 proviral DNA after the infected human cell lines treated with it, and the frequency of its excision was about 30 % without cytotoxicity. These results prove that ZFN-U3 can efficiently excise integrated HIV-1 from the human genome in infected cells. This method to delete full length HIV-1 in human genome can therefore provide a novel approach to cure HIV-infected individuals in the future.

  14. Inhibition of DNA replication of human papillomavirus by using zinc finger-single-chain FokI dimer hybrid.

    PubMed

    Mino, Takashi; Mori, Tomoaki; Aoyama, Yasuhiro; Sera, Takashi

    2014-08-01

    Previously, we reported that an artificial zinc-finger protein (AZP)-staphylococcal nuclease (SNase) hybrid (designated AZP-SNase) inhibited DNA replication of human papillomavirus type 18 (HPV-18) in mammalian cells by binding to and cleaving a specific HPV-18 ori plasmid. Although the AZP-SNase did not show any side effects under the experimental conditions, the SNase is potentially able to cleave RNA as well as DNA. In the present study, to make AZP hybrid nucleases that cleave only viral DNA, we switched the SNase moiety in the AZP-SNase to the single-chain FokI dimer (scFokI) that we had developed previously. We demonstrated that transfection with a plasmid expressing the resulting hybrid nuclease (designated AZP-scFokI) inhibited HPV-18 DNA replication in transient replication assays using mammalian cells more efficiently than AZP-SNase. Then, by linker-mediated PCR analysis, we confirmed that AZP-scFokI cleaved an HPV-18 ori plasmid around its binding site in mammalian cells. Finally, a modified MTT assay revealed that AZP-scFokI did not show any significant cytotoxicity. Thus, the newly developed AZP-scFokI hybrid is expected to serve as a novel antiviral reagent for the neutralization of human DNA viruses with less fewer potential side effects.

  15. Gene- and protein-delivered zinc finger-staphylococcal nuclease hybrid for inhibition of DNA replication of human papillomavirus.

    PubMed

    Mino, Takashi; Mori, Tomoaki; Aoyama, Yasuhiro; Sera, Takashi

    2013-01-01

    Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP-SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP-SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP-SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP-SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.

  16. Expression, purification and characterization of zinc-finger nuclease to knockout the goat beta-lactoglobulin gene.

    PubMed

    Song, Yujie; Cui, Chenchen; Zhu, Hongmei; Li, Qian; Zhao, Fan; Jin, Yaping

    2015-08-01

    Engineered zinc-finger nucleases (ZFNs) have been widely used for precise genome editing. ZFNs can induce DNA double-strand breaks at specific genomic locations and drive the introduction of an insertion or deletion of base pairs at the targeted region, consequently resulting in a loss-of-function mutation. In this study, we investigated the cloning, expression and purification of ZFN fusion proteins targeting the goat beta-lactoglobulin (BLG) gene and detected the cleavage activities of these ZFN proteins in vitro and in cells, respectively. The results showed that the pET-BLG-LFN and pET-BLG-RFN prokaryotic expression plasmids can be constructed correctly and expressed efficiently in Escherichia coli BL21 (DE3) cells to produce the 6× His-tagged ZFN proteins that can be purified by Ni-IDA-Sefinose Column. The predetermined sequence of BLG can be recognized and excised both in vitro and in goat fibroblasts by the purified ZFN fusion proteins, which demonstrated that the purified ZFN fusion proteins can be used as gene modification tools to knock out the BLG gene. Furthermore, these results lay the foundation for eliminating allergen BLG from goat milk and improving the quality of goat milk products.

  17. Selection for a Zinc-Finger Protein Contributes to Seed Oil Increase during Soybean Domestication1[OPEN

    PubMed Central

    Li, Qing-Tian; Lu, Xiang; Song, Qing-Xin; Chen, Hao-Wei; Wei, Wei; Tao, Jian-Jun; Ma, Biao; Bi, Ying-Dong; Li, Wei; Lai, Yong-Cai; Shui, Guang-Hou; Chen, Shou-Yi

    2017-01-01

    Seed oil is a momentous agronomical trait of soybean (Glycine max) targeted by domestication in breeding. Although multiple oil-related genes have been uncovered, knowledge of the regulatory mechanism of seed oil biosynthesis is currently limited. We demonstrate that the seed-preferred gene GmZF351, encoding a tandem CCCH zinc finger protein, is selected during domestication. Further analysis shows that GmZF351 facilitates oil accumulation by directly activating WRINKLED1, BIOTIN CARBOXYL CARRIER PROTEIN2, 3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE III, DIACYLGLYCEROL O-ACYLTRANSFERASE1, and OLEOSIN2 in transgenic Arabidopsis (Arabidopsis thaliana) seeds. Overexpression of GmZF351 in transgenic soybean also activates lipid biosynthesis genes, thereby accelerating seed oil accumulation. The ZF351 haplotype from the cultivated soybean group and the wild soybean (Glycine soja) subgroup III correlates well with high gene expression level, seed oil contents and promoter activity, suggesting that selection of GmZF351 expression leads to increased seed oil content in cultivated soybean. Our study provides novel insights into the regulatory mechanism for seed oil accumulation, and the manipulation of GmZF351 may have great potential in the improvement of oil production in soybean and other related crops. PMID:28184009

  18. Myeloid zinc finger 1 mediates sulindac sulfide-induced upregulation of death receptor 5 of human colon cancer cells

    PubMed Central

    Horinaka, Mano; Yoshida, Tatsushi; Tomosugi, Mitsuhiro; Yasuda, Shusuke; Sowa, Yoshihiro; Sakai, Toshiyuki

    2014-01-01

    A combined therapy of sulindac sulfide and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising strategy for the treatment of cancer. Sulindac sulfide had been shown to induce the expression of death receptor 5 (DR5), a receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism underlying the upregulation of DR5 has not yet been elucidated. We demonstrate here that myeloid zinc finger 1 (MZF1) mediates the induction of DR5 by sulindac sulfide. Sulindac sulfide induced the expression of DR5 at the protein and mRNA levels in colon cancer SW480 cells. Furthermore, sulindac sulfide increased DR5 promoter activity. We showed that sulindac sulfide stimulated DR5 promoter activity via the −301 to −253 region. This region contained a putative MZF1-binding site. Site-directed mutations in the site abrogated the enhancement in DR5 promoter activity by sulindac sulfide. MZF1 directly bound to the putative MZF1-binding site of the DR5 promoter and the binding was increased by sulindac sulfide. The expression of MZF1 was also increased by sulindac sulfide, and MZF1 siRNA attenuated the upregulation of DR5 by sulindac sulfide. These results indicate that sulindac sulfide induces the expression of DR5 by up-regulating MZF1. PMID:25102912

  19. Histone Deacetylase Inhibition Rescues Gene Knockout Levels Achieved with Integrase-Defective Lentiviral Vectors Encoding Zinc-Finger Nucleases

    PubMed Central

    Pelascini, Laetitia P.L.; Maggio, Ignazio; Liu, Jin; Holkers, Maarten; Cathomen, Toni

    2013-01-01

    Abstract Zinc-finger nucleases (ZFNs) work as dimers to induce double-stranded DNA breaks (DSBs) at predefined chromosomal positions. In doing so, they constitute powerful triggers to edit and to interrogate the function of genomic sequences in higher eukaryotes. A preferred route to introduce ZFNs into somatic cells relies on their cotransduction with two integrase-defective lentiviral vectors (IDLVs) each encoding a monomer of a functional heterodimeric pair. The episomal nature of IDLVs diminishes the risk of genotoxicity and ensures the strict transient expression profile necessary to minimize deleterious effects associated with long-term ZFN activity. However, by deploying IDLVs and conventional lentiviral vectors encoding HPRT1- or eGFP-specific ZFNs, we report that DSB formation at target alleles is limited after IDLV-mediated ZFN transfer. This IDLV-specific underperformance stems, to a great extent, from the activity of chromatin-remodeling histone deacetylases (HDACs). Importantly, the prototypic and U.S. Food and Drug Administration–approved inhibitors of metal-dependent HDACs, trichostatin A and vorinostat, respectively, did not hinder illegitimate recombination-mediated repair of targeted chromosomal DSBs. This allowed rescuing IDLV-mediated site-directed mutagenesis to levels approaching those achieved by using their isogenic chromosomally integrating counterparts. Hence, HDAC inhibition constitutes an efficacious expedient to incorporate in genome-editing strategies based on transient IDLV-mediated ZFN expression. Finally, we compared two of the most commonly used readout systems to measure targeted gene knockout activities based on restriction and mismatch-sensitive endonucleases. These experiments indicate that these enzymatic assays display a similar performance. PMID:24059449

  20. Ultrasensitive Electrochemical Detection of miRNA-21 Using a Zinc Finger Protein Specific to DNA-RNA Hybrids.

    PubMed

    Fang, Chiew San; Kim, Kwang-Sun; Yu, Byeongjun; Jon, Sangyong; Kim, Moon-Soo; Yang, Haesik

    2017-02-07

    Both high sensitivity and high specificity are crucial for detection of miRNAs that have emerged as important clinical biomarkers. Just Another Zinc finger proteins (JAZ, ZNF346) bind preferably (but nonsequence-specifically) to DNA-RNA hybrids over single-stranded RNAs, single-stranded DNAs, and double-stranded DNAs. We present an ultrasensitive and highly specific electrochemical method for miRNA-21 detection based on the selective binding of JAZ to the DNA-RNA hybrid formed between a DNA capture probe and a target miRNA-21. This enables us to use chemically stable DNA as a capture probe instead of RNA as well as to apply a standard sandwich-type assay format to miRNA detection. High signal amplification is obtained by (i) enzymatic amplification by alkaline phosphatase (ALP) coupled with (ii) electrochemical-chemical-chemical (ECC) redox cycling involving an ALP product (hydroquinone). Low nonspecific adsorption of ALP-conjugated JAZ is obtained using a polymeric self-assembled-monolayer-modified and casein-treated indium-tin oxide electrode. The detection method can discriminate between target miRNA-21 and nontarget nucleic acids (DNA-DNA hybrid, single-stranded DNA, miRNA-125b, miRNA-155, single-base mismatched miRNA, and three-base mismatched miRNA). The detection limits for miRNA-21 in buffer and 10-fold diluted serum are approximately 2 and 30 fM, respectively, indicating that the detection method is ultrasensitive. This detection method can be readily extended to multiplex detection of miRNAs with only one ALP-conjugated JAZ probe due to its nonsequence-specific binding character. We also believe that the method could offer a promising solution for point-of-care testing of miRNAs in body fluids.

  1. The role of Zic family zinc finger transcription factors in the proliferation and differentiation of retinal progenitor cells

    SciTech Connect

    Watabe, Yui; Baba, Yukihiro; Nakauchi, Hiromitsu; Mizota, Atsushi; Watanabe, Sumiko

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer Zic transcription factors expressed early retinal progenitor cells. Black-Right-Pointing-Pointer Zics sustain proliferation activity of retinal progenitor cells. Black-Right-Pointing-Pointer Overexpression of Zic in retinal progenitors perturbed rod differentiation. Black-Right-Pointing-Pointer Fate determination to rod photoreceptor was not affected. -- Abstract: Members of the Zic family of zinc finger transcription factors play critical roles in a variety of developmental processes. Using DNA microarray analysis, we found that Zics are strongly expressed in SSEA-1-positive early retinal progenitors in the peripheral region of the mouse retina. Reverse-transcription polymerase chain reaction using mRNA from the retina at various developmental stages showed that Zic1 and Zic2 are expressed in the embryonic retina and then gradually disappear during retinal development. Zic3 is also expressed in the embryonic retina; its expression level slightly decreases but it is expressed until adulthood. We overexpressed Zic1, Zic2, or Zic3 in retinal progenitors at embryonic day 17.5 and cultured the retina as explants for 2 weeks. The number of rod photoreceptors was fewer than in the control, but no other cell types showed significant differences between control and Zic overexpressing cells. The proliferation activity of normal retinal progenitors decreased after 5 days in culture, as observed in normal in vivo developmental processes. However, Zic expressing retinal cells continued to proliferate at days 5 and 7, suggesting that Zics sustain the proliferation activities of retinal progenitor cells. Since the effects of Zic1, 2, and 3 are indistinguishable in terms of differentiation and proliferation of retinal progenitors, the redundant function of Zics in retinal development is suggested.

  2. Zinc finger E-box-binding homeobox 1: its clinical significance and functional role in human thyroid cancer

    PubMed Central

    Zhang, Yan; Liu, Gang; Wu, Shihe; Jiang, Futing; Xie, Jiangping; Wang, Yuhong

    2016-01-01

    Objective Transcription factor zinc finger E-box-binding homeobox 1 (ZEB1), as one of the key inducers of epithelial-mesenchymal transition, has been reported to be regulated by microRNA-144 and Bcl-2-associated athanogene 3, which both promote thyroid cancer cell invasion. However, the involvement of ZEB1 in thyroid cancer has not been fully elucidated. In this study, we aimed to investigate the role and clinical implication of ZEB1 in this disease. Methods Immunohistochemistry was performed to examine the subcellular localization and the expression level of ZEB1 protein in 82 self-pairs of formalin-fixed and paraffin-embedded cancerous and adjacent noncancerous tissues obtained from patients with thyroid cancer. The roles of ZEB1 in thyroid cancer cell migration, invasion, and proliferation were also detected by transwell and MTT analyses, respectively. Results Immunohistochemistry showed that ZEB1 was predominantly localized in the nucleus of thyroid cancer cells. Its immunoreactive score in thyroid cancer tissues was significantly higher than that in adjacent noncancerous tissues (P=0.01). In addition, ZEB1 overexpression was significantly associated with the advanced tumor node metastasis staging (P=0.008), the positive lymph node metastasis (P=0.01) and distant metastasis (P=0.02). Furthermore, ZEB1 knockdown by siRNA could efficiently inhibit the migration, invasion, and proliferation abilities of thyroid cancer cells in vitro. Conclusion These findings indicated that ZEB1 might function as an oncogene, the overexpression of which was associated with the aggressive tumor progression of human thyroid cancer. Interestingly, ZEB1 also could promote thyroid cancer cell migration, invasion, and proliferation, suggesting that the inhibition of this protein might be a therapeutic strategy for the treatment of this malignancy. PMID:27099512

  3. The zinc finger transcription factor SlZFP2 negatively regulates abscisic acid biosynthesis and fruit ripening in tomato.

    PubMed

    Weng, Lin; Zhao, Fangfang; Li, Rong; Xu, Changjie; Chen, Kunsong; Xiao, Han

    2015-03-01

    Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. Although the ABA biosynthesis pathway in plants has been thoroughly elucidated, how ABA biosynthetic genes are regulated at the molecular level during plant development is less well understood. Here, we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2 is involved in the regulation of ABA biosynthesis during fruit development. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds, and faster seed germination, whereas down-regulation of its expression caused problematic fruit set, accelerated ripening, and inhibited seed germination. SlZFP2 represses ABA biosynthesis during fruit development through direct suppression of the ABA biosynthetic genes NOTABILIS, SITIENS, and FLACCA and the aldehyde oxidase SlAO1. We also show that SlZFP2 regulates fruit ripening through transcriptional suppression of the ripening regulator COLORLESS NON-RIPENING. Using bacterial one-hybrid screening and a selected amplification and binding assay, we identified the (A/T)(G/C)TT motif as the core binding sequence of SlZFP2. Furthermore, by RNA sequencing profiling, we found that 193 genes containing the SlZFP2-binding motifs in their promoters were differentially expressed in 2 d post anthesis fruits between the SlZFP2 RNA interference line and its nontransgenic sibling. We propose that SlZFP2 functions as a repressor to fine-tune ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening.

  4. Transient Expression of Fez Family Zinc Finger 2 Protein Regulates the Brn3b Gene in Developing Retinal Ganglion Cells.

    PubMed

    Qu, Chunsheng; Bian, Dandan; Li, Xue; Xiao, Jian; Wu, Chunping; Li, Yue; Jiang, Tian; Zhou, Xiangtian; Qu, Jia; Chen, Jie-Guang

    2016-04-01

    Retinal ganglion cells (RGCs) are projection neurons in the neural retina that relay visual information from the environment to the central nervous system. The early expression of MATH5 endows the post-mitotic precursors with RGC competence and leads to the activation ofBrn3bthat marks committed RGCs. Nevertheless, this fate commitment process and, specifically, regulation ofBrn3bremain elusive. To explore the molecular mechanisms underlying RGC generation in the mouse retina, we analyzed the expression and function of Fez family zinc finger 2 (FEZF2), a transcription factor critical for the development of projection neurons in the cerebral cortex.Fezf2mRNA and protein were transiently expressed at embryonic day 16.5 in the inner neuroblast layer and the prospective ganglion cell layer of the retina, respectively. Knockout ofFezf2in the developing retina reduced BRN3B+ cells and increased apoptotic cell markers.Fezf2knockdown by retinalin uteroelectroporation diminished BRN3B but not the coexpressed ISLET1 and BRN3A, indicating that the BRN3B decrease was the cause, not the result, of the overall reduction of BRN3B+ RGCs in theFezf2knockout retina. Moreover, the mRNA and promoter activity ofBrn3bwere increasedin vitroby FEZF2, which bound to a 5' regulatory fragment in theBrn3bgenomic locus. These results indicate that transient expression ofFezf2in the retina modulates the transcription ofBrn3band the survival of RGCs. This study improves our understanding of the transcriptional cascade required for the specification of RGCs and provides novel insights into the molecular basis of retinal development.

  5. Vascular plant one-zinc-finger protein 1/2 transcription factors regulate abiotic and biotic stress responses in Arabidopsis.

    PubMed

    Nakai, Yusuke; Nakahira, Yoichi; Sumida, Hiroki; Takebayashi, Kosuke; Nagasawa, Yumiko; Yamasaki, Kanako; Akiyama, Masako; Ohme-Takagi, Masaru; Fujiwara, Sumire; Shiina, Takashi; Mitsuda, Nobutaka; Fukusaki, Eiichiro; Kubo, Yasuyuki; Sato, Masa H

    2013-03-01

    Plants adapt to abiotic and biotic stresses by activating abscisic acid-mediated (ABA) abiotic stress-responsive and salicylic acid-(SA) or jasmonic acid-mediated (JA) biotic stress-responsive pathways, respectively. Although the abiotic stress-responsive pathway interacts antagonistically with the biotic stress-responsive pathways, the mechanisms that regulate these pathways remain largely unknown. In this study, we provide insight into the function of vascular plant one-zinc-finger proteins (VOZs) that modulate various stress responses in Arabidopsis. The expression of many stress-responsive genes was changed in the voz1voz2 double mutant under normal growth conditions. Consistent with altered stress-responsive gene expression, freezing- and drought-stress tolerances were increased in the voz1voz2 double mutant. In contrast, resistance to a fungal pathogen, Colletotrichum higginsianum, and to a bacterial pathogen, Pseudomonas syringae, was severely impaired. Thus, impairing VOZ function simultaneously conferred increased abiotic tolerance and biotic stress susceptibility. In a chilling stress condition, both the VOZ1 and VOZ2 mRNA expression levels and the VOZ2 protein level gradually decreased. VOZ2 degradation during cold exposure was completely inhibited by the addition of the 26S proteasome inhibitor, MG132, a finding that suggested that VOZ2 degradation is dependent on the ubiquitin/26S proteasome system. In voz1voz2, ABA-inducible transcription factor CBF4 expression was enhanced significantly even under normal growth conditions, despite an unchanged endogenous ABA content. A finding that suggested that VOZs negatively affect CBF4 expression in an ABA-independent manner. These results suggest that VOZs function as both negative and positive regulators of the abiotic and biotic stress-responsive pathways, and control Arabidopsis adaptation to various stress conditions.

  6. Expression of Arabidopsis FCS-Like Zinc finger genes is differentially regulated by sugars, cellular energy level, and abiotic stress.

    PubMed

    Jamsheer K, Muhammed; Laxmi, Ashverya

    2015-01-01

    Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1) signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ) gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response toward energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response.

  7. Comprehensive analysis of CCCH-type zinc finger gene family in citrus (Clementine mandarin) by genome-wide characterization.

    PubMed

    Liu, Shengrui; Khan, Muhammad Rehman Gul; Li, Yongping; Zhang, Jinzhi; Hu, Chungen

    2014-10-01

    The CCCH-type zinc finger proteins comprise a large gene family of regulatory proteins and are widely distributed in eukaryotic organisms. The CCCH proteins have been implicated in multiple biological processes and environmental responses in plants. Little information is available, however, about CCCH genes in plants, especially in woody plants such as citrus. The release of the whole-genome sequence of citrus allowed us to perform a genome-wide analysis of CCCH genes and to compare the identified proteins with their orthologs in model plants. In this study, 62 CCCH genes and a total of 132 CCCH motifs were identified, and a comprehensive analysis including the chromosomal locations, phylogenetic relationships, functional annotations, gene structures and conserved motifs was performed. Distribution mapping revealed that 54 of the 62 CCCH genes are unevenly dispersed on the nine citrus chromosomes. Based on phylogenetic analysis and gene structural features, we constructed 5 subfamilies of 62 CCCH members and integrative subfamilies from citrus, Arabidopsis, and rice, respectively. Importantly, large numbers of SNPs and InDels in 26 CCCH genes were identified from Poncirus trifoliata and Fortunella japonica using whole-genome deep re-sequencing. Furthermore, citrus CCCH genes showed distinct temporal and spatial expression patterns in different developmental processes and in response to various stress conditions. Our comprehensive analysis of CleC3Hs is a valuable resource that further elucidates the roles of CCCH family members in plant growth and development. In addition, variants and comparative genomics analyses deepen our understanding of the evolution of the CCCH gene family and will contribute to further genetics and genomics studies of citrus and other plant species.

  8. Association of a genetic variant of the ZPR1 zinc finger gene with type 2 diabetes mellitus.

    PubMed

    Tokoro, Fumitaka; Matsuoka, Reiko; Abe, Shintaro; Arai, Masazumi; Noda, Toshiyuki; Watanabe, Sachiro; Horibe, Hideki; Fujimaki, Tetsuo; Oguri, Mitsutoshi; Kato, Kimihiko; Minatoguchi, Shinya; Yamada, Yoshiji

    2015-01-01

    Various loci and genes that confer susceptibility to coronary heart disease (CHD) have been identified in Caucasian populations by genome-wide association studies (GWASs). As type 2 diabetes mellitus (DM) is an important risk factor for CHD, we hypothesized that certain polymorphisms may contribute to the genetic susceptibility to CHD through affecting the susceptibility to type 2 DM. The purpose of the present study was to examine a possible association of type 2 DM in Japanese individuals with 29 polymorphisms identified as susceptibility loci for CHD by meta-analyses of the GWASs. The study subjects comprised of 3,757 individuals (1,444 subjects with type 2 DM and 2,313 controls). The polymorphism genotypes were determined by the multiplex bead-based Luminex assay, which combines the polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology. To compensate for multiple comparisons of genotypes, the criterion of a false discovery rate (FDR) ≤0.05 was adopted for testing the statistical significance of the association. The comparisons of allele frequencies by the χ(2) test revealed that the rs964184 (C→G) of the ZPR1 zinc finger gene (ZPR1) was significantly associated (P=0.0017; FDR=0.050) with type 2 DM. Multivariable logistic regression analysis with adjustment for age, gender and body mass index revealed that rs964184 of ZPR1 was significantly associated (P=0.0012; odds ratio, 1.25; dominant model) with type 2 DM with the minor G allele representing a risk factor for this condition. Fasting plasma glucose levels (P=0.0076) and blood glycosylated hemoglobin contents (P=0.0132) significantly differed among ZPR1 genotypes with the G allele associated with increases in these parameters. ZPR1 may thus be a susceptibility locus for type 2 DM in Japanese individuals.

  9. The Proteasome Inhibitor Bortezomib Is a Potent Inducer of Zinc Finger AN1-type Domain 2a Gene Expression

    PubMed Central

    Rossi, Antonio; Riccio, Anna; Coccia, Marta; Trotta, Edoardo; La Frazia, Simone; Santoro, M. Gabriella

    2014-01-01

    The zinc finger AN1-type domain 2a gene, also known as arsenite-inducible RNA-associated protein (AIRAP), was recently identified as a novel human canonical heat shock gene strictly controlled by heat shock factor (HSF) 1. Little is known about AIRAP gene regulation in human cells. Here we report that bortezomib, a proteasome inhibitor with anticancer and antiangiogenic properties used in the clinic for treatment of multiple myeloma, is a potent inducer of AIRAP expression in human cells. Using endothelial cells as a model, we unraveled the molecular mechanism regulating AIRAP expression during proteasome inhibition. Bortezomib induces AIRAP expression at the transcriptional level early after treatment, concomitantly with polyubiquitinated protein accumulation and HSF activation. AIRAP protein is detected at high levels for at least 48 h after bortezomib exposure, together with the accumulation of HSF2, a factor implicated in differentiation and development regulation. Different from heat-mediated induction, in bortezomib-treated cells, HSF1 and HSF2 interact directly, forming HSF1-HSF2 heterotrimeric complexes recruited to a specific heat shock element in the AIRAP promoter. Interestingly, whereas HSF1 has been confirmed to be critical for AIRAP gene transcription, HSF2 was found to negatively regulate AIRAP expression after bortezomib treatment, further emphasizing an important modulatory role of this transcription factor under stress conditions. AIRAP function is still not defined. However, the fact that AIRAP is expressed abundantly in primary human cells at bortezomib concentrations comparable with plasma levels in treated patients suggests that AIRAP may participate in the regulatory network controlling proteotoxic stress during bortezomib treatment. PMID:24619424

  10. The ZNF75 zinc finger gene subfamily: Isolation and mapping of the four members in humans and great apes

    SciTech Connect

    Villa, A.; Strina, D.; Frattini, A.

    1996-07-15

    We have previously reported the characterization of the human ZNF75 gene located on Xq26, which has only limited homology (less than 65%) to other ZF genes in the databases. Here, we describe three human zinc finger genes with 86 to 95% homology to ZNF75 at the nucleotide level, which represent all the members of the human ZNF75 subfamily. One of these, ZNF75B, is a pseudogene mapped to chromosome 12q13. The other two, ZNF75A and ZNF75C, maintain on ORF in the sequenced region, and at least the latter is expressed in the U937 cell line. They were mapped to chromosomes 16 and 11, respectively. All these genes are conserved in chimpanzees, gorillas, and orangutans. The ZNF75B homologue is a pseudogene in all three great apes, and in chimpanzee it is located on chromosome 10 (phylogenetic XII), at p13 (corresponding to the human 12q13). The chimpanzee homologue of ZNF75 is also located on the Xq26 chromosome, in the same region, as detected by in situ hybridization. As expected, nucleotide changes were clearly more abundant between human and organutan than between human and chimpanzee or gorilla homologues. Members of the same class were more similar to each other than to the other homologues within the same species. This suggests that the duplication and/or retrotranscription events occurred in a common ancestor long before great ape speciation. This, together with the existance of at least two genes in cows and horses, suggests a relatively high conservation of this gene family. 20 refs., 5 figs., 1 tab.

  11. The Zinc Finger Transcription Factor SlZFP2 Negatively Regulates Abscisic Acid Biosynthesis and Fruit Ripening in Tomato1

    PubMed Central

    Weng, Lin; Zhao, Fangfang; Li, Rong; Xu, Changjie; Chen, Kunsong

    2015-01-01

    Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. Although the ABA biosynthesis pathway in plants has been thoroughly elucidated, how ABA biosynthetic genes are regulated at the molecular level during plant development is less well understood. Here, we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2 is involved in the regulation of ABA biosynthesis during fruit development. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds, and faster seed germination, whereas down-regulation of its expression caused problematic fruit set, accelerated ripening, and inhibited seed germination. SlZFP2 represses ABA biosynthesis during fruit development through direct suppression of the ABA biosynthetic genes NOTABILIS, SITIENS, and FLACCA and the aldehyde oxidase SlAO1. We also show that SlZFP2 regulates fruit ripening through transcriptional suppression of the ripening regulator COLORLESS NON-RIPENING. Using bacterial one-hybrid screening and a selected amplification and binding assay, we identified the (A/T)(G/C)TT motif as the core binding sequence of SlZFP2. Furthermore, by RNA sequencing profiling, we found that 193 genes containing the SlZFP2-binding motifs in their promoters were differentially expressed in 2 d post anthesis fruits between the SlZFP2 RNA interference line and its nontransgenic sibling. We propose that SlZFP2 functions as a repressor to fine-tune ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening. PMID:25637453

  12. Functional analysis of a breast cancer-associated FGFR2 single nucleotide polymorphism using zinc finger mediated genome editing.

    PubMed

    Robbez-Masson, Luisa J; Bödör, Csaba; Jones, J Louise; Hurst, Helen C; Fitzgibbon, Jude; Hart, Ian R; Grose, Richard P

    2013-01-01

    Genome wide association studies have identified single nucleotide polymorphisms (SNP) within fibroblast growth factor receptor 2 (FGFR2) as one of the highest ranking risk alleles in terms of development of breast cancer. The potential effect of these SNPs, in intron two, was postulated to be due to the differential binding of cis-regulatory elements, such as transcription factors, since all the SNPs in linkage disequilibrium were located in a regulatory DNA region. A Runx2 binding site was reported to be functional only in the minor, disease associated allele of rs2981578, resulting in increased expression of FGFR2 in cancers from patients homozygous for that allele. Moreover, the increased risk conferred by the minor FGFR2 allele associates most strongly in oestrogen receptor alpha positive (ERα) breast tumours, suggesting a potential interaction between ERα and FGFR signalling. Here, we have developed a human cell line model system to study the effect of the putative functional SNP, rs2981578, on cell behaviour. MCF7 cells, an ERα positive breast cancer cell line homozygous for the wild-type allele were edited using a Zinc Finger Nuclease approach. Unexpectedly, the acquisition of a single risk allele in MCF7 clones failed to affect proliferation or cell cycle progression. Binding of Runx2 to the risk allele was not observed. However FOXA1 binding, an important ERα partner, appeared decreased at the rs2981578 locus in the risk allele cells. Differences in allele specific expression (ASE) of FGFR2 were not observed in a panel of 72 ERα positive breast cancer samples. Thus, the apparent increased risk of developing ERα positive breast cancer seems not to be caused by rs2981578 alone. Rather, the observed increased risk of developing breast cancer might be the result of a coordinated effect of multiple SNPs forming a risk haplotype in the second intron of FGFR2.

  13. A classic zinc finger from friend of GATA mediates an interaction with the coiled-coil of transforming acidic coiled-coil 3.

    PubMed

    Simpson, Raina J Y; Yi Lee, Stella Hoi; Bartle, Natalie; Sum, Eleanor Y; Visvader, Jane E; Matthews, Jacqueline M; Mackay, Joel P; Crossley, Merlin

    2004-09-17

    Classic zinc finger domains (cZFs) consist of a beta-hairpin followed by an alpha-helix. They are among the most abundant of all protein domains and are often found in tandem arrays in DNA-binding proteins, with each finger contributing an alpha-helix to effect sequence-specific DNA recognition. Lone cZFs, not found in tandem arrays, have been postulated to function in protein interactions. We have studied the transcriptional co-regulator Friend of GATA (FOG), which contains nine zinc fingers. We have discovered that the third cZF of FOG contacts a coiled-coil domain in the centrosomal protein transforming acidic coiled-coil 3 (TACC3). Although FOG-ZF3 exhibited low solubility, we have used a combination of mutational mapping and protein engineering to generate a derivative that was suitable for in vitro and structural analysis. We report that the alpha-helix of FOG-ZF3 recognizes a C-terminal portion of the TACC3 coiled-coil. Remarkably, the alpha-helical surface utilized by FOG-ZF3 is the same surface responsible for the well established sequence-specific DNA-binding properties of many other cZFs. Our data demonstrate the versatility of cZFs and have implications for the analysis of many as yet uncharacterized cZF proteins.

  14. Single Amino Acid Exchanges in Separate Domains of the Drosophila Serendipity δ Zinc Finger Protein Cause Embryonic and Sex Biased Lethality

    PubMed Central

    Crozatier, M.; Kongsuwan, K.; Ferrer, P.; Merriam, J. R.; Lengyel, J. A.; Vincent, A.

    1992-01-01

    The Drosophila serendipity (sry) delta (δ) zinc finger protein is a sequence-specific DNA binding protein, maternally inherited by the embryo and present in nuclei of transcriptionally active cells throughout fly development. We report here the isolation and characterization of four ethyl methanesulfate-induced zygotic lethal mutations of different strengths in the sry δ gene. For the stronger allele, all of the lethality occurs during late embryogenesis or the first larval instar. In the cases of the three weaker alleles, most of the lethality occurs during pupation; moreover, those adult escapers that emerge are sterile males lacking partially or completely in spermatozoa bundles. Genetic analysis of sry δ thus indicates that it is an essential gene, whose continued expression throughout the life cycle, notably during embryogenesis and pupal stage, is required for viability. Phenotypic analysis of sry δ hemizygote escaper males further suggests that sry δ may be involved in regulation of two different sets of genes: genes required for viability and genes involved in gonadal development. All four sry δ alleles are fully rescued by a wild-type copy of sry δ, but not by an additional copy of the sry β gene, reinforcing the view that, although structurally related, these two genes exert distinct functions. Molecular characterization of the four sry δ mutations revealed that these mutations correspond to single amino acid replacements in the sry δ protein. Three of these replacements map to the same (third out of seven) zinc finger in the carboxy-terminal DNA binding domain; interestingly, none affects the zinc finger consensus residues. The fourth mutation is located in the NH(2)-proximal part of the protein, in a domain proposed to be involved in specific protein-protein interactions. PMID:1516821

  15. Overexpression of the zinc finger protein MZF1 inhibits hematopoietic development from embryonic stem cells: correlation with negative regulation of CD34 and c-myb promoter activity.

    PubMed Central

    Perrotti, D; Melotti, P; Skorski, T; Casella, I; Peschle, C; Calabretta, B

    1995-01-01

    Zinc finger genes encode proteins that act as transcription factors. The myeloid zinc finger 1 (MZF1) gene encodes a zinc finger protein with two DNA-binding domains that recognize two distinct consensus sequences, is preferentially expressed in hematopoietic cells, and may be involved in the transcriptional regulation of hematopoiesis-specific genes. Reverse transcription-PCR analysis of human peripheral blood CD34+ cells cultured under lineage-restricted conditions demonstrated MZF1 expression during both myeloid and erythroid differentiation. Sequence analysis of the 5'-flanking region of the CD34 and c-myb genes, which are a marker of and a transcriptional factor required for hematopoietic proliferation and differentiation, respectively, revealed closely spaced MZF1 consensus binding sites found by electrophoretic mobility shift assays to interact with recombinant MZF1 protein. Transient or constitutive MZF1 expression in different cell types resulted in specific inhibition of chloramphenicol acetyltransferase activity driven by the CD34 or c-myb 5'-flanking region. To determine whether transcriptional modulation by MZF1 activity plays a role in hematopoietic differentiation, constructs containing the MZF1 cDNA under the control of different promoters were transfected into murine embryonic stem cells which, under defined in vitro culture conditions, generate colonies of multiple hematopoietic lineages. Constitutive MZF1 expression interfered with the ability of embryonic stem cells to undergo hematopoietic commitment and erythromyeloid colony formation and prevented the induced expression of CD34 and c-myb mRNAs during differentiation of these cells. These data indicate that MZF1 plays a critical role in hematopoiesis by modulating the expression of genes involved in this process. PMID:7565760

  16. Gene targeting technologies in rats: zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats.

    PubMed

    Mashimo, Tomoji

    2014-01-01

    The laboratory rat has been widely used as an animal model in biomedical science for more than 150 years. Applying zinc-finger nucleases or transcription activator-like effector nucleases to rat embryos via microinjection is an efficient genome editing tool for generating targeted knockout rats. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonucleases have been used as an effective tool for precise and multiplex genome editing in mice and rats. In this review, the advantages and disadvantages of these site-specific nuclease technologies for genetic analysis and manipulation in rats are discussed.

  17. Generation of SNCA Cell Models Using Zinc Finger Nuclease (ZFN) Technology for Efficient High-Throughput Drug Screening

    PubMed Central

    Dansithong, Warunee; Paul, Sharan; Scoles, Daniel R.; Pulst, Stefan M.; Huynh, Duong P.

    2015-01-01

    Parkinson’s disease (PD) is a progressive neurodegenerative disorder caused by loss of dopaminergic neurons of the substantia nigra. The hallmark of PD is the appearance of neuronal protein aggregations known as Lewy bodies and Lewy neurites, of which α-synuclein forms a major component. Familial PD is rare and is associated with missense mutations of the SNCA gene or increases in gene copy number resulting in SNCA overexpression. This suggests that lowering SNCA expression could be therapeutic for PD. Supporting this hypothesis, SNCA reduction was neuroprotective in cell line and rodent PD models. We developed novel cell lines expressing SNCA fused to the reporter genes luciferase (luc) or GFP with the objective to enable high-throughput compound screening (HTS) for small molecules that can lower SNCA expression. Because SNCA expression is likely regulated by far-upstream elements (including the NACP-REP1 located at 8852 bp upstream of the transcription site), we employed zinc finger nuclease (ZFN) genome editing to insert reporter genes in-frame downstream of the SNCA gene in order to retain native SNCA expression control. This ensured full retention of known and unknown up- and downstream genetic elements controlling SNCA expression. Treatment of cells with the histone deacetylase inhibitor valproic acid (VPA) resulted in significantly increased SNCA-luc and SNCA-GFP expression supporting the use of our cell lines for identifying small molecules altering complex modes of expression control. Cells expressing SNCA-luc treated with a luciferase inhibitor or SNCA siRNA resulted in Z’-scores ≥ 0.75, suggesting the suitability of these cell lines for use in HTS. This study presents a novel use of genome editing for the creation of cell lines expressing α-synuclein fusion constructs entirely under native expression control. These cell lines are well suited for HTS for compounds that lower SNCA expression directly or by acting at long-range sites to the SNCA

  18. Genome-wide Regulatory Roles of the C2H2-type Zinc Finger Protein ZNF764 on the Glucocorticoid Receptor

    PubMed Central

    Fadda, Abeer; Syed, Najeeb; Mackeh, Rafah; Papadopoulou, Anna; Suzuki, Shigeru; Jithesh, Puthen V.; Kino, Tomoshige

    2017-01-01

    The C2H2-type zinc finger protein ZNF764 acts as an enhancer for several steroid hormone receptors, and haploinsufficiency of this gene may be responsible for tissue resistance to multiple steroid hormones including glucocorticoids observed in a patient with 16p11.2 microdeletion. We examined genome-wide regulatory actions of ZNF764 on the glucocorticoid receptor (GR) in HeLa cells as a model system. ZNF764- and GR-binding sites demonstrated similar distribution in various genomic features. They positioned predominantly around 50–500 kbs from the transcription start sites of their nearby genes, and were closely localized with each other, overlapping in ~37% of them. ZNF764 demonstrated differential on/off effects on GR-binding and subsequent mRNA expression: some genes were highly dependent on the presence/absence of ZNF764, but others were not. Pathway analysis revealed that these 3 gene groups were involved in distinct cellular activities. ZNF764 physically interacted with GR at ligand-binding domain through its KRAB domain, and both its physical interaction to GR and zinc finger domain appear to be required for ZNF764 to regulate GR transcriptional activity. Thus, ZNF764 is a cofactor directing GR transcriptional activity toward specific biologic pathways by changing GR binding and transcriptional activity on the glucocorticoid-responsive genes. PMID:28139699

  19. POZ domain transcription factor, FBI-1, represses transcription of ADH5/FDH by interacting with the zinc finger and interfering with DNA binding activity of Sp1.

    PubMed

    Lee, Dong-Kee; Suh, Dongchul; Edenberg, Howard J; Hur, Man-Wook

    2002-07-26

    The POZ domain is a protein-protein interaction motif that is found in many transcription factors, which are important for development, oncogenesis, apoptosis, and transcription repression. We cloned the POZ domain transcription factor, FBI-1, that recognizes the cis-element (bp -38 to -22) located just upstream of the core Sp1 binding sites (bp -22 to +22) of the ADH5/FDH minimal promoter (bp -38 to +61) in vitro and in vivo, as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. The ADH5/FDH minimal promoter is potently repressed by the FBI-1. Glutathione S-transferase fusion protein pull-down showed that the POZ domains of FBI-1, Plzf, and Bcl-6 directly interact with the zinc finger DNA binding domain of Sp1. DNase I footprinting assays showed that the interaction prevents binding of Sp1 to the GC boxes of the ADH5/FDH promoter. Gal4-POZ domain fusions targeted proximal to the GC boxes repress transcription of the Gal4 upstream activator sequence-Sp1-adenovirus major late promoter. Our data suggest that POZ domain represses transcription by interacting with Sp1 zinc fingers and by interfering with the DNA binding activity of Sp1.

  20. The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger.

    PubMed Central

    Kudla, B; Caddick, M X; Langdon, T; Martinez-Rossi, N M; Bennett, C F; Sibley, S; Davies, R W; Arst, H N

    1990-01-01

    The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans has been sequenced and its transcript mapped and orientated. A single ORF can encode a protein of 719 amino acids. A 52 amino acid region including a putative 'zinc finger' strongly resembles putative DNA binding regions of the major regulatory protein of erythroid cells. The derived protein sequence also contains a highly acidic region possibly involved in gene activation and 22 copies of the motif S(T)PXX, abundant in DNA binding proteins. Analysis of chromosomal rearrangements and transformation with deletion clones identified 342 N-terminal and 124 C-terminal residues as inessential and localized a C-terminal region required for nitrogen metabolite repressibility. A -1 frameshift eliminating the inessential 122 C-terminal amino acids is a surprising loss-of-function mutation. Extraordinary basicity of the replacement C terminus might explain its phenotype. Mutant sequencing also identified a polypeptide chain termination and several missense mutations, but most interesting are sequence changes associated with specificity mutations. A mutation elevating expression of some structural genes under areA control whilst reducing or not affecting expression of others is a leucine to valine change in the zinc finger loop. It reverts to a partly reciprocal phenotype by replacing the mutant valine by methionine. Images Fig.2 Fig.4 Fig.5 Fig. 8. Fig. 9. PMID:1970293

  1. WRNIP1 accumulates at laser light irradiated sites rapidly via its ubiquitin-binding zinc finger domain and independently from its ATPase domain

    SciTech Connect

    Nomura, Hironoshin; Yoshimura, Akari; Edo, Takato; Kanno, Shin-ichiro; Tada, Syusuke; Seki, Masayuki; Yasui, Akira; Enomoto, Takemi

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer WRNIP1 accumulates in laser light irradiated sites very rapidly via UBZ domain. Black-Right-Pointing-Pointer The ATPase domain of WRNIP1 is dispensable for its accumulation. Black-Right-Pointing-Pointer The accumulation of WRNIP1 seems not to be dependent on the interaction with WRN. -- Abstract: WRNIP1 (Werner helicase-interacting protein 1) was originally identified as a protein that interacts with the Werner syndrome responsible gene product. WRNIP1 contains a ubiquitin-binding zinc-finger (UBZ) domain in the N-terminal region and two leucine zipper motifs in the C-terminal region. In addition, it possesses an ATPase domain in the middle of the molecule and the lysine residues serving as ubiquitin acceptors in the entire of the molecule. Here, we report that WRNIP1 accumulates in laser light irradiated sites very rapidly via its ubiquitin-binding zinc finger domain, which is known to bind polyubiquitin and to be involved in ubiquitination of WRNIP1 itself. The accumulation of WRNIP1 in laser light irradiated sites also required the C-terminal region containing two leucine zippers, which is reportedly involved in the oligomerization of WRNIP1. Mutated WRNIP1 with a deleted ATPase domain or with mutations in lysine residues, which serve as ubiquitin acceptors, accumulated in laser light irradiated sites, suggesting that the ATPase domain of WRNIP1 and ubiquitination of WRNIP1 are dispensable for the accumulation.

  2. Zinc-fingers and homeoboxes 1 (ZHX1) binds DNA methyltransferase (DNMT) 3B to enhance DNMT3B-mediated transcriptional repression

    SciTech Connect

    Kim, Sung-Hak; Park, Jinah; Choi, Moon-Chang; Kim, Hwang-Phill; Park, Jung-Hyun; Jung, Yeonjoo; Lee, Ju-Hee; Oh, Do-Youn; Im, Seock-Ah; Bang, Yung-Jue; Kim, Tae-You; E-mail: kimty@snu.ac.kr

    2007-04-06

    DNA methyltransferases (DNMT) 3B is a de novo DNMT that represses transcription independent of DNMT activity. In order to gain a better insight into DNMT3B-mediated transcriptional repression, we performed a yeast two-hybrid analysis using DNMT3B as a bait. Of the various binding candidates, ZHX1, a member of zinc-finger and homeobox protein, was found to interact with DNMT3B in vivo and in vitro. N-terminal PWWP domain of DNMT3B was required for its interaction with homeobox motifs of ZHX1. ZHX1 contains nuclear localization signal at C-terminal homeobox motif, and both ZHX1 and DNMT3B were co-localized in nucleus. Furthermore, we found that ZHX1 enhanced the transcriptional repression mediated by DNMT3B when DNMT3B is directly targeted to DNA. These results showed for First the direct linkage between DNMT and zinc-fingers homeoboxes protein, leading to enhanced gene silencing by DNMT3B.

  3. Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA

    PubMed Central

    Minczuk, Michal; Papworth, Monika A.; Miller, Jeffrey C.; Murphy, Michael P.; Klug, Aaron

    2008-01-01

    The selective degradation of mutated mitochondrial DNA (mtDNA) molecules is a potential strategy to re-populate cells with wild-type (wt) mtDNA molecules and thereby alleviate the defective mitochondrial function that underlies mtDNA diseases. Zinc finger nucleases (ZFNs), which are nucleases conjugated to a zinc-finger peptide (ZFP) engineered to bind a specific DNA sequence, could be useful for the selective degradation of particular mtDNA sequences. Typically, pairs of complementary ZFNs are used that heterodimerize on the target DNA sequence; however, conventional ZFNs were ineffective in our system. To overcome this, we created single-chain ZFNs by conjugating two FokI nuclease domains, connected by a flexible linker, to a ZFP with an N-terminal mitochondrial targeting sequence. Here we show that these ZFNs are efficiently transported into mitochondria in cells and bind mtDNA in a sequence-specific manner discriminating between two 12-bp long sequences that differ by a single base pair. Due to their selective binding they cleave dsDNA at predicted sites adjacent to the mutation. When expressed in heteroplasmic cells containing a mixture of mutated and wt mtDNA these ZFNs selectively degrade mutated mtDNA, thereby increasing the proportion of wt mtDNA molecules in the cell. Therefore, mitochondria-targeted single-chain ZFNs are a promising candidate approach for the treatment of mtDNA diseases. PMID:18511461

  4. An A20/AN1-type zinc finger protein modulates gibberellins and abscisic acid contents and increases sensitivity to abiotic stress in rice (Oryza sativa).

    PubMed

    Zhang, Ye; Lan, Hongxia; Shao, Qiaolin; Wang, Ruqin; Chen, Hui; Tang, Haijuan; Zhang, Hongsheng; Huang, Ji

    2016-01-01

    The plant hormones gibberellins (GA) and abscisic acid (ABA) play important roles in plant development and stress responses. Here we report a novel A20/AN1-type zinc finger protein ZFP185 involved in GA and ABA signaling in the regulation of growth and stress response. ZFP185 was constitutively expressed in various rice tissues. Overexpression of ZFP185 in rice results in a semi-dwarfism phenotype, reduced cell size, and the decrease of endogenous GA3 content. By contrast, higher GA3 content was observed in RNAi plants. The application of exogenous GA3 can fully rescue the semi-dwarfism phenotype of ZFP185 overexpressing plants, suggesting the negative role of ZFP185 in GA biosynthesis. Besides GA, overexpression of ZFP185 decreased ABA content and expression of several ABA biosynthesis-related genes. Moreover, it was found that ZFP185, unlike previously known A20/AN1-type zinc finger genes, increases sensitivity to drought, cold, and salt stresses, implying the negative role of ZFP185 in stress tolerance. ZFP185 was localized in the cytoplasm and lacked transcriptional activation potential. Our study suggests that ZFP185 regulates plant growth and stress responses by affecting GA and ABA biosynthesis in rice.

  5. ZNF536, a Novel Zinc Finger Protein Specifically Expressed in the Brain, Negatively Regulates Neuron Differentiation by Repressing Retinoic Acid-Induced Gene Transcription▿

    PubMed Central

    Qin, Zhen; Ren, Fangli; Xu, Xialian; Ren, Yongming; Li, Hongge; Wang, Yinyin; Zhai, Yonggong; Chang, Zhijie

    2009-01-01

    Neuronal differentiation is tightly regulated by a variety of factors. In a search for neuron-specific genes, we identified a highly conserved novel zinc finger protein, ZNF536. We observed that ZNF536 is most abundant in the brain and, in particular, is expressed in the developing central nervous system and dorsal root ganglia and localized in the cerebral cortex, hippocampus, and hypothalamic area. During neuronal differentiation of P19 cells induced by retinoic acid (RA), ZNF536 expression is increased at an early stage, and it is maintained at a constant level in later stages. Overexpression of ZNF536 results in an inhibition of RA-induced neuronal differentiation, while depletion or mutation of the ZNF536 gene results in an enhancement of differentiation. We further demonstrated that ZNF536 inhibits expression of neuron-specific marker genes, possibly through the inhibition of RA response element-mediated transcriptional activity, as overexpression of RA receptor α can rescue the inhibitory role of ZNF536 in neuronal differentiation and neuron-specific gene expression. Our studies have identified a novel zinc finger protein that negatively regulates neuron differentiation. PMID:19398580

  6. Biz1, a Zinc Finger Protein Required for Plant Invasion by Ustilago maydis, Regulates the Levels of a Mitotic Cyclin[W

    PubMed Central

    Flor-Parra, Ignacio; Vranes, Miroslav; Kämper, Jörg; Pérez-Martín, José

    2006-01-01

    Plant invasion by pathogenic fungi involves regulated growth and highly organized fungal morphological changes. For instance, when the smut fungus Ustilago maydis infects maize (Zea mays), its dikaryotic infective filament is cell cycle arrested, and appressoria are differentiated prior to plant penetration. Once the filament enters the plant, the cell cycle block is released and fungal cells begin proliferation, suggesting a tight interaction between plant invasion and the cell cycle and morphogenesis control systems. We describe a novel factor, Biz1 (b-dependent zinc finger protein), which has two Cys2His2 zinc finger domains and nuclear localization, suggesting a transcriptional regulatory function. The deletion of biz1 shows no detectable phenotypic alterations during axenic growth. However, mutant cells show a severe reduction in appressoria formation and plant penetration, and those hyphae that invade the plant arrest their pathogenic development directly after plant penetration. biz1 is induced via the b-mating–type locus, the key control instance for pathogenic development. The gene is expressed at high levels throughout pathogenic development, which induces a G2 cell cycle arrest that is a direct consequence of the downregulation of the mitotic cyclin Clb1. Our data support a model in which Biz1 is involved in cell cycle arrest preceding plant penetration as well as in the induction of appressoria. PMID:16905655

  7. Interaction between southern rice black-streaked dwarf virus minor core protein P8 and a rice zinc finger transcription factor.

    PubMed

    Li, Jing; Cai, Nian-Jun; Xue, Jin; Yang, Jian; Chen, Jian-Ping; Zhang, Heng-Mu

    2017-01-25

    The fijivirus southern rice black-streaked dwarf virus (SRBSDV) causes one of the most serious viral diseases of rice in China and Vietnam. To better understand the molecular basis of SRBSDV infection, a yeast two-hybrid screen of a rice cDNA library was carried out using P8, a minor core protein of SRBSDV, as the bait. A rice Cys2His2-type zinc finger protein (OsZFP) was found to interact with SRBSDV P8. A strong interaction between SRBSDV P8 and OsZFP was then confirmed by pull-down assays, and bimolecular fluorescence complementation assays showed that the in vivo interaction was specifically localized in the nucleus of plant cells. Using a series of deletion mutants, it was shown that both the NTP-binding region of P8 and the first two zinc fingers of OsZFP were crucial for their interaction in plant cells. The localization in the nucleus and activation of transcription in yeast supports the notion that OsZFP is a transcription factor. SRBSDV P8 may play an important role in fijiviral infection and symptom development by interfering with the host transcription activity of OsZFP.

  8. The Kruppel-like zinc finger protein ZNF224 recruits the arginine methyltransferase PRMT5 on the transcriptional repressor complex of the aldolase A gene.

    PubMed

    Cesaro, Elena; De Cegli, Rossella; Medugno, Lina; Florio, Francesca; Grosso, Michela; Lupo, Angelo; Izzo, Paola; Costanzo, Paola

    2009-11-20

    Gene transcription in eukaryotes is modulated by the coordinated recruitment of specific transcription factors and chromatin-modulating proteins. Indeed, gene activation and/or repression is/are regulated by histone methylation status at specific arginine or lysine residues. In this work, by co-immunoprecipitation experiments, we demonstrate that PRMT5, a type II protein arginine methyltransferase that monomethylates and symmetrically dimethylates arginine residues, is physically associated with the Kruppel-like associated box-zinc finger protein ZNF224, the aldolase A gene repressor. Moreover, chromatin immunoprecipitation assays show that PRMT5 is recruited to the L-type aldolase A promoter and that methylation of the nucleosomes that surround the L-type promoter region occurs in vivo on the arginine 3 of histone H4. Consistent with its association to the ZNF224 repressor complex, the decrease of PRMT5 expression produced by RNA interference positively affects L-type aldolase A promoter transcription. Finally, the alternating occupancy of the L-type aldolase A promoter by the ZNF224-PRMT5 repression complex in proliferating and growth-arrested cells suggests that these regulatory proteins play a significant role during the cell cycle modulation of human aldolase A gene expression. Our data represent the first experimental evidence that protein arginine methylation plays a role in ZNF224-mediated transcriptional repression and provide novel insight into the chromatin modifications required for repression of gene transcription by Kruppel-like associated box-zinc finger proteins.

  9. The use of ion mobility mass spectrometry to assist protein design: a case study on zinc finger fold versus coiled coil interactions.

    PubMed

    Berezovskaya, Yana; Porrini, Massimiliano; Nortcliffe, Chris; Barran, Perdita E

    2015-04-21

    The dramatic conformational change in zinc fingers on binding metal ions for DNA recognition makes their structure-function behaviour an attractive target to mimic in de novo designed peptides. Mass spectrometry, with its high throughput and low sample consumption provides insight into how primary amino acid sequence can encode stable tertiary fold. We present here the use of ion mobility mass spectrometry (IM-MS) coupled with molecular dynamics (MD) simulations as a rapid analytical platform to inform de novo design efforts for peptide-metal and peptide-peptide interactions. A dual peptide-based synthetic system, ZiCop based on a zinc finger peptide motif, and a coiled coil partner peptide Pp, have been investigated. Titration mass spectrometry determines the relative binding affinities of different divalent metal ions as Zn(2+) > Co(2+) ≫ Ca(2+). With collision induced dissociation (CID), we probe complex stability, and establish that peptide-metal interactions are stronger and more 'specific' than those of peptide-peptide complexes, and the anticipated hetero-dimeric complex is more stable than the two homo-dimers. Collision cross-sections (CCS) measurements by IM-MS reveal increased stability with respect to unfolding of the metal-bound peptide over its apo-form, and further, larger collision cross sections for the hetero-dimeric forms suggest that dimeric species formed in the absence of metal are coiled coil like. MD supports these structural assignments, backed up by data from visible light absorbance measurements.

  10. GLIS3, a novel member of the GLIS subfamily of Krüppel-like zinc finger proteins with repressor and activation functions.

    PubMed

    Kim, Yong-Sik; Nakanishi, Gen; Lewandoski, Mark; Jetten, Anton M

    2003-10-01

    In this study, we describe the identification and characterization of a novel transcription factor GLI-similar 3 (GLIS3). GLIS3 is an 83.8 kDa nuclear protein containing five C2H2-type Krüppel-like zinc finger motifs that exhibit 93% identity with those of GLIS1, however, little homology exists outside their zinc finger domains. GLIS3 can function as a repressor and activator of transcription. Deletion mutant analysis determined that the N- and C-termini are required for optimal transcriptional activity. GLIS3 binds to the GLI-RE consensus sequence and is able to enhance GLI-RE-dependent transcription. GLIS3(DeltaC496), a dominant-negative mutant, inhibits transcriptional activation by GLIS3 and GLI1. Whole mount in situ hybridization on mouse embryos from stage E6.5 through E14.5 demonstrated that GLIS3 is expressed in specific regions in developing kidney and testis and in a highly dynamic pattern during neurulation. From E11.5 through E12.5 GLIS3 was strongly expressed in the interdigital regions, which are fated to undergo apoptosis. The temporal and spatial pattern of GLIS3 expression observed during embryonic development suggests that it may play a critical role in the regulation of a variety of cellular processes during development. Both the repressor and activation functions of GLIS3 may be involved in this control.

  11. Structure, chromosome location, and expression of the mouse zinc finger gene Krox-20: multiple gene products and coregulation with the proto-oncogene c-fos.

    PubMed Central

    Chavrier, P; Janssen-Timmen, U; Mattéi, M G; Zerial, M; Bravo, R; Charnay, P

    1989-01-01

    We have analyzed the structure and the regulation of Krox-20, a mouse zinc finger-encoding gene which is transiently activated following serum stimulation of quiescent fibroblast cells in culture. The gene is localized on chromosome 10, band B5, in the mouse, and the homologous human gene also maps to chromosome 10 (region q21.1 to q22.1). Alternative splicing of the 5'-most intron of the Krox-20 gene gives rise to mRNAs encoding putative zinc finger proteins with different N termini. The first exon contains a sequence element with strong similarity to the c-fos proto-oncogene serum response element (SRE). This element can functionally substitute for the c-fos SRE, and it binds the same nuclear protein. It is probably responsible for the serum induction of Krox-20, possibly in combination with a weaker SRE located in the 5'-flanking region of the gene. Our findings suggest that c-fos, Krox-20, and a number of immediate-early serum response genes are coregulated and that the SRE and its cognate protein are essential components of this regulatory pathway. Images PMID:2496302

  12. The Krüppel-like zinc finger protein GLIS3 transactivates neurogenin 3 for proper fetal pancreatic islet differentiation in mice

    PubMed Central

    Yang, Y.; Chang, B. H-J.; Yechoor, V.; Chen, W.; Li, L.; Tsai, M.-J.

    2011-01-01

    Aims/hypothesis Mutations in GLIS3, which encodes a Krüppel-like zinc finger transcription factor, were found to underlie sporadic neonatal diabetes. Inactivation of Glis3 by gene targeting in mice was previously shown to lead to neonatal diabetes, but the underlying mechanism remains largely unknown. We aimed to elucidate the mechanism of action of GLIS family zinc finger 3 (GLIS3) in Glis3−/− mice and to further decipher its action in in-vitro systems. Methods We created Glis3−/− mice and monitored the morphological and biochemical phenotype of their pancreatic islets at different stages of embryonic development. We combined these observations with experiments on Glis3 expressed in cultured cells, as well as in in vitro systems in the presence of other reconstituted components. Results In vivo and in vitro analyses placed Glis3 upstream of Neurog3, the endocrine pancreas lineage-defining transcription factor. We found that GLIS3 binds to specific GLIS3-response elements in the Neurog3 promoter, activating Neurog3 gene transcription both directly, and synergistically with hepatic nuclear factor 6 and forkhead box A2. Conclusions/interpretation These results indicate that GLIS3 controls fetal islet differentiation via direct transactivation of Neurog3, a perturbation that causes neonatal diabetes in mice. PMID:21786021

  13. Factor-induced Reprogramming and Zinc Finger Nuclease-aided Gene Targeting Cause Different Genome Instability in β-Thalassemia Induced Pluripotent Stem Cells (iPSCs)*

    PubMed Central

    Ma, Ning; Shan, Yongli; Liao, Baojian; Kong, Guanyi; Wang, Cheng; Huang, Ke; Zhang, Hui; Cai, Xiujuan; Chen, Shubin; Pei, Duanqing; Chen, Nansheng; Pan, Guangjin

    2015-01-01

    The generation of personalized induced pluripotent stem cells (iPSCs) followed by targeted genome editing provides an opportunity for developing customized effective cellular therapies for genetic disorders. However, it is critical to ascertain whether edited iPSCs harbor unfavorable genomic variations before their clinical application. To examine the mutation status of the edited iPSC genome and trace the origin of possible mutations at different steps, we have generated virus-free iPSCs from amniotic cells carrying homozygous point mutations in β-hemoglobin gene (HBB) that cause severe β-thalassemia (β-Thal), corrected the mutations in both HBB alleles by zinc finger nuclease-aided gene targeting, and obtained the final HBB gene-corrected iPSCs by excising the exogenous drug resistance gene with Cre recombinase. Through comparative genomic hybridization and whole-exome sequencing, we uncovered seven copy number variations, five small insertions/deletions, and 64 single nucleotide variations (SNVs) in β-Thal iPSCs before the gene targeting step and found a single small copy number variation, 19 insertions/deletions, and 340 single nucleotide variations in the final gene-corrected β-Thal iPSCs. Our data revealed that substantial but different genomic variations occurred at factor-induced somatic cell reprogramming and zinc finger nuclease-aided gene targeting steps, suggesting that stringent genomic monitoring and selection are needed both at the time of iPSC derivation and after gene targeting. PMID:25795783

  14. Band gap narrowing in zinc oxide-based semiconductor thin films

    SciTech Connect

    Kumar, Jitendra E-mail: akrsri@gmail.com; Kumar Srivastava, Amit E-mail: akrsri@gmail.com

    2014-04-07

    A simple expression is proposed for the band gap narrowing (or shrinkage) in semiconductors using optical absorption measurements of spin coated 1 at. % Ga-doped ZnO (with additional 0–1.5 at. % zinc species) thin films as ΔE{sub BGN} = Bn{sup 1/3} [1 − (n{sub c}/n){sup 1/3}], where B is the fitting parameter, n is carrier concentration, and n{sub c} is the critical density required for shrinkage onset. Its uniqueness lies in not only describing variation of ΔE{sub BGN} correctly but also allowing deduction of n{sub c} automatically for several M-doped ZnO (M: Ga, Al, In, B, Mo) systems. The physical significance of the term [1 − (n{sub c}/n){sup 1/3}] is discussed in terms of carrier separation.

  15. The ancient source of a distinct gene family encoding proteins featuring RING and C(3)H zinc-finger motifs with abundant expression in developing brain and nervous system.

    PubMed

    Gray, T A; Hernandez, L; Carey, A H; Schaldach, M A; Smithwick, M J; Rus, K; Marshall Graves, J A; Stewart, C L; Nicholls, R D

    2000-05-15

    Intronless genes can arise by germline retrotransposition of a cDNA originating as mRNA from an intron-containing source gene. Previously, we described several members of a family of intronless mammalian genes encoding a novel class of zinc-finger proteins, including one that shows imprinted expression and one that escapes X-inactivation. We report here the identification and characterization of the Makorin ring finger protein 1 gene (MKRN1), a highly transcribed, intron-containing source for this family of genes. Phylogenetic analyses clearly indicate that the MKRN1 gene is the ancestral founder of this gene family. We have identified MKRN1 orthologs from human, mouse, wallaby, chicken, fruitfly, and nematode, underscoring the age and conservation of this gene. The MKRN gene family encodes putative ribonucleoproteins with a distinctive array of zinc-finger motifs, including two to four C(3)H zinc-fingers, an unusual Cys/His arrangement that may represent a novel zinc-finger structure, and a highly conserved RING zinc-finger. To date, we have identified nine MKRN family loci distributed throughout the human genome. The human and mouse MKRN1 loci map to a conserved syntenic group near the T-cell receptor beta cluster (TCRB) in chromosome 7q34-q35 and chromosome 6A, respectively. MKRN1 is widely transcribed in mammals, with high levels in murine embryonic nervous system and adult testis. The ancient origin of MKRN1, high degree of conservation, and expression pattern suggest important developmental and functional roles for this gene and its expressed family members.

  16. Zinc finger-inspired nanohydrogels with glutathione/pH triggered degradation based on coordination substitution for highly efficient delivery of anti-cancer drugs.

    PubMed

    Zhang, Zihao; Wan, Jiaxun; Sun, Luyan; Li, Yongjing; Guo, Jia; Wang, Changchun

    2016-03-10

    Biodegradable materials used for drug delivery are of great demand due to their ability to degrade into low molecular weight species and further excrete from the body by metabolism. Herein, we report a new kind of zinc(II) crosslinked poly(methacrylic acid) nanohydrogels (ZCLNs) inspired by zinc finger proteins with dual stimuli-triggered degradation (glutathione and pH) for the first time. Compared with the disulfide bond crosslinked nanohydrogels, this new kind of ZCLNs is beneficial to the degradation of a wide range of cells, including normal cells. Ex vivo fluorescence images showed that the DOX-loaded folate-PEG conjugated zinc(II)-crosslinked polymeric nanohydrogels (FPZCLNs-15) preferentially accumulated in tumor tissue and the accumulation in normal tissues was much less compared with DOX-loaded PZCLNs-15 (non-targeted nanohydrogels) and free DOX, the FPZCLNs-15 (targeting system) delivered DOX to the tumor site with approximately 3.6- and 1.6-fold higher than free DOX and PZCLNs-15, respectively. Meanwhile, the PZCLNs-15 and FPZCLNs-15 reduced the concentration of DOX in the heart by 3.2- and 5.0-fold respectively, as compared to the free DOX. Moreover, a superior tumor growth inhibition and negligible damage to normal organs like the heart and kidney, which is reported to be vulnerable to DOX-associated side effects, are further demonstrated.

  17. Genetic analysis of human immunodeficiency virus type 1 integrase and the U3 att site: unusual phenotype of mutants in the zinc finger-like domain.

    PubMed Central

    Masuda, T; Planelles, V; Krogstad, P; Chen, I S

    1995-01-01

    Retroviral integration is the step which leads to establishment of the provirus, cis- and trans-acting regions of the human immunodeficiency type 1 (HIV-1) retrovirus genome, including the attachment site (att) at the ends of the unintegrated viral DNA and the conserved domains within the integrase (IN) protein, have been identified as being important for integration. We investigated the role of each of these regions in the context of an infectious HIV-1 molecular clone through point mutagenesis of the att site and the zinc finger-like and catalytic domains of IN. The effect of each mutation on integration activity was examined by using a single-step infection system with envelope-pseudotype virus. The relative integration efficiency was estimated by monitoring the levels of viral DNA over time in the infected cells. The integration activities of catalytic domain point mutants and att site deletion mutants were estimated to be 0.5 and 5% of wild-type activity, respectively. However, in contrast with previous in vitro cell-free integration studies, alteration of the highly conserved CA dinucleotide resulted in a mutant which still retained 40% of wild-type integration activity. The relative levels of expression of each mutant, as measured by a luciferase reporter gene, correlated with levels of integration. This observation is consistent with those of previous studies indicating that integration is an obligatory step for retroviral gene expression. Interestingly, we found that three different HIV-1 constructs bearing point mutations in the zinc finger-like domain synthesized much lower levels of viral DNA after infection, suggesting impairment of these mutants before or at the initiation of reverse transcription. Western blot (immunoblot) analysis demonstrated wild-type levels of reverse transcriptase within the mutant virions. In vitro endogenous reverse transcription assays indicated that all three mutants in the zinc finger-like domain had wild-type levels of

  18. Human KZNF Gene Catalog - A comprehensive catalog of human KRAB-associated zinc finger genes: insights into the evolutionary history of a large family of transcriptional repressors

    DOE Data Explorer

    Huntley, S; Baggott, D. M.; Hamilton, A. T.; Tran-Gyamfi, M.; Yang, S.; Kim, J.; Gordon, L.; Branscomb, E.; Stubbs, L.

    Kruppel-type zinc finger (ZNF) motifs are prevalent components of transcription factor proteins in all eukaryotes. KRAB-ZNF proteins, in which a potent repressor domain is attached to a tandem array of DNA-binding zinc-finger motifs, are specific to tetrapod vertebrates and represent the largest class of ZNF proteins in mammals. To define the full repertoire of human KRAB-ZNF proteins, we searched the genome sequence for key motifs and then constructed and manually curated gene models incorporating those sequences. The resulting gene catalog contains 423 KRAB-ZNF protein-coding loci, yielding alternative transcripts that altogether predict at least 742 structurally distinct proteins. Active rounds of segmental duplication, involving single genes or larger regions and including both tandem and distributed duplication events, have driven the expansion of this mammalian gene family. Comparisons between the human genes and ZNF loci mined from the draft mouse, dog, and chimpanzee genomes not only identified 103 KRAB-ZNF genes that are conserved in mammals but also highlighted a substantial level of lineage-specific change; at least 136 KRAB-ZNF coding genes are primate specific, including many recent duplicates. KRAB-ZNF genes are widely expressed and clustered genes are typically not coregulated, indicating that paralogs have evolved to fill roles in many different biological processes. To facilitate further study, we have developed a Web-based public resource with access to gene models, sequences, and other data, including visualization tools to provide genomic context and interaction with other public data sets. [This abstract was copied from: S Huntley, DM Baggott, AT Hamilton, M Tran-Gyamfi, S Yang, J Kim, L Gordon, E Branscomb, and L Stubbs. 2006. A comprehensive catalog of human KRAB-associated zinc finger genes: insights into the evolutionary history of a large family of transcriptional repressors, Genome Research 16(5):669 - 677] The website provides the

  19. Akt phosphorylates myc-associated zinc finger protein (MAZ), releases P-MAZ from the p53 promoter, and activates p53 transcription.

    PubMed

    Lee, Wei-Ping; Lan, Keng-Hsin; Li, Chung-Pin; Chao, Yee; Lin, Han-Chieh; Lee, Shou-Dong

    2016-05-28

    The p53 protein is a cell cycle regulator. When the cell cycle progresses, p53 plays an important role in putting a brake on the G1 phase to prevent unwanted errors during cell division. Akt is a downstream kinase of receptor tyrosine kinase. Upon activation, Akt phorphorylates IKK that then phosphorylates IκB and releases NF-κB, leading to transcriptional activation of Dmp1. Dmp1 is a transcriptional activator of Arf. It has been known that oncogene activation stabilizes p53 through transcriptional activation of Arf, which then binds and inhibits Mdm2. In the current study, we show that myc-associated zinc finger protein (MAZ) is a transcriptional repressor of the p53 promoter. Akt phosphorylates MAZ at Thr385, and the phosphorylated MAZ is released from the p53 promoter, leading to transcriptional activation of p53, a new mechanism that contributes to increased p53 protein pool during oncogene activation.

  20. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome

    PubMed Central

    DeKelver, Russell C.; Choi, Vivian M.; Moehle, Erica A.; Paschon, David E.; Hockemeyer, Dirk; Meijsing, Sebastiaan H.; Sancak, Yasemin; Cui, Xiaoxia; Steine, Eveline J.; Miller, Jeffrey C.; Tam, Phillip; Bartsevich, Victor V.; Meng, Xiangdong; Rupniewski, Igor; Gopalan, Sunita M.; Sun, Helena C.; Pitz, Kathleen J.; Rock, Jeremy M.; Zhang, Lei; Davis, Gregory D.; Rebar, Edward J.; Cheeseman, Iain M.; Yamamoto, Keith R.; Sabatini, David M.; Jaenisch, Rudolf; Gregory, Philip D.; Urnov, Fyodor D.

    2010-01-01

    Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a “safe harbor” locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells. PMID:20508142

  1. Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine.

    PubMed

    Gersbach, Charles A; Perez-Pinera, Pablo

    2014-08-01

    New technologies have recently been developed to control the expression of human genes in their native genomic context by engineering synthetic transcription factors that can be targeted to any DNA sequence. The ability to precisely regulate any gene as it occurs naturally in the genome provides a means to address a variety of diseases and disorders. This approach also circumvents some of the traditional challenges of gene therapy. In this editorial, we review the technologies that have enabled targeted human gene activation, including the engineering of transcription factors based on zinc finger proteins, transcription activator-like effectors and the CRISPR/Cas9 system. Additionally, we highlight examples in which these methods have been developed for therapeutic applications and discuss challenges and opportunities.

  2. Sequoia, a tramtrack-related zinc finger protein, functions as a pan-neural regulator for dendrite and axon morphogenesis in Drosophila.

    PubMed

    Brenman, J E; Gao, F B; Jan, L Y; Jan, Y N

    2001-11-01

    Morphological complexity of neurons contributes to their functional complexity. How neurons generate different dendritic patterns is not known. We identified the sequoia mutant from a previous screen for dendrite mutants. Here we report that Sequoia is a pan-neural nuclear protein containing two putative zinc fingers homologous to the DNA binding domain of Tramtrack. sequoia mutants affect the cell fate decision of a small subset of neurons but have global effects on axon and dendrite morphologies of most and possibly all neurons. In support of sequoia as a specific regulator of neuronal morphogenesis, microarray experiments indicate that sequoia may regulate downstream genes that are important for executing neurite development rather than altering a variety of molecules that specify cell fates.

  3. A Family of Zinc Finger Proteins Is Required forChromosome-specific Pairing and Synapsis during Meiosis in C.elegans

    SciTech Connect

    Phillips, Carolyn M.; Dernburg, Abby F.

    2006-06-07

    Homologous chromosome pairing and synapsis are prerequisitefor accurate chromosome segregation during meiosis. Here, we show that afamily of four related C2H2 zinc-finger proteins plays a central role inthese events in C. elegans. These proteins are encoded within a tandemgene cluster. In addition to the X-specific HIM-8 protein, threeadditional paralogs collectively mediate the behavior of the fiveautosomes. Each chromosome relies on a specific member of the family topair and synapse with its homolog. These "ZIM" proteins concentrate atspecial regions called meiotic pairing centers on the correspondingchromosomes. These sites are dispersed along the nuclear envelope duringearly meiotic prophase, suggesting a role analogous to thetelomere-mediated meiotic bouquet in other organisms. To gain insightinto the evolution of these components, wecharacterized homologs in C.briggsae and C. remanei, which revealed changes in copy number of thisgene family within the nematode lineage.

  4. The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid- and alkaline-expressed genes by ambient pH.

    PubMed Central

    Tilburn, J; Sarkar, S; Widdick, D A; Espeso, E A; Orejas, M; Mungroo, J; Peñalva, M A; Arst, H N

    1995-01-01

    The pH regulation of gene expression in Aspergillus nidulans is mediated by pacC, whose 678 residue-derived protein contains three putative Cys2His2 zinc fingers. Ten pacCc mutations mimicking growth at alkaline pH remove between 100 and 214 C-terminal residues, including a highly acidic region containing an acidic glutamine repeat. Nine pacC+/- mutations mimicking acidic growth conditions remove between 299 and 505 C-terminal residues. Deletion of the entire pacC coding region mimics acidity but leads additionally to poor growth and conidiation. A PacC fusion protein binds DNA with the core consensus GCCARG. At alkaline ambient pH, PacC activates transcription of alkaline-expressed genes (including pacC itself) and represses transcription of acid-expressed genes. pacCc mutations obviate the need for pH signal transduction. Images PMID:7882981

  5. The human zinc-finger protein-7 gene is located 90 kb 3' of MYC and is not expressed in Burkitt lymphoma cell lines.

    PubMed

    Feduchi, E; Gallego, M I; Lazo, P A

    1994-09-15

    The zinc-finger gene-7 (ZNF7) was located 90 kb 3' of MYC on human chromosome 8 band q24 by pulsed-field gel electrophoresis (PFGE). This position lies between the MLV14 and BVR1 loci, 2 variant translocation breakpoints in Burkitt lymphomas. The structure of the ZNF7 gene was not altered by translocations in Burkitt-lymphoma cell lines as shown by its germline-restriction map configuration. The chromosomal region surrounding the ZNF7 gene was extensively methylated. The ZNF7 gene was not expressed in 19 BL cell lines. Expression was detected only in the BL41 and BL47 cell lines and in the SW756 cervical-carcinoma cell line. The RNA in each was of a different size. We postulate that the lack of ZNF7 expression in Burkitt lymphomas might contribute to the tumor phenotype.

  6. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome.

    PubMed

    DeKelver, Russell C; Choi, Vivian M; Moehle, Erica A; Paschon, David E; Hockemeyer, Dirk; Meijsing, Sebastiaan H; Sancak, Yasemin; Cui, Xiaoxia; Steine, Eveline J; Miller, Jeffrey C; Tam, Phillip; Bartsevich, Victor V; Meng, Xiangdong; Rupniewski, Igor; Gopalan, Sunita M; Sun, Helena C; Pitz, Kathleen J; Rock, Jeremy M; Zhang, Lei; Davis, Gregory D; Rebar, Edward J; Cheeseman, Iain M; Yamamoto, Keith R; Sabatini, David M; Jaenisch, Rudolf; Gregory, Philip D; Urnov, Fyodor D

    2010-08-01

    Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a "safe harbor" locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells.

  7. Identification of the Zinc Finger Protein ZRANB2 as a Novel Maternal Lipopolysaccharide-binding Protein That Protects Embryos of Zebrafish against Gram-negative Bacterial Infections.

    PubMed

    Wang, Xia; Du, Xiaoyuan; Li, Hongyan; Zhang, Shicui

    2016-02-19

    Zinc finger ZRANB2 proteins are widespread in animals, but their functions and mechanisms remain poorly defined. Here we clearly demonstrate that ZRANB2 is a newly identified LPS-binding protein present abundantly in the eggs/embryos of zebrafish. We also show that recombinant ZRANB2 (rZRANB2) acts as a pattern recognition receptor capable of identifying the bacterial signature molecule LPS as well as binding the Gram-negative bacteria Escherichia coli, Vibrio anguilarum, and Aeromonas hydrophila and functions as an antibacterial effector molecule capable of directly killing the bacteria. Furthermore, we reveal that N-terminal residues 11-37 consisting of the first ZnF_RBZ domain are indispensable for ZRANB2 antimicrobial activity. Importantly, microinjection of rZRANB2 into early embryos significantly enhanced the resistance of the embryos against pathogenic A. hydrophila challenge, and this enhanced bacterial resistance was markedly reduced by co-injection of anti-ZRANB2 antibody. Moreover, precipitation of ZRANB2 in the embryo extracts by preincubation with anti-ZRANB2 antibody caused a marked decrease in the antibacterial activity of the extracts against the bacteria tested. In addition, the N-terminal peptide Z1/37 or Z11/37 with in vitro antibacterial activity also promoted the resistance of embryos against A. hydrophila, but the peptide Z38/198 without in vitro antibacterial activity did not. Collectively, these results indicate that ZRANB2 is a maternal LPS-binding protein that can protect the early embryos of zebrafish against pathogenic attacks, a novel role ever assigned to ZRANB2 proteins. This work also provides new insights into the immunological function of the zinc finger proteins that are widely distributed in various animals.

  8. A Comprehensive Catalog of Human KRAB-associated Zinc Finger Genes: Insights into the Evolutionary History of a Large Family of Transcriptional Repressors

    SciTech Connect

    Huntley, S; Baggott, D M; Hamilton, A T; Tran-Gyamfi, M; Yang, S; Kim, J; Gordon, L; Branscomb, E; Stubbs, L

    2005-09-30

    Krueppel-type zinc finger (ZNF) motifs are prevalent components of transcription factor proteins in all eukaryotic species. In mammals, most ZNF proteins comprise a single class of transcriptional repressors in which a chromatin interaction domain, called the Krueppel-associated box (KRAB) is attached to a tandem array of DNA-binding zinc-finger motifs. KRAB-ZNF loci are specific to tetrapod vertebrates, but have expanded dramatically in numbers through repeated rounds of segmental duplication to create a gene family with hundreds of members in mammals. To define the full repertoire of human KRAB-ZNF proteins, we searched the human genome for key motifs and used them to construct and manually curate gene models. The resulting KRAB-ZNF gene catalog includes 326 known genes, 243 of which were structurally corrected by manual annotation, and 97 novel KRAB-ZNF genes; this single family therefore comprises 20% of all predicted human transcription factor genes. Many of the genes are alternatively spliced, yielding a total of 743 distinct predicted proteins. Although many human KRAB-ZNF genes are conserved in mammals, at least 136 and potentially more than 200 genes of this type are primate-specific including many recent segmental duplicates. KRAB-ZNF genes are active in a wide variety of human tissues suggesting roles in many key biological processes, but most member genes remain completely uncharacterized. Because of their sheer numbers, wide-ranging tissue-specific expression patterns, and remarkable evolutionary divergence we predict that KRAB-ZNF transcription factors have played critical roles in crafting many aspects of human biology, including both deeply conserved and primate-specific traits.

  9. Comprehensive Evolutionary and Expression Analysis of FCS-Like Zinc finger Gene Family Yields Insights into Their Origin, Expansion and Divergence

    PubMed Central

    Jamsheer K, Muhammed; Mannully, Chanchal Thomas; Gopan, Nandu; Laxmi, Ashverya

    2015-01-01

    Plant evolution is characterized by frequent genome duplication events. Expansion of habitat resulted in the origin of many novel genes and genome duplication events which in turn resulted in the expansion of many regulatory gene families. The plant-specific FCS-Like Zinc finger (FLZ) gene family is characterized by the presence of a FCS-Like Zinc finger (FLZ) domain which mediates the protein-protein interaction. In this study, we identified that the expansion of FLZ gene family size in different species is correlated with ancestral and lineage-specific whole genome duplication events. The subsequent gene loss found to have a greater role in determining the size of this gene family in many species. However, genomic block duplications played the significant role in the expansion of FLZ gene family in some species. Comparison of Arabidopsis thaliana and Oryza sativa FLZ gene family revealed monocot and dicot specific evolutionary trends. The FLZ genes were found to be under high purifying selection. The spatiotemporal expression analyses of Arabidopsis thaliana FLZ gene family revealed that majority of the members are highly expressed in reproductive organs. FLZ genes were also found to be highly expressed during vegetative-to-reproductive phase transition which is correlated with the proposed role of this gene family in sugar signaling. The comparison of sequence, structural and expression features of duplicated genes identified lineage-specific redundancy and divergence. This extensive evolutionary analysis and expression analysis of Arabidopsis thaliana FLZ genes will pave the way for further functional analysis of FLZ genes. PMID:26252898

  10. GsZFP1, a new Cys2/His2-type zinc-finger protein, is a positive regulator of plant tolerance to cold and drought stress.

    PubMed

    Luo, Xiao; Bai, Xi; Zhu, Dan; Li, Yong; Ji, Wei; Cai, Hua; Wu, Jing; Liu, Baohui; Zhu, Yanming

    2012-06-01

    Plant acclimation to environmental stress is controlled by a complex network of regulatory genes that compose distinct stress-response regulons. The C2H2-type zinc-finger proteins (ZFPs) have been implicated in different cellular processes involved in plant development and stress responses. Through microarray analysis, an alkaline (NaHCO(3))-responsive ZFP gene GsZFP1 was identified and subsequently cloned from Glyycine soja. GsZFP1 encodes a 35.14 kDa protein with one C2H2-type zinc-finger motif. The QALGGH domain, conserved in most plant C2H2-type ZFPs, is absent in the GsZFP1 protein sequence. A subcellular localization study using a GFP fusion protein indicated that GsZFP1 is localized to the nucleus. Real-time RT-PCR analysis showed that GsZFP1 was induced in the leaf by ABA (100 μM), salt (200 mM NaCl), and cold (4°C), and in the root by ABA (100 μM), cold (4°C), and drought (30% PEG 6000). Over-expression of GsZFP1 in transgenic Arabidopsis resulted in a greater tolerance to cold and drought stress, a decreased water loss rate, and an increase in proline irrespective of environmental conditions. The over-expression of GsZFP1 also increased the expression of a number of stress-response marker genes, including CBF1, CBF2, CBF3, NCED3, COR47, and RD29A in response to cold stress and RAB18, NCED3, P5CS, RD22, and RD29A in response to drought stress, especially early during stress treatments. Our studies suggest that GsZFP1 plays a crucial role in the plant response to cold and drought stress.

  11. Gain, Loss and Divergence in Primate Zinc-Finger Genes: A Rich Resource for Evolution of Gene Regulatory Differences between Species

    PubMed Central

    Nowick, Katja; Fields, Christopher; Gernat, Tim; Caetano-Anolles, Derek; Kholina, Nadezda; Stubbs, Lisa

    2011-01-01

    The molecular changes underlying major phenotypic differences between humans and other primates are not well understood, but alterations in gene regulation are likely to play a major role. Here we performed a thorough evolutionary analysis of the largest family of primate transcription factors, the Krüppel-type zinc finger (KZNF) gene family. We identified and curated gene and pseudogene models for KZNFs in three primate species, chimpanzee, orangutan and rhesus macaque, to allow for a comparison with the curated set of human KZNFs. We show that the recent evolutionary history of primate KZNFs has been complex, including many lineage-specific duplications and deletions. We found 213 species-specific KZNFs, among them 7 human-specific and 23 chimpanzee-specific genes. Two human-specific genes were validated experimentally. Ten genes have been lost in humans and 13 in chimpanzees, either through deletion or pseudogenization. We also identified 30 KZNF orthologs with human-specific and 42 with chimpanzee-specific sequence changes that are predicted to affect DNA binding properties of the proteins. Eleven of these genes show signatures of accelerated evolution, suggesting positive selection between humans and chimpanzees. During primate evolution the most extensive re-shaping of the KZNF repertoire, including most gene additions, pseudogenizations, and structural changes occurred within the subfamily homininae. Using zinc finger (ZNF) binding predictions, we suggest potential impact these changes have had on human gene regulatory networks. The large species differences in this family of TFs stands in stark contrast to the overall high conservation of primate genomes and potentially represents a potent driver of primate evolution. PMID:21738707

  12. ATRX binds to atypical chromatin domains at the 3′ exons of zinc finger genes to preserve H3K9me3 enrichment

    PubMed Central

    Chowdhury, Asif H.; Hasson, Dan; Dyer, Michael A.

    2016-01-01

    ABSTRACT ATRX is a SWI/SNF chromatin remodeler proposed to govern genomic stability through the regulation of repetitive sequences, such as rDNA, retrotransposons, and pericentromeric and telomeric repeats. However, few direct ATRX target genes have been identified and high-throughput genomic approaches are currently lacking for ATRX. Here we present a comprehensive ChIP-sequencing study of ATRX in multiple human cell lines, in which we identify the 3′ exons of zinc finger genes (ZNFs) as a new class of ATRX targets. These 3′ exonic regions encode the zinc finger motifs, which can range from 1–40 copies per ZNF gene and share large stretches of sequence similarity. These regions often contain an atypical chromatin signature: they are transcriptionally active, contain high levels of H3K36me3, and are paradoxically enriched in H3K9me3. We find that these ZNF 3′ exons are co-occupied by SETDB1, TRIM28, and ZNF274, which form a complex with ATRX. CRISPR/Cas9-mediated loss-of-function studies demonstrate (i) a reduction of H3K9me3 at the ZNF 3′ exons in the absence of ATRX and ZNF274 and, (ii) H3K9me3 levels at atypical chromatin regions are particularly sensitive to ATRX loss compared to other H3K9me3-occupied regions. As a consequence of ATRX or ZNF274 depletion, cells with reduced levels of H3K9me3 show increased levels of DNA damage, suggesting that ATRX binds to the 3′ exons of ZNFs to maintain their genomic stability through preservation of H3K9me3. PMID:27029610

  13. Identification of the Zinc Finger Protein ZRANB2 as a Novel Maternal Lipopolysaccharide-binding Protein That Protects Embryos of Zebrafish against Gram-negative Bacterial Infections*

    PubMed Central

    Wang, Xia; Du, Xiaoyuan; Li, Hongyan; Zhang, Shicui

    2016-01-01

    Zinc finger ZRANB2 proteins are widespread in animals, but their functions and mechanisms remain poorly defined. Here we clearly demonstrate that ZRANB2 is a newly identified LPS-binding protein present abundantly in the eggs/embryos of zebrafish. We also show that recombinant ZRANB2 (rZRANB2) acts as a pattern recognition receptor capable of identifying the bacterial signature molecule LPS as well as binding the Gram-negative bacteria Escherichia coli, Vibrio anguilarum, and Aeromonas hydrophila and functions as an antibacterial effector molecule capable of directly killing the bacteria. Furthermore, we reveal that N-terminal residues 11–37 consisting of the first ZnF_RBZ domain are indispensable for ZRANB2 antimicrobial activity. Importantly, microinjection of rZRANB2 into early embryos significantly enhanced the resistance of the embryos against pathogenic A. hydrophila challenge, and this enhanced bacterial resistance was markedly reduced by co-injection of anti-ZRANB2 antibody. Moreover, precipitation of ZRANB2 in the embryo extracts by preincubation with anti-ZRANB2 antibody caused a marked decrease in the antibacterial activity of the extracts against the bacteria tested. In addition, the N-terminal peptide Z1/37 or Z11/37 with in vitro antibacterial activity also promoted the resistance of embryos against A. hydrophila, but the peptide Z38/198 without in vitro antibacterial activity did not. Collectively, these results indicate that ZRANB2 is a maternal LPS-binding protein that can protect the early embryos of zebrafish against pathogenic attacks, a novel role ever assigned to ZRANB2 proteins. This work also provides new insights into the immunological function of the zinc finger proteins that are widely distributed in various animals. PMID:26740623

  14. The natural history of the WRKY–GCM1 zinc fingers and the relationship between transcription factors and transposons

    PubMed Central

    Babu, M. Madan; Iyer, Lakshminarayan M.; Balaji, S.; Aravind, L.

    2006-01-01

    WRKY and GCM1 are metal chelating DNA-binding domains (DBD) which share a four stranded fold. Using sensitive sequence searches, we show that this WRKY–GCM1 fold is also shared by the FLYWCH Zn-finger domain and the DBDs of two classes of Mutator-like element (MULE) transposases. We present evidence that they share a stabilizing core, which suggests a possible origin from a BED finger-like intermediate that was in turn ultimately derived from a C2H2 Zn-finger domain. Through a systematic study of the phyletic pattern, we show that this WRKY–GCM1 superfamily is a widespread eukaryote-specific group of transcription factors (TFs). We identified several new members across diverse eukaryotic lineages, including potential TFs in animals, fungi and Entamoeba. By integrating sequence, structure, gene expression and transcriptional network data, we present evidence that at least two major global regulators belonging to this superfamily in Saccharomyces cerevisiae (Rcs1p and Aft2p) have evolved from transposons, and attained the status of transcription regulatory hubs in recent course of ascomycete yeast evolution. In plants, we show that the lineage-specific expansion of WRKY–GCM1 domain proteins acquired functional diversity mainly through expression divergence rather than by protein sequence divergence. We also use the WRKY–GCM1 superfamily as an example to illustrate the importance of transposons in the emergence of new TFs in different lineages. PMID:17130173

  15. Study of structural and optical properties of chemically synthesized nanostructured cadmium zinc sulphide films for band gap tunability

    NASA Astrophysics Data System (ADS)

    Mochahari, P. K.; Sarma, K. C.

    2016-01-01

    Nanostructured cadmium zinc sulphide films have been deposited onto cleaned glass substrates by chemical bath deposition method at room temperature using polyvinyl alcohol as capping agent. X-ray diffraction analysis confirms the formation of cubic-phase cadmium zinc sulphide films. Crystallite size obtained from the calculation of Scherrer's formula and Williamson-Hall plot as well as size-strain plot is found to decrease with the increase in zinc concentration. The films have very high dislocation density of the order of 1016 m-2, whereas the strain is of the order of 10-3. Scanning electron microscopic image reveals that the particles are agglomerated to form nanoclusters and energy-dispersive X-ray analysis confirms that films are composed of cadmium, zinc and sulphur. High-resolution transmission electron microscopic image reveals that the shape of the particles is nearly spherical, uniformly distributed. Selected-area electron diffraction pattern supports the formation of cubic phase of the film. Optical absorption peaks of the films shift towards lower wavelength side and their optical band gap increases with the increase in zinc concentration. The increase in zinc concentration enhances the photoluminescence emission intensity, whose emission is in the green region of visible spectrum.

  16. Zinc.

    PubMed

    Barceloux, D G

    1999-01-01

    The use of zinc in metal alloys and medicinal lotions dates back before the time of Christ. Currently, most of the commercial production of zinc involves the galvanizing of iron and the manufacture of brass. Some studies support the use of zinc gluconate lozenges to treat the common cold, but there are insufficient data at this time to recommend the routine use of these lozenges. Zinc is an essential co-factor in a variety of cellular processes including DNA synthesis, behavioral responses, reproduction, bone formation, growth, and wound healing. Zinc is a relatively common metal with an average concentration of 50 mg/kg soil and a range of 10-300 mg/kg soil. Meat, seafood, dairy products, nuts, legumes, and whole grains contain relatively high concentrations of zinc. The mobility of zinc in anaerobic environments is poor and therefore severe zinc contamination occurs primarily near points sources of zinc release. The recommended daily allowance for adults is 15 mg zinc. The ingestion of 1-2 g zinc sulfate produces emesis. Zinc compounds can produce irritation and corrosion of the gastrointestinal tract, along with acute renal tubular necrosis and interstitial nephritis. Inhalation of high concentrations of zinc chloride from smoke bombs detonated in closed spaces may cause chemical pneumonitis and adult respiratory distress syndrome. In the occupational setting inhalation of fumes from zinc oxide is the most common cause of metal fume fever (fatigue, chills, fever, myalgias, cough, dyspnea, leukocytosis, thirst, metallic taste, salivation). Zinc compounds are not suspected carcinogens. Treatment of zinc toxicity is supportive. Calcium disodium ethylenediaminetetraacetate (CaNa2EDTA) is the chelator of choice based on case reports that demonstrate normalization of zinc concentrations, but there are few clinical data to confirm the efficacy of this agent.

  17. Zinc

    MedlinePlus

    ... pill" to help remove excess water from the body. Another effect of amiloride (Midamor) is that it can increase the amount of zinc in the body. Taking zinc supplements with amiloride (Midamor) might cause ...

  18. An Arabidopsis Zinc Finger Protein Increases Abiotic Stress Tolerance by Regulating Sodium and Potassium Homeostasis, Reactive Oxygen Species Scavenging and Osmotic Potential

    PubMed Central

    Zang, Dandan; Li, Hongyan; Xu, Hongyun; Zhang, Wenhui; Zhang, Yiming; Shi, Xinxin; Wang, Yucheng

    2016-01-01

    Plant zinc finger proteins (ZFPs) comprise a large protein family and they are mainly involved in abiotic stress tolerance. Although Arabidopsis RING/FYVE/PHD ZFP At5g62460 (AtRZFP) is found to bind to zinc, whether it is involved in abiotic stress tolerance is still unknown. In the present study, we characterized the roles of AtRZFP in response to abiotic stresses. The expression of AtRZFP was induced significantly by salt and osmotic stress. AtRZFP positively mediates tolerance to salt and osmotic stress. Additionally, compared with wild-type Arabidopsis plants, plants overexpressing AtRZFP showed reduced reactive oxygen species (ROSs) accumulation, enhanced superoxide dismutase and peroxidase activity, increased soluble sugars and proline contents, reduced K+ loss, decreased Na+ accumulation, stomatal aperture and the water loss rate. Conversely, AtRZFP knockout plants displayed the opposite physiological changes when exposed to salt or osmotic stress conditions. These data suggested that AtRZFP enhances salt and osmotic tolerance through a series of physiological processes, including enhanced ROSs scavenging, maintaining Na+ and K+ homeostasis, controlling the stomatal aperture to reduce the water loss rate, and accumulating soluble sugars and proline to adjust the osmotic potential. PMID:27605931

  19. Electronic characterization of defects in narrow gap semiconductors: Comparison of electronic energy levels and formation energies in mercury cadmium telluride, mercury zinc telluride, and mercury zinc selenide

    NASA Astrophysics Data System (ADS)

    Patterson, James D.; Li, Wei-Gang

    1995-03-01

    The project has evolved to that of using Green's functions to predict properties of deep defects in narrow gap materials. Deep defects are now defined as originating from short range potentials and are often located near the middle of the energy gap. They are important because they affect the lifetime of charge carriers and hence the switching time of transistors. We are now moving into the arena of predicting formation energies of deep defects. This will also allow us to make predictions about the relative concentrations of the defects that could be expected at a given temperature. The narrow gap materials mercury cadmium telluride (MCT), mercury zinc telluride (MZT), and mercury zinc selenide (MZS) are of interest to NASA because they have commercial value for infrared detecting materials, and because there is a good possibility that they can be grown better in a microgravity environment. The uniform growth of these crystals on earth is difficult because of convection (caused by solute depletion just ahead of the growing interface, and also due to thermal gradients). In general it is very difficult to grow crystals with both radial and axial homogeneity.

  20. Electronic characterization of defects in narrow gap semiconductors: Comparison of electronic energy levels and formation energies in mercury cadmium telluride, mercury zinc telluride, and mercury zinc selenide

    NASA Technical Reports Server (NTRS)

    Patterson, James D.; Li, Wei-Gang

    1995-01-01

    The project has evolved to that of using Green's functions to predict properties of deep defects in narrow gap materials. Deep defects are now defined as originating from short range potentials and are often located near the middle of the energy gap. They are important because they affect the lifetime of charge carriers and hence the switching time of transistors. We are now moving into the arena of predicting formation energies of deep defects. This will also allow us to make predictions about the relative concentrations of the defects that could be expected at a given temperature. The narrow gap materials mercury cadmium telluride (MCT), mercury zinc telluride (MZT), and mercury zinc selenide (MZS) are of interest to NASA because they have commercial value for infrared detecting materials, and because there is a good possibility that they can be grown better in a microgravity environment. The uniform growth of these crystals on earth is difficult because of convection (caused by solute depletion just ahead of the growing interface, and also due to thermal gradients). In general it is very difficult to grow crystals with both radial and axial homogeneity.

  1. Zinc

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Zinc was recognized as an essential trace metal for humans during the studies of Iranian adolescent dwarfs in the early 1960s. Zinc metal existing as Zn2+ is a strong electron acceptor in biological systems without risks of oxidant damage to cells. Zn2+ functions in the structure of proteins and is ...

  2. A factor that regulates the class II major histocompatibility complex gene DPA is a member of a subfamily of zinc finger proteins that includes a Drosophila developmental control protein.

    PubMed Central

    Sugawara, M; Scholl, T; Ponath, P D; Strominger, J L

    1994-01-01

    A novel DNA sequence element termed the J element involved in the regulated expression of class II major histocompatibility complex genes was recently described. To study this element and its role in class II gene regulation further, a cDNA library was screened with oligonucleotide probes containing both the S element and the nearby J element of the human DPA gene. Several DNA clones were obtained by this procedure, one of which, clone 18, is reported and characterized here. It encodes a protein predicted to contain 688 amino acid residues, including 11 zinc finger motifs of the C2H2 type in the C-terminal region, that are Krüppel-like in the conservation of the H/C link sequence connecting them. The 160 N-terminal amino acids in the nonfinger region of clone 18 are highly homologous with similar regions of several other human, mouse, and Drosophila sequences, defining a subfamily of Krüppel-like zinc finger proteins termed TAB (tramtrack [ttk]-associated box) here. One of the Drosophila sequences, ttk, is a developmental control gene, while a second does not contain a zinc finger region but encodes a structure important in oocyte development. An acidic activation domain is located between the N-terminal conserved region of clone 18 and its zinc fingers. This protein appears to require both the S and J elements, which are separated by 10 bp for optimal binding. Antisense cDNA to clone 18 inhibited the expression of a reporter construct containing the DPA promoter, indicating its functional importance in the expression of this class II gene. Images PMID:7969177

  3. A20 zinc finger protein inhibits TNF-induced apoptosis and stress response early in the signaling cascades and independently of binding to TRAF2 or 14-3-3 proteins.

    PubMed

    Lademann, U; Kallunki, T; Jäättelä, M

    2001-03-01

    A20 zinc finger protein is a negative regulator of tumor necrosis factor (TNF)-induced signaling pathways leading to apoptosis, stress response and inflammation. A20 has been shown to bind to TNF-receptor-associated factor 2 (TRAF2) and 14-3-3 chaperone proteins. Our data indicate that the zinc finger domain of A20 is sufficient and that neither TRAF2 nor 14-3-3 binding is necessary for the inhibitory effects of A20. Mutations in the 14-3-3 binding site of A20 did, however, result in a partial cleavage of A20 protein suggesting that 14-3-3 chaperone proteins may stabilize A20. Furthermore, we show that A20 acts early in TNF-induced signaling cascades blocking both TNF-induced rapid activation of c-Jun N-terminal kinase and processing of the receptor-associated caspase-8. Taken together our data indicate that the zinc finger domain of A20 contains all necessary functional domains required for the inhibition of TNF signaling and that A20 may function at the level of the receptor signaling complex.

  4. Genome-wide analysis of the DNA-binding with one zinc finger (Dof) transcription factor family in bananas.

    PubMed

    Dong, Chen; Hu, Huigang; Xie, Jianghui

    2016-12-01

    DNA-binding with one finger (Dof) domain proteins are a multigene family of plant-specific transcription factors involved in numerous aspects of plant growth and development. In this study, we report a genome-wide search for Musa acuminata Dof (MaDof) genes and their expression profiles at different developmental stages and in response to various abiotic stresses. In addition, a complete overview of the Dof gene family in bananas is presented, including the gene structures, chromosomal locations, cis-regulatory elements, conserved protein domains, and phylogenetic inferences. Based on the genome-wide analysis, we identified 74 full-length protein-coding MaDof genes unevenly distributed on 11 chromosomes. Phylogenetic analysis with Dof members from diverse plant species showed that MaDof genes can be classified into four subgroups (StDof I, II, III, and IV). The detailed genomic information of the MaDof gene homologs in the present study provides opportunities for functional analyses to unravel the exact role of the genes in plant growth and development.

  5. Assignment of the human ZNF83 (HPF1) zinc finger gene to chromosome 19q13. 3-q13. 4

    SciTech Connect

    Marine, J.C.; Lecoq, P.J.; Poncelet, D.A.; Martial, J.A. ); Bellefroid, E.J.; Bourguignon, C. ); Riviere, M.; Szpirer, J.; Szpirer, C. )

    1994-05-01

    The authors have isolated a collection of human ZFPs encoding cDNAs (HPF1 to -9) by hybridization with a finger motif oligonucleotide probe. Here, they describe the localization of a chromosome 19-linked human ZFP gene (HPF1/ZNF83). They first assigned the ZNF83 gene on chromosome 19 by the screening of a human x rodent hybrid panel by DNA hybridization with a fragment of a previously cloned cDNA (data not shown). To further localize the gene within chromosome 19, the regional assignment of the ZNF83 gene was determined by fluorescence in situ hybridization and digital imaging microscopy as described elsewhere. Human metaphase spreads were hybridized with biotinylated ZNF83 cDNA, and hybridization was detected with fluorescein isothiocyanate-conjugated avidin-DCS. Chromosomes were identified by staining with 4,6-diamino-2-phenylindol dihydrochloride. The fractional length (Flpter) distance of the signal to the p arm terminus relative to the total chromosome length gave a Flpter value between 82.8 and 89.9, which is consistent with an assignment of the ZNF83 gene in ISCN region 19q13.3-q13.4. 14 refs., 1 fig.

  6. Altered carbohydrate metabolism in the storage roots of sweet potato plants overexpressing the SRF1 gene, which encodes a Dof zinc finger transcription factor.

    PubMed

    Tanaka, Masaru; Takahata, Yasuhiro; Nakayama, Hiroki; Nakatani, Makoto; Tahara, Makoto

    2009-09-01

    In order to characterize the functions of the sweetpotato SRF1 gene, which encodes a Dof zinc finger transcriptional factor preferentially expressed in the storage roots, we isolated its full length cDNA and produced transgenic sweetpotato plants with altered SRF1 expression levels. The isolated cDNA of SRF1 encoded a polypeptide of 497 amino acids and was closely related to the cyclic Dof factors of Arabidopsis and the ascorbate oxidase binding protein of pumpkin. SRF1 was most highly expressed in storage roots, although some expression was also observed in other vegetative tissue. Transgenic plants overexpressing SRF1 showed significantly higher storage root dry matter content compared to the original cultivar Kokei No. 14 or control transgenic plants. In these plants, the starch content per fresh weight of the storage roots was also higher than that of the wild-type plants, while the glucose and fructose content drastically decreased. Among the enzymes involved in the sugar metabolism, soluble acid invertase showed a decreased activity in the transgenic plants. Gene expression analysis showed that the expression of Ibbetafruct2, which encodes an isoform of vacuolar invertase, was suppressed in the transgenic plants overexpressing the SRF1 gene. These data suggest that SRF1 modulates the carbohydrate metabolism in the storage roots through negative regulation of a vacuolar invertase gene.

  7. A Zinc Finger Protein Regulates Flowering Time and Abiotic Stress Tolerance in Chrysanthemum by Modulating Gibberellin Biosynthesis[C][W][OPEN

    PubMed Central

    Yang, Yingjie; Ma, Chao; Xu, Yanjie; Wei, Qian; Imtiaz, Muhammad; Lan, Haibo; Gao, Shan; Cheng, Lina; Wang, Meiyan; Fei, Zhangjun; Hong, Bo; Gao, Junping

    2014-01-01

    Flowering time and an ability to tolerate abiotic stresses are important for plant growth and development. We characterized BBX24, a zinc finger transcription factor gene, from Chrysanthemum morifolium and found it to be associated with both flowering time and stress tolerance. Transgenic lines with suppressed expression of Cm-BBX24 (Cm-BBX24-RNAi) flowered earlier than wild-type plants and showed decreased tolerance to freezing and drought stresses. Global expression analysis revealed that genes associated with both photoperiod and gibberellin (GA) biosynthesis pathways were upregulated in Cm-BBX24-RNAi lines, relative to the wild type. By contrast, genes that were upregulated in overexpressing lines (Cm-BBX24-OX), but downregulated in Cm-BBX24-RNAi lines (both relative to the wild type), included genes related to compatible solutes and carbohydrate metabolism, both of which are associated with abiotic stress. Cm-BBX24 expression was also influenced by daylength and GA4/7 application. Under long days, changes in endogenous GA1, GA4, GA19, and GA20 levels occurred in young leaves of transgenic lines, relative to the wild type. Regulation of flowering involves the FLOWERING TIME gene, which integrates photoperiod and GA biosynthesis pathways. We postulate that Cm-BBX24 plays a dual role, modulating both flowering time and abiotic stress tolerance in chrysanthemum, at least in part by influencing GA biosynthesis. PMID:24858937

  8. In Vivo Zinc Finger Nuclease-mediated Targeted Integration of a Glucose-6-phosphatase Transgene Promotes Survival in Mice With Glycogen Storage Disease Type IA.

    PubMed

    Landau, Dustin J; Brooks, Elizabeth Drake; Perez-Pinera, Pablo; Amarasekara, Hiruni; Mefferd, Adam; Li, Songtao; Bird, Andrew; Gersbach, Charles A; Koeberl, Dwight D

    2016-04-01

    Glycogen storage disease type Ia (GSD Ia) is caused by glucose-6-phosphatase (G6Pase) deficiency in association with severe, life-threatening hypoglycemia that necessitates lifelong dietary therapy. Here we show that use of a zinc-finger nuclease (ZFN) targeted to the ROSA26 safe harbor locus and a ROSA26-targeting vector containing a G6PC donor transgene, both delivered with adeno-associated virus (AAV) vectors, markedly improved survival of G6Pase knockout (G6Pase-KO) mice compared with mice receiving the donor vector alone (P < 0.04). Furthermore, transgene integration has been confirmed by sequencing in the majority of the mice treated with both vectors. Targeted alleles were 4.6-fold more common in livers of mice with GSD Ia, as compared with normal littermates, at 8 months following vector administration (P < 0.02). This suggests a selective advantage for vector-transduced hepatocytes following ZFN-mediated integration of the G6Pase vector. A short-term experiment also showed that 3-month-old mice receiving the ZFN had significantly-improved biochemical correction, in comparison with mice that received the donor vector alone. These data suggest that the use of ZFNs to drive integration of G6Pase at a safe harbor locus might improve vector persistence and efficacy, and lower mortality in GSD Ia.

  9. A Zinc-Finger-Family Transcription Factor, AbVf19, Is Required for the Induction of a Gene Subset Important for Virulence in Alternaria brassicicola

    SciTech Connect

    Srivastava, Akhil; Ohm, Robin A.; Oxiles, Lindsay; Brooks, Fred; Lawrence, Christopher B.; Grigoriev, Igor V.; Cho, Yangrae

    2011-10-26

    Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen with a broad host range within the family Brassicaceae. It produces secondary metabolites that marginally affect virulence. Cell wall degrading enzymes (CDWE) have been considered important for pathogenesis but none of them individually have been identified as significant virulence factors in A. brassicicola. In this study, knockout mutants of a gene, AbVf19, were created and produced considerably smaller lesions than the wild type on inoculated host plants. The presence of tandem zinc-finger domains in the predicted amino acid sequence and nuclear localization of AbVf19- reporter protein suggested that it was a transcription factor. Gene expression comparisons using RNA-seq identified 74 genes being downregulated in the mutant during a late stage of infection. Among the 74 downregulated genes, 28 were putative CWDE genes. These were hydrolytic enzyme genes that composed a small fraction of genes within each family of cellulases, pectinases, cutinases, and proteinases. The mutants grew slower than the wild type on an axenic medium with pectin as a major carbon source. This study demonstrated the existence and the importance of a transcription factor that regulates a suite of genes that are important for decomposing and utilizing plant material during the late stage of plant infection.

  10. In Vivo Zinc Finger Nuclease-mediated Targeted Integration of a Glucose-6-phosphatase Transgene Promotes Survival in Mice With Glycogen Storage Disease Type IA

    PubMed Central

    Landau, Dustin J; Brooks, Elizabeth Drake; Perez-Pinera, Pablo; Amarasekara, Hiruni; Mefferd, Adam; Li, Songtao; Bird, Andrew; Gersbach, Charles A; Koeberl, Dwight D

    2016-01-01

    Glycogen storage disease type Ia (GSD Ia) is caused by glucose-6-phosphatase (G6Pase) deficiency in association with severe, life-threatening hypoglycemia that necessitates lifelong dietary therapy. Here we show that use of a zinc-finger nuclease (ZFN) targeted to the ROSA26 safe harbor locus and a ROSA26-targeting vector containing a G6PC donor transgene, both delivered with adeno-associated virus (AAV) vectors, markedly improved survival of G6Pase knockout (G6Pase-KO) mice compared with mice receiving the donor vector alone (P < 0.04). Furthermore, transgene integration has been confirmed by sequencing in the majority of the mice treated with both vectors. Targeted alleles were 4.6-fold more common in livers of mice with GSD Ia, as compared with normal littermates, at 8 months following vector administration (P < 0.02). This suggests a selective advantage for vector-transduced hepatocytes following ZFN-mediated integration of the G6Pase vector. A short-term experiment also showed that 3-month-old mice receiving the ZFN had significantly-improved biochemical correction, in comparison with mice that received the donor vector alone. These data suggest that the use of ZFNs to drive integration of G6Pase at a safe harbor locus might improve vector persistence and efficacy, and lower mortality in GSD Ia. PMID:26865405

  11. Functions of the CCCH type zinc finger protein OsGZF1 in regulation of the seed storage protein GluB-1 from rice.

    PubMed

    Chen, Yi; Sun, Aijun; Wang, Mei; Zhu, Zhen; Ouwerkerk, Pieter B F

    2014-04-01

    Glutelins are the most abundant storage proteins in rice grain and can make up to 80 % of total protein content. The promoter region of GluB-1, one of the glutelin genes in rice, has been intensively used as a model to understand regulation of seed-storage protein accumulation. In this study, we describe a zinc finger gene of the Cys3His1 (CCCH or C3H) class, named OsGZF1, which was identified in a yeast one-hybrid screening using the core promoter region of GluB-1 as bait and cDNA expression libraries prepared from developing rice panicles and grains as prey. The OsGZF1 protein binds specifically to the bait sequence in yeast and this interaction was confirmed in vitro. OsGZF1 is predominantly expressed in a confined domain surrounding the scutellum of the developing embryo and is localised in the nucleus. Transient expression experiments demonstrated that OsGZF1 can down-regulate a GluB-1-GUS (β-glucuronidase) reporter and OsGZF1 was also able to significantly reduce activation conferred by RISBZ1 which is a known strong GluB-1 activator. Furthermore, down-regulation of OsGZF1 by an RNAi approach increased grain nitrogen concentration. We propose that OsGZF1 has a function in regulating the GluB-1 promoter and controls accumulation of glutelins during grain development.

  12. Homology-driven genome editing in hematopoietic stem and progenitor cells using zinc finger nuclease mRNA and AAV6 donors

    PubMed Central

    Wang, Jianbin; Exline, Colin M.; DeClercq, Joshua J.; Llewellyn, G. Nicholas; Hayward, Samuel B.; Li, Patrick Wai-Lun; Shivak, David A.; Surosky, Richard T.; Gregory, Philip D.; Holmes, Michael C.; Cannon, Paula M

    2016-01-01

    Genome editing with targeted nucleases and DNA donor templates homologous to the break site has proven challenging in human hematopoietic stem and progenitor cells (HSPCs), and particularly in the most primitive, long-term repopulating cell population. Here we report that combining electroporation of zinc finger nuclease (ZFN) mRNA with donor template delivery by AAV serotype 6 vectors directs efficient genome editing in HSPCs, achieving site-specific insertion of a GFP cassette at the CCR5 and AAVS1 loci in mobilized peripheral blood CD34+ HSPCs at mean frequencies of 17% and 26%, respectively, and in fetal liver HSPCs at 19% and 43%, respectively. Notably, this approach modified the CD34+CD133+CD90+ cell population, a minor component of CD34+ cells that contains long-term repopulating hematopoietic stem cells (HSCs). Genome-edited HSPCs also engrafted in immune deficient mice long-term, confirming that HSCs are targeted by this approach. Our results provide a strategy for more robust application of genome editing technologies in HSPCs. PMID:26551060

  13. Correction of Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients Through Genomic Excision of Exon 51 by Zinc Finger Nucleases

    PubMed Central

    Ousterout, David G; Kabadi, Ami M; Thakore, Pratiksha I; Perez-Pinera, Pablo; Brown, Matthew T; Majoros, William H; Reddy, Timothy E; Gersbach, Charles A

    2015-01-01

    Duchenne muscular dystrophy (DMD) is caused by genetic mutations that result in the absence of dystrophin protein expression. Oligonucleotide-induced exon skipping can restore the dystrophin reading frame and protein production. However, this requires continuous drug administration and may not generate complete skipping of the targeted exon. In this study, we apply genome editing with zinc finger nucleases (ZFNs) to permanently remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript. This approach can restore the dystrophin reading frame in ~13% of DMD patient mutations. Transfection of two ZFNs targeted to sites flanking the exon 51 splice acceptor into DMD patient myoblasts led to deletion of this genomic sequence. A clonal population was isolated with this deletion and following differentiation we confirmed loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin protein expression. Furthermore, transplantation of corrected cells into immunodeficient mice resulted in human dystrophin expression localized to the sarcolemmal membrane. Finally, we quantified ZFN toxicity in human cells and mutagenesis at predicted off-target sites. This study demonstrates a powerful method to restore the dystrophin reading frame and protein expression by permanently deleting exons. PMID:25492562

  14. A conserved function of the zinc finger transcription factor Sp8/9 in allometric appendage growth in the milkweed bug Oncopeltus fasciatus.

    PubMed

    Schaeper, Nina D; Prpic, Nikola-Michael; Wimmer, Ernst A

    2009-08-01

    The genes encoding the closely related zinc finger transcription factors Buttonhead (Btd) and D-Sp1 are expressed in the developing limb primordia of Drosophila melanogaster and are required for normal growth of the legs. The D-Sp1 homolog of the red flour beetle Tribolium castaneum, Sp8 (appropriately termed Sp8/9), is also required for the proper growth of the leg segments. Here we report on the isolation and functional study of the Sp8/9 gene from the milkweed bug Oncopeltus fasciatus. We show that Sp8/9 is expressed in the developing appendages throughout development and that the downregulation of Sp8/9 via RNAi leads to antennae, rostrum, and legs with shortened and fused segments. This supports a conserved role of Sp8/9 in allometric leg segment growth. However, all leg segments including the claws are present and the expression of the leg genes Distal-less, dachshund, and homothorax are proportionally normal, thus providing no evidence for a role of Sp8/9 in appendage specification.

  15. The Zinc-Finger Protein Slug Causes Desmosome Dissociation, an Initial and Necessary Step for Growth Factor–induced Epithelial–Mesenchymal Transition

    PubMed Central

    Savagner, Pierre; Yamada, Kenneth M.; Thiery, Jean Paul

    1997-01-01

    Epithelial–mesenchymal transition (EMT) is an essential morphogenetic process during embryonic development. It can be induced in vitro by hepatocyte growth factor/scatter factor (HGF/SF), or by FGF-1 in our NBT-II cell model for EMT. We tested for a central role in EMT of a zinc-finger protein called Slug. Slug mRNA and protein levels were increased transiently in FGF-1–treated NBT-II cells. Transient or stable transfection of Slug cDNA in NBT-II cells resulted in a striking disappearance of the desmosomal markers desmoplakin and desmoglein from cell–cell contact areas, mimicking the initial steps of FGF-1 or HGF/SF- induced EMT. Stable transfectant cells expressed Slug protein and were less epithelial, with increased cell spreading and cell–cell separation in subconfluent cultures. Interestingly, NBT-II cells transfected with antisense Slug cDNA were able to resist EMT induction by FGF-1 or even HGF/SF. This antisense effect was suppressed by retransfection with Slug sense cDNA. Our results indicate that Slug induces the first phase of growth factor–induced EMT, including desmosome dissociation, cell spreading, and initiation of cell separation. Moreover, the antisense inhibition experiments suggest that Slug is also necessary for EMT. PMID:9182671

  16. The human homolog of a mouse-imprinted gene, Peg3, maps to a zinc finger gene-rich region of human chromosome 19q13.4.

    PubMed

    Kim, J; Ashworth, L; Branscomb, E; Stubbs, L

    1997-05-01

    Peg3 (paternally expressed gene 3) is the first imprinted gene detected in the proximal region of mouse chromosome 7. Because imprinting is a trait that is generally conserved among mammals, and imprinted domains generally encompass several adjacent genes, expression patterns and chromosomal environment of the human counterpart of Peg3 are of special interest. In this study we have localized human PEG3 approximately 2 Mb proximal of the telomere of chromosome 19q, within a region known to carry large numbers of tandemly clustered Krüppel-type zinc finger-containing (ZNF) genes. Peg3 also encodes a Krüppel-type ZNF protein but one that is distinguished from other ZNF gene products by the fact that it carries two novel proline-rich motifs. Comparison between mouse Peg3 and partial human PEG3 gene sequences revealed a high level of conservation between the two species, despite the fact that one of the two proline-rich repeats is absent from the human gene. Our data demonstrate that the human gene is expressed at highest levels in ovary and placenta; mouse Peg3, by contrast, is transcribed at highest levels in the adult brain. These comparative mapping, sequencing, and expression data provide the first clues to the potential activities of PEG3, and generate new tools to aid in the analysis of structure and function of a potentially new imprinted domain located in human chromosome 19q13.4 and mouse chromosome 7.

  17. Nuclear hormone receptors involved in neoplasia: erb A exhibits a novel DNA sequence specificity determined by amino acids outside of the zinc-finger domain.

    PubMed Central

    Chen, H; Smit-McBride, Z; Lewis, S; Sharif, M; Privalsky, M L

    1993-01-01

    The erb A oncogene is a dominant negative allele of a thyroid hormone receptor gene and acts in the cancer cell by encoding a transcriptional repressor. We demonstrate here that the DNA sequence recognition properties of the oncogenic form of the erb A protein are significantly altered from those of the normal thyroid hormone receptors and more closely resemble those of the retinoic acid receptors; this alteration appears to play an important role in defining the targets of erb A action in neoplasia. Unexpectedly, the novel DNA recognition properties of erb A are encoded by an N-terminal region not previously implicated as playing this function in current models of receptor-DNA interaction. Two N-terminal erb A amino acids in particular, histidine 12 and cysteine 32, contribute to this phenomenon, acting in conjunction with amino acids in the zinc finger domain. The effects of the N-terminal domain can be observed at the level of both DNA binding and transcriptional modulation. Our results indicate that unanticipated determinants within the nuclear hormone receptors participate in DNA sequence recognition and may contribute to the differential target gene specificity displayed by different receptor forms. Images PMID:8096060

  18. PML-associated repressor of transcription (PAROT), a novel KRAB-zinc finger repressor, is regulated through association with PML nuclear bodies

    SciTech Connect

    Fleischer, Sandra; Wiemann, Stefan; Will, Hans; Hofmann, Thomas G. . E-mail: t.hofmann@dkfz.de

    2006-04-01

    Promyelocytic leukemia nuclear bodies (PML-NBs) are implicated in transcriptional regulation. Here we identify a novel transcriptional repressor, PML-associated repressor of transcription (PAROT), which is regulated in its repressor activity through recruitment to PML-NBs. PAROT is a Krueppel-associated box ( KRAB) zinc-finger (ZNF) protein, which comprises an amino terminal KRAB-A and KRAB-B box, a linker domain and 8 tandemly repeated C{sub 2}H{sub 2}-ZNF motifs at its carboxy terminus. Consistent with its domain structure, when tethered to DNA, PAROT represses transcription, and this is partially released by the HDAC inhibitor trichostatin A. PAROT colocalizes with members of the heterochromatin protein 1 (HP1) family and with transcriptional intermediary factor-1{beta}/KRAB-associated protein 1 (TIF-1{beta}/KAP1), a transcriptional corepressor for the KRAB-ZNF family. Interestingly, PML isoform IV, in contrast to PML-III, efficiently recruits PAROT and TIF-1{beta} from heterochromatin to PML-NBs. PML-NB recruitment of PAROT partially releases its transcriptional repressor activity, indicating that PAROT can be regulated through subnuclear compartmentalization. Taken together, our data identify a novel transcriptional repressor and provide evidence for its regulation through association with PML-NBs.

  19. Regions outside the DNA-binding domain are critical for proper in vivo specificity of an archetypal zinc finger transcription factor.

    PubMed

    Burdach, Jon; Funnell, Alister P W; Mak, Ka Sin; Artuz, Crisbel M; Wienert, Beeke; Lim, Wooi F; Tan, Lit Yeen; Pearson, Richard C M; Crossley, Merlin

    2014-01-01

    Transcription factors (TFs) are often regarded as being composed of a DNA-binding domain (DBD) and a functional domain. The two domains are considered separable and autonomous, with the DBD directing the factor to its target genes and the functional domain imparting transcriptional regulation. We examined an archetypal zinc finger (ZF) TF, Krüppel-like factor 3 with an N-terminal domain that binds the corepressor CtBP and a DBD composed of three ZFs at its C-terminus. We established a system to compare the genomic occupancy profile of wild-type Krüppel-like factor 3 with two mutants affecting the N-terminal functional domain: a mutant unable to contact the cofactor CtBP and a mutant lacking the entire N-terminal domain, but retaining the ZFs intact. Chromatin immunoprecipitation followed by sequencing was used to assess binding across the genome in murine embryonic fibroblasts. Unexpectedly, we observe that mutations in the N-terminal domain generally reduced binding, but there were also instances where binding was retained or even increased. These results provide a clear demonstration that the correct localization of TFs to their target genes is not solely dependent on their DNA-contact domains. This informs our understanding of how TFs operate and is of relevance to the design of artificial ZF proteins.

  20. Role of Promyelocytic Leukemia Zinc Finger (PLZF) in Cell Proliferation and Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) Gene Repression*

    PubMed Central

    Choi, Won-Il; Kim, Min-Young; Jeon, Bu-Nam; Koh, Dong-In; Yun, Chae-Ok; Li, Yan; Lee, Choong-Eun; Oh, Jiyoung; Kim, Kunhong; Hur, Man-Wook

    2014-01-01

    Promyelocytic leukemia zinc finger (PLZF) is a transcription repressor that was initially isolated as a fusion protein with retinoic acid receptor α. PLZF is aberrantly overexpressed in various human solid tumors, such as clear cell renal carcinoma, glioblastoma, and seminoma. PLZF causes cellular transformation of NIH3T3 cells and increases cell proliferation in several cell types. PLZF also increases tumor growth in the mouse xenograft tumor model. PLZF may stimulate cell proliferation by controlling expression of the genes of the p53 pathway (ARF, TP53, and CDKN1A). We found that PLZF can directly repress transcription of CDKN1A encoding p21, a negative regulator of cell cycle progression. PLZF binds to the proximal Sp1-binding GC-box 5/6 and the distal p53-responsive elements of the CDKN1A promoter to repress transcription. Interestingly, PLZF interacts with Sp1 or p53 and competes with Sp1 or p53. PLZF interacts with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylates Ac-H3 and Ac-H4 histones at the CDKN1A promoter, which indicated the involvement of the corepressor·HDACs complex in transcription repression by PLZF. Also, PLZF represses transcription of TP53 and also decreases p53 protein stability by ubiquitination. PLZF may act as a potential proto-oncoprotein in various cell types. PMID:24821727

  1. Effects of Static Cold Storage and Hypothermic Machine Perfusion on Oxidative Stress Factors, Adhesion Molecules, and Zinc Finger Transcription Factor Proteins Before and After Liver Transplantation.

    PubMed

    Zhao, De-Fang; Dong, Qin; Zhang, Tong

    2017-02-17

    BACKGROUND This study aimed to investigate the effects of static cold storage (SCS) and hypothermic machine perfusion (HMP) on the oxidative stress factors (OSF), adhesion molecules (AM), and zinc finger transcription factor (Snail) before and after liver transplantation. MATERIAL AND METHODS Experimental dogs were randomly divided into donor (group A), SCS (group B), and HMP (group C) (n=30) groups. Livers retrieved from group A were transplanted into group B after SCS, and the livers sampled from group B were transplanted into group C after HMP. The dogs in group A were euthanized and discarded, and the livers sampled from group C were used for other experiments. Twenty dogs with successful liver transplants were randomly selected from groups B and C for analysis. RESULTS During the liver sampling process, the levels of OSF, AM, and Snail between the 2 groups showed no significant differences (P>0.05); before the transplantation, the levels of chemokine CXCL14 and Snail between the 2 groups showed no significant differences (P>0.05), and compared with group B, HIF-1α and P-selectin in group C were lower (P<0.01); 60 min after the transplantation, HIF-1α, chemokine CXCL14, P-selectin, and Snail in group C were lower than that in group B (P<0.01). CONCLUSIONS HMP can significantly reduce the levels of OSF and inflammatory factors, which is conducive for liver transplantation.

  2. Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma

    PubMed Central

    Beane, Joal D; Lee, Gary; Zheng, Zhili; Mendel, Matthew; Abate-Daga, Daniel; Bharathan, Mini; Black, Mary; Gandhi, Nimisha; Yu, Zhiya; Chandran, Smita; Giedlin, Martin; Ando, Dale; Miller, Jeff; Paschon, David; Guschin, Dmitry; Rebar, Edward J; Reik, Andreas; Holmes, Michael C; Gregory, Philip D; Restifo, Nicholas P; Rosenberg, Steven A; Morgan, Richard A; Feldman, Steven A

    2015-01-01

    Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9–84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500–2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted. PMID:25939491

  3. Homologous recombination contributes to the repair of zinc-finger-nuclease induced double strand breaks in pig primary cells and facilitates recombination with exogenous DNA.

    PubMed

    Klymiuk, Nikolai; Fezert, Pauline; Wünsch, Annegret; Kurome, Mayuko; Kessler, Barbara; Wolf, Eckhard

    2014-05-10

    Site-specific nucleases have become powerful tools for genome editing by the introduction of end-joining-mediated mutations, but it is unclear to which extent induced double strand breaks will also facilitate homologous recombination with exogenous DNA. This question is, however, of particular importance for somatic cells, which have to be modified for the generation of large animal models, but, on the other hand, have also been described to be reluctant to recombination-based DNA repair. Here, we examined zinc-finger nucleases for their potential to introduce modifications in pig somatic cells via end-joining or recombination. We found that co-transfection with nuclease-encoding plasmids resulted in a dramatic boost of recombination with different targeting vectors, suggesting a much more prominent role of this repair pathway in somatic cells than was previously thought. Although recombination with any of the vectors even occurred on both alleles of the target gene, we found also evidence for distinct properties of the used vectors regarding their preference for mono-allelic or bi-allelic modification. Thus, we show that the combined usage of site-specific nucleases and targeting vectors does not only promote homologous recombination in somatic cells but might also resemble a promising tool for detailed examination of DNA repair pathways.

  4. Generation of mastitis resistance in cows by targeting human lysozyme gene to β-casein locus using zinc-finger nucleases.

    PubMed

    Liu, Xu; Wang, Yongsheng; Tian, Yuchen; Yu, Yuan; Gao, Mingqing; Hu, Guangdong; Su, Feng; Pan, Shaohui; Luo, Yan; Guo, Zekun; Quan, Fusheng; Zhang, Yong

    2014-04-07

    Mastitis costs the dairy industry billions of dollars annually and is the most consequential disease of dairy cattle. Transgenic cows secreting an antimicrobial peptide demonstrated resistance to mastitis. The combination of somatic cell gene targeting and nuclear transfer provides a powerful method to produce transgenic animals. Recent studies found that a precisely placed double-strand break induced by engineered zinc-finger nucleases (ZFNs) stimulated the integration of exogenous DNA stretches into a pre-determined genomic location, resulting in high-efficiency site-specific gene addition. Here, we used ZFNs to target human lysozyme (hLYZ) gene to bovine β-casein locus, resulting in hLYZ knock-in of approximately 1% of ZFN-treated bovine fetal fibroblasts (BFFs). Gene-targeted fibroblast cell clones were screened by junction PCR amplification and Southern blot analysis. Gene-targeted BFFs were used in somatic cell nuclear transfer. In vitro assays demonstrated that the milk secreted by transgenic cows had the ability to kill Staphylococcus aureus. We report the production of cloned cows carrying human lysozyme gene knock-in β-casein locus using ZFNs. Our findings open a unique avenue for the creation of transgenic cows from genetic engineering by providing a viable tool for enhancing resistance to disease and improving the health and welfare of livestock.

  5. MusaSAP1, a A20/AN1 zinc finger gene from banana functions as a positive regulator in different stress responses.

    PubMed

    Sreedharan, Shareena; Shekhawat, Upendra K Singh; Ganapathi, Thumballi R

    2012-11-01

    A20/AN1 zinc finger domain containing Stress Associated Proteins (SAP) are involved in diverse stress response pathways in plants. In the present study, a novel banana SAP gene, MusaSAP1, was identified from banana EST database and was subsequently characterized by overexpression in transgenic banana plants. Expression profiling in native banana plants showed that MusaSAP1 was up-regulated by drought, salt, cold, heat and oxidative stress as well as by treatment with abscisic acid. Cellular localization assay carried out by making a MusaSAP1::GFP fusion protein indicated that MusaSAP1 is incompletely translocated to nucleus. Copy number analysis performed using real time PCR and Southern blotting indicated that MusaSAP1 occurs in the banana genome in a single copy per 11 chromosome set. Transgenic banana plants constitutively overexpressing MusaSAP1 displayed better stress endurance characteristics as compared to controls in both in vitro and ex vivo assays. Lesser membrane damage as indicated by reduced malondialdehyde levels in transgenic leaves subjected to drought, salt or oxidative stress pointed towards significant role for MusaSAP1 in stress amelioration pathways of banana. Strong up-regulation of a polyphenol oxidase (PPO) coding transcript in MusaSAP1 overexpressing plants together with induction of MusaSAP1 by wounding and methyl jasmonate treatment indicated possible involvement of MusaSAP1 in biotic stress responses where PPOs perform major functions in multiple defense pathways.

  6. Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases.

    PubMed

    Ousterout, David G; Kabadi, Ami M; Thakore, Pratiksha I; Perez-Pinera, Pablo; Brown, Matthew T; Majoros, William H; Reddy, Timothy E; Gersbach, Charles A

    2015-03-01

    Duchenne muscular dystrophy (DMD) is caused by genetic mutations that result in the absence of dystrophin protein expression. Oligonucleotide-induced exon skipping can restore the dystrophin reading frame and protein production. However, this requires continuous drug administration and may not generate complete skipping of the targeted exon. In this study, we apply genome editing with zinc finger nucleases (ZFNs) to permanently remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript. This approach can restore the dystrophin reading frame in ~13% of DMD patient mutations. Transfection of two ZFNs targeted to sites flanking the exon 51 splice acceptor into DMD patient myoblasts led to deletion of this genomic sequence. A clonal population was isolated with this deletion and following differentiation we confirmed loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin protein expression. Furthermore, transplantation of corrected cells into immunodeficient mice resulted in human dystrophin expression localized to the sarcolemmal membrane. Finally, we quantified ZFN toxicity in human cells and mutagenesis at predicted off-target sites. This study demonstrates a powerful method to restore the dystrophin reading frame and protein expression by permanently deleting exons.

  7. Drosophila HP1c isoform interacts with the zinc-finger proteins WOC and Relative-of-WOC to regulate gene expression.

    PubMed

    Font-Burgada, Joan; Rossell, David; Auer, Herbert; Azorín, Fernando

    2008-11-01

    Heterochromatin protein 1 (HP1) proteins are conserved in eukaryotes, with most species containing several isoforms. Based on the properties of Drosophila HP1a, it was proposed that HP1s bind H3K9me2,3 and recruit factors involved in heterochromatin assembly and silencing. Yet, it is unclear whether this general picture applies to all HP1 isoforms and functional contexts. Here, we report that Drosophila HP1c regulates gene expression, as (1) it localizes to active chromatin domains, where it extensively colocalizes with the poised form of RNApolymerase II (RNApol II), Pol IIo(ser5), and H3K4me3, suggesting a contribution to transcriptional regulation; (2) its targeting to a reporter gene does not induce silencing but, on the contrary, increases its expression, and (3) it interacts with the zinc-finger proteins WOC (without children) and Relative-of-WOC (ROW), which are putative transcription factors. Here, we also show that, although HP1c efficiently binds H3K9me2,3 in vitro, its binding to chromatin strictly depends on both WOC and ROW. Moreover, expression profiling indicates that HP1c, WOC, and ROW regulate a common gene expression program that, in part, is executed in the context of the nervous system. From this study, which unveils the essential contribution of DNA-binding proteins to HP1c functionality and recruitment, HP1 proteins emerge as an increasingly diverse family of chromatin regulators.

  8. A large protein containing zinc finger domains binds to related sequence elements in the enhancers of the class I major histocompatibility complex and kappa immunoglobulin genes.

    PubMed Central

    Baldwin, A S; LeClair, K P; Singh, H; Sharp, P A

    1990-01-01

    A cDNA from a B-cell library was previously isolated that encodes a sequence-specific DNA-binding protein with affinities for related sites in a class I major histocompatibility complex (MHC) and kappa immunoglobulin gene enhancers. We report here approximately 6.5 kilobases of sequence of the MBP-1 (MHC enhancer binding protein 1) cDNA. MBP-1 protein has a molecular weight predicted to be greater than 200,000. A DNA-binding domain with high affinity for the MHC enhancer sequence TGGGGATTCCCCA was localized to an 118-amino-acid protein fragment containing two zinc fingers of the class Cys2-X12-His2. Analysis of expression of MBP-1 mRNA revealed relatively high expression in HeLa cells and in a human retinal cell line, with lower levels in Jurkat T cells and in two B-cell lines. Interestingly, expression of MBP-1 mRNA was inducible by mitogen and phorbol ester treatment of Jurkat T cells and by serum treatment of confluent serum-deprived human fibroblasts. Images PMID:2108316

  9. The Human CCHC-type Zinc Finger Nucleic Acid-Binding Protein Binds G-Rich Elements in Target mRNA Coding Sequences and Promotes Translation.

    PubMed

    Benhalevy, Daniel; Gupta, Sanjay K; Danan, Charles H; Ghosal, Suman; Sun, Hong-Wei; Kazemier, Hinke G; Paeschke, Katrin; Hafner, Markus; Juranek, Stefan A

    2017-03-21

    The CCHC-type zinc finger nucleic acid-binding protein (CNBP/ZNF9) is conserved in eukaryotes and is essential for embryonic development in mammals. It has been implicated in transcriptional, as well as post-transcriptional, gene regulation; however, its nucleic acid ligands and molecular function remain elusive. Here, we use multiple systems-wide approaches to identify CNBP targets and function. We used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to identify 8,420 CNBP binding sites on 4,178 mRNAs. CNBP preferentially bound G-rich elements in the target mRNA coding sequences, most of which were previously found to form G-quadruplex and other stable structures in vitro. Functional analyses, including RNA sequencing, ribosome profiling, and quantitative mass spectrometry, revealed that CNBP binding did not influence target mRNA abundance but rather increased their translational efficiency. Considering that CNBP binding prevented G-quadruplex structure formation in vitro, we hypothesize that CNBP is supporting translation by resolving stable structures on mRNAs.

  10. Zinc-finger transcription factors are associated with guanine quadruplex motifs in human, chimpanzee, mouse and rat promoters genome-wide.

    PubMed

    Kumar, Pankaj; Yadav, Vinod Kumar; Baral, Aradhita; Kumar, Parveen; Saha, Dhurjhoti; Chowdhury, Shantanu

    2011-10-01

    Function of non-B DNA structures are poorly understood though several bioinformatics studies predict role of the G-quadruplex DNA structure in transcription. Earlier, using transcriptome profiling we found evidence of widespread G-quadruplex-mediated gene regulation. Herein, we asked whether potential G-quadruplex (PG4) motifs associate with transcription factors (TF). This was analyzed using 220 position weight matrices [designated as transcription factor binding sites (TFBS)], representing 187 unique TF, in >75,000 genes in human, chimpanzee, mouse and rat. Results show binding sites of nine TFs, including that of AP-2, SP1, MAZ and VDR, occurred significantly within 100 bases of the PG4 motif (P < 1.24E-10). PG4-TFBS combinations were conserved in 'orthologously' related promoters across all four organisms and were associated with >850 genes in each genome. Remarkably, seven of the nine TFs were zinc-finger binding proteins indicating a novel characteristic of PG4 motifs. To test these findings, transcriptome profiles from human cell lines treated with G-quadruplex-specific molecules were used; 66 genes were significantly differentially expressed across both cell-types, which also harbored conserved PG4 motifs along with one/more of the nine TFBS. In addition, genes regulated by PG4-TFBS combinations were found to be co-regulated in human tissues, further emphasizing the regulatory significance of the associations.

  11. Human and mouse ZFY genes produce a conserved testis-specific transcript encoding a zinc finger protein with a short acidic domain and modified transactivation potential.

    PubMed

    Decarpentrie, Fanny; Vernet, Nadège; Mahadevaiah, Shantha K; Longepied, Guy; Streichemberger, Eric; Aknin-Seifer, Isabelle; Ojarikre, Obah A; Burgoyne, Paul S; Metzler-Guillemain, Catherine; Mitchell, Michael J

    2012-06-15

    Mammalian ZFY genes are located on the Y chromosome, and code putative transcription factors with 12-13 zinc fingers preceded by a large acidic (activating) domain. In mice, there are two genes, Zfy1 and Zfy2, which are expressed mainly in the testis. Their transcription increases in germ cells as they enter meiosis, both are silenced by meiotic sex chromosome inactivation (MSCI) during pachytene, and Zfy2 is strongly reactivated later in spermatids. Recently, we have shown that mouse Zfy2, but not Zfy1, is involved in triggering the apoptotic elimination of specific types of sex chromosomally aberrant spermatocytes. In humans, there is a single widely transcribed ZFY gene, and there is no evidence for a specific role in the testis. Here, we characterize ZFY transcription during spermatogenesis in mice and humans. In mice, we define a variety of Zfy transcripts, among which is a Zfy2 transcript that predominates in spermatids, and a Zfy1 transcript, lacking an exon encoding approximately half of the acidic domain, which predominates prior to MSCI. In humans, we have identified a major testis-specific ZFY transcript that encodes a protein with the same short acidic domain. This represents the first evidence that ZFY has a conserved function during human spermatogenesis. We further show that, in contrast to the full acidic domain, the short domain does not activate transcription in yeast, and we hypothesize that this explains the functional difference observed between Zfy1 and Zfy2 during mouse meiosis.

  12. Zinc finger protein genes from Cucurbita pepo are promising tools for conferring non-Cucurbitaceae plants with ability to accumulate persistent organic pollutants.

    PubMed

    Inui, Hideyuki; Hirota, Matashi; Goto, Junya; Yoshihara, Ryouhei; Kodama, Noriko; Matsui, Tomomi; Yamazaki, Kiyoshi; Eun, Heesoo

    2015-03-01

    Some cultivars of cucumbers, melons, pumpkins, and zucchini, which are members of the Cucurbitaceae family, are uniquely subject to contamination by hydrophobic pollutants such as the organohalogen insecticides DDT. However, the molecular mechanisms for the accumulation of these pollutants in cucurbits have not been determined. Here, cDNA subtraction analysis of Cucurbita pepo cultivars that are low and high accumulators of hydrophobic contaminants revealed that a gene for zinc finger proteins (ZFPs) are preferentially expressed in high accumulators. The cloned CpZFP genes were classified into 2 types: (1) the PBG type, which were expressed in C. pepo cultivars Patty Green, Black Beauty, and Gold Rush, and (2) the BG type, which were expressed in Black Beauty and Gold Rush. Expression of these CpZFP genes in transgenic tobacco plants carrying an aryl hydrocarbon receptor-based inducible gene expression system significantly induced β-glucuronidase activity when the plants were treated with a polychlorinated biphenyl (PCB) compound, indicating that highly hydrophobic PCBs accumulated in the plants. In transgenic tobacco plants carrying CpZFPs, accumulation of dioxins and dioxin-like compounds increased in their aerial parts when they were cultivated in the dioxin-contaminated soil. In summary, we propose that addition of CpZFP genes is a promising tool for conferring noncucurbits with the ability to accumulate hydrophobic contaminants.

  13. Identification of a Cis-Acting Element of ART1, a C2H2-Type Zinc-Finger Transcription Factor for Aluminum Tolerance in Rice1[OA

    PubMed Central

    Tsutsui, Tomokazu; Yamaji, Naoki; Feng Ma, Jian

    2011-01-01

    Rice (Oryza sativa) is one of the most aluminum (Al)-tolerant species among small-grain cereals. Recent identification of a transcription factor AL RESISTANCE TRANSCRIPTION FACTOR1 (ART1) revealed that this high Al tolerance in rice is achieved by multiple genes involved in detoxification of Al at different cellular levels. ART1 is a C2H2-type zinc-finger transcription factor and regulates the expression of 31 genes in the downstream. In this study, we attempted to identify a cis-acting element of ART1. We used the promoter region of SENSITIVE TO AL RHIZOTOXICITY1, an Al tolerance gene in the downstream of ART1. With the help of gel-shift assay, we were able to identify the cis-acting element as GGN(T/g/a/C)V(C/A/g)S(C/G). This element was found in the promoter region of 29 genes among 31 ART1-regulated genes. To confirm this cis-acting element in vivo, we transiently introduced this element one or five times tandemly repeated sequence with 35S minimal promoter and green fluorescent protein reporter together with or without ART1 gene in the tobacco (Nicotiana tabacum) mesophyll protoplasts. The results showed that the expression of green fluorescent protein reporter responded to ART1 expression. Furthermore, the expression increased with repetition of the cis-acting element. Our results indicate that the five nucleotides identified are the target DNA-binding sequence of ART1. PMID:21502187

  14. The Arabidopsis Zinc Finger-Homeodomain Genes Encode Proteins with Unique Biochemical Properties That Are Coordinately Expressed during Floral Development1

    PubMed Central

    Tan, Queenie K.-G.; Irish, Vivian F.

    2006-01-01

    Arabidopsis (Arabidopsis thaliana) contains approximately 100 homeobox genes, many of which have been shown to play critical roles in various developmental processes. Here we characterize the zinc finger-homeodomain (ZF-HD) subfamily of homeobox genes, consisting of 14 members in Arabidopsis. We demonstrate that the HDs of the ZF-HD proteins share some similarities with other known HDs in Arabidopsis, but they contain distinct features that cluster them as a unique class of plant HD-containing proteins. We have carried out mutational analyses to show that the noncanonical residues present in the HDs of this family of proteins are important for function. Yeast (Saccharomyces cerevisiae) two-hybrid matrix analyses of the ZF-HD proteins reveal that these proteins both homo- and heterodimerize, which may contribute to greater selectivity in DNA binding. These assays also show that most of these proteins do not contain an intrinsic activation domain, suggesting that interactions with other factors are required for transcriptional activation. We also show that the family members are all expressed predominantly or exclusively in floral tissue, indicating a likely regulatory role during floral development. Furthermore, we have identified loss-of-function mutations for six of these genes that individually show no obvious phenotype, supporting the idea that the encoded proteins have common roles in floral development. Based on these results, we propose the ZF-HD gene family encodes a group of transcriptional regulators with unique biochemical activities that play overlapping regulatory roles in Arabidopsis floral development. PMID:16428600

  15. The zinc fingers of the SR-like protein ZRANB2 are single-stranded RNA-binding domains that recognize 5′ splice site-like sequences

    SciTech Connect

    Loughlin, Fionna E.; Mansfield, Robyn E.; Vaz, Paula M.; McGrath, Aaron P.; Setiyaputra, Surya; Gamsjaeger, Roland; Chen, Eva S.; Morris, Brian J.; Guss, J. Mitchell; Mackay, Joel P.

    2009-09-02

    The alternative splicing of mRNA is a critical process in higher eukaryotes that generates substantial proteomic diversity. Many of the proteins that are essential to this process contain arginine/serine-rich (RS) domains. ZRANB2 is a widely-expressed and highly-conserved RS-domain protein that can regulate alternative splicing but lacks canonical RNA-binding domains. Instead, it contains 2 RanBP2-type zinc finger (ZnF) domains. We demonstrate that these ZnFs recognize ssRNA with high affinity and specificity. Each ZnF binds to a single AGGUAA motif and the 2 domains combine to recognize AGGUAA(N{sub x})AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5{prime} splice site. We show using X-ray crystallography that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine 'ladder.' A number of other human proteins that function in RNA processing also contain RanBP2 ZnFs in which the RNA-binding residues of ZRANB2 are conserved. The ZnFs of ZRANB2 therefore define another class of RNA-binding domain, advancing our understanding of RNA recognition and emphasizing the versatility of ZnF domains in molecular recognition.

  16. Phylogenic analysis revealed an expanded C₂H₂-homeobox subfamily and expression profiles of C₂H₂ zinc finger gene family in Verticillium dahliae.

    PubMed

    Xiong, Dianguang; Wang, Yonglin; Deng, Chenglin; Hu, Ruowen; Tian, Chengming

    2015-05-15

    C2H2 zinc finger (CZF) proteins are a major class of transcription factors that play crucial roles in fungal growth, development, various stress responses, and virulence. Little genome-wide data is available regarding the roles of CZF proteins in Verticillium dahliae, a destructive pathogen that causes vascular wilt disease in more than 200 plant species. We identified a total of 79 typical CZF genes in V. dahliae. Comparative analysis revealed that four plant pathogenic fungi, V. dahliae, Fusarium oxysporum, Magnaporthe oryzae, and Botrytis cinerea, have comparable numbers of predicted CZF genes with similar characteristics. Phylogenetic analysis identified a C2H2-homeobox subfamily in V. dahliae containing seven genes with similar gene structures. V. dahliae and F. oxysporum (Hypocreomycetidae) have more genes of this subfamily than M. oryzae (Sordariomycetidae) and B. cinerea (Leotiomycetes). Furthermore, gene-expression analysis of the smoke tree wilt fungus V. dahliae strain XS11 using digital gene-expression profiling and RT-qPCR revealed that a number of CZF genes were differentially expressed during microsclerotia formation, nutritional starvation, and simulated in planta conditions. Furthermore, the expression profiles revealed that some CZF genes were overrepresented during multiple stages, indicating that they might play diverse roles. Our results provide useful information concerning the functions of CZF genes in microsclerotia formation, nutritional stress responses, and pathogenicity in V. dahliae, and form a basis for future functional studies of these genes.

  17. Sequence and expression of GLN3, a positive nitrogen regulatory gene of Saccharomyces cerevisiae encoding a protein with a putative zinc finger DNA-binding domain.

    PubMed Central

    Minehart, P L; Magasanik, B

    1991-01-01

    The GLN3 gene of Saccharomyces cerevisiae is required for the activation of transcription of a number of genes in response to the replacement of glutamine by glutamate as source of nitrogen. We cloned the GLN3 gene and constructed null alleles by gene disruption. GLN3 is not essential for growth, but increased copies of GLN3 lead to a drastic decrease in growth rate. The complete nucleotide sequence of the GLN3 gene was determined, revealing one open reading frame encoding a polypeptide of 730 amino acids, with a molecular weight of approximately 80,000. The GLN3 protein contains a single putative Cys2/Cys2 zinc finger which has homology to the Neurospora crassa NIT2 protein, the Aspergillus nidulans AREA protein, and the erythroid-specific transcription factor GATA-1. Immunoprecipitation experiments indicated that the GLN3 protein binds the nitrogen upstream activation sequence of GLN1, the gene encoding glutamine synthetase. Neither control of transcription nor control of initiation of translation of GLN3 is important for regulation in response to glutamine availability. Images PMID:1682800

  18. Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA

    PubMed Central

    Watanabe, Masahito; Nakano, Kazuaki; Matsunari, Hitomi; Matsuda, Taisuke; Maehara, Miki; Kanai, Takahiro; Kobayashi, Mirina; Matsumura, Yukina; Sakai, Rieko; Kuramoto, Momoko; Hayashida, Gota; Asano, Yoshinori; Takayanagi, Shuko; Arai, Yoshikazu; Umeyama, Kazuhiro; Nagaya, Masaki; Hanazono, Yutaka; Nagashima, Hiroshi

    2013-01-01

    Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration. PMID:24130776

  19. A G-Rich Motif in the lncRNA Braveheart Interacts with a Zinc-Finger Transcription Factor to Specify the Cardiovascular Lineage.

    PubMed

    Xue, Zhihong; Hennelly, Scott; Doyle, Boryana; Gulati, Arune A; Novikova, Irina V; Sanbonmatsu, Karissa Y; Boyer, Laurie A

    2016-10-06

    Long non-coding RNAs (lncRNAs) are an emerging class of transcripts that can modulate gene expression; however, their mechanisms of action remain poorly understood. Here, we experimentally determine the secondary structure of Braveheart (Bvht) using chemical probing methods and show that this ∼590 nt transcript has a modular fold. Using CRISPR/Cas9-mediated editing of mouse embryonic stem cells, we find that deletion of 11 nt in a 5' asymmetric G-rich internal loop (AGIL) of Bvht (bvht(dAGIL)) dramatically impairs cardiomyocyte differentiation. We demonstrate a specific interaction between AGIL and cellular nucleic acid binding protein (CNBP/ZNF9), a zinc-finger protein known to bind single-stranded G-rich sequences. We further show that CNBP deletion partially rescues the bvht(dAGIL) mutant phenotype by restoring differentiation capacity. Together, our work shows that Bvht functions with CNBP through a well-defined RNA motif to regulate cardiovascular lineage commitment, opening the door for exploring broader roles of RNA structure in development and disease.

  20. Engineering drought tolerant tomato plants over-expressing BcZAT12 gene encoding a C₂H₂ zinc finger transcription factor.

    PubMed

    Rai, Avinash Chandra; Singh, Major; Shah, Kavita

    2013-01-01

    Efficient genetic transformation of cotyledonary explants of tomato (Solanum lycopersicum, cv. H-86, Kashi vishesh) was obtained. Disarmed Agrobacterium tumifaciens strain GV 3101 was used in conjugation with binary vector pBinAR containing a construct consisting of the coding sequence of the BcZAT12 gene under the regulatory control of the stress inducible Bclea1a promoter. ZAT12 encodes a C₂H₂ zinc finger protein which confers multiple abiotic stress tolerance to plants. Integration of ZAT12 gene into nuclear genome of individual kanamycin resistant transformed T₀ tomato lines was confirmed by Southern blot hybridization with segregation analysis of T(1) plants showing Mendelian inheritance of the transgene. Expression of ZAT12 in drought-stressed transformed tomato lines was verified in T₂ generation plants using RT-PCR. Of the six transformed tomato lines (ZT1-ZT6) the transformants ZT1 and ZT5 showed maximum expression of BcZAT12 gene transcripts when exposed to 7 days drought stress. Analysis of relative water content (RWC), electrolyte leakage (EL), chlorophyll colour index (CCI), H₂O₂ level and catalase activity suggested that tomato BcZAT12 transformants ZT1 and ZT5 have significantly increased levels of drought tolerance. These results suggest that BcZAT12 transformed tomato cv. H-86 has real potential for molecular breeding programs aimed at augmenting yield of tomato in regions affected with drought stress.

  1. Zinc finger protein 91 (ZFP91) activates HIF-1α via NF-κB/p65 to promote proliferation and tumorigenesis of colon cancer

    PubMed Central

    Ma, Juan; Mi, Chunliu; Wang, Ke Si; Lee, Jung Joon; Jin, Xuejun

    2016-01-01

    Zinc finger protein 91 (ZFP91) has been reported to be involved in various biological processes. However, the clinical significance and biological role of ZFP91 in colon cancer remains unknown. Here, we show that ZFP91 expression is upregulated in patients with colon cancer. We found that ZFP91 upregulated HIF-1α at the levels of promoter and protein in colon cancer cells. Using chromatin immunoprecipitation, electrophoretic mobility shift assay and luciferase reporter gene assay, we found that NF-κB/p65 is required for the binding of ZFP91 to the HIF-1α promoter at −197/−188 base pairs and for the transcriptional activation of HIF-1α gene mediated by ZFP91. Flow cytometry, 5-ethynyl-2′-deoxyuridine (EdU) incorporation and tumor xenograft assay demonstrated that ZFP91 enhanced cell proliferation of colon cancer through upregulating HIF-1α in vitro and in vivo. Furthermore, ZFP91 is positively associated with HIF-1α in human colon cancer. Thus, we concluded that ZFP91 activates transcriptional coregulatory protein HIF-1α through transcription factor NF-κB/p65 in the promotion of proliferation and tumorigenesis in colon cancer cell. ZFP91 may serve as a driver gene to activate HIF-1α transcription in the development of cancer. PMID:27144516

  2. Identification of “safe harbor” loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair

    PubMed Central

    Cantos, Christian; Francisco, Perigio; Trijatmiko, Kurniawan R.; Slamet-Loedin, Inez; Chadha-Mohanty, Prabhjit K.

    2014-01-01

    Zinc-finger nucleases (ZFNs) have proved to be successful tools for targeted genome manipulation in several organisms. Their main property is the induction of double-strand breaks (DSBs) at specific sites, which are further repaired through homologous recombination (HR) or non-homologous end joining (NHEJ). However, for the appropriate integration of genes at specific chromosomal locations, proper sites for gene integration need to be identified. These regions, hereby named safe harbor loci, must be localized in non-coding regions and possess high gene expression. In the present study, three different ZFN constructs (pZFN1, pZFN2, pZFN3), harboring β-glucuronidase (GUS) as a reporter gene, were used to identify safe harbor loci on rice chromosomes. The constructs were delivered into IR64 rice by using an improved Agrobacterium-mediated transformation protocol, based on the use of immature embryos. Gene expression was measured by histochemical GUS activity and the flanking regions were determined through thermal-asymmetric interlaced polymerase chain reaction (TAIL PCR). Following sequencing, 28 regions were identified as putative sites for safe integration, but only one was localized in a non-coding region and also possessed high GUS expression. These findings have significant applicability to create crops with new and valuable traits, since the site can be subsequently used to stably introduce one or more genes in a targeted manner. PMID:25018764

  3. Gene correction by homologous recombination with zinc finger nucleases in primary cells from a mouse model of a generic recessive genetic disease.

    PubMed

    Connelly, Jon P; Barker, Jenny C; Pruett-Miller, Shondra; Porteus, Matthew H

    2010-06-01

    Zinc Finger nucleases (ZFNs) have been used to create precise genome modifications at frequencies that might be therapeutically useful in gene therapy. We created a mouse model of a generic recessive genetic disease to establish a preclinical system to develop the use of ZFN-mediated gene correction for gene therapy. We knocked a mutated GFP gene into the ROSA26 locus in murine embryonic stem (ES) cells and used these cells to create a transgenic mouse. We used ZFNs to determine the frequency of gene correction by gene targeting in different primary cells from this model. We achieved targeting frequencies from 0.17 to 6% in different cell types, including primary fibroblasts and astrocytes. We demonstrate that ex vivo gene-corrected fibroblasts can be transplanted back into a mouse where they retained the corrected phenotype. In addition, we achieved targeting frequencies of over 1% in ES cells, and the targeted ES cells retained the ability to differentiate into cell types from all three germline lineages. In summary, potentially therapeutically relevant frequencies of ZFN-mediated gene targeting can be achieved in a variety of primary cells and these cells can then be transplanted back into a recipient.

  4. Engineered Zinc-Finger Proteins Can Compensate Genetic Haploinsufficiency by Transcriptional Activation of the Wild-Type Allele: Application to Willams-Beuren Syndrome and Supravalvular Aortic Stenosis

    PubMed Central

    Zhang, Pei; Huang, Angela; Morales-Ruiz, Manuel; Starcher, Barry C.; Huang, Yan; Sessa, William C.; Niklason, Laura E.

    2012-01-01

    Abstract Williams-Beuren syndrome (WBS) and supravalvular aortic stenosis (SVAS) are genetic syndromes marked by the propensity to develop severe vascular stenoses. Vascular lesions in both syndromes are caused by haploinsufficiency of the elastin gene. We used these distinct genetic syndromes as models to evaluate the feasibility of using engineered zinc-finger protein transcription factors (ZFPs) to achieve compensatory expression of haploinsufficient genes by inducing augmented expression from the remaining wild-type allele. For complex genes with multiple splice variants, this approach could have distinct advantages over cDNA-based gene replacement strategies. Targeting the elastin gene, we show that transcriptional activation by engineered ZFPs can induce compensatory expression from the wild-type allele in the setting of classic WBS and SVAS genetic mutations, increase elastin expression in wild-type cells, induce expression of the major elastin splice variants, and recapitulate their natural stoichiometry. Further, we establish that transcriptional activation of the mutant allele in SVAS does not overcome nonsense-mediated decay, and thus ZFP-mediated transcriptional activation is not likely to induce production of a mutant protein, a crucial consideration. Finally, we show in bioengineered blood vessels that ZFP-mediated induction of elastin expression is capable of stimulating functional elastogenesis. Haploinsufficiency is a common mechanism of genetic disease. These findings have significant implications for WBS and SVAS, and establish that haploinsufficiency can be overcome by targeted transcriptional activation without inducing protein expression from the mutant allele. PMID:22891920

  5. Overexpression of a novel chrysanthemum Cys2/His2-type zinc finger protein gene DgZFP3 confers drought tolerance in tobacco.

    PubMed

    Liu, Qing-Lin; Xu, Ke-Dong; Zhong, Ming; Pan, Yuan-Zhi; Jiang, Bei-Bei; Liu, Guang-Li; Jia, Yin; Zhang, Hai-Qing

    2013-11-01

    A drought stress-responsive Cys2/His2-type zinc finger protein gene DgZFP3 was previously isolated (Liu et al., Afr J Biotechnol 11:7781-7788, 2012b) from chrysanthemum. To assess roles of DgZFP3 in plant drought stress responses, we performed gain-of-function experiment. The DgZFP3-overexpression tobacco plants showed significant drought tolerance over the wild type (WT). The transgenic lines exhibited less accumulation of H2O2 under drought stress, more accumulation of proline and greater activities of peroxidase (POD) and superoxide dismutase than the WT under both control conditions and drought stress. In addition, there was greater up-regulation of the ROS-related enzyme genes (NtSOD and NtPOD) and stress-related genes (NtLEA5 and NtDREB) in transgenic lines under normal or drought conditons. Thus DgZFP3 probably plays a positive regulatory role in drought stress response and has the potential to be utilized in transgenic breeding to improve drought stress tolerance in plants.

  6. FILAMENTOUS FLOWER, a meristem and organ identity gene of Arabidopsis, encodes a protein with a zinc finger and HMG-related domains.

    PubMed

    Sawa, S; Watanabe, K; Goto, K; Liu, Y G; Shibata, D; Kanaya, E; Morita, E H; Okada, K

    1999-05-01

    Distinctive from that of the animal system, the basic plan of the plant body is the continuous formation of a structural unit, composed of a stem with a meristem at the top and lateral organs continuously forming at the meristem. Therefore, mechanisms controlling the formation, maintenance, and development of a meristem will be a key to understanding the body plan of higher plants. Genetic analyses of filamentous flower (fil) mutants have indicated that FIL is required for the maintenance and growth of inflorescence and floral meristems, and of floral organs of Arabidopsis thaliana. FIL encodes a protein carrying a zinc finger and a HMG box-like domain, which is known to work as a transcription regulator. As expected, the FIL protein was shown to have a nuclear location. In situ hybridization clearly demonstrated that FIL is expressed only at the abaxial side of primordia of leaves and floral organs. Transgenic plants, ectopically expressing FIL, formed filament-like leaves with randomly arranged cells at the leaf margin. Our results indicate that cells at the abaxial side of the lateral organs are responsible for the normal development of the organs as well as for maintaining the activity of meristems.

  7. RelB NF-kappaB represses estrogen receptor alpha expression via induction of the zinc finger protein Blimp1.

    PubMed

    Wang, Xiaobo; Belguise, Karine; O'Neill, Christine F; Sánchez-Morgan, Nuria; Romagnoli, Mathilde; Eddy, Sean F; Mineva, Nora D; Yu, Ziyang; Min, Chengyin; Trinkaus-Randall, Vickery; Chalbos, Dany; Sonenshein, Gail E

    2009-07-01

    Aberrant constitutive expression of NF-kappaB subunits, reported in more than 90% of breast cancers and multiple other malignancies, plays pivotal roles in tumorigenesis. Higher RelB subunit expression was demonstrated in estrogen receptor alpha (ERalpha)-negative breast cancers versus ERalpha-positive ones, due in part to repression of RelB synthesis by ERalpha signaling. Notably, RelB promoted a more invasive phenotype in ERalpha-negative cancers via induction of the BCL2 gene. We report here that RelB reciprocally inhibits ERalpha synthesis in breast cancer cells, which contributes to a more migratory phenotype. Specifically, RelB is shown for the first time to induce expression of the zinc finger repressor protein Blimp1 (B-lymphocyte-induced maturation protein), the critical mediator of B- and T-cell development, which is transcribed from the PRDM1 gene. Blimp1 protein repressed ERalpha (ESR1) gene transcription. Commensurately higher Blimp1/PRDM1 expression was detected in ERalpha-negative breast cancer cells and primary breast tumors. Induction of PRDM1 gene expression was mediated by interaction of Bcl-2, localized in the mitochondria, with Ras. Thus, the induction of Blimp1 represents a novel mechanism whereby the RelB NF-kappaB subunit mediates repression, specifically of ERalpha, thereby promoting a more migratory phenotype.

  8. RelB NF-κB Represses Estrogen Receptor α Expression via Induction of the Zinc Finger Protein Blimp1▿ ‡

    PubMed Central

    Wang, Xiaobo; Belguise, Karine; O'Neill, Christine F.; Sánchez-Morgan, Nuria; Romagnoli, Mathilde; Eddy, Sean F.; Mineva, Nora D.; Yu, Ziyang; Min, Chengyin; Trinkaus-Randall, Vickery; Chalbos, Dany; Sonenshein, Gail E.

    2009-01-01

    Aberrant constitutive expression of NF-κB subunits, reported in more than 90% of breast cancers and multiple other malignancies, plays pivotal roles in tumorigenesis. Higher RelB subunit expression was demonstrated in estrogen receptor alpha (ERα)-negative breast cancers versus ERα-positive ones, due in part to repression of RelB synthesis by ERα signaling. Notably, RelB promoted a more invasive phenotype in ERα-negative cancers via induction of the BCL2 gene. We report here that RelB reciprocally inhibits ERα synthesis in breast cancer cells, which contributes to a more migratory phenotype. Specifically, RelB is shown for the first time to induce expression of the zinc finger repressor protein Blimp1 (B-lymphocyte-induced maturation protein), the critical mediator of B- and T-cell development, which is transcribed from the PRDM1 gene. Blimp1 protein repressed ERα (ESR1) gene transcription. Commensurately higher Blimp1/PRDM1 expression was detected in ERα-negative breast cancer cells and primary breast tumors. Induction of PRDM1 gene expression was mediated by interaction of Bcl-2, localized in the mitochondria, with Ras. Thus, the induction of Blimp1 represents a novel mechanism whereby the RelB NF-κB subunit mediates repression, specifically of ERα, thereby promoting a more migratory phenotype. PMID:19433448

  9. Control of dissected leaf morphology by a Cys(2)His(2) zinc finger transcription factor in the model legume Medicago truncatula

    PubMed Central

    Yu, Jianbin; Ge, Liangfa; Wang, Hongliang; Berbel, Ana; Liu, Yu; Chen, Yuhui; Li, Guangming; Tadege, Million; Wen, Jiangqi; Cosson, Viviane; Mysore, Kirankumar S.; Ratet, Pascal; Madueño, Francisco; Bai, Guihua; Chen, Rujin

    2010-01-01

    Plant leaves are diverse in their morphology, reflecting to a large degree the plant diversity in the natural environment. How different leaf morphology is determined is not yet understood. The leguminous plant Medicago truncatula exhibits dissected leaves with three leaflets at the tip. We show that development of the trifoliate leaves is determined by the Cys(2)His(2) zinc finger transcription factor PALM1. Loss-of-function mutants of PALM1 develop dissected leaves with five leaflets clustered at the tip. We demonstrate that PALM1 binds a specific promoter sequence and down-regulates the expression of the M. truncatula LEAFY/UNIFOLIATA orthologue SINGLE LEAFLET1 (SGL1), encoding an indeterminacy factor necessary for leaflet initiation. Our data indicate that SGL1 is required for leaflet proliferation in the palm1 mutant. Interestingly, ectopic expression of PALM1 effectively suppresses the lobed leaf phenotype from overexpression of a class 1 KNOTTED1-like homeobox protein in Arabidopsis plants. Taken together, our results show that PALM1 acts as a determinacy factor, regulates the spatial-temporal expression of SGL1 during leaf morphogenesis and together with the LEAFY/UNIFOLIATA orthologue plays an important role in orchestrating the compound leaf morphology in M. truncatula. PMID:20498057

  10. Tamarix hispida zinc finger protein ThZFP1 participates in salt and osmotic stress tolerance by increasing proline content and SOD and POD activities.

    PubMed

    Zang, Dandan; Wang, Chao; Ji, Xiaoyu; Wang, Yucheng

    2015-06-01

    Zinc finger proteins (ZFPs) are a large family that play important roles in various biological processes, such as signal transduction, RNA binding, morphogenesis, transcriptional regulation, abiotic or biotic stress response. However, the functions of ZFPs involved in abiotic stress are largely not known. In the present study, we cloned and functionally characterized a ZFP gene, ThZFP1, from Tamarix hispida. The expression of ThZFP1 is highly induced by NaCl, mannitol or ABA treatment. To study the function of ThZFP1 involved in abiotic stress response, transgenic T. hispida plants with overexpression or knockdown of ThZFP1 were generated using a transient transformation system. Gain- and loss-of-function studies of ThZFP1 suggested that ThZFP1 can induce the expression of a series of genes, including delta-pyrroline-5-carboxylate synthetase (P5CS), peroxidase (POD) and superoxide dismutase (SOD), leading to accumulation of proline and enhanced activities of SOD and POD. These physiological changes enhanced proline content and reactive oxygen species (ROS) scavenging capability when exposed to salt or osmotic stress. All the results obtained from T. hispida plants were further confirmed by analyses of the transgenic Arabidopsis plants overexpressing ThZFP1. These data together suggested that ThZFP1 positively regulates proline accumulation and activities of SOD and POD under salt and osmotic stress conditions.

  11. Lyar, a cell growth-regulating zinc finger protein, was identified to be associated with cytoplasmic ribosomes in male germ and cancer cells.

    PubMed

    Yonezawa, Kahori; Sugihara, Yoshihiko; Oshima, Kenzi; Matsuda, Tsukasa; Nadano, Daita

    2014-10-01

    Translational control is a basic mechanism for gene regulation in cells and important for tissue growth and development in mammals. Deregulation of the mechanism thus causes diseases such as cancer. Considering the importance of the ribosome as a factory of polypeptide synthesis, some new factors have been expected to be associated with the ribosome and involved in translational control. Our proteomic survey for these factors identified a zinc finger protein, Lyar, in cytoplasmic ribosomes of the rodent testis. Subcellular fractionation of the testis provided data supporting association of Lyar with ribosomes. Lyar was then suggested to be included in the 60S large subunit, but not in polysomes, by ultracentrifugation of testicular ribosomes. While analysis of tissue distribution of Lyar has indicated its testis-predominant expression, Lyar mRNA was expressed in the cancer cells originated from tissues other than testis, and Lyar promoted proliferation of NIH-3T3 cells. Furthermore, translation was increased by Lyar in vitro, pointing out the first experimental link between this protein and translation. Taken together, Lyar seems to be a new player in translational control and a potential target for cancer therapy.

  12. Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma.

    PubMed

    Beane, Joal D; Lee, Gary; Zheng, Zhili; Mendel, Matthew; Abate-Daga, Daniel; Bharathan, Mini; Black, Mary; Gandhi, Nimisha; Yu, Zhiya; Chandran, Smita; Giedlin, Martin; Ando, Dale; Miller, Jeff; Paschon, David; Guschin, Dmitry; Rebar, Edward J; Reik, Andreas; Holmes, Michael C; Gregory, Philip D; Restifo, Nicholas P; Rosenberg, Steven A; Morgan, Richard A; Feldman, Steven A

    2015-08-01

    Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9-84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500-2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.

  13. Loss of c-Kit and bone marrow failure upon conditional removal of the GATA-2 C-terminal zinc finger domain in adult mice.

    PubMed

    Li, Haiyan S; Jin, Jin; Liang, Xiaoxuan; Matatall, Katie A; Ma, Ying; Zhang, Huiyuan; Ullrich, Stephen E; King, Katherine Y; Sun, Shao-Cong; Watowich, Stephanie S

    2016-09-01

    Heterozygous mutations in the transcriptional regulator GATA-2 associate with multilineage immunodeficiency, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML). The majority of these mutations localize in the zinc finger (ZnF) domains, which mediate GATA-2 DNA binding. Deregulated hematopoiesis with GATA-2 mutation frequently develops in adulthood, yet GATA-2 function in the bone marrow remains unresolved. To investigate this, we conditionally deleted the GATA-2 C-terminal ZnF (C-ZnF) coding sequences in adult mice. Upon Gata2 C-ZnF deletion, we observed rapid peripheral cytopenia, bone marrow failure, and decreased c-Kit expression on hematopoietic progenitors. Transplant studies indicated GATA-2 has a cell-autonomous role in bone marrow hematopoiesis. Moreover, myeloid lineage populations were particularly sensitive to Gata2 hemizygosity, while molecular assays indicated GATA-2 regulates c-Kit expression in multilineage progenitor cells. Enforced c-Kit expression in Gata2 C-ZnF-deficient hematopoietic progenitors enhanced myeloid colony activity, suggesting GATA-2 sustains myelopoiesis via a cell intrinsic role involving maintenance of c-Kit expression. Our results provide insight into mechanisms regulating hematopoiesis in bone marrow and may contribute to a better understanding of immunodeficiency and bone marrow failure associated with GATA-2 mutation.

  14. Stress-responsive mitogen-activated protein kinases interact with the EAR motif of a poplar zinc finger protein and mediate its degradation through the 26S proteasome.

    PubMed

    Hamel, Louis-Philippe; Benchabane, Meriem; Nicole, Marie-Claude; Major, Ian T; Morency, Marie-Josée; Pelletier, Gervais; Beaudoin, Nathalie; Sheen, Jen; Séguin, Armand

    2011-11-01

    Mitogen-activated protein kinases (MAPKs) contribute to the establishment of plant disease resistance by regulating downstream signaling components, including transcription factors. In this study, we identified MAPK-interacting proteins, and among the newly discovered candidates was a Cys-2/His-2-type zinc finger protein named PtiZFP1. This putative transcription factor belongs to a family of transcriptional repressors that rely on an ERF-associated amphiphilic repression (EAR) motif for their repression activity. Amino acids located within this repression motif were also found to be essential for MAPK binding. Close examination of the primary protein sequence revealed a functional bipartite MAPK docking site that partially overlaps with the EAR motif. Transient expression assays in Arabidopsis (Arabidopsis thaliana) protoplasts suggest that MAPKs promote PtiZFP1 degradation through the 26S proteasome. Since features of the MAPK docking site are conserved among other EAR repressors, our study suggests a novel mode of defense mechanism regulation involving stress-responsive MAPKs and EAR repressors.

  15. Structures of three members of Pfam PF02663 (FmdE) implicated in microbial methanogenesis reveal a conserved α+β core domain and an auxiliary C-terminal treble-clef zinc finger

    PubMed Central

    Axelrod, Herbert L.; Das, Debanu; Abdubek, Polat; Astakhova, Tamara; Bakolitsa, Constantina; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Farr, Carol L.; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Lam, Winnie W.; Marciano, David; McMullan, Daniel; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; van den Bedem, Henry; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    Examination of the genomic context for members of the FmdE Pfam family (PF02663), such as the protein encoded by the fmdE gene from the methanogenic archaeon Methanobacterium thermoautotrophicum, indicates that 13 of them are co-transcribed with genes encoding subunits of molybdenum formylmethanofuran dehydrogenase (EC 1.2.99.5), an enzyme that is involved in microbial methane production. Here, the first crystal structures from PF02663 are described, representing two bacterial and one archaeal species: B8FYU2_DESHY from the anaerobic dehalogenating bacterium Desulfito­bacterium hafniense DCB-2, Q2LQ23_SYNAS from the syntrophic bacterium Syntrophus aciditrophicus SB and Q9HJ63_THEAC from the thermoacidophilic archaeon Thermoplasma acidophilum. Two of these proteins, Q9HJ63_THEAC and Q2LQ23_SYNAS, contain two domains: an N-terminal thioredoxin-like α+β core domain (NTD) consisting of a five-stranded, mixed β-sheet flanked by several α-helices and a C-terminal zinc-finger domain (CTD). B8FYU2_DESHY, on the other hand, is composed solely of the NTD. The CTD of Q9HJ63_THEAC and Q2LQ23_SYNAS is best characterized as a treble-clef zinc finger. Two significant structural differences between Q9HJ63_THEAC and Q2LQ23_SYNAS involve their metal binding. First, zinc is bound to the putative active site on the NTD of Q9HJ63_THEAC, but is absent from the NTD of Q2LQ23_SYNAS. Second, whereas the structure of the CTD of Q2LQ23_SYNAS shows four Cys side chains within coordination distance of the Zn atom, the structure of Q9HJ63_THEAC is atypical for a treble-cleft zinc finger in that three Cys side chains and an Asp side chain are within coordination distance of the zinc. PMID:20944230

  16. Electronic Characterization of Defects in Narrow Gap Semiconductors-Comparison of Electronic Energy Levels and Formation Energies in Mercury Cadmium Telluride, Mercury Zinc Telluride, and Mercury Zinc Selenide

    NASA Technical Reports Server (NTRS)

    Patterson, James D.

    1996-01-01

    We have used a Green's function technique to calculate the energy levels and formation energy of deep defects in the narrow gap semiconductors mercury cadmium telluride (MCT), mercury zinc telluride (MZT) and mercury zinc selenide (MZS). The formation energy is calculated from the difference between the total energy with an impurity cluster and the total energy for the perfect crystal. Substitutional (including antisite), interstitial (self and foreign), and vacancy deep defects are considered. Relaxation effects are calculated (with molecular dynamics). By use of a pseudopotential, we generalize the ideal vacancy model so as to be able to consider relaxation for vacancies. Different charge states are considered and the charged state energy shift (as computed by a modified Haldane-Anderson model) can be twice that due to relaxation. Different charged states for vacancies were not calculated to have much effect on the formation energy. For all cases we find deep defects in the energy gap only for cation site s-like orbitals or anion site p-like orbitals, and for the substitutional case only the latter are appreciably effected by relaxation. For most cases for MCT, MZT, MZS, we consider x (the concentration of Cd or Zn) in the range appropriate for a band gap of 0.1 eV. For defect energy levels, the absolute accuracy of our results is limited, but the precision is good, and hence chemical trends are accurately predicted. For the same reason, defect formation energies are more accurately predicted than energy level position. We attempt, in Appendix B, to calculate vacancy formation energies using relatively simple chemical bonding ideas due to Harrison. However, these results are only marginally accurate for estimating vacancy binding energies. Appendix C lists all written reports and publications produced for the grant. We include abstracts and a complete paper that summarizes our work which is not yet available.

  17. Finger pain

    MedlinePlus

    Pain - finger ... Nearly everyone has had finger pain at some time. You may have: Tenderness Burning Stiffness Numbness Tingling Coldness Swelling Change in skin color Redness Many conditions, such ...

  18. Down-regulation of the zinc-finger homeobox protein TSHZ2 releases GLI1 from the nuclear repressor complex to restore its transcriptional activity during mammary tumorigenesis

    PubMed Central

    Riku, Miho; Inaguma, Shingo; Ito, Hideaki; Tsunoda, Takumi; Ikeda, Hiroshi; Kasai, Kenji

    2016-01-01

    Although breast cancer is one of the most common malignancies, the molecular mechanisms underlying its development and progression are not fully understood. To identify key molecules involved, we screened publicly available microarray datasets for genes differentially expressed between breast cancers and normal mammary glands. We found that three of the genes predicted in this analysis were differentially expressed among human mammary tissues and cell lines. Of these genes, we focused on the role of the zinc-finger homeobox protein TSHZ2, which is down-regulated in breast cancer cells. We found that TSHZ2 is a nuclear protein harboring a bipartite nuclear localization signal, and we confirmed its function as a C-terminal binding protein (CtBP)-dependent transcriptional repressor. Through comprehensive screening, we identified TSHZ2-suppressing genes such as AEBP1 and CXCR4, which are conversely up-regulated by GLI1, the downstream transcription factor of Hedgehog signaling. We found that GLI1 forms a ternary complex with CtBP2 in the presence of TSHZ2 and that the transcriptional activity of GLI1 is suppressed by TSHZ2 in a CtBP-dependent manner. Indeed, knockdown of TSHZ2 increases the expression of AEBP1 and CXCR4 in TSHZ2-expressing immortalized mammary duct epithelium. Concordantly, immunohistochemical staining of mammary glands revealed that normal duct cells expresses GLI1 in the nucleus along with TSHZ2 and CtBP2, whereas invasive ductal carcinoma cells, which does not express TSHZ2, show the increase in the expression of AEBP1 and CXCR4 and in the cytoplasmic localization of GLI1. Thus, we propose that down-regulation of TSHZ2 is crucial for mammary tumorigenesis via the activation of GLI1. PMID:26744317

  19. HYPERSENSITIVE TO RED AND BLUE 1, a ZZ-type zinc finger protein, regulates phytochrome B-mediated red and cryptochrome-mediated blue light responses.

    PubMed

    Kang, Xiaojun; Chong, Jason; Ni, Min

    2005-03-01

    Plant photoreceptors that regulate photomorphogenic development include red/far-red-light-absorbing phytochromes and blue/UV-A-light-absorbing cryptochromes. We have undertaken a genetic screen to identify additional components downstream of the photoreceptors in Arabidopsis thaliana. We identified a short hypocotyl mutant under red and blue light, hypersensitive to red and blue 1 (hrb1). Mutation in HRB1 also enhances the end-of-day far-red light response, inhibits leaf expansion and petiole elongation, and attenuates the expression of CAB3 and CHS. Double mutant analysis indicates that phyB is epistatic to hrb1 under red light, and cry1 cry2 is epistatic to hrb1 under blue light for both hypocotyl growth and light-regulated gene expression responses. HRB1 localizes to the nucleus and belongs to a protein family of Drought induced 19 (Di19). HRB1 and all other family members contain a ZZ-type zinc finger domain, which in other organisms is implicated in protein-protein interactions between dystrophin and calmodulin and between transcriptional adaptors and activators. HRB1 activity is also required for red and blue light-induced expression of PHYTOCHROME INTERACTING FACTOR 4 (PIF4). pif4 shows a very similar hypersensitive response as hrb1 to both red light and blue light and is epistatic to hrb1 in control of light-regulated gene expression responses. Thus, the roles of HRB1 and PIF4 together in regulating both red and blue light responses may represent points where red light signaling and blue light signaling intersect.

  20. In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases.

    PubMed

    Sebastiano, Vittorio; Maeder, Morgan L; Angstman, James F; Haddad, Bahareh; Khayter, Cyd; Yeo, Dana T; Goodwin, Mathew J; Hawkins, John S; Ramirez, Cherie L; Batista, Luis F Z; Artandi, Steven E; Wernig, Marius; Joung, J Keith

    2011-11-01

    The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications.

  1. In-Depth Mutational Analysis of the Promyelocytic Leukemia Zinc Finger BTB/POZ Domain Reveals Motifs and Residues Required for Biological and Transcriptional Functions

    PubMed Central

    Melnick, Ari; Ahmad, K. Farid; Arai, Sally; Polinger, Adam; Ball, Helen; Borden, Katherine L.; Carlile, Graeme W.; Prive, Gilbert G.; Licht, Jonathan D.

    2000-01-01

    The promyelocytic leukemia zinc finger (PLZF) protein is a transcription factor disrupted in patients with t(11;17)(q23;q21)-associated acute promyelocytic leukemia. PLZF contains an N-terminal BTB/POZ domain which is required for dimerization, transcriptional repression, formation of high-molecular-weight DNA-protein complexes, nuclear sublocalization, and growth suppression. X-ray crystallographic data show that the PLZF BTB/POZ domain forms an obligate homodimer via an extensive interface. In addition, the dimer possesses several highly conserved features, including a charged pocket, a hydrophobic monomer core, an exposed hydrophobic surface on the floor of the dimer, and two negatively charged surface patches. To determine the role of these structures, mutational analysis of the BTB/POZ domain was performed. We found that point mutations in conserved residues that disrupt the dimer interface or the monomer core result in a misfolded nonfunctional protein. Mutation of key residues from the exposed hydrophobic surface suggests that these are also important for the stability of PLZF complexes. The integrity of the charged-pocket region was crucial for proper folding of the BTB/POZ domain. In addition, the pocket was critical for the ability of the BTB/POZ domain to repress transcription. Alteration of charged-pocket residue arginine 49 to a glutamine (mutant R49Q) yields a domain that can still dimerize but activates rather than represses transcription. In the context of full-length PLZF, a properly folded BTB/POZ domain was required for all PLZF functions. However, PLZF with the single pocket mutation R49Q repressed transcription, while the double mutant D35N/R49Q could not, despite its ability to dimerize. These results indicate that PLZF requires the BTB/POZ domain for dimerization and the charged pocket for transcriptional repression. PMID:10938130

  2. The BTB-zinc Finger Transcription Factor Abrupt Acts as an Epithelial Oncogene in Drosophila melanogaster through Maintaining a Progenitor-like Cell State

    PubMed Central

    Turkel, Nezaket; Sahota, Virender K.; Bolden, Jessica E.; Goulding, Karen R.; Doggett, Karen; Willoughby, Lee F.; Blanco, Enrique; Martin-Blanco, Enrique; Corominas, Montserrat; Ellul, Jason; Aigaki, Toshiro; Richardson, Helena E.; Brumby, Anthony M.

    2013-01-01

    The capacity of tumour cells to maintain continual overgrowth potential has been linked to the commandeering of normal self-renewal pathways. Using an epithelial cancer model in Drosophila melanogaster, we carried out an overexpression screen for oncogenes capable of cooperating with the loss of the epithelial apico-basal cell polarity regulator, scribbled (scrib), and identified the cell fate regulator, Abrupt, a BTB-zinc finger protein. Abrupt overexpression alone is insufficient to transform cells, but in cooperation with scrib loss of function, Abrupt promotes the formation of massive tumours in the eye/antennal disc. The steroid hormone receptor coactivator, Taiman (a homologue of SRC3/AIB1), is known to associate with Abrupt, and Taiman overexpression also drives tumour formation in cooperation with the loss of Scrib. Expression arrays and ChIP-Seq indicates that Abrupt overexpression represses a large number of genes, including steroid hormone-response genes and multiple cell fate regulators, thereby maintaining cells within an epithelial progenitor-like state. The progenitor-like state is characterised by the failure to express the conserved Eyes absent/Dachshund regulatory complex in the eye disc, and in the antennal disc by the failure to express cell fate regulators that define the temporal elaboration of the appendage along the proximo-distal axis downstream of Distalless. Loss of scrib promotes cooperation with Abrupt through impaired Hippo signalling, which is required and sufficient for cooperative overgrowth with Abrupt, and JNK (Jun kinase) signalling, which is required for tumour cell migration/invasion but not overgrowth. These results thus identify a novel cooperating oncogene, identify mammalian family members of which are also known oncogenes, and demonstrate that epithelial tumours in Drosophila can be characterised by the maintenance of a progenitor-like state. PMID:23874226

  3. Phylogenetic distribution and evolution of the linked RNA-binding and NOT1-binding domains in the tristetraprolin family of tandem CCCH zinc finger proteins.

    PubMed

    Blackshear, Perry J; Perera, Lalith

    2014-04-01

    In humans, the tristetraprolin or TTP family of CCCH tandem zinc finger (TZF) proteins comprises 3 members, encoded by the genes ZFP36, ZFP36L1, and ZFP36L2. These proteins have direct orthologues in essentially all vertebrates studied, with the exception of birds, which appear to lack a version of ZFP36. Additional family members are found in rodents, amphibians, and fish. In general, the encoded proteins contain 2 critical macromolecular interaction domains: the CCCH TZF domain, which is necessary for high-affinity binding to AU-rich elements in mRNA; and an extreme C-terminal domain that, in the case of TTP, interacts with NOT1, the scaffold of a large multi-protein complex that contains deadenylases. TTP and its related proteins act by first binding to AU-rich elements in mRNA, and then recruiting deadenylases to the mRNA, where they can processively remove the adenosine residues from the poly(A) tail. Highly conserved TZF domains have been found in unicellular eukaryotes such as yeasts, and these domains can bind AU-rich elements that resemble those bound by the mammalian proteins. However, certain fungi appear to lack proteins with intact TZF domains, and the TTP family proteins that are expressed in other fungi often lack the characteristic C-terminal NOT1 binding domain found in the mammalian proteins. For these reasons, we investigated the phylogenetic distribution of the relevant sequences in available databases. Both domains are present in family member proteins from most lineages of eukaryotes, suggesting their mutual presence in a common ancestor. However, the vertebrate type of NOT1-binding domain is missing in most fungi, and the TZF domain itself has disappeared or degenerated in recently evolved fungi. Nonetheless, both domains are present together in the proteins from several unicellular eukaryotes, including at least 1 fungus, and they seem to have remained together during the evolution of metazoans.

  4. Diversity of Prdm9 Zinc Finger Array in Wild Mice Unravels New Facets of the Evolutionary Turnover of this Coding Minisatellite

    PubMed Central

    Buard, Jérôme; Rivals, Eric; Dunoyer de Segonzac, Denis; Garres, Charlotte; Caminade, Pierre; de Massy, Bernard; Boursot, Pierre

    2014-01-01

    In humans and mice, meiotic recombination events cluster into narrow hotspots whose genomic positions are defined by the PRDM9 protein via its DNA binding domain constituted of an array of zinc fingers (ZnFs). High polymorphism and rapid divergence of the Prdm9 gene ZnF domain appear to involve positive selection at DNA-recognition amino-acid positions, but the nature of the underlying evolutionary pressures remains a puzzle. Here we explore the variability of the Prdm9 ZnF array in wild mice, and uncovered a high allelic diversity of both ZnF copy number and identity with the caracterization of 113 alleles. We analyze features of the diversity of ZnF identity which is mostly due to non-synonymous changes at codons −1, 3 and 6 of each ZnF, corresponding to amino-acids involved in DNA binding. Using methods adapted to the minisatellite structure of the ZnF array, we infer a phylogenetic tree of these alleles. We find the sister species Mus spicilegus and M. macedonicus as well as the three house mouse (Mus musculus) subspecies to be polyphyletic. However some sublineages have expanded independently in Mus musculus musculus and M. m. domesticus, the latter further showing phylogeographic substructure. Compared to random genomic regions and non-coding minisatellites, none of these patterns appears exceptional. In silico prediction of DNA binding sites for each allele, overlap of their alignments to the genome and relative coverage of the different families of interspersed repeated elements suggest a large diversity between PRDM9 variants with a potential for highly divergent distributions of recombination events in the genome with little correlation to evolutionary distance. By compiling PRDM9 ZnF protein sequences in Primates, Muridae and Equids, we find different diversity patterns among the three amino-acids most critical for the DNA-recognition function, suggesting different diversification timescales. PMID:24454780

  5. Effect of Over-Expression of Zinc-Finger Protein (ZFX) on Self-Renewal and Drug-Resistance of Hepatocellular Carcinoma

    PubMed Central

    Zhang, Shuhong; Shu, Ronghua; Yue, Meng; Zhang, Shuhong

    2016-01-01

    Background X-chromosome-coupled zinc finger protein (ZFX) in the Zfy protein family is abundantly expressed in both embryonic and hematopoietic stem cells (HSCs). ZFX exist in various tumor cells and is correlated with proliferation and survival of tumor cells. As a malignant tumor with high invasiveness, hepatocellular carcinoma (HCC) may present resistance against chemotherapy and features of stem cells. This study aimed to explore the expression of ZFX in HCC cells, in an attempt to illustrate the role of ZFX in tumorigenesis. Material/Methods The expression of ZFX in tumor tissues was quantified by RT-PCR. The ZFX expression was then silenced to evaluate the stem cell-like features of HCC cells, including self-renewal, colony formation, and cell cycle, along with the sensitivity to cisplatin. Xenograft of ZFX-overexpressed HCC on nude mice was performed to evaluate the in vivo effect of ZFX on tumor growth. Results Quantitative RT-PCR showed over-expression of ZFX in 51.8% of HCC tumors. The silencing of ZFX gene inhibited the self-renewal, colony formation, and proliferation ability of HCC cells (p<0.05 in all cases) via the cell cycle arrest at G0/G1 phase, in addition to the elevated sensitivity of tumor cells to cisplatin (p<0.001). Further studies showed that binding between ZFX and promoter regions of Nanog or SOX-2 regulatory factor initiate their expression in HCC cells. The xenograft experiment indicated the potentiation of tumor growth by ZFX over-expression. Conclusions ZFX is over-expressed in HCC cells, and correlates with stem cell-like features and pleiotropic characteristics. PMID:27566731

  6. Development of promyelocytic leukemia zinc finger-expressing innate CD4 T cells requires stronger T-cell receptor signals than conventional CD4 T cells.

    PubMed

    Qiao, Yu; Zhu, Lingqiao; Sofi, Hanief; Lapinski, Philip E; Horai, Reiko; Mueller, Kristen; Stritesky, Gretta L; He, Xi; Teh, Hung-Sia; Wiest, David L; Kappes, Dietmar J; King, Philip D; Hogquist, Kristin A; Schwartzberg, Pamela L; Sant'Angelo, Derek B; Chang, Cheong-Hee

    2012-10-02

    MHC class II-expressing thymocytes and thymic epithelial cells can mediate CD4 T-cell selection resulting in functionally distinct thymocyte-selected CD4 (T-CD4) and epithelial-selected CD4 (E-CD4) T cells, respectively. However, little is known about how T-cell receptor (TCR) signaling influences the development of these two CD4 T-cell subsets. To study TCR signaling for T-CD4 T-cell development, we used a GFP reporter system of Nur77 in which GFP intensity directly correlates with TCR signaling strength. T-CD4 T cells expressed higher levels of GFP than E-CD4 T cells, suggesting that T-CD4 T cells received stronger TCR signaling than E-CD4 T cells during selection. Elimination of Ras GTPase-activating protein enhanced E-CD4 but decreased T-CD4 T-cell selection efficiency, suggesting a shift to negative selection. Conversely, the absence of IL-2-inducible T-cell kinase that causes poor E-CD4 T-cell selection due to insufficient TCR signaling improved T-CD4 T-cell generation, consistent with rescue from negative selection. Strong TCR signaling during T-CD4 T-cell development correlates with the expression of the transcription factor promyelocytic leukemia zinc finger protein. However, although modulation of the signaling strength affected the efficiency of T-CD4 T-cell development during positive and negative selection, the signaling strength is not as important for the effector function of T-CD4 T cells. These findings indicate that innate T-CD4 T cells, together with invariant natural killer T cells and γδ T cells, receive strong TCR signals during their development and that signaling requirements for the development and the effector functions are distinct.

  7. ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) Interacts with FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) Linking Iron Deficiency and Oxidative Stress Responses.

    PubMed

    Le, Cham Thi Tuyet; Brumbarova, Tzvetina; Ivanov, Rumen; Stoof, Claudia; Weber, Eva; Mohrbacher, Julia; Fink-Straube, Claudia; Bauer, Petra

    2016-01-01

    Plants grown under iron (Fe)-deficient conditions induce a set of genes that enhance the efficiency of Fe uptake by the roots. In Arabidopsis (Arabidopsis thaliana), the central regulator of this response is the basic helix-loop-helix transcription factor FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT). FIT activity is regulated by protein-protein interactions, which also serve to integrate external signals that stimulate and possibly inhibit Fe uptake. In the search of signaling components regulating FIT function, we identified ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12), an abiotic stress-induced transcription factor. ZAT12 interacted with FIT, dependent on the presence of the ethylene-responsive element-binding factor-associated amphiphilic repression motif. ZAT12 protein was found expressed in the root early differentiation zone, where its abundance was modulated in a root layer-specific manner. In the absence of ZAT12, FIT expression was upregulated, suggesting a negative effect of ZAT12 on Fe uptake. Consistently, zat12 loss-of-function mutants had higher Fe content than the wild type at sufficient Fe. We found that under Fe deficiency, hydrogen peroxide (H2O2) levels were enhanced in a FIT-dependent manner. FIT protein, in turn, was stabilized by H2O2 but only in the presence of ZAT12, showing that H2O2 serves as a signal for Fe deficiency responses. We propose that oxidative stress-induced ZAT12 functions as a negative regulator of Fe acquisition. A model where H2O2 mediates the negative regulation of plant responses to prolonged stress might be applicable to a variety of stress conditions.

  8. Generation of GFAP::GFP astrocyte reporter lines from human adult fibroblast-derived iPS cells using zinc-finger nuclease technology.

    PubMed

    Zhang, Ping-Wu; Haidet-Phillips, Amanda M; Pham, Jacqueline T; Lee, Youngjin; Huo, Yuqing; Tienari, Pentti J; Maragakis, Nicholas J; Sattler, Rita; Rothstein, Jeffrey D

    2016-01-01

    Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100β, NFIA and ALDH1L1. In addition, mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy.

  9. The zinc finger and C-terminal domains of MTA proteins are required for FOG-2-mediated transcriptional repression via the NuRD complex.

    PubMed

    Roche, Andrea E; Bassett, Brett J; Samant, Sadhana A; Hong, Wei; Blobel, Gerd A; Svensson, Eric C

    2008-02-01

    FOG-2 is a transcriptional co-regulator that is required for cardiac morphogenesis as mice deficient in this factor die during mid-gestation of cardiac malformations. FOG-2 interacts with GATA4 to attenuate GATA4-dependent gene expression. The first 12 amino acids of FOG-2 (the FOG Repression Motif) are necessary to mediate this repression. To determine the mechanism by which the FOG Repression Motif functions, we identified 7 polypeptides from rat cardiac nuclear extracts that co-purified with a GST-FOG-2 fusion protein. All proteins identified are members of the NuRD nucleosome remodeling complex. Using in vitro binding and co-immunoprecipitation assays, we demonstrate that Metastasis-Associated proteins (MTA)-1, 2 and 3 and Retinoblastoma binding proteins RbAp46 and RbAp48 interact with FOG-2, but not with a mutant form of FOG-2 that is unable to repress transcription. Furthermore, we define a novel domain located in the C-terminal portion of MTA-1 that mediates the FOG-2/MTA-1 interaction. We also demonstrate that knockdown of MTA protein expression dramatically impairs the ability of FOG-2 to repress GATA4 activity. Finally, we show that the zinc finger domain of MTA-1 is required for FOG-2-mediated transcriptional repression and that this domain interacts with RbAp46 and RbAp48 subunits of the NuRD complex. Together, these results demonstrate the importance of FOG-2/MTA/RbAp interactions for FOG-2-mediated transcriptional repression and further define the molecular interactions between the FOG Repression Motif and the NuRD complex.

  10. Inhibition of NF-kappaB by ZAS3, a zinc-finger protein that also binds to the kappaB motif.

    PubMed

    Hong, Joung-Woo; Allen, Carl E; Wu, Lai-Chu

    2003-10-14

    The ZAS proteins are large zinc-finger transcriptional proteins implicated in growth, signal transduction, and lymphoid development. Recombinant ZAS fusion proteins containing one of the two DNA-binding domains have been shown to bind specifically to the kappaB motif, but the endogenous ZAS proteins or their physiological functions are largely unknown. The kappaB motif, GGGACTTTCC, is a gene regulatory element found in promoters and enhancers of genes involved in immunity, inflammation, and growth. The Rel family of NF-kappaB, predominantly p65.p50 and p50.p50, are transcription factors well known for inducing gene expression by means of interaction with the kappaB motif during acute-phase responses. A functional link between ZAS and NF-kappaB, two distinct families of kappaB-binding proteins, stems from our previous in vitro studies that show that a representative member, ZAS3, associates with TRAF2, an adaptor molecule in tumor necrosis factor signaling, to inhibit NF-kappaB activation. Biochemical and genetic evidence presented herein shows that ZAS3 encodes major kappaB-binding proteins in B lymphocytes, and that NF-kappaB is constitutively activated in ZAS3-deficient B cells. The data suggest that ZAS3 plays crucial functions in maintaining cellular homeostasis, at least in part by inhibiting NF-kappaB by means of three mechanisms: inhibition of nuclear translocation of p65, competition for kappaB gene regulatory elements, and repression of target gene transcription.

  11. Zinc-Finger Transcription Factor ZAT6 Positively Regulates Cadmium Tolerance through the Glutathione-Dependent Pathway in Arabidopsis1[OPEN

    PubMed Central

    Chen, Jian; Yan, Xingxing; Liu, Yunlei; Wang, Ren; Fan, Tingting; Ren, Yongbing; Tang, Xiaofeng; Xiao, Fangming

    2016-01-01

    Cadmium (Cd) is an environmental pollutant with high toxicity to animals and plants. It has been established that the glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway is one of the most important mechanisms contributing to Cd accumulation and tolerance in plants. However, the transcription factors involved in regulating GSH-dependent PC synthesis pathway remain largely unknown. Here, we identified an Arabidopsis (Arabidopsis thaliana) Cd-resistant mutant xcd2-D (XVE system-induced cadmium-tolerance2) using a forward genetics approach. The mutant gene underlying xcd2-D mutation was revealed to encode a known zinc-finger transcription factor, ZAT6. Transgenic plants overexpressing ZAT6 showed significant increase of Cd tolerance, whereas loss of function of ZAT6 led to decreased Cd tolerance. Increased Cd accumulation and tolerance in ZAT6-overexpressing lines was GSH dependent and associated with Cd-activated synthesis of PC, which was correlated with coordinated activation of PC-synthesis related gene expression. By contrast, loss of function of ZAT6 reduced Cd accumulation and tolerance, which was accompanied by abolished PC synthesis and gene expression. Further analysis revealed that ZAT6 positively regulates the transcription of GSH1, GSH2, PCS1, and PCS2, but ZAT6 is capable of specifically binding to GSH1 promoter in vivo. Consistently, overexpression of GSH1 has been shown to restore Cd sensitivity in the zat6-1 mutant, suggesting that GSH1 is a key target of ZAT6. Taken together, our data provide evidence that ZAT6 coordinately activates PC synthesis-related gene expression and directly targets GSH1 to positively regulate Cd accumulation and tolerance in Arabidopsis. PMID:26983992

  12. Two paths for stabilization of ERG in prostate carcinogenesis: TMPRSS2-ERG fusions and speckle-type pox virus and zinc finger protein mutations

    PubMed Central

    Pascal, Laura E; Wang, Zhou

    2016-01-01

    Speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) is an E3 ubiquitin ligase adaptor protein that specifically promotes the ubiquitination and proteasome degradation of proteins. SPOP mutations are frequent in prostate cancer, and in a previous study, An et al. demonstrated that SPOP induced the degradation of the androgen receptor (AR) suggesting that SPOP is important in maintaining prostate homeostasis. In this current highlighted report, An and colleagues showed that ERG, which has been implicated as an oncoprotein in prostate cancer, contains putative SPOP-binding consensus (SBC) motifs 42ASSSS46 and 423VTSSS427 in the N- and C-terminal of ERG, respectively. The authors went on to demonstrate that SPOP promotes the ubiquitination and degradation of ERG through binding to the degron/SBC motif at the ERG N-terminus. SPOP mutations in the MATH domain prevented recognition and targeting of ERG for ubiquitination and degradation. In addition, N-terminal truncated ERG proteins encoded by the most frequently identified TMPRSS2-ERG rearrangements in prostate cancer (T1-E4 and T1-E5) were resistant to SPOP-mediated degradation, resulting in the stabilization of truncated ERG proteins. Stabilization of ERG protein through either SPOP mutation or TMPRSS2-ERG fusions induced proliferation and invasion in prostate cancer cells. This study along with a recently published similar report provides two previously unrecognized mechanisms for the upregulation of ERG proteins frequently observed in prostate cancers. These findings generate great enthusiasm for the development of targeted therapeutic strategies designed to eliminate ERG protein in prostate cancer cells. PMID:26763545

  13. Association between the MLX Interacting Protein-Like, BUD13 Homolog and Zinc Finger Protein 259 Gene Polymorphisms and Serum Lipid Levels

    PubMed Central

    Aung, Lynn-Htet-Htet; Yin, Rui-Xing; Wu, Jin-Zhen; Wu, Dong-Feng; Wang, Wei; Li, Hui

    2014-01-01

    This study aimed to detect the association between the MLX interacting protein-like (MLXIPL), BUD13 homolog (BUD13) and zinc finger protein 259 (ZNF259) single nucleotide polymorphisms (SNPs) and serum lipid levels in the Chinese Mulao and Han populations. Genotyping of 9 SNPs was performed in 825 Mulao and 781 Han participants. The genotype and allele frequencies of ZNF259 rs2075290 and rs964184, and BUD13 rs10790162 SNPs were different between the Mulao and Han populations (P < 0.001). The SNPs of ZNF259 rs2075290 and BUD13 rs10790162 were associated with serum total cholesterol levels; ZNF259 rs2075290 and rs964184, BUD13 rs10790162, and MLXIPL rs3812316 and rs13235543 were associated with triglyceride (TG); and MLXIPL rs35332062 was associated with apolipoprotein (Apo) A1 in the Mulaos (P < 0.006–0.001). However, in the Hans, the SNPs of ZNF259 rs2075290 and BUD13 rs10790162 were associated with serum TG levels; ZNF259 rs2075290 was associated with low-density lipoprotein cholesterol and the ApoA1/ApoB ratio (P < 0.006–0.001). Significant linkage disequilibria were noted among ZNF259 rs2075290 and rs964184 and BUD13 rs10790162, and between MLXIPL rs3812316 and rs13235543 (r2 > 0.05, P < 0.001). The haplotypes of A-C-G-A-C (rs2075290A-rs964184C-rs10790162G-rs17119975A-rs11556024C) and C-C-C-C (rs799161C-rs35332062C-rs3812316C-rs13235543C) accounted for over half of the % haplotype of each ethnic group. PMID:24989072

  14. Tuberculate fruit gene Tu encodes a C2 H2 zinc finger protein that is required for the warty fruit phenotype in cucumber (Cucumis sativus L.).

    PubMed

    Yang, Xuqin; Zhang, Weiwei; He, Huanle; Nie, Jingtao; Bie, Beibei; Zhao, Junlong; Ren, Guoliang; Li, Yue; Zhang, Dabing; Pan, Junsong; Cai, Run

    2014-06-01

    Cucumber fruits that have tubercules and spines (trichomes) are known to possess a warty (Wty) phenotype. In this study, the tuberculate fruit gene Tu was identified by map-based cloning, and was found to encode a transcription factor (TF) with a single C2 H2 zinc finger domain. Tu was identified in all 38 Wty lines examined, and was completely absent from all 56 non-warty (nWty) lines. Cucumber plants transgenic for Tu (TCP) revealed that Tu was required for the Wty fruit phenotype. Subcellular localization showed that the fusion protein GFP-Tu was localized mainly to the nucleus. Based on analyses of semi-quantitative and quantitative reverse transcription polymerase chain reaction (RT-PCR), and mRNA in situ hybridization, we found that Tu was expressed specifically in fruit spine cells during development of fruit tubercules. Moreover, cytokinin (CTK) content measurements and cytological observations in Wty and nWty fruits revealed that the Wty fruit phenotype correlated with high endogenous CTK concentrations. As a result of further analyses on the transcriptomic profile of the nWty fruit epidermis and TCP fruit warts, expression of CTK-associated genes, and hormone content in nWty fruit epidermis, Wty fruit warts and epidermis, and TCP fruit warts and epidermis, we found that Tu probably promoted CTK biosynthesis in fruit warts. Here we show that Tu could not be expressed in the glabrous and tubercule-free mutant line gl that contained Tu, this result that futher confirmed the epistatic effect of the trichome (spine) gene Gl over Tu. Taken together, these data led us to propose a genetic pathway for the Wty fruit trait that could guide future mechanistic studies.

  15. Evaluation of zinc finger E-box binding homeobox 1 and transforming growth factor-beta2 expression in bladder cancer tissue in comparison with healthy adjacent tissue

    PubMed Central

    Mahdavinezhad, Ali; Yadegarazari, Reza; Mousavi-Bahar, Seyed Habibollah; Poorolajal, Jalal; Jafari, Mohammad; Amirzargar, Mohammad Ali; Effatpanah, Hosein

    2017-01-01

    Purpose The fifth most common cancer is allocated to bladder cancer (BC) worldwide. Understanding the molecular mechanisms of BC invasion and metastasis to identify target therapeutic strategies will improve disease survival. So the aim of this study was to measure expression rate of zinc finger E-box binding homeobox 1 (ZEB1) and transforming growth factor-beta2 (TGF-β2) mRNA in tissue samples of patients with BC and its healthy adjacent tissue samples and their association with muscle invasion, size and grade of the tumor. Materials and Methods Tissue samples were collected from 35 newly diagnosed untreated patients with BC from 2013 to 2014. Total RNA was extracted from about 50-mg tissue samples using TRIzol reagent. TAKARA SYBR Premix EX Tag II was applied to determine the rate of mRNA expression by real-time polymerase chain reaction (PCR). To obtain final validation, PCR product of ZEB1 and TGF-β2 were sequenced. STATA 11 software was used to analyze the data. Results The expression level of ZEB1 in tumor samples was significantly more than of in healthy adjacent tissue samples. Up-regulation of TGF-β2 showed a strong association with muscle invasion (p=0.017). There was also demonstrated a relationship between over expression of ZEB1 with the tumor size (p=0.050). Conclusions It looks ZEB1 and TGF-β2 had a role in BC patients. In this study ZEB1 expression was higher in BC tissues than that of in healthy control tissues. There was demonstrated a markedly association between overexpression of TGF-β2 and muscle invasion. Therefore, they are supposed to be candidate as potential biomarkers for early detection and progression of BC. PMID:28261684

  16. High expression of Zinc-finger protein X-linked is associated with reduced E-cadherin expression and unfavorable prognosis in nasopharyngeal carcinoma.

    PubMed

    Li, Yin; Yan, Xuebing; Yan, Leilei; Shan, Zezhi; Liu, Sihong; Chen, Xiaojuan; Zou, Jianyin; Zhang, Weitian; Jin, Zhiming

    2015-01-01

    Zinc-finger protein X-linked (ZFX), a novel transcription factor required for self-renewal of embryonic stem cells, has recently been implicated in the initiation and progression of various human malignancies. However, its clinical significance in cancer patients remains largely inconclusive and its role in nasopharyngeal carcinoma (NPC) has never been reported. In this study, quantitative real-time polymerase chain reaction, Western blot and Immunohistochemistry were performed to detect ZFX expression in NPC and normal nasopharyngeal tissues. As a result, we found ZFX expression was significantly elevated in NPC tissues compared with that in normal nasopharyngeal tissues. The statistical analysis based on immunohistochemical staining demonstrated that ZFX expression was significantly correlated with lymph node stage and clinical stage. Furthermore, we found NPC patients with high ZFX expression had lower 5-year overall survival rates, progression-free survival rates, loco-regional relapse-free survival rates and distant metastasis-free survival rates than those with low ZFX expression (all P<0.05). The multivariate analysis indicated that ZFX expression was an independent prognostic factor for patients with NPC. More importantly, we also detected E-cadherin expression in NPC tissues and found it was inversely correlated with ZFX expression in NPC tissues, suggesting a potential involvement of ZFX in Epithelial-mesenchymal transition (EMT). Therefore, it is speculated that ZFX may promote NPC progression partly by regulating EMT. In summary, our study not only for the first time identified that ZFX could serve as an effective prognostic biomarker for NPC patients, but also suggested that targeting ZFX might be a novel therapeutic strategy for preventing NPC progression and metastasis.

  17. Effects of HAb18G/CD147 knockout on hepatocellular carcinoma cells in vitro using a novel zinc-finger nuclease-targeted gene knockout approach.

    PubMed

    Li, Hong-Wei; Yang, Xiang-Min; Tang, Juan; Wang, Shi-Jie; Chen, Zhi-Nan; Jiang, Jian-Li

    2015-03-01

    HAb18G/CD147 belongs to the immunoglobulin superfamily and predominantly functions as an inducer of matrix metalloproteinase secretion for tumor invasion and metastasis. This study was designed to investigate the effects of HAb18G/CD147 knockout on hepatocellular carcinoma cells using zinc-finger nuclease (ZFNs)-targeted gene knockout approach. The HCC cell line SMMC-7721 was used for ZFNs-targeted cleavage of the HAb18G/CD147 gene. RT-PCR and Western blot assays were used to detect HAb18G/CD147 expression. HAb18G phenotypic changes following HAb18G/CD147 knockout in SMMC-K7721 cells were assessed using tumor cell adhesion, invasion, migration and colony formation and flow cytometric assays. These data demonstrated that tumor cell adhesion, invasion, migration, and colony formation capabilities of SMMC-K7721 were significantly reduced compared to parental cells or SMMC-7721 with re-expression of HAb18G/CD147 protein transfected with HAb18G/CD147 cDNA. Moreover, knockout of HAb18G/CD147 expression also induced SMMC-K7721 cells to undergo apoptosis compared to SMMC-7721 and SMMC-R7721 (P < 0.01). Molecularly, protein expression of p53 was induced in these cells, but re-expression of HAb18G/CD147 reduced p53 levels in SMMC-R7721 cells, possibly through inhibition of the PI3K-Akt-MDM2 signaling pathway. The findings provide a novel insight into the mechanisms underlying HAb18G/CD147-induced progression of HCC cells.

  18. Zinc-finger protein ZFP318 is essential for expression of IgD, the alternatively spliced Igh product made by mature B lymphocytes.

    PubMed

    Enders, Anselm; Short, Alanna; Miosge, Lisa A; Bergmann, Hannes; Sontani, Yovina; Bertram, Edward M; Whittle, Belinda; Balakishnan, Bhavani; Yoshida, Kaoru; Sjollema, Geoff; Field, Matthew A; Andrews, T Daniel; Hagiwara, Hiromi; Goodnow, Christopher C

    2014-03-25

    IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells.

  19. Molecular dynamics of zinc-finger ubiquitin binding domains: a comparative study of histone deacetylase 6 and ubiquitin-specific protease 5.

    PubMed

    Dos Santos Passos, Carolina; Simões-Pires, Claudia A; Carrupt, Pierre-Alain; Nurisso, Alessandra

    2016-12-01

    HDAC6 is a unique cytoplasmic histone deacetylase characterized by two deacetylase domains, and by a zinc-finger ubiquitin binding domain (ZnF-UBP) able to recognize ubiquitin (Ub). The latter has recently been demonstrated to be involved in the progression of neurodegenerative diseases and in mediating infection by the influenza A virus. Nowadays, understanding the dynamic and energetic features of HDAC6 ZnF-UBP-Ub recognition is considered as a crucial step for the conception of HDAC6 potential modulators. In this study, the atomic, solvent-related, and thermodynamic features behind HDAC6 ZnF-UBP-Ub recognition have been analyzed through molecular dynamics simulations. The behavior was then compared to the prototypical ZnF-UBP from ubiquitin-specific protease 5 (USP5) in order to spot relevant differences useful for selective drug design. Principal component analysis highlighted flapping motions of the L2A loop which were lowered down upon Ub binding in both systems. While polar and nonpolar interactions involving Ub G75 and G76 residues were also common features stabilizing both complexes, salt bridges showed a different pattern, more significant in HDAC6 ZnF-UBP-Ub, whose energetic contribution in USP5 ZnF-UBP-Ub was compensated by the presence of a more stable bridging water molecule. Whereas molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) free energies of binding were comparable for both systems, in agreement with experiments, computational alanine scanning and free energy decomposition data revealed that HDAC6 E1141 and D1178 are potential hotspots for the design of selective HDAC6 modulators.

  20. cAMP-dependent posttranscriptional regulation of steroidogenic acute regulatory (STAR) protein by the zinc finger protein ZFP36L1/TIS11b.

    PubMed

    Duan, Haichuan; Cherradi, Nadia; Feige, Jean-Jacques; Jefcoate, Colin

    2009-04-01

    Star is expressed in steroidogenic cells as 3.5- and 1.6-kb transcripts that differ only in their 3'-untranslated regions (3'-UTR). In mouse MA10 testis and Y-1 adrenal lines, Br-cAMP preferentially stimulates 3.5-kb mRNA. ACTH is similarly selective in primary bovine adrenocortical cells. The 3.5-kb form harbors AU-rich elements (AURE) in the extended 3'-UTR, which enhance turnover. After peak stimulation of 3.5-kb mRNA, degradation is seen. Star mRNA turnover is enhanced by the zinc finger protein ZFP36L1/TIS11b, which binds to UAUUUAUU repeats in the extended 3'-UTR. TIS11b is rapidly stimulated in each cell type in parallel with Star mRNA. Cotransfection of TIS11b selectively decreases cytomegalovirus-promoted Star mRNA and luciferase-Star 3'-UTR reporters harboring the extended 3'-UTR. Direct complex formation was demonstrated between TIS11b and the extended 3'-UTR of the 3.5-kb Star. AURE mutations revealed that TIS11b-mediated destabilization required the first two UAUUUAUU motifs. HuR, which also binds AURE, did not affect Star expression. Targeted small interfering RNA knockdown of TIS11b specifically enhanced stimulation of 3.5-kb Star mRNA in bovine adrenocortical cells, MA-10, and Y-1 cells but did not affect the reversals seen after peak stimulation. Direct transfection of Star mRNA demonstrated that Br-cAMP stimulated a selective turnover of 3.5-kb mRNA independent of AURE, which may correspond to these reversal processes. Steroidogenic acute regulatory (STAR) protein induction was halved by TIS11b knockdown, concomitant with decreased cholesterol metabolism. TIS11b suppression of 3.5-kb mRNA is therefore surprisingly coupled to enhanced Star translation leading to increased cholesterol metabolism.

  1. Three zinc-finger RNA-binding proteins in cabbage (Brassica rapa) play diverse roles in seed germination and plant growth under normal and abiotic stress conditions.

    PubMed

    Park, Ye Rin; Choi, Min Ji; Park, Su Jung; Kang, Hunseung

    2017-01-01

    Despite the increasing understanding of the stress-responsive roles of zinc-finger RNA-binding proteins (RZs) in several plant species, such as Arabidopsis thaliana, wheat (Triticum aestivum) and rice (Oryza sativa), the functions of RZs in cabbage (Brassica rapa) have not yet been elucidated. In this study, the functional roles of the three RZ family members present in the cabbage genome, designated as BrRZ1, BrRZ2 and BrRZ3, were investigated in transgenic Arabidopsis under normal and environmental stress conditions. Subcellular localization analysis revealed that all BrRZ proteins were exclusively localized in the nucleus. The expression levels of each BrRZ were markedly increased by cold, drought or salt stress and by abscisic acid (ABA) treatment. Expression of BrRZ3 in Arabidopsis retarded seed germination and stem growth and reduced seed yield of Arabidopsis plants under normal growth conditions. Germination of BrRZ2- or BrRZ3-expressing Arabidopsis seeds was delayed compared with that of wild-type seeds under dehydration or salt stress conditions and cold stress conditions, respectively. Seedling growth of BrRZ3-expressing transgenic Arabidopsis plants was significantly inhibited under salt, dehydration or cold stress conditions. Notably, seedling growth of all three BrRZ-expressing transgenic Arabidopsis plants was inhibited upon ABA treatment. Importantly, all BrRZs possessed RNA chaperone activity. Taken together, these results indicate that the three cabbage BrRZs harboring RNA chaperone activity play diverse roles in seed germination and seedling growth of plants under abiotic stress conditions as well as in the presence of ABA.

  2. Positional cloning of zinc finger domain transcription factor Zfp69, a candidate gene for obesity-associated diabetes contributed by mouse locus Nidd/SJL.

    PubMed

    Scherneck, Stephan; Nestler, Matthias; Vogel, Heike; Blüher, Matthias; Block, Marcel-Dominique; Berriel Diaz, Mauricio; Herzig, Stephan; Schulz, Nadja; Teichert, Marko; Tischer, Sina; Al-Hasani, Hadi; Kluge, Reinhart; Schürmann, Annette; Joost, Hans-Georg

    2009-07-01

    Polygenic type 2 diabetes in mouse models is associated with obesity and results from a combination of adipogenic and diabetogenic alleles. Here we report the identification of a candidate gene for the diabetogenic effect of a QTL (Nidd/SJL, Nidd1) contributed by the SJL, NON, and NZB strains in outcross populations with New Zealand Obese (NZO) mice. A critical interval of distal chromosome 4 (2.1 Mbp) conferring the diabetic phenotype was identified by interval-specific congenic introgression of SJL into diabetes-resistant C57BL/6J, and subsequent reporter cross with NZO. Analysis of the 10 genes in the critical interval by sequencing, qRT-PCR, and RACE-PCR revealed a striking allelic variance of Zfp69 encoding zinc finger domain transcription factor 69. In NZO and C57BL/6J, a retrotransposon (IAPLTR1a) in intron 3 disrupted the gene by formation of a truncated mRNA that lacked the coding sequence for the KRAB (Krüppel-associated box) and Znf-C2H2 domains of Zfp69, whereas the diabetogenic SJL, NON, and NZB alleles generated a normal mRNA. When combined with the B6.V-Lep(ob) background, the diabetogenic Zfp69(SJL) allele produced hyperglycaemia, reduced gonadal fat, and increased plasma and liver triglycerides. mRNA levels of the human orthologue of Zfp69, ZNF642, were significantly increased in adipose tissue from patients with type 2 diabetes. We conclude that Zfp69 is the most likely candidate for the diabetogenic effect of Nidd/SJL, and that retrotransposon IAPLTR1a contributes substantially to the genetic heterogeneity of mouse strains. Expression of the transcription factor in adipose tissue may play a role in the pathogenesis of type 2 diabetes.

  3. Gli-similar (Glis) Krüppel-like zinc finger proteins: insights into their physiological functions and critical roles in neonatal diabetes and cystic renal disease

    PubMed Central

    Kang, Hong Soon; ZeRuth, Gary; Lichti-Kaiser, Kristin; Vasanth, Shivakumar; Yin, Zhengyu; Kim, Yong-Sik; Jetten, Anton M.

    2010-01-01

    Summary GLI-similar (Glis)1–3 proteins constitute a subfamily of the Krüppel-like zinc finger transcription factors that are closely related to the Gli family. Glis1–3 play critical roles in the regulation of a number of physiological processes and have been implicated in several pathologies. Mutations in GLIS2 have been linked to nephronophthisis, an autosomal recessive cystic kidney disease. Loss of Glis2 function leads to renal atrophy and fibrosis that involves epithelial-mesenchymal transition (EMT) of renal tubule epithelial cells. Mutations in human GLIS3 have been implicated in a syndrome characterized by neonatal diabetes and congenital hypothyroidism (NDH) and in some patients accompanied by polycystic kidney disease, glaucoma, and liver fibrosis. In addition, the GLIS3 gene has been identified as a susceptibility locus for the risk of type 1 and 2 diabetes. Glis3 plays a key role in pancreatic development, particularly in the generation of β-cells and in the regulation of insulin gene expression. Glis2 and Glis3 proteins have been demonstrated to localize to the primary cilium, a signaling organelle that has been implicated in several pathologies, including cystic renal diseases. This association suggests that Glis2/3 are part of primary cilium-associated signaling pathways that control the activity of Glis proteins. Upon activation in the primary cilium, Glis proteins may translocate to the nucleus where they subsequently regulate gene transcription by interacting with Glis-binding sites in the promoter regulatory region of target genes. In this review, we discuss the current knowledge of the Glis signaling pathways, their physiological functions, and their involvement in several human pathologies. PMID:20865670

  4. Zinc-finger protein 91 plays a key role in LIGHT-induced activation of non-canonical NF-{kappa}B pathway

    SciTech Connect

    Jin, Hong Ri; Jin, Xuejun; Lee, Jung Joon

    2010-10-01

    Research highlights: {yields} LIGHT induces ZFP91expression and nuclear translocation of p65, p52, and RelB in LT{beta}R signaling. {yields} ZFP91 knock-down abolishes DNA-binding activity of p52 and RelB but not of p65. {yields} ZFP91 regulates LIGHT-induced stabilization and activation of NIK. {yields} ZFP91 is required for the expression of non-canonical NF-{kappa}B target genes. -- Abstract: LIGHT is a member of tumor necrosis factor (TNF) superfamily, and its function is mediated through lymphotoxin-{beta} receptor (LT{beta}R), which is known to play important roles in inflammatory and immune responses through activation of NF-{kappa}B signaling pathways. However, molecular mechanism of LT{beta}R ligation-induced NF-{kappa}B signaling remains incompletely understood. In this report we demonstrate that a novel zinc-finger protein 91 (ZFP91) is a critical regulator in LIGHT-induced activation of non-canonical NF-{kappa}B pathway. ZFP91 appears to be required for NF-{kappa}B2 (p100) processing to p52, nuclear translocation of p52 and RelB, and DNA-binding activity of NF-{kappa}B in LIGHT-induced activation of non-canonical NF-{kappa}B pathway. Furthermore, ZFP91 knock-down by RNA interference blocks the LIGHT-induced accumulation of NIK and p100 processing, as well as the expression of non-canonical NF-{kappa}B target genes. These data clearly indicate that ZFP91 is a key regulator in LIGHT-induced activation of non-canonical NF-{kappa}B pathway in LT{beta}R signaling.

  5. Diversity of Prdm9 zinc finger array in wild mice unravels new facets of the evolutionary turnover of this coding minisatellite.

    PubMed

    Buard, Jérôme; Rivals, Eric; Dunoyer de Segonzac, Denis; Garres, Charlotte; Caminade, Pierre; de Massy, Bernard; Boursot, Pierre

    2014-01-01

    In humans and mice, meiotic recombination events cluster into narrow hotspots whose genomic positions are defined by the PRDM9 protein via its DNA binding domain constituted of an array of zinc fingers (ZnFs). High polymorphism and rapid divergence of the Prdm9 gene ZnF domain appear to involve positive selection at DNA-recognition amino-acid positions, but the nature of the underlying evolutionary pressures remains a puzzle. Here we explore the variability of the Prdm9 ZnF array in wild mice, and uncovered a high allelic diversity of both ZnF copy number and identity with the caracterization of 113 alleles. We analyze features of the diversity of ZnF identity which is mostly due to non-synonymous changes at codons -1, 3 and 6 of each ZnF, corresponding to amino-acids involved in DNA binding. Using methods adapted to the minisatellite structure of the ZnF array, we infer a phylogenetic tree of these alleles. We find the sister species Mus spicilegus and M. macedonicus as well as the three house mouse (Mus musculus) subspecies to be polyphyletic. However some sublineages have expanded independently in Mus musculus musculus and M. m. domesticus, the latter further showing phylogeographic substructure. Compared to random genomic regions and non-coding minisatellites, none of these patterns appears exceptional. In silico prediction of DNA binding sites for each allele, overlap of their alignments to the genome and relative coverage of the different families of interspersed repeated elements suggest a large diversity between PRDM9 variants with a potential for highly divergent distributions of recombination events in the genome with little correlation to evolutionary distance. By compiling PRDM9 ZnF protein sequences in Primates, Muridae and Equids, we find different diversity patterns among the three amino-acids most critical for the DNA-recognition function, suggesting different diversification timescales.

  6. The long zinc finger domain of PRDM9 forms a highly stable and long-lived complex with its DNA recognition sequence.

    PubMed

    Striedner, Yasmin; Schwarz, Theresa; Welte, Thomas; Futschik, Andreas; Rant, Ulrich; Tiemann-Boege, Irene

    2017-02-02

    PR domain containing protein 9 (PRDM9) is a meiosis-specific, multi-domain protein that regulates the location of recombination hotspots by targeting its DNA recognition sequence for double-strand breaks (DSBs). PRDM9 specifically recognizes DNA via its tandem array of zinc fingers (ZnFs), epigenetically marks the local chromatin by its histone methyltransferase activity, and is an important tether that brings the DNA into contact with the recombination initiation machinery. A strong correlation between PRDM9-ZnF variants and specific DNA motifs at recombination hotspots has been reported; however, the binding specificity and kinetics of the ZnF domain are still obscure. Using two in vitro methods, gel mobility shift assays and switchSENSE, a quantitative biophysical approach that measures binding rates in real time, we determined that the PRDM9-ZnF domain forms a highly stable and long-lived complex with its recognition sequence, with a dissociation halftime of many hours. The ZnF domain exhibits an equilibrium dissociation constant (K D) in the nanomolar (nM) range, with polymorphisms in the recognition sequence directly affecting the binding affinity. We also determined that alternative sequences (15-16 nucleotides in length) can be specifically bound by different subsets of the ZnF domain, explaining the binding plasticity of PRDM9 for different sequences. Finally, longer binding targets are preferred than predicted from the numbers of ZnFs contacting the DNA. Functionally, a long-lived complex translates into an enzymatically active PRDM9 at specific DNA-binding sites throughout meiotic prophase I that might be relevant in stabilizing the components of the recombination machinery to a specific DNA target until DSBs are initiated by Spo11.

  7. The zinc finger gene ZIC2 has features of an oncogene and its over- expression correlates strongly with the clinical course of epithelial ovarian cancer

    PubMed Central

    Marchini, Sergio; Poynor, Elizabeth; Barakat, Richard R; Clivio, Luca; Cinquini, Michela; Fruscio, Robert; Porcu, Luca; Bussani, Cecilia; D’Incalci, Maurizio; Erba, Eugenio; Romano, Michela; Cattoretti, Giorgio; Katsaros, Dionyssios; Koff, Andrew; Luzzatto, Lucio

    2015-01-01

    Purpose Epithelial ovarian tumors (EOTs) are amongst the most lethal of malignancies in women. We have previously identified ZIC2 as expressed at a higher level in samples of a malignant form (MAL) of EOT than in samples of a form with low malignant potential (LMP). We have now investigated the role of ZIC2 in driving tumor growth and its association with clinical outcomes. Experimental Design ZIC2 expression levels were analysed in two independent tumor tissue collections of LMP and MAL. In vitro experiments aimed to test the role of ZIC2 as a transforming gene. Cox models were used to correlate ZIC2 expression with clinical endpoints. Results ZIC2 expression was about 40-fold in terms of mRNA and about 17-fold in terms of protein in MAL (n = 193) versus LMP (n = 39) tumors. ZIC2 mRNA levels were high in MAL cell lines, but undetectable in LMP cell lines. Over-expression of ZIC2 was localized to the nucleus. ZIC2 over-expression increases the growth rate and foci formation of NIH 3T3 cells, and stimulates anchorage-independent colony formation; down-regulation of ZIC2 decreases the growth rate of MAL cell lines. Zinc finger domains 1 and 2 are required for transforming activity. In stage I MAL ZIC2 expression was significantly associated with overall survival in both univariate (p = 0.046), and multivariate model (p = 0.049). Conclusions ZIC2, a transcription factor related to the sonic hedgehog pathway, is a strong discriminant between MAL and LMP tumors: it may be a major determinant of outcome of EOT. PMID:22733541

  8. Gli-similar (Glis) Krüppel-like zinc finger proteins: insights into their physiological functions and critical roles in neonatal diabetes and cystic renal disease.

    PubMed

    Kang, Hong Soon; ZeRuth, Gary; Lichti-Kaiser, Kristin; Vasanth, Shivakumar; Yin, Zhengyu; Kim, Yong-Sik; Jetten, Anton M

    2010-11-01

    GLI-similar (Glis) 1-3 proteins constitute a subfamily of the Krüppel-like zinc finger transcription factors that are closely related to the Gli family. Glis1-3 play critical roles in the regulation of a number of physiological processes and have been implicated in several pathologies. Mutations in GLIS2 have been linked to nephronophthisis, an autosomal recessive cystic kidney disease. Loss of Glis2 function leads to renal atrophy and fibrosis that involves epithelial-mesenchymal transition (EMT) of renal tubule epithelial cells. Mutations in human GLIS3 have been implicated in a syndrome characterized by neonatal diabetes and congenital hypothyroidism (NDH) and in some patients accompanied by polycystic kidney disease, glaucoma, and liver fibrosis. In addition, the GLIS3 gene has been identified as a susceptibility locus for the risk of type 1 and 2 diabetes. Glis3 plays a key role in pancreatic development, particularly in the generation of ß-cells and in the regulation of insulin gene expression. Glis2 and Glis3 proteins have been demonstrated to localize to the primary cilium, a signaling organelle that has been implicated in several pathologies, including cystic renal diseases. This association suggests that Glis2/3 are part of primary cilium-associated signaling pathways that control the activity of Glis proteins. Upon activation in the primary cilium, Glis proteins may translocate to the nucleus where they subsequently regulate gene transcription by interacting with Glis-binding sites in the promoter regulatory region of target genes. In this review, we discuss the current knowledge of the Glis signaling pathways, their physiological functions, and their involvement in several human pathologies.

  9. Myeloid zinc finger (MZF)-like, Kruppel-like and Ets families of transcription factors determine the cell-specific expression of mouse extracellular superoxide dismutase.

    PubMed Central

    Zelko, Igor N; Folz, Rodney J

    2003-01-01

    Extracellular superoxide dismutase (EC-SOD or SOD3) is an important protective enzyme against the toxicity of superoxide radicals that are produced under both physiological and pathophysiological conditions. We have isolated and characterized over 11 kb of the mouse EC-SOD gene and its 5'- and 3'-flanking regions. The gene consists of two exons, with the entire coding region located within exon 2. In order to study the mechanism of cell-specific gene regulation for mouse EC-SOD, we characterized 2500 bp of its 5'-flanking region using cultured cells derived from mouse lung fibroblasts (MLg), kidney medulla (mIMCD3) and hepatocytes (Hepa 1-6). Real-time PCR showed that basal expression of EC-SOD was considerably higher in MLg cells compared with the other cell types. Reporter-gene assays revealed that the proximal promoter region was sufficient to support this high expression in MLg cells. Although no obvious TATA box was identified, our results show that a highly purine-rich region from -208 to +104 contains active binding sites for both the Kruppel-like and Ets families of transcription factors. Using electrophoretic mobility shift, DNase footprinting and reporter gene assays, we identified myeloid zinc finger 1 and gut-enriched Kruppel-like-factor-like nuclear transcription factors as repressors of EC-SOD expression, whereas nuclear transcription factors from the Ets family, such as Elf-1 and GA-binding protein alpha and beta, were potent activators of EC-SOD transcription. We propose a model that highlights competition between Ets activators and Kruppel-like repressors within the proximal promoter region that determines the level of EC-SOD expression in a particular cell type. PMID:12374566

  10. Phylogenetic Distribution and Evolution of the Linked RNA-Binding and NOT1-Binding Domains in the Tristetraprolin Family of Tandem CCCH Zinc Finger Proteins

    PubMed Central

    Perera, Lalith

    2014-01-01

    In humans, the tristetraprolin or TTP family of CCCH tandem zinc finger (TZF) proteins comprises 3 members, encoded by the genes ZFP36, ZFP36L1, and ZFP36L2. These proteins have direct orthologues in essentially all vertebrates studied, with the exception of birds, which appear to lack a version of ZFP36. Additional family members are found in rodents, amphibians, and fish. In general, the encoded proteins contain 2 critical macromolecular interaction domains: the CCCH TZF domain, which is necessary for high-affinity binding to AU-rich elements in mRNA; and an extreme C-terminal domain that, in the case of TTP, interacts with NOT1, the scaffold of a large multi-protein complex that contains deadenylases. TTP and its related proteins act by first binding to AU-rich elements in mRNA, and then recruiting deadenylases to the mRNA, where they can processively remove the adenosine residues from the poly(A) tail. Highly conserved TZF domains have been found in unicellular eukaryotes such as yeasts, and these domains can bind AU-rich elements that resemble those bound by the mammalian proteins. However, certain fungi appear to lack proteins with intact TZF domains, and the TTP family proteins that are expressed in other fungi often lack the characteristic C-terminal NOT1 binding domain found in the mammalian proteins. For these reasons, we investigated the phylogenetic distribution of the relevant sequences in available databases. Both domains are present in family member proteins from most lineages of eukaryotes, suggesting their mutual presence in a common ancestor. However, the vertebrate type of NOT1-binding domain is missing in most fungi, and the TZF domain itself has disappeared or degenerated in recently evolved fungi. Nonetheless, both domains are present together in the proteins from several unicellular eukaryotes, including at least 1 fungus, and they seem to have remained together during the evolution of metazoans. PMID:24697206

  11. ATL9, a RING Zinc Finger Protein with E3 Ubiquitin Ligase Activity Implicated in Chitin- and NADPH Oxidase-Mediated Defense Responses

    PubMed Central

    Berrocal-Lobo, Marta; Stone, Sophia; Yang, Xin; Antico, Jay; Callis, Judy; Ramonell, Katrina M.; Somerville, Shauna

    2010-01-01

    Pathogen associated molecular patterns (PAMPs) are signals detected by plants that activate basal defenses. One of these PAMPs is chitin, a carbohydrate present in the cell walls of fungi and in insect exoskeletons. Previous work has shown that chitin treatment of Arabidopsis thaliana induced defense-related genes in the absence of a pathogen and that the response was independent of the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling pathways. One of these genes is ATL9 ( = ATL2G), which encodes a RING zinc-finger like protein. In the current work we demonstrate that ATL9 has E3 ubiquitin ligase activity and is localized to the endoplasmic reticulum. The expression pattern of ATL9 is positively correlated with basal defense responses against Golovinomyces cichoracearum, a biotrophic fungal pathogen. The basal levels of expression and the induction of ATL9 by chitin, in wild type plants, depends on the activity of NADPH oxidases suggesting that chitin-mediated defense response is NADPH oxidase dependent. Although ATL9 expression is not induced by treatment with known defense hormones (SA, JA or ET), full expression in response to chitin is compromised slightly in mutants where ET- or SA-dependent signaling is suppressed. Microarray analysis of the atl9 mutant revealed candidate genes that appear to act downstream of ATL9 in chitin-mediated defenses. These results hint at the complexity of chitin-mediated signaling and the potential interplay between elicitor-mediated signaling, signaling via known defense pathways and the oxidative burst. PMID:21203445

  12. Improvement of acetic acid tolerance of Saccharomyces cerevisiae using a zinc-finger-based artificial transcription factor and identification of novel genes involved in acetic acid tolerance.

    PubMed

    Ma, Cui; Wei, Xiaowen; Sun, Cuihuan; Zhang, Fei; Xu, Jianren; Zhao, Xinqing; Bai, Fengwu

    2015-03-01

    Acetic acid is present in cellulosic hydrolysate as a potent inhibitor, and the superior acetic acid tolerance of Saccharomyces cerevisiae ensures good cell viability and efficient ethanol production when cellulosic raw materials are used as substrates. In this study, a mutant strain of S. cerevisiae ATCC4126 (Sc4126-M01) with improved acetic acid tolerance was obtained through screening strains transformed with an artificial zinc finger protein transcription factor (ZFP-TF) library. Further analysis indicated that improved acetic acid tolerance was associated with improved catalase (CAT) activity. The ZFP coding sequence associated with the improved phenotype was identified, and real-time RT-PCR analysis revealed that three of the possible genes involved in the enhanced acetic acid tolerance regulated by this ZFP-TF, namely YFL040W, QDR3, and IKS1, showed decreased transcription levels in Sc4126-M01 in the presence of acetic acid, compared to those in the control strain. Sc4126-M01 mutants having QDR3 and IKS1 deletion (ΔQDR3 and ΔIKS1) exhibited higher acetic acid tolerance than the wild-type strain under acetic acid treatment. Glucose consumption rate and ethanol productivity in the presence of 5 g/L acetic acid were improved in the ΔQDR3 mutant compared to the wild-type strain. Our studies demonstrated that the synthetic ZFP-TF library can be used to improve acetic acid tolerance of S. cerevisiae and that the employment of an artificial transcription factor can facilitate the exploration of novel functional genes involved in stress tolerance of S. cerevisiae.

  13. Bio-inspired band gap engineering of zinc oxide by intracrystalline incorporation of amino acids.

    PubMed

    Brif, Anastasia; Ankonina, Guy; Drathen, Christina; Pokroy, Boaz

    2014-01-22

    Bandgap engineering of zinc oxide semiconductors can be achieved using a bio-inspired method. During a bioInspired crystallization process, incorporation of amino acids into the crystal structure of ZnO induces lattice strain that leads to linear bandgap shifts. This allows for fine tuning of the bandgap in a bio-inspired route.

  14. New Insights on the Burstein-Moss Shift and Band Gap Narrowing in Indium-Doped Zinc Oxide Thin Films.

    PubMed

    Saw, K G; Aznan, N M; Yam, F K; Ng, S S; Pung, S Y

    2015-01-01

    The Burstein-Moss shift and band gap narrowing of sputtered indium-doped zinc oxide (IZO) thin films are investigated as a function of carrier concentrations. The optical band gap shifts below the carrier concentration of 5.61 × 1019 cm-3 are well-described by the Burstein-Moss model. For carrier concentrations higher than 8.71 × 1019 cm-3 the shift decreases, indicating that band gap narrowing mechanisms are increasingly significant and are competing with the Burstein-Moss effect. The incorporation of In causes the resistivity to decrease three orders of magnitude. As the mean-free path of carriers is less than the crystallite size, the resistivity is probably affected by ionized impurities as well as defect scattering mechanisms, but not grain boundary scattering. The c lattice constant as well as film stress is observed to increase in stages with increasing carrier concentration. The asymmetric XPS Zn 2p3/2 peak in the film with the highest carrier concentration of 7.02 × 1020 cm-3 suggests the presence of stacking defects in the ZnO lattice. The Raman peak at 274 cm-1 is attributed to lattice defects introduced by In dopants.

  15. New Insights on the Burstein-Moss Shift and Band Gap Narrowing in Indium-Doped Zinc Oxide Thin Films

    PubMed Central

    Saw, K. G.; Aznan, N. M.; Yam, F. K.; Ng, S. S.; Pung, S. Y.

    2015-01-01

    The Burstein-Moss shift and band gap narrowing of sputtered indium-doped zinc oxide (IZO) thin films are investigated as a function of carrier concentrations. The optical band gap shifts below the carrier concentration of 5.61 × 1019 cm-3 are well-described by the Burstein-Moss model. For carrier concentrations higher than 8.71 × 1019 cm-3 the shift decreases, indicating that band gap narrowing mechanisms are increasingly significant and are competing with the Burstein-Moss effect. The incorporation of In causes the resistivity to decrease three orders of magnitude. As the mean-free path of carriers is less than the crystallite size, the resistivity is probably affected by ionized impurities as well as defect scattering mechanisms, but not grain boundary scattering. The c lattice constant as well as film stress is observed to increase in stages with increasing carrier concentration. The asymmetric XPS Zn 2p3/2 peak in the film with the highest carrier concentration of 7.02 × 1020 cm-3 suggests the presence of stacking defects in the ZnO lattice. The Raman peak at 274 cm-1 is attributed to lattice defects introduced by In dopants. PMID:26517364

  16. The effect of induced strains on the optical band gaps in lanthanum-doped zinc ferrite nanocrystalline powders

    NASA Astrophysics Data System (ADS)

    Hamed, Fathalla; Ramachandran, Tholkappiyan; Kurapati, Vishista

    2016-07-01

    ZnFe1.96La0.04O4 nanocrystalline powders were synthesized by auto-combustion with the aid of glycine as fuel. The synthesized powders were subjected to heat treatment in air at constant temperatures (600-970∘C) for a period of 2 h. The annealed powders were characterized by X-ray diffraction (XRD), Fourier transformation infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and UV-Vis-NIR spectroscopy. The as-synthesized and annealed powders formed spongy porous network structure with voids and pores. All the powders were found to be single phase nanomaterial with cubic spinel crystal structure and the desired composition; however, they contained strains, dislocations and lattice distortions. Some of these strains and dislocations are relaxed as a function of annealing temperature. The powders displayed direct and indirect optical band gaps. The energies of these band gaps were found to vary as a function of the induced strains and dislocations. It is suggested that the energy of the optical band gap in lanthanum-doped zinc ferrite nanocrystalline powders can be varied as a function of induced strains if the initial preparation conditions and the following heat treatments are controlled.

  17. The 91-205 amino acid region of AcMNPV ORF34 (Ac34), which comprises a potential C3H zinc finger, is required for its nuclear localization and optimal virus multiplication.

    PubMed

    Qiu, Jianxiang; Tang, Zhimin; Yuan, Meijin; Wu, Wenbi; Yang, Kai

    2017-01-15

    During baculovirus infection, most viral proteins must be imported to the nucleus to support virus multiplication. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf34 (ac34) is an alphabaculovirus unique gene that is required for optimal virus production. Ac34 distributes in both the cytoplasm and the nuclei of virus-infected Sf9 cells, but contains no conventional nuclear localization signal (NLS). In this study, we investigated the nuclear targeting domains in Ac34. Transient expression assays showed that Ac34 localized in both the cytoplasm and the nuclei of Sf9 cells, indicating that no viral protein is required for Ac34 nuclear localization. Subcellular localization analysis of Ac34 truncations and internal deletions fused with green fluorescent protein in plasmid-transfected Sf9 cells identified that the 91-205 amino acid (aa) region is required for Ac34 nuclear localization. Mutations in a potential C3H zinc finger (aa 116-131) in Ac34 resulted in exclusive cytoplasmic distribution of GFP:Ac34, suggesting that the zinc finger is required for Ac34 nuclear localization. To assess the functional importance of Ac34 in the nucleus during virus replication, recombinant AcMNPV bacmids containing a series of Ac34 truncations, internal deletions, or site mutations fused with HA tags were constructed. Subcellular localization analysis showed that Ac34 with internal deletions in aa 91-205 or site mutations in the potential zinc finger was predominantly distributed in the cytoplasm. Viral plaque assays and virus growth curves indicated that disruption of Ac34 nuclear localization significantly impaired virus replication. Taken together, our findings demonstrated that the nuclear localization of Ac34 requires the 91-205 aa region and its nuclear localization is essential for optimal virus replication.

  18. AtC3H17, a Non-Tandem CCCH Zinc Finger Protein, Functions as a Nuclear Transcriptional Activator and Has Pleiotropic Effects on Vegetative Development, Flowering and Seed Development in Arabidopsis.

    PubMed

    Seok, Hye-Yeon; Woo, Dong-Hyuk; Park, Hee-Yeon; Lee, Sun-Young; Tran, Huong T; Lee, Eun-Hye; Vu Nguyen, Linh; Moon, Yong-Hwan

    2016-03-01

    Despite increasing reports that CCCH zinc finger proteins function in plant development and stress responses, the functions and molecular aspects of many CCCH zinc finger proteins remain uncharacterized. Here, we characterized the biological and molecular functions of AtC3H17, a unique Arabidopsis gene encoding a non-tandem CCCH zinc finger protein. AtC3H17 was ubiquitously expressed throughout the life cycle of Arabidopsis plants and their organs. The rate and ratio of seed germination of atc3h17 mutants were slightly slower and lower, respectively, than those of the wild type (WT), whereas AtC3H17-overexpressing transgenic plants (OXs) showed an enhanced germination rate. atc3h17 mutant seedlings were smaller and lighter than WT seedlings while AtC3H17 OX seedlings were larger and heavier. In regulation of flowering time, atc3h17 mutants showed delayed flowering, whereas AtC3H17 OXs showed early flowering compared with the WT. In addition, overexpression of AtC3H17 affected seed development, displaying abnormalities compared with the WT. AtC3H17 protein was localized to the nucleus and showed transcriptional activation activity in yeast and Arabidopsis protoplasts. The N-terminal region of AtC3H17, containing a conserved EELR-like motif, was necessary for transcriptional activation activity, and the two conserved glutamate residues in the EELR-like motif played an important role in transcriptional activation activity. Real-time PCR and transactivation analyses showed that AtC3H17 might be involved in seed development via transcriptional activation of OLEO1, OLEO2 and CRU3. Our results suggest that AtC3H17 has pleiotropic effects on vegetative development such as seed germination and seedling growth, flowering and seed development, and functions as a nuclear transcriptional activator in Arabidopsis.

  19. Zinc Finger Nuclease Mediated Knockout of ADP-Dependent Glucokinase in Cancer Cell Lines: Effects on Cell Survival and Mitochondrial Oxidative Metabolism

    PubMed Central

    Richter, Susan; Morrison, Shona; Connor, Tim; Su, Jiechuang; Print, Cristin G.; Ronimus, Ron S.; McGee, Sean L.; Wilson, William R.

    2013-01-01

    Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines. PMID:23799003

  20. C2H2 zinc finger proteins of the SP/KLF, Wilms tumor, EGR, Huckebein, and Klumpfuss families in metazoans and beyond

    PubMed Central

    Pei, Jimin; Grishin, Nick V.

    2015-01-01

    Specificity proteins (SPs) and Krüppel-like factors (KLFs) are C2H2-type Zinc finger transcription factors that play essential roles in differentiation, development, proliferation and cell death. SP/KLF proteins, similarly to Wilms tumor protein 1 (WT1), Early Growth Response (EGR), Huckebein, and Klumpfuss, prefer to bind GC-rich sequences such as GC-box and CACCC-box (GT-box). We searched various genomes and transcriptomes of metazoans and single-cell holozoans for members of these families. Seven groups of KLFs (KLFA–G) and three groups of SPs (SPA–C) were identified in the three lineages of Bilateria (Deuterostomia, Ecdysozoa, and Lophotrochozoa). The last ancestor of jawed vertebrates was inferred to have at least 18 KLFs (group A: KLF1/2/4/17, group B: KLF3/8/12; group C: KLF5/5l; group D: KLF6/7; group E: KLF9/13/16; group F: KLF10/KLF11; group G: KLF15/15l) and 10 SPs (group A: SP1/2/3/4; group B: SP5/5l; group C: SP6/7/8/9), since they were found in both cartilaginous and boned fishes. Placental mammals have added KLF14 (group E) and KLF18 (group A), and lost KLF5l (KLF5-like) and KLF15l (KLF15-like). Multiple KLF members were found in basal metazoans (Ctenophora, Porifera, Placozoa, and Cnidaria). Ctenophora has the least number of KLFs and no SPs, which could be attributed to its proposed sister group relationship to other metazoans or gene loss. While SP, EGR and Klumpfuss were only detected in metazoans, KLF, WT1, and Huckebein are present in nonmetazoan holozoans. Of the seven metazoan KLF groups, only KLFG, represented by KLF15 in human, was found in nonmetazoans. In addition, two nonmetazoan groups of KLFs are present in Choanoflagellatea and Filasterea. WT1 could be evolutionarily the earliest among these GC/GT-box-binding families due to its sole presence in Ichthyosporea. PMID:26187067

  1. Use of zinc-finger nucleases to knock out the WAS gene in K562 cells: a human cellular model for Wiskott-Aldrich syndrome.

    PubMed

    Toscano, Miguel G; Anderson, Per; Muñoz, Pilar; Lucena, Gema; Cobo, Marién; Benabdellah, Karim; Gregory, Philip D; Holmes, Michael C; Martin, Francisco

    2013-03-01

    Mutations in the WAS gene cause Wiskott-Aldrich syndrome (WAS), which is characterized by eczema, immunodeficiency and microthrombocytopenia. Although the role of WASP in lymphocytes and myeloid cells is well characterized, its role on megakaryocyte (MK) development is poorly understood. In order to develop a human cellular model that mimics the megakaryocytic-derived defects observed in WAS patients we used K562 cells, a well-known model for study of megakaryocytic development. We knocked out the WAS gene in K562 cells using a zinc-finger nuclease (ZFN) pair targeting the WAS intron 1 and a homologous donor DNA that disrupted WASP expression. Knockout of WASP on K562 cells (K562WASKO cells) resulted in several megakaryocytic-related defects such as morphological alterations, lower expression of CD41, lower increments in F-actin polymerization upon stimulation, reduced CD43 expression and increased phosphatidylserine exposure. All these defects have been previously described either in WAS-knockout mice or in WAS patients, validating K562WASKO as a cell model for WAS. However, K562WASPKO cells showed also increased basal F-actin and adhesion, increased expression of CD61 and reduced expression of TGFβ and Factor VIII, defects that have never been described before for WAS-deficient cells. Interestingly, these phenotypic alterations correlate with different roles for WASP in megakaryocytic differentiation. All phenotypic alterations observed in K562WASKO cells were alleviated upon expression of WAS following lentiviral transduction, confirming the role of WASP in these phenotypes. In summary, in this work we have validated a human cellular model, K562WASPKO, that mimics the megakaryocytic-related defects found in WAS-knockout mice and have found evidences for a role of WASP as regulator of megakaryocytic differentiation. We propose the use of K562WASPKO cells as a tool to study the molecular mechanisms involved in the megakaryocytic-related defects observed in WAS

  2. Use of zinc-finger nucleases to knock out the WAS gene in K562 cells: a human cellular model for Wiskott-Aldrich syndrome

    PubMed Central

    Toscano, Miguel G.; Anderson, Per; Muñoz, Pilar; Lucena, Gema; Cobo, Marién; Benabdellah, Karim; Gregory, Philip D.; Holmes, Michael C.; Martin, Francisco

    2013-01-01

    SUMMARY Mutations in the WAS gene cause Wiskott-Aldrich syndrome (WAS), which is characterized by eczema, immunodeficiency and microthrombocytopenia. Although the role of WASP in lymphocytes and myeloid cells is well characterized, its role on megakaryocyte (MK) development is poorly understood. In order to develop a human cellular model that mimics the megakaryocytic-derived defects observed in WAS patients we used K562 cells, a well-known model for study of megakaryocytic development. We knocked out the WAS gene in K562 cells using a zinc-finger nuclease (ZFN) pair targeting the WAS intron 1 and a homologous donor DNA that disrupted WASP expression. Knockout of WASP on K562 cells (K562WASKO cells) resulted in several megakaryocytic-related defects such as morphological alterations, lower expression of CD41ɑ, lower increments in F-actin polymerization upon stimulation, reduced CD43 expression and increased phosphatidylserine exposure. All these defects have been previously described either in WAS-knockout mice or in WAS patients, validating K562WASKO as a cell model for WAS. However, K562WASPKO cells showed also increased basal F-actin and adhesion, increased expression of CD61 and reduced expression of TGFβ and Factor VIII, defects that have never been described before for WAS-deficient cells. Interestingly, these phenotypic alterations correlate with different roles for WASP in megakaryocytic differentiation. All phenotypic alterations observed in K562WASKO cells were alleviated upon expression of WAS following lentiviral transduction, confirming the role of WASP in these phenotypes. In summary, in this work we have validated a human cellular model, K562WASPKO, that mimics the megakaryocytic-related defects found in WAS-knockout mice and have found evidences for a role of WASP as regulator of megakaryocytic differentiation. We propose the use of K562WASPKO cells as a tool to study the molecular mechanisms involved in the megakaryocytic-related defects observed

  3. Evolutionary conservation of zinc finger transcription factor binding sites in promoters of genes co-expressed with WT1 in prostate cancer

    PubMed Central

    Eisermann, Kurtis; Tandon, Sunpreet; Bazarov, Anton; Brett, Adina; Fraizer, Gail; Piontkivska, Helen

    2008-01-01

    Background Gene expression analyses have led to a better understanding of growth control of prostate cancer cells. We and others have identified the presence of several zinc finger transcription factors in the neoplastic prostate, suggesting a potential role for these genes in the regulation of the prostate cancer transcriptome. One of the transcription factors (TFs) identified in the prostate cancer epithelial cells was the Wilms tumor gene (WT1). To rapidly identify coordinately expressed prostate cancer growth control genes that may be regulated by WT1, we used an in silico approach. Results Evolutionary conserved transcription factor binding sites (TFBS) recognized by WT1, EGR1, SP1, SP2, AP2 and GATA1 were identified in the promoters of 24 differentially expressed prostate cancer genes from eight mammalian species. To test the relationship between sequence conservation and function, chromatin of LNCaP prostate cancer and kidney 293 cells were tested for TF binding using chromatin immunoprecipitation (ChIP). Multiple putative TFBS in gene promoters of placental mammals were found to be shared with those in human gene promoters and some were conserved between genomes that diverged about 170 million years ago (i.e., primates and marsupials), therefore implicating these sites as candidate binding sites. Among those genes coordinately expressed with WT1 was the kallikrein-related peptidase 3 (KLK3) gene commonly known as the prostate specific antigen (PSA) gene. This analysis located several potential WT1 TFBS in the PSA gene promoter and led to the rapid identification of a novel putative binding site confirmed in vivo by ChIP. Conversely for two prostate growth control genes, androgen receptor (AR) and vascular endothelial growth factor (VEGF), known to be transcriptionally regulated by WT1, regulatory sequence conservation was observed and TF binding in vivo was confirmed by ChIP. Conclusion Overall, this targeted approach rapidly identified important candidate

  4. Zinc-Finger Nuclease Knockout of Dual-Specificity Protein Phosphatase-5 Enhances the Myogenic Response and Autoregulation of Cerebral Blood Flow in FHH.1BN Rats

    PubMed Central

    Fan, Fan; Geurts, Aron M.; Pabbidi, Mallikarjuna R.; Smith, Stanley V.; Harder, David R.; Jacob, Howard; Roman, Richard J.

    2014-01-01

    We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background. We identified 4 single nucleotide polymorphisms in the Dusp5 gene in FHH as compared with BN rats, two of which altered CpG sites and another that caused a G155R mutation. To determine whether Dusp5 contributes to the impaired myogenic response in FHH rats, we created a Dusp5 knockout (KO) rat in the FHH.1BN genetic background using a zinc-finger nuclease that introduced an 11 bp frame-shift deletion and a premature stop codon at AA121. The expression of Dusp5 was decreased and the levels of its substrates, phosphorylated ERK1/2 (p-ERK1/2), were enhanced in the KO rats. The diameter of the MCA decreased to a greater extent in Dusp5 KO rats than in FHH.1BN and FHH rats when the perfusion pressure was increased from 40 to 140 mmHg. CBF increased markedly in FHH rats when MAP was increased from 100 to 160 mmHg, and CBF was better autoregulated in the Dusp5 KO and FHH.1BN rats. The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats. These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats. PMID:25397684

  5. Mallet finger - aftercare

    MedlinePlus

    Baseball finger - aftercare; Drop finger - aftercare; Avulsion fracture - mallet finger - aftercare ... away from the rest of the bone (avulsion fracture) Mallet finger most often occurs when something hits ...

  6. DPL-1 DP, LIN-35 Rb and EFL-1 E2F act with the MCD-1 zinc-finger protein to promote programmed cell death in Caenorhabditis elegans.

    PubMed

    Reddien, Peter W; Andersen, Erik C; Huang, Michael C; Horvitz, H Robert

    2007-04-01

    The genes egl-1, ced-9, ced-4, and ced-3 play major roles in programmed cell death in Caenorhabditis elegans. To identify genes that have more subtle activities, we sought mutations that confer strong cell-death defects in a genetically sensitized mutant background. Specifically, we screened for mutations that enhance the cell-death defects caused by a partial loss-of-function allele of the ced-3 caspase gene. We identified mutations in two genes not previously known to affect cell death, dpl-1 and mcd-1 (modifier of cell death). dpl-1 encodes the C. elegans homolog of DP, the human E2F-heterodimerization partner. By testing genes known to interact with dpl-1, we identified roles in cell death for four additional genes: efl-1 E2F, lin-35 Rb, lin-37 Mip40, and lin-52 dLin52. mcd-1 encodes a novel protein that contains one zinc finger and that is synthetically required with lin-35 Rb for animal viability. dpl-1 and mcd-1 act with efl-1 E2F and lin-35 Rb to promote programmed cell death and do so by regulating the killing process rather than by affecting the decision between survival and death. We propose that the DPL-1 DP, MCD-1 zinc finger, EFL-1 E2F, LIN-35 Rb, LIN-37 Mip40, and LIN-52 dLin52 proteins act together in transcriptional regulation to promote programmed cell death.

  7. Rearrangements of the retinoic acid receptor alpha and promyelocytic leukemia zinc finger genes resulting from t(11;17)(q23;q21) in a patient with acute promyelocytic leukemia.

    PubMed Central

    Chen, S J; Zelent, A; Tong, J H; Yu, H Q; Wang, Z Y; Derré, J; Berger, R; Waxman, S; Chen, Z

    1993-01-01

    Cytogenetic study of a patient with acute promyelocytic leukemia (APL) showed an unusual karyotype 46,xy,t(11;17) (q23;21) without apparent rearrangement of chromosome 15. Molecular studies showed rearrangements of the retinoic acid receptor alpha (RAR alpha) gene but no rearrangement of the promyelocytic leukemia gene consistent with the cytogenetic data. Similar to t(15;17) APL, all-trans retinoic acid treatment in this patient produced an early leukocytosis which was followed by a myeloid maturation, but the patient died too early to achieve remission. Further molecular analysis of this patient showed a rearrangement between the RAR alpha gene and a newly discovered zinc finger gene named PLZF (promyelocytic leukemia zinc finger). The fusion PLZF-RAR alpha gene found in this case, was not found in DNA obtained from the bone marrow of normals, APL with t(15;17) and in one patient with AML-M2 with a t(11;17). Fluorescence in situ hybridization using a PLZF specific probe localized the PLZF gene to chromosomal band 11q23.1. Partial exon/intron structure of the PLZF gene flanking the break point on chromosome 11 was also established and the breakpoint within the RAR alpha gene was mapped approximately 2 kb downstream of the exon encoding the 5' untranslated region and the unique A2 domain of the RAR alpha 2 isoform. Images PMID:8387545

  8. Trigger finger

    MedlinePlus

    ... Redness in your cut or hand Swelling or warmth in your cut or hand Yellow or green drainage from the cut Hand pain or discomfort Fever If your trigger finger returns, call your surgeon. You may need another surgery.

  9. Xenopus laevis zygote arrest 2 (zar2) encodes a zinc finger RNA-binding protein that binds to the translational control sequence in the maternal Wee1 mRNA and regulates translation.

    PubMed

    Charlesworth, Amanda; Yamamoto, Tomomi M; Cook, Jonathan M; Silva, Kevin D; Kotter, Cassandra V; Carter, Gwendolyn S; Holt, Justin W; Lavender, Heather F; MacNicol, Angus M; Ying Wang, Yi; Wilczynska, Anna

    2012-09-15

    Zygote arrest (Zar) proteins are crucial for early embryonic development, but their molecular mechanism of action is unknown. The Translational Control Sequence (TCS) in the 3' untranslated region (UTR) of the maternal mRNA, Wee1, mediates translational repression in immature Xenopus oocytes and translational activation in mature oocytes, but the protein that binds to the TCS and mediates translational control is not known. Here we show that Xenopus laevis Zar2 (encoded by zar2) binds to the TCS in maternal Wee1 mRNA and represses translation in immature oocytes. Using yeast 3 hybrid assays and electrophoretic mobility shift assays, Zar2 was shown to bind specifically to the TCS in the Wee1 3'UTR. RNA binding required the presence of Zn(2+) and conserved cysteines in the C-terminal domain, suggesting that Zar2 contains a zinc finger. Consistent with regulating maternal mRNAs, Zar2 was present throughout oogenesis, and endogenous Zar2 co-immunoprecipitated endogenous Wee1 mRNA from immature oocytes, demonstrating the physiological significance of the protein-RNA interaction. Interestingly, Zar2 levels decreased during oocyte maturation. Dual luciferase reporter tethered assays showed that Zar2 repressed translation in immature oocytes. Translational repression was relieved during oocyte maturation and this coincided with degradation of Zar2 during maturation. This is the first report of a molecular function of zygote arrest proteins. These data show that Zar2 contains a zinc finger and is a trans-acting factor for the TCS in maternal mRNAs in immature Xenopus oocytes.

  10. Negative protein 1, which is required for function of the chicken lysozyme gene silencer in conjunction with hormone receptors, is identical to the multivalent zinc finger repressor CTCF.

    PubMed Central

    Burcin, M; Arnold, R; Lutz, M; Kaiser, B; Runge, D; Lottspeich, F; Filippova, G N; Lobanenkov, V V; Renkawitz, R

    1997-01-01

    The transcriptional repressor negative protein 1 (NeP1) binds specifically to the F1 element of the chicken lysozyme gene silencer and mediates synergistic repression by v-ERBA, thyroid hormone receptor, or retinoic acid receptor. Another protein, CCCTC-binding factor (CTCF), specifically binds to 50-bp-long sequences that contain repetitive CCCTC elements in the vicinity of vertebrate c-myc genes. Previously cloned chicken, mouse, and human CTCF cDNAs encode a highly conserved 11-Zn-finger protein. Here, NeP1 was purified and DNA bases critical for NeP1-F1 interaction were determined. NeP1 is found to bind a 50-bp stretch of nucleotides without any obvious sequence similarity to known CTCF binding sequences. Despite this remarkable difference, these two proteins are identical. They have the same molecular weight, and NeP1 contains peptide sequences which are identical to sequences in CTCF. Moreover, NeP1 and CTCF specifically recognize each other's binding DNA sequence and induce identical conformational alterations in the F1 DNA. Therefore, we propose to replace the name NeP1 with CTCF. To analyze the puzzling sequence divergence in CTCF binding sites, we studied the DNA binding of 12 CTCF deletions with serially truncated Zn fingers. While fingers 4 to 11 are indispensable for CTCF binding to the human c-myc P2 promoter site A, a completely different combination of fingers, namely, 1 to 8 or 5 to 11, was sufficient to bind the lysozyme silencer site F1. Thus, CTCF is a true multivalent factor with multiple repressive functions and multiple sequence specificities. PMID:9032255

  11. Requirement of the C3HC4 zinc RING finger of the Arabidopsis PEX10 for photorespiration and leaf peroxisome contact with chloroplasts.

    PubMed

    Schumann, Uwe; Prestele, Jakob; O'Geen, Henriette; Brueggeman, Robert; Wanner, Gerhard; Gietl, Christine

    2007-01-16

    Plant peroxisomes perform multiple vital metabolic processes including lipid mobilization in oil-storing seeds, photorespiration, and hormone biosynthesis. Peroxisome biogenesis requires the function of peroxin (PEX) proteins, including PEX10, a C(3)HC(4) Zn RING finger peroxisomal membrane protein. Loss of function of PEX10 causes embryo lethality at the heart stage. We investigated the function of PEX10 with conditional sublethal mutants. Four T-DNA insertion lines expressing pex10 with a dysfunctional RING finger were created in an Arabidopsis WT background (DeltaZn plants). They could be normalized by growth in an atmosphere of high CO(2) partial pressure, indicating a defect in photorespiration. beta-Oxidation in mutant glyoxysomes was not affected. However, an abnormal accumulation of the photorespiratory metabolite glyoxylate, a lowered content of carotenoids and chlorophyll a and b, and a decreased quantum yield of photosystem II were detected under normal atmosphere, suggesting impaired leaf peroxisomes. Light and transmission electron microscopy demonstrated leaf peroxisomes of the DeltaZn plants to be more numerous, multilobed, clustered, and not appressed to the chloroplast envelope as in WT. We suggest that inactivation of the RING finger domain in PEX10 has eliminated protein interaction required for attachment of peroxisomes to chloroplasts and movement of metabolites between peroxisomes and chloroplasts.

  12. Finger Multiplication

    ERIC Educational Resources Information Center

    Holmes, Bill

    2010-01-01

    The author has been prompted to write this article about finger multiplication for a number of reasons. Firstly there are a number of related articles in past issues of "Mathematics Teaching" ("MT") which have connections to this algorithm. Secondly, very few of his primary teaching students and professional colleagues appear to be aware of the…

  13. Finger Foods for Babies

    MedlinePlus

    ... Feeding Your 1- to 2-Year-Old Finger Foods for Babies KidsHealth > For Parents > Finger Foods for ... will accept a new food. previous continue