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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. Ubiquitin interactions of NZF zinc fingers

    PubMed Central

    Alam, Steven L; Sun, Ji; Payne, Marielle; Welch, Brett D; Blake, B Kelly; Davis, Darrell R; Meyer, Hemmo H; Emr, Scott D; Sundquist, Wesley I

    2004-01-01

    Ubiquitin (Ub) functions in many different biological pathways, where it typically interacts with proteins that contain modular Ub recognition domains. One such recognition domain is the Npl4 zinc finger (NZF), a compact zinc-binding module found in many proteins that function in Ub-dependent processes. We now report the solution structure of the NZF domain from Npl4 in complex with Ub. The structure reveals that three key NZF residues (13TF14/M25) surrounding the zinc coordination site bind the hydrophobic ‘Ile44' surface of Ub. Mutations in the 13TF14/M25 motif inhibit Ub binding, and naturally occurring NZF domains that lack the motif do not bind Ub. However, substitution of the 13TF14/M25 motif into the nonbinding NZF domain from RanBP2 creates Ub-binding activity, demonstrating the versatility of the NZF scaffold. Finally, NZF mutations that inhibit Ub binding by the NZF domain of Vps36/ESCRT-II also inhibit sorting of ubiquitylated proteins into the yeast vacuole. Thus, the NZF is a versatile protein recognition domain that is used to bind ubiquitylated proteins during vacuolar protein sorting, and probably many other biological processes. PMID:15029239

  3. 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

  4. 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

  5. RNA binding by the Wilms tumor suppressor zinc finger proteins.

    PubMed Central

    Caricasole, A; Duarte, A; Larsson, S H; Hastie, N D; Little, M; Holmes, G; Todorov, I; Ward, A

    1996-01-01

    The Wilms tumor suppressor gene WT1 is implicated in the ontogeny of genito-urinary abnormalities, including Denys-Drash syndrome and Wilms tumor of the kidney. WT1 encodes Kruppel-type zinc finger proteins that can regulate the expression of several growth-related genes, apparently by binding to specific DNA sites located within 5' untranslated leader regions as well as 5' promoter sequences. Both WT1 and a closely related early growth response factor, EGR1, can bind the same DNA sequences from the mouse gene encoding insulin-like growth factor 2 (Igf-2). We report that WT1, but not EGR1, can bind specific Igf-2 exonic RNA sequences, and that the zinc fingers are required for this interaction. WT1 zinc finger 1, which is not represented in EGR1, plays a more significant role in RNA binding than zinc finger 4, which does have a counterpart in EGR1. Furthermore, the normal subnuclear localization of WT1 proteins is shown to be RNase, but not DNase, sensitive. Therefore, WT1 might, like the Kruppel-type zinc finger protein TFIIIA, regulate gene expression by both transcriptional and posttranscriptional mechanisms. Images Fig. 1 Fig. 2 Fig. 3 PMID:8755514

  6. 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

  7. Light-Inducible Gene Regulation with Engineered Zinc Finger Proteins

    PubMed Central

    Polstein, Lauren R.; Gersbach, Charles A.

    2014-01-01

    The coupling of light-inducible protein-protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to the DNA-binding site of a highly customizable engineered zinc finger protein. This chapter provides methods for modifying LITEZ to target new DNA sequences, engineering a programmable LED array to illuminate cell cultures, and using the modified LITEZ system to achieve spatiotemporal control of transgene expression in mammalian cells. PMID:24718797

  8. 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

  9. 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.

  10. 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.

  11. The interaction of DNA with multi-Cys2His2 zinc finger proteins

    NASA Astrophysics Data System (ADS)

    Liu, Lei; Heermann, Dieter W.

    2015-02-01

    The multi-Cys2His2 (mC2H2) zinc finger protein, like CTCF, plays a central role in the three-dimensional organization of chromatin and gene regulation. The interaction between DNA and mC2H2 zinc finger proteins becomes crucial to better understand how CTCF dynamically shapes the chromatin structure. Here, we study a coarse-grained model of the mC2H2 zinc finger proteins in complexes with DNA, and in particular, we study how a mC2H2 zinc finger protein binds to and searches for its target DNA loci. On the basis of coarse-grained molecular dynamics simulations, we present several interesting kinetic conformational properties of the proteins, such as the rotation-coupled sliding, the asymmetrical roles of different zinc fingers and the partial binding partial dangling mode. In addition, two kinds of studied mC2H2 zinc finger proteins, of CG-rich and AT-rich binding motif each, were able to recognize their target sites and slide away from their non-target sites, which shows a proper sequence specificity in our model and the derived force field for mC2H2-DNA interaction. A further application to CTCF shows that the protein binds to a specific DNA duplex only with its central zinc fingers. The zinc finger domains of CTCF asymmetrically bend the DNA, but do not form a DNA loop alone in our simulations.

  12. 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.

  13. Correlation between functional and binding activities of designer zinc-finger proteins

    PubMed Central

    Kang, Jong Seok

    2006-01-01

    Rapid progress in the ability to develop and utilize zinc-finger proteins with customized sequence specificity have led to their increasing use as tools for modulation of target gene transcription in the post-genomic era. In the present paper, a series of in vitro binding assays and in vivo reporter analyses were used to demonstrate that a zinc-finger protein can effectively specify a base at each position of the target site in vivo and that functional activity of the zinc-finger protein as either a transcriptional repressor or activator is positively correlated with its binding affinity. In addition, this correlation can be extended to artificial engineered zinc-finger proteins. These data suggest that the binding affinity of designer zinc-finger proteins with novel specificity might be a determinant for their ability to regulate transcription of a gene of interest. PMID:17176251

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

    DOE PAGESBeta

    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 chosenmore » parts performed insufficiently to create oscillations, but we include future directions for improvement upon our work presented here.« less

  15. Inhibition of virus DNA replication by artificial zinc finger proteins.

    PubMed

    Sera, Takashi

    2005-02-01

    Prevention of virus infections is a major objective in agriculture and human health. One attractive approach to the prevention is inhibition of virus replication. To demonstrate this concept in vivo, an artificial zinc finger protein (AZP) targeting the replication origin of the Beet severe curly top virus (BSCTV), a model DNA virus, was created. In vitro DNA binding assays indicated that the AZP efficiently blocked binding of the viral replication protein (Rep), which initiates virus replication, to the replication origin. All of the transgenic Arabidopsis plants expressing the AZP showed phenotypes strongly resistant to virus infection, and 84% of the transgenic plants showed no symptom. Southern blot analysis demonstrated that BSCTV replication was completely suppressed in the transgenic plants. Since the mechanism of viral DNA replication is well conserved among plants and mammals, this approach could be applied not only to agricultural crop protection but also to the prevention of virus infections in humans. PMID:15681461

  16. Modular Synthetic Inverters from Zinc Finger Proteins and Small RNAs

    PubMed Central

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

    2016-01-01

    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. Our chosen parts performed insufficiently to create oscillations, but we include future directions for improvement upon our work presented here. PMID:26886888

  17. A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity

    PubMed Central

    Blindauer, Claudia A.; Harrison, Mark D.; Parkinson, John A.; Robinson, Andrea K.; Cavet, Jennifer S.; Robinson, Nigel J.; Sadler, Peter J.

    2001-01-01

    Zinc is essential for many cellular processes, including DNA synthesis, transcription, and translation, but excess can be toxic. A zinc-induced gene, smtA, is required for normal zinc-tolerance in the cyanobacterium Synechococcus PCC 7942. Here we report that the protein SmtA contains a cleft lined with Cys-sulfur and His-imidazole ligands that binds four zinc ions in a Zn4Cys9His2 cluster. The thiolate sulfurs of five Cys ligands provide bridges between the two ZnCys4 and two ZnCys3His sites, giving two fused six-membered rings with distorted boat conformations. The inorganic core strongly resembles the Zn4Cys11 cluster of mammalian metallothionein, despite different amino acid sequences, a different linear order of the ligands, and presence of histidine ligands. Also, SmtA contains elements of secondary structure not found in metallothioneins. One of the two Cys4-coordinated zinc ions in SmtA readily exchanges with exogenous metal (111Cd), whereas the other is inert. The thiolate sulfur ligands bound to zinc in this site are buried within the protein. Regions of β-strand and α-helix surround the inert site to form a zinc finger resembling the zinc fingers in GATA and LIM-domain proteins. Eukaryotic zinc fingers interact specifically with other proteins or DNA and an analogous interaction can therefore be anticipated for prokaryotic zinc fingers. SmtA now provides structural proof for the existence of zinc fingers in prokaryotes, and sequences related to the zinc finger motif can be identified in several bacterial genomes. PMID:11493688

  18. 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.

  19. Crystallographicand Biochemical Analysis of the Ran-Binding Zinc Finger Domain

    SciTech Connect

    Partridge, J.; Schwartz, T

    2009-01-01

    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.

  20. HRT, a novel zinc finger, transcriptional repressor from barley.

    PubMed

    Raventós, D; Skriver, K; Schlein, M; Karnahl, K; Rogers, S W; Rogers, J C; Mundy, J

    1998-09-01

    A barley gene encoding a novel DNA-binding protein (HRT) was identified by southwestern screening with baits containing a gibberellin phytohormone response element from an alpha-amylase promoter. The HRT gene contains two introns, the larger of which (5722 base pairs (bp)) contains a 3094-bp LINE-like element with homology to maize Colonist1. In vitro mutagenesis and zinc- and DNA-binding assays demonstrate that HRT contains three unusual zinc fingers with a CX8-9CX10CX2H consensus sequence. HRT is targeted to nuclei, and homologues are expressed in other plants. In vivo, functional tests in plant cells indicate that full-length HRT can repress expression from certain promoters including the Amy1/6-4 and Amy2/32 alpha-amylase promoters. In contrast, truncated forms of HRT containing DNA-binding domains can activate, or derepress, transcription from these promoters. Northern hybridizations indicate that HRT mRNA accumulates to low levels in various tissues. Roles for HRT in mediating developmental and phytohormone-responsive gene expression are discussed. PMID:9722564

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

    DOE PAGESBeta

    Bach, Christian; Sherman, William; Pallis, Jani; Patra, Prabir; 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 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

  2. Sequence Discrimination by Alternatively Spliced Isoforms of a DNA Binding Zinc Finger Domain

    NASA Astrophysics Data System (ADS)

    Gogos, Joseph A.; Hsu, Tien; Bolton, Jesse; Kafatos, Fotis C.

    1992-09-01

    Two major developmentally regulated isoforms of the Drosophila chorion transcription factor CF2 differ by an extra zinc finger within the DNA binding domain. The preferred DNA binding sites were determined and are distinguished by an internal duplication of TAT in the site recognized by the isoform with the extra finger. The results are consistent with modular interactions between zinc fingers and trinucleotides and also suggest rules for recognition of AT-rich DNA sites by zinc finger proteins. The results show how modular finger interactions with trinucleotides can be used, in conjunction with alternative splicing, to alter the binding specificity and increase the spectrum of sites recognized by a DNA binding domain. Thus, CF2 may potentially regulate distinct sets of target genes during development.

  3. 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

  4. Comparative Analysis of Zinc Finger Proteins Involved in Plant Disease Resistance

    PubMed Central

    Gupta, Santosh Kumar; Rai, Amit Kumar; Kanwar, Shamsher Singh; Sharma, Tilak R.

    2012-01-01

    A meta-analysis was performed to understand the role of zinc finger domains in proteins of resistance (R) genes cloned from different crops. We analyzed protein sequences of seventy R genes of various crops in which twenty six proteins were found to have zinc finger domains along with nucleotide binding sites - leucine rice repeats (NBS-LRR) domains. We identified thirty four zinc finger domains in the R proteins of nine crops and were grouped into 19 types of zinc fingers. The size of individual zinc finger domain within the R genes varied from 11 to 84 amino acids, whereas the size of proteins containing these domains varied from 263 to 1305 amino acids. The biophysical analysis revealed that molecular weight of Pi54 zinc finger was lowest whereas the highest one was found in rice Pib zinc finger named as Transposes Transcription Factor (TTF). The instability (R2 = 0.95) and the aliphatic (R2 = 0.94) indices profile of zinc finger domains follows the polynomial distribution pattern. The pairwise identity analysis showed that the Lin11, Isl-1 & Mec-3 (LIM) zinc finger domain of rice blast resistance protein pi21 have 12.3% similarity with the nuclear transcription factor, X-box binding-like 1 (NFX) type zinc finger domain of Pi54 protein. For the first time, we reported that Pi54 (Pi-kh-Tetep), a rice blast resistance (R) protein have a small zinc finger domain of NFX type located on the C-terminal in between NBS and LRR domains of the R-protein. Compositional analysis depicted by the helical wheel diagram revealed the presence of a hydrophobic region within this domain which might help in exposing the LRR region for a possible R-Avr interaction. This domain is unique among all other cloned plant disease resistance genes and might play an important role in broad-spectrum nature of rice blast resistance gene Pi54. PMID:22916136

  5. Comparative analysis of zinc finger proteins involved in plant disease resistance.

    PubMed

    Gupta, Santosh Kumar; Rai, Amit Kumar; Kanwar, Shamsher Singh; Sharma, Tilak R

    2012-01-01

    A meta-analysis was performed to understand the role of zinc finger domains in proteins of resistance (R) genes cloned from different crops. We analyzed protein sequences of seventy R genes of various crops in which twenty six proteins were found to have zinc finger domains along with nucleotide binding sites - leucine rice repeats (NBS-LRR) domains. We identified thirty four zinc finger domains in the R proteins of nine crops and were grouped into 19 types of zinc fingers. The size of individual zinc finger domain within the R genes varied from 11 to 84 amino acids, whereas the size of proteins containing these domains varied from 263 to 1305 amino acids. The biophysical analysis revealed that molecular weight of Pi54 zinc finger was lowest whereas the highest one was found in rice Pib zinc finger named as Transposes Transcription Factor (TTF). The instability (R(2) =0.95) and the aliphatic (R(2) =0.94) indices profile of zinc finger domains follows the polynomial distribution pattern. The pairwise identity analysis showed that the Lin11, Isl-1 & Mec-3 (LIM) zinc finger domain of rice blast resistance protein pi21 have 12.3% similarity with the nuclear transcription factor, X-box binding-like 1 (NFX) type zinc finger domain of Pi54 protein. For the first time, we reported that Pi54 (Pi-k(h)-Tetep), a rice blast resistance (R) protein have a small zinc finger domain of NFX type located on the C-terminal in between NBS and LRR domains of the R-protein. Compositional analysis depicted by the helical wheel diagram revealed the presence of a hydrophobic region within this domain which might help in exposing the LRR region for a possible R-Avr interaction. This domain is unique among all other cloned plant disease resistance genes and might play an important role in broad-spectrum nature of rice blast resistance gene Pi54. PMID:22916136

  6. [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. PMID:24010359

  7. 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. PMID:23106502

  8. Targeted chromosomal deletions in human cells using zinc finger nucleases.

    PubMed

    Lee, Hyung Joo; Kim, Eunji; Kim, Jin-Soo

    2010-01-01

    We present a novel approach for generating targeted deletions of genomic segments in human and other eukaryotic cells using engineered zinc finger nucleases (ZFNs). We found that ZFNs designed to target two different sites in a human chromosome could introduce two concurrent DNA double-strand breaks (DSBs) in the chromosome and give rise to targeted deletions of the genomic segment between the two sites. Using this method in human cells, we were able to delete predetermined genomic DNA segments in the range of several-hundred base pairs (bp) to 15 mega-bp at frequencies of 10(-3) to 10(-1). These high frequencies allowed us to isolate clonal populations of cells, in which the target chromosomal segments were deleted, by limiting dilution. Sequence analysis revealed that many of the deletion junctions contained small insertions or deletions and microhomologies, indicative of DNA repair via nonhomologous end-joining. Unlike other genome engineering tools such as recombinases and meganucleases, ZFNs do not require preinsertion of target sites into the genome and allow precise manipulation of endogenous genomic scripts in animal and plant cells. Thus, ZFN-induced genomic deletions should be broadly useful as a novel method in biomedical research, biotechnology, and gene therapy. PMID:19952142

  9. Dopamine receptor regulating factor, DRRF: a zinc finger transcription factor.

    PubMed

    Hwang, C K; D'Souza, U M; Eisch, A J; Yajima, S; Lammers, C H; Yang, Y; Lee, S H; Kim, Y M; Nestler, E J; Mouradian, M M

    2001-06-19

    Dopamine receptor genes are under complex transcription control, determining their unique regional distribution in the brain. We describe here a zinc finger type transcription factor, designated dopamine receptor regulating factor (DRRF), which binds to GC and GT boxes in the D1A and D2 dopamine receptor promoters and effectively displaces Sp1 and Sp3 from these sequences. Consequently, DRRF can modulate the activity of these dopamine receptor promoters. Highest DRRF mRNA levels are found in brain with a specific regional distribution including olfactory bulb and tubercle, nucleus accumbens, striatum, hippocampus, amygdala, and frontal cortex. Many of these brain regions also express abundant levels of various dopamine receptors. In vivo, DRRF itself can be regulated by manipulations of dopaminergic transmission. Mice treated with drugs that increase extracellular striatal dopamine levels (cocaine), block dopamine receptors (haloperidol), or destroy dopamine terminals (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) show significant alterations in DRRF mRNA. The latter observations provide a basis for dopamine receptor regulation after these manipulations. We conclude that DRRF is important for modulating dopaminergic transmission in the brain. PMID:11390978

  10. Sequence-specific DNA binding by glucocorticoid receptor "zinc finger peptides".

    PubMed

    Archer, T K; Hager, G L; Omichinski, J G

    1990-10-01

    Steroid hormone receptors can activate or repress transcription from responsive loci by binding to DNA. We have examined the mechanism of DNA binding by individually synthesizing the putative "zinc finger peptides" from the rat glucocorticoid receptor. Atomic absorption studies show that the peptides will bind zinc on an equimolar basis, and circular dichroism experiments demonstrate a significant alteration in secondary structure in the presence of zinc. The results from a series of experiments establish that metal ion is required for binding to DNA and that the amino-terminal zinc finger shows a significantly greater affinity for glucocorticoid response element-containing DNA over control DNA. These observations indicate that a single synthetic "zinc finger peptide" is able to bind to DNA in a sequence-specific manner. PMID:2120703

  11. 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

  12. A gene encoding 22 highly related zinc fingers is expressed in lymphoid cell lines.

    PubMed Central

    Lovering, R; Trowsdale, J

    1991-01-01

    A cDNA was isolated from a T cell library using an oligonucleotide probe corresponding to a sequence conserved in proteins with multiple zinc fingers of the C2H2 type. The predicted protein structure of this cDNA (ZNF43) showed that it contained 22 of the Krüppel type of zinc finger motifs in tandem. The amino acid sequence was strongly conserved between each of the finger domains of this cDNA, except for variable residue positions within the putative DNA binding site. Within the zinc finger domain the amino acid sequence of the four zinc fingers 6 to 9 was very similar to the amino acid sequence of fingers 10 to 13, the DNA sequence bound by this group of eight fingers may include a short repeat. Southern blotting showed that ZNF43 was one of a closely related family of proteins with 10 to 20 members. The members of the ZNF43 family did not appear to be clustered at the chromosomal level. The transcription of many members of this gene family was increased in lymphoid cell lines. After in vitro induced terminal differentiation of the human HL60 cell line the expression of the ZNF43 family was reduced. The expression of the ZNF43 gene was mainly limited to T and B cell lines. The gene was differentially spliced and different cell lines expressed different combinations of transcripts. Images PMID:1711675

  13. Disruption of the RAG2 zinc finger motif impairs protein stability and causes immunodeficiency.

    PubMed

    Xu, Ke; Liu, Haifeng; Shi, Zhubing; Song, Guangrong; Zhu, Xiaoyan; Jiang, Yuzhang; Zhou, Zhaocai; Liu, Xiaolong

    2016-04-01

    Although the RAG2 core domain is the minimal region required for V(D)J recombination, the noncore region also plays important roles in the regulation of recombination, and mutations in this region are often related to severe combined immunodeficiency. A complete understanding of the functions of the RAG2 noncore region and the potential contributions of its individual residues has not yet been achieved. Here, we show that the zinc finger motif within the noncore region of RAG2 is indispensable for maintaining the stability of the RAG2 protein. The zinc finger motif in the noncore region of RAG2 is highly conserved from zebrafish to humans. Knock-in mice carrying a zinc finger mutation (C478Y) exhibit decreased V(D)J recombination efficiency and serious impairment in T/B-cell development due to RAG2 instability. Further studies also reveal the importance of the zinc finger motif for RAG2 stability. Moreover, mice harboring a RAG2 noncore region mutation (N474S), which is located near C478 but is not zinc-binding, exhibit no impairment in either RAG2 stability or T/B-cell development. Taken together, our findings contribute to defining critical functions of the RAG2 zinc finger motif and provide insights into the relationships between the mutations within this motif and immunodeficiency diseases. PMID:26692406

  14. Deep Vertebrate Roots for Mammalian Zinc Finger Transcription Factor Subfamilies

    PubMed Central

    Liu, Hui; Chang, Li-Hsin; Sun, Younguk; Lu, Xiaochen; Stubbs, Lisa

    2014-01-01

    While many vertebrate transcription factor (TF) families are conserved, the C2H2 zinc finger (ZNF) family stands out as a notable exception. In particular, novel ZNF gene types have arisen, duplicated, and diverged independently throughout evolution to yield many lineage-specific TF genes. This evolutionary dynamic not only raises many intriguing questions but also severely complicates identification of those ZNF genes that remain functionally conserved. To address this problem, we searched for vertebrate “DNA binding orthologs” by mining ZNF loci from eight sequenced genomes and then aligning the patterns of DNA-binding amino acids, or “fingerprints,” extracted from the encoded ZNF motifs. Using this approach, we found hundreds of lineage-specific genes in each species and also hundreds of orthologous groups. Most groups of orthologs displayed some degree of fingerprint divergence between species, but 174 groups showed fingerprint patterns that have been very rigidly conserved. Focusing on the dynamic KRAB-ZNF subfamily—including nearly 400 human genes thought to possess potent KRAB-mediated epigenetic silencing activities—we found only three genes conserved between mammals and nonmammalian groups. These three genes, members of an ancient familial cluster, encode an unusual KRAB domain that functions as a transcriptional activator. Evolutionary analysis confirms the ancient provenance of this activating KRAB and reveals the independent expansion of KRAB-ZNFs in every vertebrate lineage. Most human ZNF genes, from the most deeply conserved to the primate-specific genes, are highly expressed in immune and reproductive tissues, indicating that they have been enlisted to regulate evolutionarily divergent biological traits. PMID:24534434

  15. 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

  16. Quantum chemical modelling of reactivity and selectivity of 1,2-dithiolanes towards retroviral and cellular zinc fingers

    NASA Astrophysics Data System (ADS)

    Topol, Igor A.; Nemukhin, Alexander V.; Burt, Stanley K.

    Interactions of 1,2-dithiolane species with zinc-containing sites, which mimic the zinc finger domains of retroviral and the cellular zinc finger proteins, have been investigated by quantum chemistry tools. According to the calculations, the immediate domains of zinc binding sites in the cellular and retroviral zinc fingers interact differently with such agents of the disulphide family. Thus, when approaching the model cellular-type domains, the molecules of 1,2-dithiolanes experience considerable potential barriers along the reaction path. However, these species react practically barrier-less with the model retroviral-type domains at the correlated DFT level. The results of the quantum chemical modelling provide firm support to the selectivity of 1,2-dithiolanes towards retroviral and cellular zinc fingers. This can be of great practical importance for the design of therapeutics that accomplish functional inactivation of the zinc fingers of the human immunodeficiency virus (HIV-1) retroviral type nucleocapsid protein NCp7.

  17. Crystal Structure of an Unusual Thioredoxin Protein with a Zinc Finger Domain

    SciTech Connect

    Ye, J.; Cho, S; Fuselier, J; Li, W; Beckwith, J; Rapoport, T

    2007-01-01

    Many Gram-negative bacteria have two cytoplasmic thioredoxins, thioredoxin-1 and -2, encoded by the trxA and trxC genes, respectively. Both thioredoxins have the highly conserved WCGPC motif and function as disulfide-bond reductases. However, thioredoxin-2 has unique features: it has an N-terminal motif that binds a zinc ion, and its transcription is under the control of OxyR, which allows it to be up-regulated under oxidative stress. Here, we report the crystal structure of thioredoxin-2 from Rhodobacter capsulatus. The C-terminal region of thioredoxin-2 forms a canonical thioredoxin fold with a central {beta}-sheet consisting of five strands and four flanking {alpha}-helices on either side. The N-terminal zinc finger is composed of four short {beta}-strands (S1-S4) connected by three short loops (L1-L3). The four cysteines are at loops L1 and L3 and form a tetragonal binding site for a zinc ion. The zinc finger is close to the first {beta}-strand and first {alpha}-helix of the thioredoxin fold. Nevertheless, the zinc finger may not directly affect the oxidoreductase activity of thioredoxin-2 because the zinc finger is not near the active site of a protomer and because thioredoxin-2 is a monomer in solution. On the basis of structural similarity to the zinc fingers in Npl4 and Vps36, we propose that the N-terminal zinc finger of thioredoxin-2 mediates protein-protein interactions, possibly with its substrates or chaperones.

  18. Potential application of FoldX force field based protein modeling in zinc finger nucleases design.

    PubMed

    He, ZuYong; Mei, Gui; Zhao, ChunPeng; Chen, YaoSheng

    2011-05-01

    Engineered sequence-specific zinc finger nucleases (ZFNs) make the highly efficient modification of eukaryotic genomes possible. However, most current strategies for developing zinc finger nucleases with customized sequence specificities require the construction of numerous tandem arrays of zinc finger proteins (ZFPs), and subsequent largescale in vitro validation of their DNA binding affinities and specificities via bacterial selection. The labor and expertise required in this complex process limits the broad adoption of ZFN technology. An effective computational assisted design strategy will lower the complexity of the production of a pair of functional ZFNs. Here we used the FoldX force field to build 3D models of 420 ZFP-DNA complexes based on zinc finger arrays developed by the Zinc Finger Consortium using OPEN (oligomerized pool engineering). Using nonlinear and linear regression analysis, we found that the calculated protein-DNA binding energy in a modeled ZFP-DNA complex strongly correlates to the failure rate of the zinc finger array to show significant ZFN activity in human cells. In our models, less than 5% of the three-finger arrays with calculated protein-DNA binding energies lower than -13.132 kcal mol(-1) fail to form active ZFNs in human cells. By contrast, for arrays with calculated protein-DNA binding energies higher than -5 kcal mol(-1), as many as 40% lacked ZFN activity in human cells. Therefore, we suggest that the FoldX force field can be useful in reducing the failure rate and increasing efficiency in the design of ZFNs. PMID:21455692

  19. A Xenopus zinc finger protein that specifically binds dsRNA and RNA-DNA hybrids.

    PubMed

    Finerty, P J; Bass, B L

    1997-08-15

    Proteins containing C2H2 type zinc finger motifs represent one of the largest classes of nucleic acid-binding proteins found in nature. We describe a novel zinc finger protein, dsRBP-ZFa, isolated by screening an expression library with dsRNA. The dsRBP-ZFa cDNA encodes a protein containing seven zinc finger motifs and an acidic C-terminal domain. Mobility shift experiments demonstrate that dsRBP-ZFa binds dsRNA and RNA-DNA hybrids with nanomolar dissociation constants and in a sequence independent manner. We also show that DNA and single stranded RNA fail to compete with dsRNA for binding suggesting dsRBP-ZFa prefers to bind an A-form helix. Using western analyses we have localized dsRBP-ZFa primarily to the nucleus of Xenopus laevis oocytes. The identification of dsRBP-ZFa provides the first example of a zinc finger protein that is specific for dsRNA. In addition, dsRBP-ZFa does not contain the previously described dsRNA binding motif, suggesting certain zinc fingers may provide an alternative way to recognize the A-form helix. PMID:9268652

  20. Multiple bacteria encode metallothioneins and SmtA-like zinc fingers.

    PubMed

    Blindauer, Claudia A; Harrison, Mark D; Robinson, Andrea K; Parkinson, John A; Bowness, Peter W; Sadler, Peter J; Robinson, Nigel J

    2002-09-01

    Zinc is essential but toxic in excess. Bacterial metallothionein, SmtA from Synechococcus PCC 7942, sequesters and detoxifies four zinc ions per molecule and contains a zinc finger structurally similar to eukaryotic GATA. The dearth of other reported bacterial metallothioneins has been surprising. Here we describe related bacterial metallothioneins (BmtA) from Anabaena PCC 7120, Pseudomonas aeruginosa and Pseudomonas putida that bind multiple zinc ions with high stability towards protons. Thiol modification demonstrates that cysteine coordinates zinc in all of these proteins. Additionally, (111)Cd-NMR, and (111)Cd-edited (1)H-NMR, identified histidine ligands in Anabaena PCC 7120 BmtA, analogous to SmtA. A related Escherichia coli protein bound only a single zinc ion, via four cysteine residues, with low stability towards protons; (111)Cd-NMR and (111)Cd-edited (1)H-NMR confirmed exclusive cysteine-coordination, and these cysteine residues reacted rapidly with 5,5'-dithiobis-(2-nitrobenzoic acid). (1)H-NMR of proteins from P. aeruginosa, Anabaena PCC 7120 and E. coli generated fingerprints diagnostic for the GATA-like zinc finger fold of SmtA. These studies reveal first the existence of multiple bacterial metallothioneins, and second proteins with SmtA-like lone zinc fingers, devoid of a cluster,and designated GatA. We have identified 12 smtA-like genes in sequence databases including four of the gatA type. PMID:12207707

  1. Purification of proteins containing zinc finger domains using Immobilized Metal Ion Affinity Chromatography

    PubMed Central

    Voráčková, Irena; Suchanová, Šárka; Ulbrich, Pavel; Diehl, William E.; Ruml, Tomáš

    2011-01-01

    Heterologous proteins are frequently purified by Immobilized Metal Ion Affinity Chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these approaches, we took an advantage of natural interaction of zinc finger domains with metal ions to purify functionally similar retroviral proteins from two different retroviruses. We found that these proteins exhibited significantly different affinities to the immobilized metal ions, despite that both contain the same type of zinc finger motif (i.e. CCHC). While zinc finger proteins may differ in biochemical properties, the multitude of IMAC platforms should allow relatively simple yet specific method for their isolation in native state. PMID:21600288

  2. Somatic human ZBTB7A zinc finger mutations promote cancer progression.

    PubMed

    Liu, X-S; Liu, Z; Gerarduzzi, C; Choi, D E; Ganapathy, S; Pandolfi, P P; Yuan, Z-M

    2016-06-01

    We recently reported that ZBTB7A is a bona fide transcription repressor of key glycolytic genes and its downregulation in human cancer contributes to tumor metabolism. As reduced expression of ZBTB7A is found only in a subset of human cancers, we explored alternative mechanisms of its inactivation by mining human cancer genome databases. We discovered recurrent somatic mutations of ZBTB7A in multiple types of human cancers with a marked enrichment of mutations within the zinc finger domain. Functional characterization of the mutants demonstrated that mutations within the zinc finger region of ZBTB7A invariably resulted in loss of function. As a consequence, the glycolytic genes were markedly upregulated in cancer cells harboring ZBTB7A zinc finger mutation, leading to increased glycolysis and proliferation. Our study uncovers the loss-of-function mutation in ZBTB7A as a novel mechanism causing elevated glycolysis in human cancer, which carries important therapeutic implication. PMID:26455326

  3. 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. PMID:27238756

  4. 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.

  5. Role and Regulation of Myeloid Zinc Finger Protein 1 in Cancer.

    PubMed

    Eguchi, Taka; Prince, Thomas; Wegiel, Barbara; Calderwood, Stuart K

    2015-10-01

    Myeloid zinc finger 1 (MZF1) belongs to the SCAN-Zinc Finger (SCAN-ZF) transcription factor family that has recently been implicated in a number of types of cancer. Although the initial studies concentrated on the role of MZF1 in myeloid differentiation and leukemia, the factor now appears to be involved in the etiology of major solid tumors such as lung, cervical, breast, and colorectal cancer. Here we discuss the regulation of MZF1 that mediated its recruitment and activation in cancer, concentrating on posttranslational modification by phosphorylation, and sumoylation, formation of promyelocytic leukemia nuclear bodies and its association with co-activators and co-repressors. PMID:25903835

  6. Proteomic analysis of MCF-7 cell lines expressing the zinc-finger or the proline-rich domain of retinoblastoma-interacting-zinc-finger protein.

    PubMed

    Chambery, Angela; Farina, Annarita; Di Maro, Antimo; Rossi, Mariangela; Abbondanza, Ciro; Moncharmont, Bruno; Malorni, Livia; Cacace, Giuseppina; Pocsfalvi, Gabriella; Malorni, Antonio; Parente, Augusto

    2006-05-01

    To identify a growth-promoting activity related to retinoblastoma-interacting-zinc-finger (RIZ) protein, differential protein expression of MCF-7 cell lines expressing the zinc-finger or the proline-rich domain of RIZ protein was analyzed by a robust bottom-up mass-spectrometry proteomic approach. Spots corresponding to qualitative and quantitative differences in protein expression have been selected and identified. Some of these proteins have been previously reported as being associated with different types of carcinomas or involved in cell proliferation and differentiation. Knowledge of specific differentially expressed proteins by MCF-7-derived cell lines expressing RIZ different domains will provide the basis for identifying a growth-promoting activity related to RIZ gene products. PMID:16674107

  7. Herbivory responsive C2H2 zinc finger transcription factor protein StZFP2 from potato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While C2H2 zinc finger transcription factors are often regulated by abiotic stress, their role during insect infestation has been overlooked. This study demonstrates that the transcripts of the zinc finger transcription factors StZFP1 and StZFP2 are induced in potato (Solanum tuberosum) upon infesta...

  8. Expression and RNA-binding of human zinc-finger antiviral protein

    SciTech Connect

    Jeong, Mi Suk; Kim, Eun Jung; Jang, Se Bok

    2010-06-04

    Zinc-finger antiviral protein (ZAP) is a recently isolated host antiviral factor that inhibits the replication of many viruses such as Moloney murine leukemia virus (MLV) and Sindbis virus (SIN) by preventing the accumulation of viral mRNA in the cytoplasm. ZAP comprises four CCCH zinc-finger motifs, the second and fourth of which are responsible for protein activity based on their integrity. Thus far, there have been no reports on whether or not ZAP expressed in Escherichia coli is soluble. Therefore, we expressed N-terminal ZAP (NZAP, 254 amino acids) in E. coli as a fusion protein with several different cleavage sites and protein tags. Cleaved ZAP in soluble form strongly bound to RNA through its four CCCH zinc-finger motifs. Here, we provide evidence indicating that ZAP directly interacted with viral RNA. Each conserved zinc-finger motif of ZAP coordinates a zinc ion using three cysteines and one histidine. These findings suggest that ZAP recruits the cellular RNA degradation machinery for the degradation of viral RNA.

  9. C terminal retroviral-type zinc finger domain from the HIV-1 nucleocapsid protein is structurally similar to the N-terminal zinc finger domain

    SciTech Connect

    South, T.L.; Blake, P.R. ); Hare, D.R.; Summers, M.F. )

    1991-06-25

    Two-dimensional NMR spectroscopic and computational methods were employed for the structure determination of an 18-residue peptide with the amino acid sequence of the C-terminal retriviral-type (r.t.) zinc finger domain from the nucleocapsid protein (NCP) of HIV-1 (Zn(HIV1-F2)). Unlike results obtained for the first retroviral-type zinc finger peptide, Zn (HIV1-F1) broad signals indicative of confomational lability were observed in the {sup 1}H NMR spectrum of An(HIV1-F2) at 25 C. The NMR signals narrowed upon cooling to {minus}2 C, enabling complete {sup 1}H NMR signal assignment via standard two-dimensional (2D) NMR methods. Distance restraints obtained from qualitative analysis of 2D nuclear Overhauser effect (NOESY) data were sued to generate 30 distance geometry (DG) structures with penalties in the range 0.02-0.03 {angstrom}{sup 2}. All structures were qualitatively consistent with the experimental NOESY spectrum based on comparisons with 2D NOESY back-calculated spectra. These results indicate that the r.t. zinc finger sequences observed in retroviral NCPs, simple plant virus coat proteins, and in a human single-stranded nucleic acid binding protein share a common structural motif.

  10. Recruitment of mRNA-destabilizing protein TIS11 to stress granules is mediated by its zinc finger domain

    SciTech Connect

    Murata, Tomiyasu; Morita, Noriyoshi; Hikita, Kiyomi; Kiuchi, Kiyomi; Kiuchi, Kazutoshi; Kaneda, Norio . E-mail: nkaneda@ccmfs.meijo-u.ac.jp

    2005-02-15

    TIS11, a member of the CCCH zinc finger protein family, was found to be distributed throughout cells with a preferential cytoplasmic localization when transiently expressed in COS-7 cells. Upon treatment with heat shock, TIS11 became localized in discrete particles in the cytoplasm of the transfectants. We showed the TIS11-positive particles to be stress granules (SGs), which are known to be formed in the cytoplasm of eukaryotic cells in response to environmental stresses. By deletion studies using the green fluorescent protein fusion system, we mapped a functional stress granule (SG) localization signal to a region containing two tandem repeats of the zinc finger motif of TIS11. Site-directed mutations of Tyr105/Tyr113, Gly109/Gly 114, and Phe119 in the first zinc finger motif diminished the ability of this TIS11 domain to direct SG localization. Importantly, when the zinc-chelating Cys residues in either the first or second zinc finger were mutated to Ala residues, the recruitment of the TIS11 zinc finger region to SG was significantly inhibited by the mutation and was completely abolished by the mutation in both zinc fingers. These results suggest that recruitment of TIS11 to heat shock-induced SG is governed by the tandem zinc finger domains of this protein.

  11. 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

  12. Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish

    PubMed Central

    Zhu, Cong; Smith, Tom; McNulty, Joseph; Rayla, Amy L.; Lakshmanan, Abirami; Siekmann, Arndt F.; Buffardi, Matthew; Meng, Xiangdong; Shin, Jimann; Padmanabhan, Arun; Cifuentes, Daniel; Giraldez, Antonio J.; Look, A. Thomas; Epstein, Jonathan A.; Lawson, Nathan D.; Wolfe, Scot A.

    2011-01-01

    Zinc-finger nucleases (ZFNs) allow targeted gene inactivation in a wide range of model organisms. However, construction of target-specific ZFNs is technically challenging. Here, we evaluate a straightforward modular assembly-based approach for ZFN construction and gene inactivation in zebrafish. From an archive of 27 different zinc-finger modules, we assembled more than 70 different zinc-finger cassettes and evaluated their specificity using a bacterial one-hybrid assay. In parallel, we constructed ZFNs from these cassettes and tested their ability to induce lesions in zebrafish embryos. We found that the majority of zinc-finger proteins assembled from these modules have favorable specificities and nearly one-third of modular ZFNs generated lesions at their targets in the zebrafish genome. To facilitate the application of ZFNs within the zebrafish community we constructed a public database of sites in the zebrafish genome that can be targeted using this archive. Importantly, we generated new germline mutations in eight different genes, confirming that this is a viable platform for heritable gene inactivation in vertebrates. Characterization of one of these mutants, gata2a, revealed an unexpected role for this transcription factor in vascular development. This work provides a resource to allow targeted germline gene inactivation in zebrafish and highlights the benefit of a definitive reverse genetic strategy to reveal gene function. PMID:21937602

  13. Mutant and overexpression analysis of a C2H2 single zinc finger gene of Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The goal of this work was to characterize an Arabidopsis C2H2 single zinc finger gene, named AtZFP11, that is similar to SUPERMAN (SUP) and RABBIT EARS (RBE). No altered phenotype was observed in mutants analyzed that were derived through TILLING, nor a T-DNA insertion into the exon of AtZFP11. Pl...

  14. Alteration of zif268 zinc-finger motifs gives rise to non-native zinc-co-ordination sites but preserves wild-type DNA recognition.

    PubMed Central

    Green, A; Sarkar, B

    1998-01-01

    Zinc fingers are among the major structural motifs found in proteins that are involved in eukaryotic gene regulation. Many of these zinc-finger domains are involved in DNA binding. This study investigated whether the zinc-co-ordinating (Cys)2(His)2 motif found in the three zinc fingers of zif268 could be replaced by a (Cys)4 motif while still preserving DNA recognition. (Cys)2(His)2-to-(Cys)4 mutations were generated in each of the three zinc fingers of zif268 individually, as well as in fingers 1 and 3, and fingers 2 and 3 together. Whereas finger 1 and finger 3 tolerate the switch, such an alteration in finger 2 renders the polypeptide incapable of DNA recognition. The protein-DNA interaction was examined in greater detail by using a methylation-interference assay. The mutant polypeptides containing the (Cys)4 motif in fingers 1 or 3 recognize DNA in a manner identical to the wild-type protein, suggesting that the (Cys)4 motif appears to give rise to a properly folded finger. Additional results indicate that a zif268 variant containing a (Cys)2(His)(Ala) arrangement in finger 1 is also capable of DNA recognition in a manner identical to the wild-type polypeptide. This appears to be the first time that such alterations, in the context of an intact DNA-binding domain, have still allowed for specific DNA recognition. Taken together, the work presented here enhances our understanding of the relationship between metal ligation and DNA-binding by zinc fingers. PMID:9639566

  15. Rapid Mutation of Endogenous Zebrafish Genes Using Zinc Finger Nucleases Made by Oligomerized Pool ENgineering (OPEN)

    PubMed Central

    Maeder, Morgan L.; Reyon, Deepak; Sander, Jeffry D.; Peterson, Randall T.; Joung, J. Keith

    2009-01-01

    Background Customized zinc finger nucleases (ZFNs) form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-joining-mediated repair of ZFN-induced DNA double-stranded breaks. The Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology, recently described the first rapid, highly effective, and publicly available method for engineering zinc finger arrays. The Consortium has previously used this new method (known as OPEN for Oligomerized Pool ENgineering) to generate high quality ZFN pairs that function in human and plant cells. Methodology/Principal Findings Here we show that OPEN can also be used to generate ZFNs that function efficiently in zebrafish. Using OPEN, we successfully engineered ZFN pairs for five endogenous zebrafish genes: tfr2, dopamine transporter, telomerase, hif1aa, and gridlock. Each of these ZFN pairs induces targeted insertions and deletions with high efficiency at its endogenous gene target in somatic zebrafish cells. In addition, these mutations are transmitted through the germline with sufficiently high frequency such that only a small number of fish need to be screened to identify founders. Finally, in silico analysis demonstrates that one or more potential OPEN ZFN sites can be found within the first three coding exons of more than 25,000 different endogenous zebrafish gene transcripts. Conclusions and Significance In summary, our study nearly triples the total number of endogenous zebrafish genes successfully modified using ZFNs (from three to eight) and suggests that OPEN provides a reliable

  16. Structure and Ubiquitin Interactions of the Conserved Zinc Finger Domain of Npl4*

    PubMed Central

    Wang, Bin; Alam, Steven L.; Meyer, Hemmo H.; Payne, Marielle; Stemmler, Timothy L.; Davis, Darrell R.; Sundquist, Wesley I.

    2012-01-01

    Ubiquitylated proteins are directed into a large number of different cellular pathways through interactions with effector proteins that contain conserved ubiquitin binding motifs. Here, we report the solution structure and ubiquitin binding properties of one such motif, the Npl4 zinc finger or RanBP2/Nup358 zinc finger (NZF) domain. Npl4 NZF forms a compact module composed of four antiparallel β-strands linked by three ordered loops. A single zinc ion is coordinated by four conserved cysteines from the first and third loops, which form two rubredoxin knuckles. Npl4 NZF binds specifically, but weakly, to free ubiquitin using a conserved 13TF14 dipeptide to interact with the “Ile-44” surface of ubiquitin. Our studies reveal the structure of this versatile class of protein binding domains and provide a means for identifying the subset of NZF domains likely to bind ubiquitin. PMID:12644454

  17. EOR-2 Is an Obligate Binding Partner of the BTB–Zinc Finger Protein EOR-1 in Caenorhabditis elegans

    PubMed Central

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

    2010-01-01

    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. PMID:20065070

  18. Exploration of the Zinc Finger Motif in Controlling Activity of Matrix Metalloproteinases

    PubMed Central

    2015-01-01

    Discovering ways to control the activity of matrix metalloproteinases (MMPs), zinc-dependent enzymes capable of degrading extracellular matrix proteins, is an important field of cancer research. We report here a novel strategy for assembling MMP inhibitors on the basis of oligopeptide ligands by exploring the pattern known as the zinc finger motif. Advanced molecular modeling tools were used to characterize the structural binding motifs of experimentally tested MMP inhibitors, as well as those of newly proposed peptidomimetics, in their zinc-containing active sites. The results of simulations based on the quantum mechanics/molecular mechanics (QM/MM) approach and Car–Parrinello molecular dynamics with QM/MM potentials demonstrate that, upon binding of Regasepin1, a known MMP-9 inhibitor, the Zn2+(His3) structural element is rearranged to the Zn2+(Cys2His2) zinc finger motif, in which two Cys residues are borrowed from the ligand. Following consideration of the crystal structure of MMP-2 with its inhibitor, the oligopeptide APP-IP, we proposed a new peptidomimetic with two replacements in the substrate, Tyr3Cys and Asp6Cys. Simulations show that this peptide variant blocks an enzyme active site by the Zn2+(Cys2His2) zinc finger construct. Similarly, a natural substrate of MMP-2, Ace-Gln-Gly ∼ Ile-Ala-Gly-Nme, can be converted to an inhibiting compound by two replacements, Ile by Cys and Gly by the d isomer of Cys, favoring formation of the zinc finger motif. PMID:25375834

  19. Lead neurotoxicity: exploring the potential impact of lead substitution in zinc-finger proteins on mental health.

    PubMed

    Ordemann, Jacqueline Michelle; Austin, Rachel Narehood

    2016-06-01

    Childhood lead poisoning is a costly and largely preventable public health problem that lowers IQs, decreases attention spans, and leads to the development of other childhood intellectual disabilities. Furthermore, recent evidence links developmental lead poisoning with the etiology of disorders that appear much later in life, such as Alzheimer's disease, Parkinson's disease, and schizophrenia. Little is known about how lead influences the onset of these disorders. This paper reviews the evidence that lead substitution for zinc in zinc-finger proteins contributes to the development of Alzheimer's disease, Parkinson's disease, and schizophrenia. The zinc-finger proteins potentially impacted by lead include DNA methyltransferase 1 (DNMT1) and Presenilin 1 and 2 (PSEN1/2) in Alzheimer's disease, the dopamine receptor in Parkinson's disease, and the NMDA receptor, zinc-finger protein 804A (ZNF804A), and disrupted-in-schizophrenia 1 (DISC1)-binding zinc-finger (DBZ) in schizophrenia. PMID:26745006

  20. The third to fifth zinc fingers play an essential role in the binding of ZFP809 to the MLV-derived PBS.

    PubMed

    Ichida, Yu; Utsunomiya, Yuko; Onodera, Masafumi

    2016-01-15

    Members of the kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family mediate a number of cellular processes through binding to target DNA sequences via zinc fingers. Generally, zinc fingers recognize three-nucleotide sequences; however, this rule is not universally applicable. Zinc finger protein 809 (ZFP809) belongs to the KRAB-ZFP family and functions in repressing the expression of Moloney murine leukemia virus (MoMLV) via sequence-specific binding to the primer-binding site (PBS) located downstream of the MoMLV-long terminal repeat (LTR) and the induction of epigenetic modifications at LTR, such as repressive histone modifications and de novo DNA methylation. Previously, we demonstrated the role of the first to fifth zinc fingers of ZFP809 in binding to MLV PBS, indicating these zinc fingers do not recognize MLV PBS as a three-nucleotide sequence. Therefore, in the present study, we constructed truncated and mutated zinc fingers and examined their ability to bind to MLV PBS. The third to fifth zinc fingers of ZFP809 were found to be essential for binding to MLV PBS. Furthermore, the results of the present study indicate that other zinc fingers, which were not directly involved in binding to MLV PBS, may function in potentiating binding and stable protein expression. Further characterization of the amino acid sequences of zinc fingers will help further elucidate the functions and features of KRAB-ZFP and other zinc finger proteins. PMID:26692479

  1. Fear-of-intimacy-mediated zinc transport controls the function of zinc-finger transcription factors involved in myogenesis.

    PubMed

    Carrasco-Rando, Marta; Atienza-Manuel, Alexandra; Martín, Paloma; Burke, Richard; Ruiz-Gómez, Mar

    2016-06-01

    Zinc is a component of one-tenth of all human proteins. Its cellular concentration is tightly regulated because its dyshomeostasis has catastrophic health consequences. Two families of zinc transporters control zinc homeostasis in organisms, but there is little information about their specific developmental roles. We show that the ZIP transporter Fear-of-intimacy (Foi) is necessary for the formation of Drosophila muscles. In foi mutants, myoblasts segregate normally, but their specification is affected, leading to the formation of a misshapen muscle pattern and distorted midgut. The observed phenotypes could be ascribed to the inactivation of specific zinc-finger transcription factors (ZFTFs), supporting the hypothesis that they are a consequence of intracellular depletion of zinc. Accordingly, foi phenotypes can be rescued by mesodermal expression of other ZIP members with similar subcellular localization. We propose that Foi acts mostly as a transporter to regulate zinc intracellular homeostasis, thereby impacting on the activity of ZFTFs that control specific developmental processes. Our results additionally suggest a possible explanation for the presence of large numbers of zinc transporters in organisms based on differences in ion transport specificity and/or degrees of activity among transporters. PMID:27068109

  2. TBC-Domain GAPs for Rab GTPases Accelerate GTP Hydrolysis by a Dual-Finger Mechanism

    SciTech Connect

    Pan,X.; Eathiraj, S.; Lambright, D.

    2006-01-01

    Rab GTPases regulate membrane trafficking by cycling between inactive (GDP-bound) and active (GTP-bound) conformations. The duration of the active state is limited by GTPase-activating proteins (GAPs), which accelerate the slow intrinsic rate of GTP hydrolysis. Proteins containing TBC (Tre-2, Bub2 and Cdc16) domains are broadly conserved in eukaryotic organisms and function as GAPs for Rab GTPases as well as GTPases that control cytokinesis. An exposed arginine residue is a critical determinant of GAP activity in vitro and in vivo. It has been expected that the catalytic mechanism of TBC domains would parallel that of Ras and Rho family GAPs. Here we report crystallographic, mutational and functional analyses of complexes between Rab GTPases and the TBC domain of Gyp1p. In the crystal structure of a TBC-domain-Rab-GTPase-aluminium fluoride complex, which approximates the transition-state intermediate for GTP hydrolysis, the TBC domain supplies two catalytic residues in trans, an arginine finger analogous to Ras/Rho family GAPs and a glutamine finger that substitutes for the glutamine in the DxxGQ motif of the GTPase. The glutamine from the Rab GTPase does not stabilize the transition state as expected but instead interacts with the TBC domain. Strong conservation of both catalytic fingers indicates that most TBC-domain GAPs may accelerate GTP hydrolysis by a similar dual-finger mechanism.

  3. Embryonic Neural Inducing Factor Churchill is not a DNA-Binding Zinc Finger Protein

    PubMed Central

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

    2007-01-01

    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 β-sheet. Hydrogen-deuterium exchange and backbone relaxation measurements reveals 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 β-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. PMID:17610897

  4. 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

  5. Zinc fingers as protein recognition motifs: Structural basis for the GATA-1/Friend of GATA interaction

    PubMed Central

    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.

    2005-01-01

    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. PMID:15644435

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

    PubMed

    Sidik, Harwin; Talbot, William S

    2015-12-01

    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

  7. Identification and preliminary characterization of a protein motif related to the zinc finger.

    PubMed Central

    Lovering, R; Hanson, I M; Borden, K L; Martin, S; O'Reilly, N J; Evan, G I; Rahman, D; Pappin, D J; Trowsdale, J; Freemont, P S

    1993-01-01

    We have identified a protein motif, related to the zinc finger, which defines a newly discovered family of proteins. The motif was found in the sequence of the human RING1 gene, which is proximal to the major histocompatibility complex region on chromosome six. We propose naming this motif the "RING finger" and it is found in 27 proteins, all of which have putative DNA binding functions. We have synthesized a peptide corresponding to the RING1 motif and examined a number of properties, including metal and DNA binding. We provide evidence to support the suggestion that the RING finger motif is the DNA binding domain of this newly defined family of proteins. Images Fig. 1 Fig. 4 PMID:7681583

  8. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases

    PubMed Central

    Chen, Fuqiang; Pruett-Miller, Shondra M; Huang, Yuping; Gjoka, Monika; Duda, Katarzyna; Taunton, Jack; Collingwood, Trevor N; Frodin, Morten; Davis, Gregory D

    2011-01-01

    Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point mutation, (ii) targeted genomic deletion of up to 100 kb and (iii) targeted insertion of small genetic elements concomitant with large genomic deletions. PMID:21765410

  9. An improved predictive recognition model for Cys2-His2 zinc finger proteins

    PubMed Central

    Gupta, Ankit; Christensen, Ryan G.; Bell, Heather A.; Goodwin, Mathew; Patel, Ronak Y.; Pandey, Manishi; Enuameh, Metewo Selase; Rayla, Amy L.; Zhu, Cong; Thibodeau-Beganny, Stacey; Brodsky, Michael H.; Joung, J. Keith; Wolfe, Scot A.; Stormo, Gary D.

    2014-01-01

    Cys2-His2 zinc finger proteins (ZFPs) are the largest family of transcription factors in higher metazoans. They also represent the most diverse family with regards to the composition of their recognition sequences. Although there are a number of ZFPs with characterized DNA-binding preferences, the specificity of the vast majority of ZFPs is unknown and cannot be directly inferred by homology due to the diversity of recognition residues present within individual fingers. Given the large number of unique zinc fingers and assemblies present across eukaryotes, a comprehensive predictive recognition model that could accurately estimate the DNA-binding specificity of any ZFP based on its amino acid sequence would have great utility. Toward this goal, we have used the DNA-binding specificities of 678 two-finger modules from both natural and artificial sources to construct a random forest-based predictive model for ZFP recognition. We find that our recognition model outperforms previously described determinant-based recognition models for ZFPs, and can successfully estimate the specificity of naturally occurring ZFPs with previously defined specificities. PMID:24523353

  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 structural approach reveals how neighbouring C2H2 zinc fingers influence DNA binding specificity

    PubMed Central

    Garton, Michael; Najafabadi, Hamed S.; Schmitges, Frank W.; Radovani, Ernest; Hughes, Timothy R.; Kim, Philip M.

    2015-01-01

    Development of an accurate protein–DNA recognition code that can predict DNA specificity from protein sequence is a central problem in biology. C2H2 zinc fingers constitute by far the largest family of DNA binding domains and their binding specificity has been studied intensively. However, despite decades of research, accurate prediction of DNA specificity remains elusive. A major obstacle is thought to be the inability of current methods to account for the influence of neighbouring domains. Here we show that this problem can be addressed using a structural approach: we build structural models for all C2H2-ZF–DNA complexes with known binding motifs and find six distinct binding modes. Each mode changes the orientation of specificity residues with respect to the DNA, thereby modulating base preference. Most importantly, the structural analysis shows that residues at the domain interface strongly and predictably influence the binding mode, and hence specificity. Accounting for predicted binding mode significantly improves prediction accuracy of predicted motifs. This new insight into the fundamental behaviour of C2H2-ZFs has implications for both improving the prediction of natural zinc finger-binding sites, and for prioritizing further experiments to complete the code. It also provides a new design feature for zinc finger engineering. PMID:26384429

  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. The Arabidopsis SUPERMAN protein is able to specifically bind DNA through its single Cys2-His2 zinc finger motif.

    PubMed

    Dathan, Nina; Zaccaro, Laura; Esposito, Sabrina; Isernia, Carla; Omichinski, James G; Riccio, Andrea; Pedone, Carlo; Di Blasio, Benedetto; Fattorusso, Roberto; Pedone, Paolo V

    2002-11-15

    The Arabidopsis SUPERMAN (SUP) gene has been shown to be important in maintaining the boundary between stamens and carpels, and is presumed to act by regulating cell proliferation. In this work, we show that the SUP protein, which contains a single Cys2-His2 zinc finger domain including the QALGGH sequence, highly conserved in the plant zinc finger proteins, binds DNA. Using a series of deletion mutants, it was determined that the minimal domain required for specific DNA binding (residues 15-78) includes the single zinc finger and two basic regions located on either side of this motif. Furthermore, amino acid substitutions in the zinc finger or in the basic regions, including a mutation that knocks out the function of the SUP protein in vivo (glycine 63 to aspartate), have been found to abolish the activity of the SUP DNA-binding domain. These results strongly suggest that the SUP protein functions in vivo by acting as a DNA-binding protein, likely involved in transcriptional regulation. The association of both an N-terminal and a C-terminal basic region with a single Cys2-His2 zinc finger represents a novel DNA-binding motif suggesting that the mechanism of DNA recognition adopted by the SUP protein is different from that described so far in other zinc finger proteins. PMID:12433998

  14. The Promyelocytic Leukemia Zinc Finger Transcription Factor Is Critical for Human Endometrial Stromal Cell Decidualization

    PubMed Central

    Kommagani, Ramakrishna; Szwarc, Maria M.; Vasquez, Yasmin M.; Peavey, Mary C.; Mazur, Erik C.; Gibbons, William E.; Lanz, Rainer B.; DeMayo, Francesco J.; Lydon, John P.

    2016-01-01

    Progesterone, via the progesterone receptor (PGR), is essential for endometrial stromal cell decidualization, a cellular transformation event in which stromal fibroblasts differentiate into decidual cells. Uterine decidualization supports embryo implantation and placentation as well as subsequent events, which together ensure a successful pregnancy. Accordingly, impaired decidualization results not only in implantation failure or early fetal miscarriage, but also may lead to potential adverse outcomes in all three pregnancy trimesters. Transcriptional reprogramming on a genome-wide scale underlies progesterone dependent decidualization of the human endometrial stromal cell (hESC). However, identification of the functionally essential signals encoded by these global transcriptional changes remains incomplete. Importantly, this knowledge-gap undercuts future efforts to improve diagnosis and treatment of implantation failure based on a dysfunctional endometrium. By integrating genome-wide datasets derived from decidualization of hESCs in culture, we reveal that the promyelocytic leukemia zinc finger (PLZF) transcription factor is rapidly induced by progesterone and that this induction is indispensable for progesterone-dependent decidualization. Chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) identified at least ten progesterone response elements within the PLZF gene, indicating that PLZF may act as a direct target of PGR signaling. The spatiotemporal expression profile for PLZF in both the human and mouse endometrium offers further support for stromal PLZF as a mediator of the progesterone decidual signal. To identify functional targets of PLZF, integration of PLZF ChIP-Seq and RNA Pol II RNA-Seq datasets revealed that the early growth response 1 (EGR1) transcription factor is a PLZF target for which its level of expression must be reduced to enable progesterone dependent hESC decidualization. Apart from furnishing essential insights

  15. The Promyelocytic Leukemia Zinc Finger Transcription Factor Is Critical for Human Endometrial Stromal Cell Decidualization.

    PubMed

    Kommagani, Ramakrishna; Szwarc, Maria M; Vasquez, Yasmin M; Peavey, Mary C; Mazur, Erik C; Gibbons, William E; Lanz, Rainer B; DeMayo, Francesco J; Lydon, John P

    2016-04-01

    Progesterone, via the progesterone receptor (PGR), is essential for endometrial stromal cell decidualization, a cellular transformation event in which stromal fibroblasts differentiate into decidual cells. Uterine decidualization supports embryo implantation and placentation as well as subsequent events, which together ensure a successful pregnancy. Accordingly, impaired decidualization results not only in implantation failure or early fetal miscarriage, but also may lead to potential adverse outcomes in all three pregnancy trimesters. Transcriptional reprogramming on a genome-wide scale underlies progesterone dependent decidualization of the human endometrial stromal cell (hESC). However, identification of the functionally essential signals encoded by these global transcriptional changes remains incomplete. Importantly, this knowledge-gap undercuts future efforts to improve diagnosis and treatment of implantation failure based on a dysfunctional endometrium. By integrating genome-wide datasets derived from decidualization of hESCs in culture, we reveal that the promyelocytic leukemia zinc finger (PLZF) transcription factor is rapidly induced by progesterone and that this induction is indispensable for progesterone-dependent decidualization. Chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) identified at least ten progesterone response elements within the PLZF gene, indicating that PLZF may act as a direct target of PGR signaling. The spatiotemporal expression profile for PLZF in both the human and mouse endometrium offers further support for stromal PLZF as a mediator of the progesterone decidual signal. To identify functional targets of PLZF, integration of PLZF ChIP-Seq and RNA Pol II RNA-Seq datasets revealed that the early growth response 1 (EGR1) transcription factor is a PLZF target for which its level of expression must be reduced to enable progesterone dependent hESC decidualization. Apart from furnishing essential insights

  16. 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

  17. A phenyl-thiadiazolylidene-amine derivative ejects zinc from retroviral nucleocapsid zinc fingers and inactivates HIV virions

    PubMed Central

    2012-01-01

    Background Sexual acquisition of the human immunodeficiency virus (HIV) through mucosal transmission may be prevented by using topically applied agents that block HIV transmission from one individual to another. Therefore, virucidal agents that inactivate HIV virions may be used as a component in topical microbicides. Results Here, we have identified 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine (WDO-217) as a low-molecular-weight molecule that inactivates HIV particles. Both HIV-1 and HIV-2 virions pretreated with this compound were unable to infect permissive cells. Moreover, WDO-217 was able to inhibit infections of a wide spectrum of wild-type and drug-resistant HIV-1, including clinical isolates, HIV-2 and SIV strains. Whereas the capture of virus by DC-SIGN was unaffected by the compound, it efficiently prevented the transmission of DC-SIGN-captured virus to CD4+ T-lymphocytes. Interestingly, exposure of virions to WDO-217 reduced the amount of virion-associated genomic RNA as measured by real-time RT-qPCR. Further mechanism-of-action studies demonstrated that WDO-217 efficiently ejects zinc from the zinc fingers of the retroviral nucleocapsid protein NCp7 and inhibits the cTAR destabilization properties of this protein. Importantly, WDO-217 was able to eject zinc from both zinc fingers, even when NCp7 was bound to oligonucleotides, while no covalent interaction between NCp7 and WDO-217 could be observed. Conclusion This compound is a new lead structure that can be used for the development of a new series of NCp7 zinc ejectors as candidate topical microbicide agents. PMID:23146561

  18. DNA binding specificities of the long zinc-finger recombination protein PRDM9

    PubMed Central

    2013-01-01

    Background Meiotic recombination ensures proper segregation of homologous chromosomes and creates genetic variation. In many organisms, recombination occurs at limited sites, termed 'hotspots', whose positions in mammals are determined by PR domain member 9 (PRDM9), a long-array zinc-finger and chromatin-modifier protein. Determining the rules governing the DNA binding of PRDM9 is a major issue in understanding how it functions. Results Mouse PRDM9 protein variants bind to hotspot DNA sequences in a manner that is specific for both PRDM9 and DNA haplotypes, and that in vitro binding parallels its in vivo biological activity. Examining four hotspots, three activated by Prdm9Cst and one activated by Prdm9Dom2, we found that all binding sites required the full array of 11 or 12 contiguous fingers, depending on the allele, and that there was little sequence similarity between the binding sites of the three Prdm9Cst activated hotspots. The binding specificity of each position in the Hlx1 binding site, activated by Prdm9Cst, was tested by mutating each nucleotide to its three alternatives. The 31 positions along the binding site varied considerably in the ability of alternative bases to support binding, which also implicates a role for additional binding to the DNA phosphate backbone. Conclusions These results, which provide the first detailed mapping of PRDM9 binding to DNA and, to our knowledge, the most detailed analysis yet of DNA binding by a long zinc-finger array, make clear that the binding specificities of PRDM9, and possibly other long-array zinc-finger proteins, are unusually complex. PMID:23618393

  19. Noncanonical Myo9b-RhoGAP Accelerates RhoA GTP Hydrolysis by a Dual-Arginine-Finger Mechanism.

    PubMed

    Yi, Fengshuang; Kong, Ruirui; Ren, Jinqi; Zhu, Li; Lou, Jizhong; Wu, Jane Y; Feng, Wei

    2016-07-31

    The GTP hydrolysis activities of Rho GTPases are stimulated by GTPase-activating proteins (GAPs), which contain a RhoGAP domain equipped with a characteristic arginine finger and an auxiliary asparagine for catalysis. However, the auxiliary asparagine is missing in the RhoGAP domain of Myo9b (Myo9b-RhoGAP), a unique motorized RhoGAP that specifically targets RhoA for controlling cell motility. Here, we determined the structure of Myo9b-RhoGAP in complex with GDP-bound RhoA and magnesium fluoride. Unexpectedly, Myo9b-RhoGAP contains two arginine fingers at its catalytic site. The first arginine finger resembles the one within the canonical RhoGAP domains and inserts into the nucleotide-binding pocket of RhoA, whereas the second arginine finger anchors the Switch I loop of RhoA and interacts with the nucleotide, stabilizing the transition state of GTP hydrolysis and compensating for the lack of the asparagine. Mutating either of the two arginine fingers impaired the catalytic activity of Myo9b-RhoGAP and affected the Myo9b-mediated cell migration. Our data indicate that Myo9b-RhoGAP accelerates RhoA GTP hydrolysis by a previously unknown dual-arginine-finger mechanism, which may be shared by other noncanonical RhoGAP domains lacking the auxiliary asparagine. PMID:27363609

  20. Arabidopsis thaliana tandem zinc finger 1 (AtTZF1) protein in RNA binding and decay

    PubMed Central

    Qu, Jie; Kang, Shin Gene; Wang, Wei; Musier-Forsyth, Karin; Jang, Jyan-Chyun

    2014-01-01

    SUMMARY Arabidopsis thaliana Tandem Zinc Finger 1 (AtTZF1) protein is characterized by two tandem-arrayed CCCH type zinc fingers. We have previously found that AtTZF1 affects hormone-mediated growth, stress and gene expression responses. While much has been learned at the genetic and physiological level, the molecular mechanisms underlying the effects of AtTZF1 on gene expression remain obscure. A human TZF protein, hTTP, is known to bind and trigger the degradation of mRNAs containing AU-rich elements (AREs) at the 3′ untranslated regions. However, while the TZF motif of hTTP is characterized by CX8CX5CX3H-X18-CX8CX5CX3H, AtTZF1 contains an atypical motif of CX7CX5CX3H-X16-CX5CX4CX3H. Moreover, the TZF motif of AtTZF1 is preceded by a plant-unique arginine-rich (RR) region. Using fluorescence anisotropy and electrophoretic mobility shift binding assays, we have demonstrated that AtTZF1 binds to RNA molecules with specificity and the interaction is dependent on the presence of zinc. Compared to hTTP in which TZF is solely responsible for RNA binding, both TZF and arginine-rich (RR) regions of AtTZF1 are required to achieve high affinity RNA binding. Moreover, zinc finger integrity is vital for RNA binding. Using a plant protoplast transient expression analysis, we have further revealed that AtTZF1 can trigger the decay of ARE-containing mRNAs in vivo. Taken together, our results support the notion that AtTZF1 is involved in RNA turnover. PMID:24635033

  1. 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...

  2. Structural basis for the indispensable role of a unique zinc finger motif in LNX2 ubiquitination

    PubMed Central

    Nayak, Digant; Sivaraman, J.

    2015-01-01

    LNX (Ligand of Numb Protein-X) proteins, LNX1 and LNX2, are RING- and PDZ-based E3-ubiquitin ligases known to interact with Numb. Silencing of LNX2 has been reported to down-regulate WNT and NOTCH, two key signaling pathways in tumorigenesis. Here we report the identification of the domain boundary of LNX2 to confer its ubiquitination activity, its crystal structure along with functional studies. We show that the RING domain in LNX2 is flanked by two Zinc-binding motifs (Zn-RING-Zn), in which the N-terminal Zinc-binding motif adopts novel conformation. Although this motif follows the typical Cys2His2-type zinc finger configuration, it is devoid of any secondary structure and forms an open circle conformation, which has not been reported yet. This unique N-terminal Zn-finger motif is indispensable for the activity and stability of LNX2, as verified using mutational studies. The Zn-RING-Zn domain of LNX2 is a dimer and assumes a rigid elongated structure that undergoes autoubiquitination and undergoes N-terminal polyubiquitination. The ubiquitin chains consist of all seven possible isopeptide linkages. These results were validated using full-length LNX2. Moreover we have demonstrated the ubiquitination of cell fate determinant protein, Numb by LNX2. Our study provides a structural basis for the functional machinery of LNX2 and thus provides the opportunity to investigate suitable drug targets against LNX2. PMID:26451611

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

    PubMed Central

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

    1991-01-01

    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. Images PMID:2027750

  4. 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

  5. The Zinc Finger Protein ZNF658 Regulates the Transcription of Genes Involved in Zinc Homeostasis and Affects Ribosome Biogenesis through the Zinc Transcriptional Regulatory Element

    PubMed Central

    Ogo, Ogo A.; Tyson, John; Cockell, Simon J.; Howard, Alison; Valentine, Ruth A.

    2015-01-01

    We previously identified the ZTRE (zinc transcriptional regulatory element) in genes involved in zinc homeostasis and showed that it mediates transcriptional repression in response to zinc. We now report that ZNF658 acts at the ZTRE. ZNF658 was identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry of a band excised after electrophoretic mobility shift assay using a ZTRE probe. The protein contains a KRAB domain and 21 zinc fingers. It has similarity with ZAP1 from Saccharomyces cerevisiae, which regulates the response to zinc restriction, including a conserved DNA binding region we show to be functional also in ZNF658. Small interfering RNA (siRNA) targeted to ZNF658 abrogated the zinc-induced, ZTRE-dependent reduction in SLC30A5 (ZnT5 gene), SLC30A10 (ZnT10 gene), and CBWD transcripts in human Caco-2 cells and the ability of zinc to repress reporter gene expression from corresponding promoter-reporter constructs. Microarray analysis of the effect of reducing ZNF658 expression by siRNA uncovered a large decrease in rRNA. We find that ZTREs are clustered within the 45S rRNA precursor. We also saw effects on expression of multiple ribosomal proteins. ZNF658 thus links zinc homeostasis with ribosome biogenesis, the most active transcriptional, and hence zinc-demanding, process in the cell. ZNF658 is thus a novel transcriptional regulator that plays a fundamental role in the orchestrated cellular response to zinc availability. PMID:25582195

  6. 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

  7. DNA-binding specificity of NGFI-A and related zinc finger transcription factors.

    PubMed Central

    Swirnoff, A H; Milbrandt, J

    1995-01-01

    NGFI-A is the prototypic member of a family of immediate-early gene-encoded transcription factors which includes NGFI-C, Egr3, and Krox20. These proteins possess highly homologous DNA-binding domains, composed of three Cys2-His2 zinc fingers, and all bind to and activate transcription from the sequence GCGGGGGCG. We used a PCR-mediated random site selection protocol to determine whether other sites could be bound by these proteins and the extent to which their binding site preferences are similar or different. The high-affinity consensus sites generated from the selection data are similar, and the combined consensus sequence is T-G-C-G-T/g-G/A-G-G-C/a/t-G-G/T (lowercase letters indicate bases selected less frequently). Using gel shift assays, we found that sequences that diverge from the consensus were bound by NGFI-A, confirming that there is greater variability in binding sites than has generally been acknowledged. We also provide evidence that protein-DNA interactions not noted, or whose importance was not apparent from the X-ray cocrystal structure of the NGFI-A zinc fingers complexed with DNA, contribute significantly to the binding energy of these proteins and confirm that an optimal site is at least 10 instead of 9 nucleotides in length. In contrast to the similarities in binding specificity among these proteins we found that while NGFI-A, Egr3, and Krox20 have comparable DNA binding affinities and kinetics of dissociation, the affinity of NGFI-C is more than threefold lower. This could result in differential regulation of target genes in cells where NGFI-C and the other proteins are coexpressed. Furthermore, we show that this affinity difference is a property not of the zinc fingers themselves but rather of the protein context of the DNA-binding domain. PMID:7891721

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

    PubMed

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

    2016-09-15

    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

  9. 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. PMID:8468560

  10. 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.

  11. 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

  12. Zinc Finger Structures in the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Facilitate Efficient Minus- and Plus-Strand Transfer

    PubMed Central

    Guo, Jianhui; Wu, Tiyun; Anderson, Jada; Kane, Bradley F.; Johnson, Donald G.; Gorelick, Robert J.; Henderson, Louis E.; Levin, Judith G.

    2000-01-01

    The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) has two zinc fingers, each containing the invariant metal ion binding residues CCHC. Recent reports indicate that mutations in the CCHC motifs are deleterious for reverse transcription in vivo. To identify reverse transcriptase (RT) reactions affected by such changes, we have probed zinc finger functions in NC-dependent RT-catalyzed HIV-1 minus- and plus-strand transfer model systems. Our approach was to examine the activities of wild-type NC and a mutant in which all six cysteine residues were replaced by serine (SSHS NC); this mutation severely disrupts zinc coordination. We find that the zinc fingers contribute to the role of NC in complete tRNA primer removal from minus-strand DNA during plus-strand transfer. Annealing of the primer binding site sequences in plus-strand strong-stop DNA [(+) SSDNA] to its complement in minus-strand acceptor DNA is not dependent on NC zinc fingers. In contrast, the rate of annealing of the complementary R regions in (−) SSDNA and 3′ viral RNA during minus-strand transfer is approximately eightfold lower when SSHS NC is used in place of wild-type NC. Moreover, unlike wild-type NC, SSHS NC has only a small stimulatory effect on minus-strand transfer and is essentially unable to block TAR-induced self-priming from (−) SSDNA. Our results strongly suggest that NC zinc finger structures are needed to unfold highly structured RNA and DNA strand transfer intermediates. Thus, it appears that in these cases, zinc finger interactions are important components of NC nucleic acid chaperone activity. PMID:10982342

  13. Human proviral mRNAs down regulated in choriocarcinoma encode a zinc finger protein related to Krüppel.

    PubMed Central

    Kato, N; Shimotohno, K; VanLeeuwen, D; Cohen, M

    1990-01-01

    RNA transcripts of the HERV-R (ERV3) human provirus that are abundant in placenta but absent in choriocarcinoma contain nonproviral genomic sequences at their 3' ends. We report here the isolation of cDNA clones of these genomic sequences. The transcripts encode a Krüppel-related zinc finger protein consisting of a unique leader region and more than 12 28-amino-acid finger motifs. Images PMID:2115127

  14. Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly

    PubMed Central

    Kim, Hye Joo; Lee, Hyung Joo; Kim, Hyojin; Cho, Seung Woo; Kim, Jin-Soo

    2009-01-01

    Broad applications of zinc finger nuclease (ZFN) technology—which allows targeted genome editing—in research, medicine, and biotechnology are hampered by the lack of a convenient, rapid, and publicly available method for the synthesis of functional ZFNs. Here we describe an efficient and easy-to-practice modular-assembly method using publicly available zinc fingers to make ZFNs that can modify the DNA sequences of predetermined genomic sites in human cells. We synthesized and tested hundreds of ZFNs to target dozens of different sites in the human CCR5 gene—a co-receptor required for HIV infection—and found that many of these nucleases induced site-specific mutations in the CCR5 sequence. Because human cells that harbor CCR5 null mutations are functional and normal, these ZFNs might be used for (1) knocking out CCR5 to produce T-cells that are resistant to HIV infection in AIDS patients or (2) inserting therapeutic genes at “safe sites” in gene therapy applications. PMID:19470664

  15. 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.

  16. 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. PMID:26089249

  17. ZXDC, a novel zinc finger protein that binds CIITA and activates MHC gene transcription

    PubMed Central

    Al-Kandari, Wafa; Jambunathan, Srikarthika; Navalgund, Vandana; Koneni, Rupa; Freer, Margot; Parimi, Neeta; Mudhasani, Rajini; Fontes, Joseph D.

    2006-01-01

    The class II trans-activator (CIITA) is recognized as the master regulator of major histocompatibility complex (MHC) class II gene transcription and contributes to the transcription of MHC class I genes. To better understand the function of CIITA, we performed yeast two-hybrid with the C-terminal 807 amino acids of CIITA, and cloned a novel human cDNA named zinc finger, X-linked, duplicated family member C (ZXDC). The 858 amino acid ZXDC protein contains 10 zinc fingers and a transcriptional activation domain, and was found to interact with the region of CIITA containing leucine-rich repeats. Over-expression of ZXDC in human cell lines resulted in super-activation of MHC class I and class II promoters by CIITA. Conversely, silencing of ZXDC expression reduced the ability of CIITA to activate transcription of MHC class II genes. Given the specific interaction between the ZXDC and CIITA proteins, as well as the effect of ZXDC on MHC gene transcription, it appears that ZXDC is an important regulator of both MHC class I and class II transcription. PMID:16600381

  18. Expression of Zinc Finger Protein 105 in the Testis and its Role in Male Fertility

    PubMed Central

    Zhou, Huaxin; Liu, Lan-Hsin; Zhang, Heng; Lei, Zhenmin; Lan, Zi-Jian

    2011-01-01

    Using an in silico approach, we identified a putative zinc finger domain-containing transcription factor (zinc finger protein 105, ZFP105) that was enriched in the adult mouse testis. RT-PCR analyses showed that Zfp105 was indeed highly expressed in adult mouse testis and that its expression was regulated during postnatal development. To further characterize Zfp105 expression, we generated a Zfp105:β-galactosidase (LacZ) knock-in reporter mouse line (Zfp105LacZ/+) in which a Zfp105:LacZ fusion gene was expressed. Whole-mount LacZ analyses of adult Zfp105LacZ/+ tissues showed robust LacZ staining in the testis, very weak staining in the ovary and no staining in the spleen, liver, kidney, heart, lung, thymus, adrenal gland, uterus or oviduct. Sectional LacZ staining showed that ZFP105 was highly expressed in pachytene spermatocytes. ZNF35, the human ortholog of ZFP105, was also expressed in male germ cells of normal human testis. More importantly, reduced male fertility and sloughed spermatogenic cells were observed in adult Zfp105LacZ/LacZ mice. Taken together, our results suggest that ZFP105 is a male germ-cell factor and plays a role in male reproduction. PMID:20186958

  19. 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

  20. Inhibition of Human Telomerase Activity by an Engineered Zinc Finger Protein that Binds G-Quadruplexes†

    PubMed Central

    Patel, Sachin D.; Isalan, Mark; Gavory, Gérald; Ladame, Sylvain; Choo, Yen; Balasubramanian, Shankar

    2007-01-01

    The G-quadruplex nucleic acid structural motif is a target for designing molecules that could potentially modulate telomere length or have anticancer properties. We have recently described an engineered zinc finger protein (Gq1) that binds with specificity to the intramolecular G-quadruplex formed by the human telomeric sequence 5′-(GGTTAG)5-3′ (Isalan et al. (2001) Biochemistry 40, 830-836). Here, we report that Gq1 is able to arrest the action of a DNA polymerase on a template-containing telomeric sequence. Inhibition occurs in a concentration-dependent manner, probably by forming a stabilized G-quadruplex·protein complex. Furthermore, Gq1 inhibits the apparent activity of the enzyme telomerase in vitro, with an IC50 value of 74.3 ± 11.1 nM. Possible molecular mechanisms of inhibition are discussed, together with the potential for using engineered zinc fingers to interfere with the cellular processes associated with telomere function. PMID:15491152

  1. Characterization of Drosophila OVO protein DNA binding specificity using random DNA oligomer selection suggests zinc finger degeneration.

    PubMed

    Lee, S; Garfinkel, M D

    2000-02-01

    The Drosophila melanogaster ovo locus codes for several tissue- and stage-specific proteins that all possess a common C-terminal array of four C(2)H(2)zinc fingers. Three fingers conform to the motif framework and are evolutionarily conserved; the fourth diverges considerably. The ovo genetic function affects germ cell viability, sex identity and oogenesis, while the overlapping svb function is a key selector for epidermal structures under the control of wnt and EGF receptor signaling. We isolated synthetic DNA oligomers bound by the OVO zinc finger array from a high complexity starting population and derived a statistically significant 9 bp long DNA consensus sequence, which is nearly identical to a consensus derived from several Drosophila genes known or suspected of being regulated by the ovo function in vivo. The DNA consensus recognized by Drosophila OVO protein is atypical for zinc finger proteins in that it does not conform to many of the 'rules' for the interaction of amino acid contact residues and DNA bases. Additionally, our results suggest that only three of the OVO zinc fingers contribute to DNA-binding specificity. PMID:10637336

  2. The solution structure of ZNF593 from Homo sapiens reveals a zinc finger in a predominately unstructured protein

    PubMed Central

    Hayes, Paulette L.; Lytle, Betsy L.; Volkman, Brian F.; Peterson, Francis C.

    2008-01-01

    Here, we report the solution structure of ZNF593, a protein identified in a functional study as a negative modulator of the DNA-binding activity of the Oct-2 transcription factor. ZNF593 contains a classic C2H2 zinc finger domain flanked by about 40 disordered residues on each terminus. Although the protein contains a high degree of intrinsic disorder, the structure of the zinc finger domain was resolved by NMR spectroscopy without a need for N- or C-terminal truncations. The tertiary structure of the zinc finger domain is composed of a β-hairpin that positions the cysteine side chains for zinc coordination, followed by an atypical kinked α-helix containing the two histidine side chain ligands. The structural topology of ZNF593 is similar to a fragment of the double-stranded RNA-binding protein Zfa and the C-terminal zinc finger of MBP-1, a human enhancer binding protein. The structure presented here will provide a guide for future functional studies of how ZNF593 negatively modulates the DNA-binding activity of Oct-2, a POU domain-containing transcription factor. Our work illustrates the unique capacity of NMR spectroscopy for structural analysis of folded domains in a predominantly disordered protein. PMID:18287285

  3. Crystallization and preliminary X-ray crystallographic analysis of Aart, a designed six-finger zinc-finger peptide, bound to DNA

    SciTech Connect

    Crotty, Justin W.; Etzkorn, Christopher; Barbas, Carlos F. III; Segal, David J.; Horton, Nancy C.

    2005-06-01

    Preliminary data analysis of crystals of a designed six-finger zinc-finger protein bound to DNA is presented. The cell likely contains two protein–DNA complexes and diffracts to 2.95 Å. Crystals of a designed six-finger zinc-finger protein, Aart, bound to a 22-base-pair duplex DNA containing a consensus binding site have been obtained. Crystals grew by hanging-drop vapor diffusion from solutions containing polyethylene glycol 4000 as the precipitating agent. The irregularly shaped crystals belong to space group P1, with unit-cell parameters a = 41.95, b = 71.76, c = 74.73 Å, α = 100.87, β = 96.22, γ = 106.33°. There are most likely to be two protein–DNA complexes in the asymmetric unit. A complete native data set has been collected from a high-energy synchrotron source to a resolution of 2.95 Å at 100 K, with an R{sub merge} of 9.3%.

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

    PubMed

    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

  5. 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

  6. Comprehensive analysis of CCCH zinc finger family in poplar (Populus trichocarpa)

    PubMed Central

    2012-01-01

    Background CCCH zinc finger proteins contain a typical motif of three cysteines and one histidine residues and serve regulatory functions at all stages of mRNA metabolism. In plants, CCCH type zinc finger proteins comprise a large gene family represented by 68 members in Arabidopsis and 67 in rice. These CCCH proteins have been shown to play diverse roles in plant developmental processes and environmental responses. However, this family has not been studied in the model tree species Populus to date. Results In the present study, a comprehensive analysis of the genes encoding CCCH zinc finger family in Populus was performed. Using a thorough annotation approach, a total of 91 full-length CCCH genes were identified in Populus, of which most contained more than one CCCH motif and a type of non-conventional C-X11-C-X6-C-X3-H motif was unique for Populus. All of the Populus CCCH genes were phylogeneticly clustered into 13 distinct subfamilies. In each subfamily, the gene structure and motif composition were relatively conserved. Chromosomal localization of these genes revealed that most of the CCCHs (81 of 90, 90 %) are physically distributed on the duplicated blocks. Thirty-four paralogous pairs were identified in Populus, of which 22 pairs (64.7 %) might be created by the whole genome segment duplication, whereas 4 pairs seem to be resulted from tandem duplications. In 91 CCCH proteins, we also identified 63 putative nucleon-cytoplasm shuttling proteins and 3 typical RNA-binding proteins. The expression profiles of all Populus CCCH genes have been digitally analyzed in six tissues across different developmental stages, and under various drought stress conditions. A variety of expression patterns of CCCH genes were observed during Populus development, of which 34 genes highly express in root and 22 genes show the highest level of transcript abundance in differentiating xylem. Quantitative real-time RT-PCR (RT-qPCR) was further performed to confirm the tissue

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

    PubMed Central

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

    2015-01-01

    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. PMID:26190255

  8. Herbivory responsive C2H2 zinc finger transcription factor protein StZFP2 from potato.

    PubMed

    Lawrence, Susan D; Novak, Nicole G; Jones, Richard W; Farrar, Robert R; Blackburn, Michael B

    2014-07-01

    While C2H2 zinc finger transcription factors (TF) are often regulated by abiotic stress, their role during insect infestation has been overlooked. This study demonstrates that the transcripts of the zinc finger transcription factors StZFP1 and StZFP2 are induced in potato (Solanum tuberosum L.) upon infestation by either the generalist tobacco hornworm (THW, Manduca sexta L.) or the specialist Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). StZFP1 has been previously characterized as conferring salt tolerance to transgenic tobacco and its transcript is induced by Phytophthora infestans and several abiotic stresses. StZFP2 has not been characterized previously, but contains the hallmarks of a C2H2 zinc finger TF, with two conserved zinc finger domains and DLN motif, which encodes a transcriptional repressor domain. Expression studies demonstrate that StZFP2 transcript is also induced by tobacco hornworm and Colorado potato beetle. These observations expand the role of the C2H2 transcription factor in potato to include the response to chewing insect pests. PMID:24811678

  9. A single zinc finger optimizes the DNA interactions of the nucleocapsid protein of the yeast retrotransposon Ty3

    PubMed Central

    Chaurasiya, Kathy R.; Geertsema, Hylkje; Cristofari, Gaël; Darlix, Jean-Luc; Williams, Mark C.

    2012-01-01

    Reverse transcription in retroviruses and retrotransposons requires nucleic acid chaperones, which drive the rearrangement of nucleic acid conformation. The nucleic acid chaperone properties of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC) protein have been extensively studied, and nucleic acid aggregation, duplex destabilization and rapid binding kinetics have been identified as major components of its activity. However, the properties of other nucleic acid chaperone proteins, such as retrotransposon Ty3 NC, a likely ancestor of HIV-1 NC, are not well understood. In addition, it is unclear whether a single zinc finger is sufficient to optimize the properties characteristic of HIV-1 NC. We used single-molecule DNA stretching as a method for detailed characterization of Ty3 NC chaperone activity. We found that wild type Ty3 NC aggregates single- and double-stranded DNA, weakly stabilizes dsDNA, and exhibits rapid binding kinetics. Single-molecule studies in the presence of Ty3 NC mutants show that the N-terminal basic residues and the unique zinc finger at the C-terminus are required for optimum chaperone activity in this system. While the single zinc finger is capable of optimizing Ty3 NC's DNA interaction kinetics, two zinc fingers may be necessary in order to facilitate the DNA destabilization exhibited by HIV-1 NC. PMID:21917850

  10. A single zinc finger optimizes the DNA interactions of the nucleocapsid protein of the yeast retrotransposon Ty3.

    PubMed

    Chaurasiya, Kathy R; Geertsema, Hylkje; Cristofari, Gaël; Darlix, Jean-Luc; Williams, Mark C

    2012-01-01

    Reverse transcription in retroviruses and retrotransposons requires nucleic acid chaperones, which drive the rearrangement of nucleic acid conformation. The nucleic acid chaperone properties of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC) protein have been extensively studied, and nucleic acid aggregation, duplex destabilization and rapid binding kinetics have been identified as major components of its activity. However, the properties of other nucleic acid chaperone proteins, such as retrotransposon Ty3 NC, a likely ancestor of HIV-1 NC, are not well understood. In addition, it is unclear whether a single zinc finger is sufficient to optimize the properties characteristic of HIV-1 NC. We used single-molecule DNA stretching as a method for detailed characterization of Ty3 NC chaperone activity. We found that wild type Ty3 NC aggregates single- and double-stranded DNA, weakly stabilizes dsDNA, and exhibits rapid binding kinetics. Single-molecule studies in the presence of Ty3 NC mutants show that the N-terminal basic residues and the unique zinc finger at the C-terminus are required for optimum chaperone activity in this system. While the single zinc finger is capable of optimizing Ty3 NC's DNA interaction kinetics, two zinc fingers may be necessary in order to facilitate the DNA destabilization exhibited by HIV-1 NC. PMID:21917850

  11. 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

  12. 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

    2015-01-01

    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. PMID:26190255

  13. Expression of a novel Krüpple-like zinc-finger gene, ZNF382, in human heart.

    PubMed

    Luo, Kaimei; Yuan, Wuzhou; Zhu, Chuanbin; Li, Yongqing; Wang, Yuequn; Zeng, Weiqi; Jiao, Wei; Liu, Mingyao; Wu, Xiushan

    2002-12-13

    With the aim of identifying genes involved in human heart development and disease, we have isolated a novel KRAB-related zinc-finger gene named ZNF382 from heart cDNA library. The ZNF382 gene has a predicted 548-amino acid open reading frame, encoding a putative 64kDa zinc-finger protein. The N-terminus of the ZNF382 coding region has a well-conserved Krüpple-associated box domain that consists of KRAB boxes A and B, whereas the C-terminus contains a Krüpple-type zinc-finger domain possessing nine C(2)H(2) zinc-finger motifs in tandem arrays. The ZNF382 gene is mapped to chromosome 19q13.13. Northern blot analysis indicates that a 2.9-kb transcript specific for ZNF382 is expressed at very early embryonic stage of human (at least earlier than gestation 34 day) and widely in human embryo tissues. At the adult stage, ZNF382 expression is restricted largely to heart tissue suggesting a potential role in heart development and function. PMID:12459182

  14. 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...

  15. Recognition of distinct RNA motifs by the clustered CCCH zinc fingers of neuronal protein Unkempt.

    PubMed

    Murn, Jernej; Teplova, Marianna; Zarnack, Kathi; Shi, Yang; Patel, Dinshaw J

    2016-01-01

    Unkempt is an evolutionarily conserved RNA-binding protein that regulates translation of its target genes and is required for the establishment of the early bipolar neuronal morphology. Here we determined the X-ray crystal structure of mouse Unkempt and show that its six CCCH zinc fingers (ZnFs) form two compact clusters, ZnF1-3 and ZnF4-6, that recognize distinct trinucleotide RNA substrates. Both ZnF clusters adopt a similar overall topology and use distinct recognition principles to target specific RNA sequences. Structure-guided point mutations reduce the RNA binding affinity of Unkempt both in vitro and in vivo, ablate Unkempt's translational control and impair the ability of Unkempt to induce a bipolar cellular morphology. Our study unravels a new mode of RNA sequence recognition by clusters of CCCH ZnFs that is critical for post-transcriptional control of neuronal morphology. PMID:26641712

  16. Recognition of distinct RNA motifs by the clustered CCCH zinc fingers of neuronal protein Unkempt

    PubMed Central

    Zarnack, Kathi; Shi, Yang; Patel, Dinshaw J

    2015-01-01

    Unkempt is an evolutionarily conserved RNA-binding protein that regulates translation of its target genes and is required for the establishment of the early bipolar neuronal morphology. Here we determined the X-ray crystal structure of mouse Unkempt and show that its six CCCH zinc fingers (ZnFs) form two compact clusters, ZnF–3 and ZnF4–6, that recognize distinct trinucleotide RNA substrates. Both ZnF clusters adopt a similar overall topology and use distinct recognition principles to target specific RNA sequences. Structure-guided point mutations reduce the RNA binding affinity of Unkempt both in vitro and in vivo, ablate Unkempt’s translational control and impair the ability of Unkempt to induce a bipolar cellular morphology. Our study unravels a new mode of RNA sequence recognition by clusters of CCCH ZnFs that is critical for post-transcriptional control of neuronal morphology. PMID:26641712

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

    PubMed

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

    2016-10-01

    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

  18. Control of a neuronal morphology program by an RNA-binding zinc finger protein, Unkempt.

    PubMed

    Murn, Jernej; Zarnack, Kathi; Yang, Yawei J; Durak, Omer; Murphy, Elisabeth A; Cheloufi, Sihem; Gonzalez, Dilenny M; Teplova, Marianna; Curk, Tomaž; Zuber, Johannes; Patel, Dinshaw J; Ule, Jernej; Luscombe, Nicholas M; Tsai, Li-Huei; Walsh, Christopher A; Shi, Yang

    2015-03-01

    Cellular morphology is an essential determinant of cellular function in all kingdoms of life, yet little is known about how cell shape is controlled. Here we describe a molecular program that controls the early morphology of neurons through a metazoan-specific zinc finger protein, Unkempt. Depletion of Unkempt in mouse embryos disrupts the shape of migrating neurons, while ectopic expression confers neuronal-like morphology to cells of different nonneuronal lineages. We found that Unkempt is a sequence-specific RNA-binding protein and identified its precise binding sites within coding regions of mRNAs linked to protein metabolism and trafficking. RNA binding is required for Unkempt-induced remodeling of cellular shape and is directly coupled to a reduced production of the encoded proteins. These findings link post-transcriptional regulation of gene expression with cellular shape and have general implications for the development and disease of multicellular organisms. PMID:25737280

  19. Targeted integration in rat and mouse embryos with zinc-finger nucleases.

    PubMed

    Cui, Xiaoxia; Ji, Diana; Fisher, Daniel A; Wu, Yumei; Briner, David M; Weinstein, Edward J

    2011-01-01

    Gene targeting is indispensible for reverse genetics and the generation of animal models of disease. The mouse has become the most commonly used animal model system owing to the success of embryonic stem cell-based targeting technology, whereas other mammalian species lack convenient tools for genome modification. Recently, microinjection of engineered zinc-finger nucleases (ZFNs) in embryos was used to generate gene knockouts in the rat and the mouse by introducing nonhomologous end joining (NHEJ)-mediated deletions or insertions at the target site. Here we use ZFN technology in embryos to introduce sequence-specific modifications (knock-ins) by means of homologous recombination in Sprague Dawley and Long-Evans hooded rats and FVB mice. This approach enables precise genome engineering to generate modifications such as point mutations, accurate insertions and deletions, and conditional knockouts and knock-ins. The same strategy can potentially be applied to many other species for which genetic engineering tools are needed. PMID:21151125

  20. ZNF307, a novel zinc finger gene suppresses p53 and p21 pathway

    SciTech Connect

    Li Jing; Wang Yuequn; Fan Xiongwei; Mo Xiaoyang; Wang Zequn; Li Yongqing; Yin Zhaochu; Deng Yun; Luo Na; Zhu Chuanbing; Liu Mingyao; Ma Qian; Ocorr, Karen Yuan Wuzhou Wu Xiushan

    2007-11-30

    We have cloned a novel KRAB-related zinc finger gene, ZNF307, encoding a protein of 545 aa. ZNF307 is conserved across species in evolution and is differentially expressed in human adult and fetal tissues. The fusion protein of EGFP-ZNF307 localizes in the nucleus. Transcriptional activity assays show ZNF307 suppresses transcriptional activity of L8G5-luciferase. Overexpressing ZNF307 in different cell lines also inhibits the transcriptional activities of p53 and p21. Moreover, ZNF307 works by reducing the p53 protein level and p53 protein reduction is achieved by increasing transcription of MDM2 and EP300. ZNF307 might suppress p53-p21 pathway through activating MDM2 and EP300 expression and inducing p53 degradation.

  1. Promyelocytic Leukemia Zinc Finger Protein Regulates Interferon-Mediated Innate Immunity

    PubMed Central

    Xu, Dakang; Holko, Michelle; Sadler, Anthony J.; Scott, Bernadette; Higashiyama, Shigeki; Berkofsky-Fessler, Windy; McConnell, Melanie J.; Pandolfi, Pier Paolo; Licht, Jonathan D.; Williams, Bryan R.G.

    2009-01-01

    Summary Interferons (IFNs) direct innate and acquired immune responses and, accordingly, are used therapeutically to treat a number of diseases, yet the diverse effects they elicit are not fully understood. Here we identify the promyelocytic leukemia zinc finger (PLZF) protein as a previously unrecognized component of the IFN response. IFN stimulates an association between PLZF, the promyelocytic leukemia protein and histone deacetylase 1, to induce a decisive subset of IFN-stimulated genes (ISGs). Consequently, PLZF-deficient mice have a specific ISG defect and as a result are more susceptible to viral infection. This susceptibility correlates with a marked decrease in the expression of the key antiviral mediators and an impaired IFN-mediated induction of natural killer cell function. These results provide new insights into the regulatory mechanisms of IFN signaling and the induction of innate antiviral immunity. PMID:19523849

  2. Intrinsic cell permeability of the GAGA zinc finger protein into HeLa cells.

    PubMed

    Negi, Shigeru; Terada, Yuka; Suzuyama, Misato; Matsumoto, Haruka; Honbo, Akino; Amagase, Yoko; Mizukawa, Yumiko; Kiriyama, Akiko; Iga, Katsumi; Urushidaini, Tetsuro; Sugiura, Yukio

    2015-09-01

    We examined the intrinsic cell permeability of a GAGA zinc finger obtained from the Drosophila melanogaster transcription factor and analyzed its mechanism of cellular uptake using confocal microscopy and flow cytometry. HeLa cells were treated with the Cy5-labeld GAGA peptides (containing a fluorescent chromophore) to detect fluorescence signals from the fluorescent labeling peptides by confocal microscopy. The results clearly indicated that GAGA peptides possess intrinsic cell permeability for HeLa cells. Based on the results of the flow cytometry analysis and the theoretical net positive charge of the GAGA peptides, the efficiency of cellular uptake of the GAGA peptides was predicted to depend on the net positive charge of the GAGA peptide as well as the cationic component ratio of Arg residues to Lys residues. PMID:26187668

  3. Inhibition of tomato yellow leaf curl virus replication by artificial zinc-finger proteins.

    PubMed

    Takenaka, Kosuke; Koshino-Kimura, Yoshihiro; Aoyama, Yasuhiro; Sera, Takashi

    2007-01-01

    Previously, we designed an artificial zinc-finger protein (AZP) for blocking a replication protein (Rep) of beet severe curly top virus (BSCTV) from binding to its replication origin and demonstrated that transgenic Arabidopsis plants expressing the AZP are completely resistant to the virus infection. Here we applied the AZP technology to tomato yellow leaf curl virus (TYLCV) infective to an important agricultural crop, tomato. We designed an AZP binding to the direct repeat to block the TYLCV Rep binding and confirmed in gel shift assays that the designed AZP has a higher affinity to the replication origin than that of Rep. Furthermore, we demonstrated in competitive binding assays that the AZP effectively inhibited the Rep binding in vitro. We discuss properties of the AZP for inhibition of TYLCV replication in detail. PMID:18029770

  4. Control of a neuronal morphology program by an RNA-binding zinc finger protein, Unkempt

    PubMed Central

    Zarnack, Kathi; Durak, Omer; Murphy, Elisabeth A.; Cheloufi, Sihem; Gonzalez, Dilenny M.; Teplova, Marianna; Curk, Tomaž; Zuber, Johannes; Patel, Dinshaw J.; Ule, Jernej; Luscombe, Nicholas M.; Tsai, Li-Huei; Walsh, Christopher A.

    2015-01-01

    Cellular morphology is an essential determinant of cellular function in all kingdoms of life, yet little is known about how cell shape is controlled. Here we describe a molecular program that controls the early morphology of neurons through a metazoan-specific zinc finger protein, Unkempt. Depletion of Unkempt in mouse embryos disrupts the shape of migrating neurons, while ectopic expression confers neuronal-like morphology to cells of different nonneuronal lineages. We found that Unkempt is a sequence-specific RNA-binding protein and identified its precise binding sites within coding regions of mRNAs linked to protein metabolism and trafficking. RNA binding is required for Unkempt-induced remodeling of cellular shape and is directly coupled to a reduced production of the encoded proteins. These findings link post-transcriptional regulation of gene expression with cellular shape and have general implications for the development and disease of multicellular organisms. PMID:25737280

  5. An engineered split M.HhaI-zinc finger fusion lacks the intended methyltransferase specificity

    SciTech Connect

    Meister, Glenna E.; Chandrasegaran, Srinivasan; Ostermeier, Marc

    2008-12-05

    The ability to site-specifically methylate DNA in vivo would have wide applicability to the study of basic biomedical problems as well as enable studies on the potential of site-specific DNA methylation as a therapeutic strategy for the treatment of diseases. Natural DNA methyltransferases lack the specificity required for these applications. Nomura and Barbas [W. Nomura, C.F. Barbas 3rd, In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase, J. Am. Chem. Soc. 129 (2007) 8676-8677] have reported that an engineered DNA methyltransferase comprised of fragments of M.HhaI methyltransferase and zinc finger proteins has very high specificity for the chosen target site. Our analysis of this engineered enzyme shows that the fusion protein methylates target and non-target sites with similar efficiency.

  6. 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

  7. 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. PMID:26365095

  8. The Drosophila melanogaster developmental gene g1 encodes a variant zinc-finger-motif protein.

    PubMed

    Bouchard, M L; Côté, S

    1993-03-30

    In Drosophila melanogaster, the mechanisms involved in the pattern formation of complex internal organs are still largely unknown. However, the identity of the molecular determinants that control the development of these specific tissues is emerging from the combined use of genetic and molecular approaches. We have cloned a gene that is expressed in the mesoderm, one of the fundamental embryonic germ layers which gives rise to internal structures, such as the musculature. Here, we describe the molecular characterization of this gene, designated as g1. The nucleotide (nt) sequence of its cDNA shows an open reading frame of 852 nt, which encodes a 32-kDa protein with two putative zinc fingers, and a serine/glutamine/proline-rich region. These features indicate a functional role for g1, which remains to be elucidated, in regulating gene expression during mesoderm formation. PMID:8462875

  9. High-resolution three-dimensional structure of a single zinc finger from a human enhancer binding protein in solution.

    PubMed

    Omichinski, J G; Clore, G M; Appella, E; Sakaguchi, K; Gronenborn, A M

    1990-10-01

    The three-dimensional structure of a 30-residue synthetic peptide containing the carboxy-terminal "zinc finger" motif of a human enhancer binding protein has been determined by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. The structure determination is based on 487 approximate interproton distance and 63 torsion angle (phi, psi, and chi 1) restraints. A total of 40 simulated annealing structures were calculated, and the atomic rms distribution about the mean coordinate positions (excluding residues 29 and 30 which are ill-defined) is 0.4 A for the backbone atoms, 0.8 A for all atoms, and 0.41 A for all atoms excluding the lysine and arginine side chains, which are disordered. The solution structure of the zinc finger consists of two irregular antiparallel beta-strands connected by an atypical turn (residues 3-12) and a classical alpha-helix (residues 14-24). The zinc is tetrahedrally coordinated to the sulfur atoms of two cysteines (Cys-5 and Cys-8) and to the N epsilon 2 atoms of two histidines (His-21 and His-27). The two cysteine residues are located in the turn connecting the two beta-strands (residues 5-8); one of the histidine ligands (His-21) is in the alpha-helix, while the second histidine (His-27) is at the end of a looplike structure (formed by the end of the alpha-helix and a turn). The general architecture is qualitatively similar to two previously determined low-resolution Cys2-His2 zinc finger structures, although distinct differences can be observed in the beta-strands and turn and in the region around the two histidines coordinated to zinc. Comparison of the overall polypeptide fold of the enhancer binding protein zinc finger with known structures in the crystallographic data base reveals a striking similarity to one region (residues 23-44) of the X-ray structure of proteinase inhibitor domain III of Japanese quail ovomucoid [Papamokos, E., Weber, E., Bode

  10. Characterization of the tandem CWCH2 sequence motif: a hallmark of inter-zinc finger interactions

    PubMed Central

    2010-01-01

    Background The C2H2 zinc finger (ZF) domain is widely conserved among eukaryotic proteins. In Zic/Gli/Zap1 C2H2 ZF proteins, the two N-terminal ZFs form a single structural unit by sharing a hydrophobic core. This structural unit defines a new motif comprised of two tryptophan side chains at the center of the hydrophobic core. Because each tryptophan residue is located between the two cysteine residues of the C2H2 motif, we have named this structure the tandem CWCH2 (tCWCH2) motif. Results Here, we characterized 587 tCWCH2-containing genes using data derived from public databases. We categorized genes into 11 classes including Zic/Gli/Glis, Arid2/Rsc9, PacC, Mizf, Aebp2, Zap1/ZafA, Fungl, Zfp106, Twincl, Clr1, and Fungl-4ZF, based on sequence similarity, domain organization, and functional similarities. tCWCH2 motifs are mostly found in organisms belonging to the Opisthokonta (metazoa, fungi, and choanoflagellates) and Amoebozoa (amoeba, Dictyostelium discoideum). By comparison, the C2H2 ZF motif is distributed widely among the eukaryotes. The structure and organization of the tCWCH2 motif, its phylogenetic distribution, and molecular phylogenetic analysis suggest that prototypical tCWCH2 genes existed in the Opisthokonta ancestor. Within-group or between-group comparisons of the tCWCH2 amino acid sequence identified three additional sequence features (site-specific amino acid frequencies, longer linker sequence between two C2H2 ZFs, and frequent extra-sequences within C2H2 ZF motifs). Conclusion These features suggest that the tCWCH2 motif is a specialized motif involved in inter-zinc finger interactions. PMID:20167128

  11. Expression of zinc finger protein 105 in the testis and its role in male fertility.

    PubMed

    Zhou, Huaxin; Liu, Lan-Hsin; Zhang, Heng; Lei, Zhenmin; Lan, Zi-Jian

    2010-06-01

    Using an in silico approach, we identified a putative zinc finger domain-containing transcription factor (zinc finger protein 105, ZFP105) enriched in the adult mouse testis. RT-PCR analyses showed that Zfp105 was indeed highly expressed in adult mouse testis and that its expression was regulated during postnatal development. To further characterize Zfp105 expression, we generated a Zfp105:beta-galactosidase (LacZ) knock-in reporter mouse line (Zfp105(LacZ/+)) in which a Zfp105:LacZ fusion gene was expressed. Whole-mount LacZ analyses of adult Zfp105(LacZ/+) tissues showed robust LacZ staining in the testis, very weak staining in the ovary, and no staining in the spleen, liver, kidney, heart, lung, thymus, adrenal gland, uterus, or oviduct. Sectional LacZ staining showed that ZFP105 was highly expressed in pachytene spermatocytes. ZNF35, the human ortholog of Zfp105, was also highly expressed in human testis. Immunofluorescence analysis showed that ZNF35 was located primarily in the cytoplasm of male germ cells. More importantly, reduced male fertility was observed in adult Zfp105(LacZ/LacZ) mice. Histological studies showed the presence of undifferentiated spermatogenic cells in the lumen of seminiferous tubules at stage VII and in the epididymal lumen of adult Zfp105(LacZ/LacZ) mice. Taken together, our results suggest that ZFP105 is a male germ-cell factor and plays a role in male reproduction. PMID:20186958

  12. The functional significance of common polymorphisms in zinc finger transcription factors.

    PubMed

    Lockwood, Sarah H; Guan, Anna; Yu, Abigail S; Zhang, Chi; Zykovich, Artem; Korf, Ian; Rannala, Bruce; Segal, David J

    2014-09-01

    Variants that alter the DNA-binding specificity of transcription factors could affect the specificity for and expression of potentially many target genes, as has been observed in several tumor-derived mutations. Here we examined if such trans expression quantitative trait loci (trans-eQTLs) could similarly result from common genetic variants. We chose to focus on the Cys2-His2 class of zinc finger transcription factors because they are the most abundant superfamily of transcription factors in human and have well-characterized DNA binding interactions. We identified 430 SNPs that cause missense substitutions in the DNA-contacting residues. Fewer common missense SNPs were found at DNA-contacting residues compared with non-DNA-contacting residues (P = 0.00006), consistent with possible functional selection against SNPs at DNA-contacting positions. Functional predictions based on zinc finger transcription factor (ZNF) DNA binding preferences also suggested that many common substitutions could potentially alter binding specificity. However, Hardy-Weinberg Equilibrium analysis and examination of seven orthologs within the primate lineage failed to find evidence of trans-eQTLs associated with the DNA-contacting positions or evidence of a different selection pressure on a contemporary and evolutionary timescales. The overall conclusion was that common SNPs that alter the DNA-contacting residues of these factors are unlikely to produce strong trans-eQTLs, consistent with the observations by others that trans-eQTLs in humans tend to be few and weak. Some rare SNPs might alter specificity and remained rare due to purifying selection. The study also underscores the need for large-scale eQTLs mapping efforts that might provide experimental evidence for SNPs that alter the choice of transcription factor binding sites. PMID:24970883

  13. The Functional Significance of Common Polymorphisms in Zinc Finger Transcription Factors

    PubMed Central

    Lockwood, Sarah H.; Guan, Anna; Yu, Abigail S.; Zhang, Chi; Zykovich, Artem; Korf, Ian; Rannala, Bruce; Segal, David J.

    2014-01-01

    Variants that alter the DNA-binding specificity of transcription factors could affect the specificity for and expression of potentially many target genes, as has been observed in several tumor-derived mutations. Here we examined if such trans expression quantitative trait loci (trans-eQTLs) could similarly result from common genetic variants. We chose to focus on the Cys2-His2 class of zinc finger transcription factors because they are the most abundant superfamily of transcription factors in human and have well-characterized DNA binding interactions. We identified 430 SNPs that cause missense substitutions in the DNA-contacting residues. Fewer common missense SNPs were found at DNA-contacting residues compared with non-DNA-contacting residues (P = 0.00006), consistent with possible functional selection against SNPs at DNA-contacting positions. Functional predictions based on zinc finger transcription factor (ZNF) DNA binding preferences also suggested that many common substitutions could potentially alter binding specificity. However, Hardy-Weinberg Equilibrium analysis and examination of seven orthologs within the primate lineage failed to find evidence of trans-eQTLs associated with the DNA-contacting positions or evidence of a different selection pressure on a contemporary and evolutionary timescales. The overall conclusion was that common SNPs that alter the DNA-contacting residues of these factors are unlikely to produce strong trans-eQTLs, consistent with the observations by others that trans-eQTLs in humans tend to be few and weak. Some rare SNPs might alter specificity and remained rare due to purifying selection. The study also underscores the need for large-scale eQTLs mapping efforts that might provide experimental evidence for SNPs that alter the choice of transcription factor binding sites. PMID:24970883

  14. Mutations in zinc finger 407 [ZNF407] cause a unique autosomal recessive cognitive impairment syndrome

    PubMed Central

    2014-01-01

    Background A consanguineous Arab family is affected by an apparently novel autosomal recessive disorder characterized by cognitive impairment, failure-to-thrive, hypotonia and dysmorphic features including bilateral ptosis and epicanthic folds, synophrys, midface hypoplasia, downturned mouth corners, thin upper vermillion border and prominent ears, bilateral 5th finger camptodactyly, bilateral short 4th metatarsal bones, and limited knee mobility bilaterally. Methods The family was studied by homozygosity mapping, candidate gene mutation screening and whole Exome Next Generation Sequencing of a single affected member to identify the offending gene and mutation. The mutated gene product was studied by structural bioinformatics methods. Results A damaging c.C5054G mutation affecting an evolutionary highly conserved amino acid p.S1685W was identified in the ZNF407 gene at 18q23. The Serine to Tryptophane mutation affects two of the three ZNF407 isoforms and is located in the last third of the protein, in a linker peptide adjoining two zinc-finger domains. Structural analyses of this mutation shows disruption of an H-bond that locks the relative spatial position of the two fingers, leading to a higher flexibility of the linker and thus to a decreased probability of binding to the target DNA sequence essentially eliminating the functionality of downstream domains and interfering with the expression of various genes under ZNF407 control during fetal brain development. Conclusions ZNF407 is a transcription factor with an essential role in brain development. When specific and limited in number homozygosity intervals exist that harbor the offending gene in consanguineous families, Whole Exome Sequencing of a single affected individual is an efficient approach to gene mapping and mutation identification. PMID:24907849

  15. 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. PMID:22675124

  16. Hydrophilic Residues Are Crucial for Ribosomal Protein L11 (RPL11) Interaction with Zinc Finger Domain of MDM2 and p53 Protein Activation*

    PubMed Central

    Zhang, Qi; Xiao, Hui; Chai, Sergio C.; Hoang, Quyen Q.; Lu, Hua

    2011-01-01

    Ribosomal protein L11 (RPL11) has been shown to activate p53 by binding to MDM2 and negating its p53 suppression activity in response to ribosomal stress. Although a mutation at Cys-305 within the zinc finger domain of MDM2 has been shown to drastically impair MDM2 interaction with RPL11 and thus escapes the inhibition by this ribosomal protein, it still remains elusive whether RPL11 inactivates MDM2 via direct action on this zinc finger domain and what is the chemical nature of this specific interaction. To define the roles of the MDM2 zinc finger in association with RPL11, we conducted hydrogen-deuterium exchange mass spectrometry, computational modeling, circular dichroism, and mutational analyses of the zinc finger domain of MDM2 and human RPL11. Our study reveals that RPL11 forms a stable complex with MDM2 in vitro through direct contact with its zinc finger. This binding is disrupted by single mutations of non-cysteine amino acids within the zinc finger domain of MDM2. Basic residues in RPL11 are crucial for the stable binding and RPL11 suppression of MDM2 activity toward p53. These results provide the first line of evidence for the specific interaction between RPL11 and the zinc finger of MDM2 via hydrophilic residues as well as a molecular foundation for better understanding RPL11 inhibition of MDM2 function. PMID:21903592

  17. Chalcogen bonding interactions between reducible sulfur and selenium compounds and models of zinc finger proteins.

    PubMed

    Lutz, Patricia B; Bayse, Craig A

    2016-04-01

    Reducible sulfur and selenium (r-S/Se) compounds, defined as sulfur and selenium compounds not in the lowest -2 oxidation state (e.g., -1 to +6), release Zn(2+) from zinc-sulfur proteins such as zinc fingers (ZFs) and metallothionein. A series of density functional theory calculations was performed on donor-acceptor complexes between r-S/Se compounds and models of the Cys2His2, Cys3His and Cys4 ZF sites. These S⋯S/Se chalcogen bonding interactions consist of the donation of electron density from a S lone pair on the ZF model to a S/Se-X antibonding molecular orbital of the r-S/Se compound. The strength of the interaction was shown to be dependent upon the Lewis basicity of the ZF model (Cys4>Cys3His>Cys2His2) and the Lewis acidity of the r-S/Se compound as measured by the energy of the S/Se-X antibonding orbital. Interactions with the softer r-Se compounds were stronger than the r-S compounds, consistent with the greater reactivity of the former with ZF proteins. PMID:26877152

  18. [Cloning and functional analysis of SCTF-1 encoding a C2H2-type Zinc finger protein from soybean].

    PubMed

    Song, Bing; Wang, Pi-Wu; Fu, Yong-Ping; Fan, Xu-Hong; Xia, Hai-Feng; Gao, Wei; Hong, Yang; Wang, He; Zhang, Zhuo; Ma, Jian

    2012-06-01

    The zinc finger protein is one of the proteins with finger-like domain. Some of them are transcription factors which play important role in plant growth and plant resistance to abiotic stresses. In this paper, a novel C2H2-type zinc finger protein gene SCTF-1 (GenBank accession number JQ692081) was isolated from soybean (Glycine max (L.) Merr.) This gene has a 699 bp ORF (open reading frame) with no intron and encodes a 24.9 kDa protein with 233 amino acids. Its isoelectric point (pI) is 8.33. The SCTF-1 protein contains two typical C2H2-type zinc finger domains. Both of them have highly conserved amino acid sequence-QALGGH which is a particular characteristic of plant. Transient expression of the GFP-SCTF-1 protein in onion epidermal cell showed that SCTF-1 was localized in cell nuclei. RT-PCR results showed that SCTF-1 gene was expressed with high levels in flowers and leaves in soybean, but low in roots and stems. The expression of SCTF-1 gene was strongly induced by low temperature in the soybean seedlings. Overexpression of SCTF-1 enhanced cold tolerance of transgenic tobacco (Nicotiana tabacum L.) compared to the control. PMID:22698747

  19. 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

    2016-04-22

    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. PMID:26936488

  20. Interaction between the transcription factor SPBP and the positive cofactor RNF4. An interplay between protein binding zinc fingers.

    PubMed

    Lyngsø, C; Bouteiller, G; Damgaard, C K; Ryom, D; Sanchez-Muñoz, S; Nørby, P L; Bonven, B J; Jørgensen, P

    2000-08-25

    The activator of stromelysin 1 gene transcription, SPBP, interacts with the RING finger protein RNF4. Both proteins are ubiquitously expressed and localized in the nucleus. RNF4 facilitates accumulation of specific SPBP-DNA complexes in vitro and acts as a positive cofactor in SPBP-mediated transactivation. SPBP harbors an internal zinc finger of the PHD/LAP type. This domain can form intra-chain protein-protein contacts in SPBP resulting in negative modulation of SPBP-RNF4 interaction. PMID:10849425

  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. 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

  3. 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.

  4. Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence

    SciTech Connect

    Ono, Yoshitaka; Fujii, Tomoko; Igarashi, Koichi; Kuno, Takayoshi; Tanaka, Chikako; Kikkawa, Ushio; Nishizuka, Yasutomi )

    1989-07-01

    Protein kinase C normally has a tandem repeat of a characteristic cysteine-rich sequence in C{sub 1}, the conserved region of the regulatory domain. These sequences resemble the DNA-binding zinc finger domain. For the {gamma} subspecies of rat brain protein kinase C, various deletion and point mutants in this domain were constructed, and the mutated proteins were expressed in Escherichia coli by using the T7 expression system. Radioactive phorbol 12,13-dibutyrate binding analysis indicated that a cysteine-rich zinc-finger-like sequence was essential for protein kinase C to bind phorbol ester and that one of the two sequences was sufficient for the phorbol ester binding. Conserved region C{sub 2}, another region in the regulatory domain, was apparently needed for the enzyme to require Ca{sup 2+} for phorbol ester binding activity.

  5. 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

  6. A new family of zinc finger proteins in petunia: structure, DNA sequence recognition, and floral organ-specific expression.

    PubMed

    Takatsuji, H; Nakamura, N; Katsumoto, Y

    1994-07-01

    We have previously cloned a gene for a zinc finger protein (EPF1) that is expressed specifically in petals and interacts with the promoter region of the 5-enolpyruvylshikimate-3-phosphate synthase gene in petunia. In an attempt to isolate genes encoding additional factors that interact with this promoter, we cloned four novel genes encoding zinc finger proteins (EPF2-5a, EPF2-5b, EPF2-4, and EPF2-7). Sequence analyses revealed that overall similarity between the EPF1 and the EPF2 protein family, except in the zinc finger motifs and the basic amino acid cluster, was very low, suggesting that the two groups belong to different subfamilies. DNA binding specificities of EPF1, EPF2-5, and EPF2-4 were very similar, as expected from the conserved zinc finger motifs. However, EPF2-7 showed no binding to the probes tested in spite of having the conserved motifs. DNA binding studies using a series of spacing mutant probes suggested a binding mechanism in which the EPF proteins recognize spacings in target DNA. RNA gel blot analyses and histochemical analyses with a promoter and beta-glucuronidase fusion revealed that expression of the EPF2-5 gene (EPF2-5) was petal and stamen specific. Expression of the EPF2-7 gene (EPF2-7) was sepal and petal specific and localized in vascular tissues. The preferential expression in two adjacent floral organs raises the possibility that these genes are downstream transcription factors of floral homeotic genes. PMID:8069106

  7. C2H2 type of zinc finger transcription factors in foxtail millet define response to abiotic stresses.

    PubMed

    Muthamilarasan, Mehanathan; Bonthala, Venkata Suresh; Mishra, Awdhesh Kumar; Khandelwal, Rohit; Khan, Yusuf; Roy, Riti; Prasad, Manoj

    2014-09-01

    C2H2 type of zinc finger transcription factors (TFs) play crucial roles in plant stress response and hormone signal transduction. Hence considering its importance, genome-wide investigation and characterization of C2H2 zinc finger proteins were performed in Arabidopsis, rice and poplar but no such study was conducted in foxtail millet which is a C4 Panicoid model crop well known for its abiotic stress tolerance. The present study identified 124 C2H2-type zinc finger TFs in foxtail millet (SiC2H2) and physically mapped them onto the genome. The gene duplication analysis revealed that SiC2H2s primarily expanded in the genome through tandem duplication. The phylogenetic tree classified these TFs into five groups (I-V). Further, miRNAs targeting SiC2H2 transcripts in foxtail millet were identified. Heat map demonstrated differential and tissue-specific expression patterns of these SiC2H2 genes. Comparative physical mapping between foxtail millet SiC2H2 genes and its orthologs of sorghum, maize and rice revealed the evolutionary relationships of C2H2 type of zinc finger TFs. The duplication and divergence data provided novel insight into the evolutionary aspects of these TFs in foxtail millet and related grass species. Expression profiling of candidate SiC2H2 genes in response to salinity, dehydration and cold stress showed differential expression pattern of these genes at different time points of stresses. PMID:24915771

  8. Conserved zinc fingers mediate multiple functions of ZFP100, a U7snRNP associated protein

    PubMed Central

    Wagner, Eric J.; Ospina, Jason K.; Hu, Yue; Dundr, Miroslav; Matera, A. Gregory; Marzluff, William F.

    2006-01-01

    Formation of the 3′ end of replication-dependent histone mRNAs is most robust during S phase and is mediated by both the stem–loop binding protein (SLBP) and the U7 snRNP. We previously identified a 100-kDa zinc finger protein (ZFP100) as a component of U7 snRNP that interacts with the SLBP/pre-mRNA complex. Here, we show that myc- or GFP-tagged ZFP100 overexpressed after transfection is concentrated in Cajal bodies (CBs), and unlike components of the spliceosomal snRNPs, photobleaching experiments demonstrate that ZFP100 is stably associated with CBs. Of the 18 zinc fingers contained within ZFP100, the region encompassing fingers 2–6 is sufficient to maintain CB localization. Zn fingers 5–10 are required for maximal binding of ZFP100 to a 20-amino-acid region of Lsm11, a U7 snRNP core protein. Expression of ZFP100 stimulates histone mRNA processing in vivo, assayed by activation of a reporter gene that encodes a GFP mRNA ending in a histone 3′ end. Importantly, the domain that is required for CB localization and Lsm11 binding is also sufficient to stimulate histone pre-mRNA processing in vivo. Comparisons with other mammalian ZFP100 orthologs show that the central Zn fingers sufficient for in vivo activity are most highly conserved, whereas the number and sequence of the Zn fingers in the N- and C-terminal domains vary. PMID:16714279

  9. Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells

    PubMed Central

    Morton, Jason; Davis, M. Wayne; Jorgensen, Erik M.; Carroll, Dana

    2006-01-01

    Zinc-finger nucleases are chimeric proteins consisting of engineered zinc-finger DNA-binding motifs attached to an endonuclease domain. These proteins can induce site-specific DNA double-strand breaks in genomic DNA, which are then substrates for cellular repair mechanisms. Here, we demonstrate that engineered zinc-finger nucleases function effectively in somatic cells of the nematode Caenorhabditis elegans. Although gene-conversion events were indistinguishable from uncut DNA in our assay, nonhomologous end joining resulted in mutations at the target site. A synthetic target on an extrachromosomal array was targeted with a previously characterized nuclease, and an endogenous genomic sequence was targeted with a pair of specifically designed nucleases. In both cases, ≈20% of the target sites were mutated after induction of the corresponding nucleases. Alterations in the extrachromosomal targets were largely products of end-filling and blunt ligation. By contrast, alterations in the chromosomal target were mostly deletions. We interpret these differences to reflect the abundance of homologous templates present in the extrachromosomal arrays versus the paucity of such templates for repair of chromosomal breaks. In addition, we find evidence for the involvement of error-prone DNA synthesis in both homologous and nonhomologous pathways of repair. DNA ligase IV is required for efficient end joining, particularly of blunt ends. In its absence, a secondary end-joining pathway relies more heavily on microhomologies in producing deletions. PMID:17060623

  10. 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

  11. Effect of His-Tag on Expression, Purification, and Structure of Zinc Finger Protein, ZNF191(243-368)

    PubMed Central

    Huang, Zhongxian

    2016-01-01

    Zinc finger proteins are associated with hereditary diseases and cancers. To obtain an adequate amount of zinc finger proteins for studying their properties, structure, and functions, many protein expression systems are used. ZNF191(243-368) is a zinc finger protein and can be fused with His-tag to generate fusion proteins such as His6-ZNF191(243-368) and ZNF191(243-368)-His8. The purification of His-tag protein using Ni-NTA resin can overcome the difficulty of ZNF191(243-368) separation caused by inclusion body formation. The influences of His-tag on ZNF191(243-368) properties and structure were investigated using spectrographic techniques and hydrolase experiment. Our findings suggest that insertion of a His-tag at the N-terminal or C-terminal end of ZNF191(243-368) has different effects on the protein. Therefore, an expression system should be considered based on the properties and structure of the protein. Furthermore, the hydrolase activity of ZNF191(243-368)-His8 has provided new insights into the design of biological functional molecules. PMID:27524954

  12. Site-specific DNA cleavage by artificial zinc finger-type nuclease with cerium-binding peptide

    SciTech Connect

    Nakatsukasa, Takako; Shiraishi, Yasuhisa; Negi, Shigeru; Imanishi, Miki; Futaki, Shiroh; Sugiura, Yukio . E-mail: sugiura@scl.kyoto-u.ac.jp

    2005-04-29

    The addition of a new function to native proteins is one of the most attractive protein-based designs. In this study, we have converted a C{sub 2}H{sub 2}-type zinc finger as a DNA-binding motif into a novel zinc finger-type nuclease by connecting two distinct zinc finger proteins (Sp1 and GLI) with a functional linker possessing DNA cleavage activity. As a DNA cleavage domain, we chose an analogue of the metal-binding loop (12 amino acid residues), peptide P1, which has been reported to exhibit a strong binding affinity for a lanthanide ion and DNA cleavage ability in the presence of Ce(IV). Our newly designed nucleases, Sp1(P1)GLI and Sp1(P1G)GLI, can strongly bind to a lanthanide ion and show a unique DNA cleavage pattern, in which certain positions between the two DNA-binding sites are specifically cleaved. The present result provides useful information for expanding the design strategy for artificial nucleases.

  13. A new Cys2/His2 zinc finger gene, rKr1, expressed in oligodendrocytes and neurons.

    PubMed

    Pott, U; Colello, R J; Schwab, M E

    1996-05-01

    The myelination of nerve fibers is essential for the function of the vertebrate nervous system as a prerequisite for fast saltatory conduction of action potentials. In the central nervous system (CNS), myelin is produced by oligodendrocytes. In order to identify gene regulatory proteins involved in the differentiation of this glial cell type or in the expression of myelin-specific genes, we have constructed a cDNA library from a highly enriched population of rat oligodendrocytes and screened this library for members of the Krüppel family of Cys2/His2 zinc finger proteins. One of the identified clones, named rKr1, encodes a novel protein of 650 amino acids which contains 12 carboxy-terminal zinc finger domains and an amino-terminal acidic domain. On Northern blots, a single rKr1 mRNA of 4.3 kb is detected. This message is present in all adult rat tissues tested, with the highest levels found in the CNS and testis. In situ hybridization on the P15 brain revealed that the transcript is expressed in differentiated oligodendrocytes and in subtypes of neurons. Particularly high message levels are found in motor neurons of the brainstem and the spinal cord. The modular structure of the rKr1 protein, in which a potential DNA binding region (the zinc fingers) is combined with a putative activation domain (the acidic region), suggests a function as sequence-specific transcriptional activator. PMID:8737674

  14. Zinc fingers 1, 2, 5 and 6 of transcriptional regulator, PRDM4, are required for its nuclear localisation.

    PubMed

    Tunbak, Hale; Georgiou, Christiana; Guan, Cui; Richardson, William David; Chittka, Alexandra

    2016-05-27

    PRDM4 is a member of the PRDM family of transcriptional regulators which control various aspects of cellular differentiation and proliferation. PRDM proteins exert their biological functions both in the cytosol and the nucleus of cells. All PRDM proteins are characterised by the presence of two distinct structural motifs, the PR/SET domain and the zinc finger (ZF) motifs. We previously observed that deletion of all six zinc fingers found in PRDM4 leads to its accumulation in the cytosol, whereas overexpressed full length PRDM4 is found predominantly in the nucleus. Here, we investigated the requirements for single zinc fingers in the nuclear localisation of PRDM4. We demonstrate that ZF's 1, 2, 5 and 6 contribute to the accumulation of PRDM4 in the nucleus. Their effect is additive as deleting either ZF1-2 or ZF 5-6 redistributes PRDM4 protein from being almost exclusively nuclear to cytosolic and nuclear. We investigated the potential mechanism of nuclear shuttling of PRDM4 via the importin α/β-mediated pathway and find that PRDM4 nuclear targeting is independent of α/β-mediated nuclear import. PMID:27125459

  15. The transition to endoreduplication in trophoblast giant cells is regulated by the mSNA zinc finger transcription factor.

    PubMed

    Nakayama, H; Scott, I C; Cross, J C

    1998-07-01

    Terminal cell differentiation is usually associated with cell cycle exit. In some lineages, however, cells undergo continued rounds of DNA synthesis without intervening mitoses (endoreduplication) resulting in polyploid nuclei. This is striking in rodent trophoblast giant cells which contain up to 1000N of DNA. In Drosophila, the Escargot gene has been implicated in regulating the transition from mitotic cell cycles to endocycles during development. We found that a murine homologue, mSna, was expressed in mouse trophoblast and was downregulated during giant cell differentiation. The mSNA zinc finger protein bound to E-box DNA elements and, in transfected C3H10T1/2 fibroblasts, acted as a transcriptional repressor. The maximal repressive effect was dependent on both the zinc finger DNA-binding domain and the N-terminal, seven-amino-acid SNAG domain. Misexpression experiments in Rcho-1 trophoblast cells revealed that mSna regulates the transition from replicating precursor cells to committed giant cells: overexpression blocked, whereas antisense RNA-mediated underexpression promoted trophoblast giant cell differentiation. Overexpression of mSna in precursor cells had no effect on cell cycle kinetics, but did increase cyclin A and B levels, implying actions during G2. These effects were dependent on both the zinc finger and SNAG domains. Together, these data suggest that mSNA has an ESCARGOT-like function to repress the transcription of genes that promote the transition from mitotic to endoreduplicative cell cycles in rodent trophoblast. PMID:9676199

  16. The Arabidopsis SUPERMAN protein is able to specifically bind DNA through its single Cys2–His2 zinc finger motif

    PubMed Central

    Dathan, Nina; Zaccaro, Laura; Esposito, Sabrina; Isernia, Carla; Omichinski, James G.; Riccio, Andrea; Pedone, Carlo; Di Blasio, Benedetto; Fattorusso, Roberto; Pedone, Paolo V.

    2002-01-01

    The Arabidopsis SUPERMAN (SUP) gene has been shown to be important in maintaining the boundary between stamens and carpels, and is presumed to act by regulating cell proliferation. In this work, we show that the SUP protein, which contains a single Cys2–His2 zinc finger domain including the QALGGH sequence, highly conserved in the plant zinc finger proteins, binds DNA. Using a series of deletion mutants, it was determined that the minimal domain required for specific DNA binding (residues 15–78) includes the single zinc finger and two basic regions located on either side of this motif. Furthermore, amino acid substitutions in the zinc finger or in the basic regions, including a mutation that knocks out the function of the SUP protein in vivo (glycine 63 to aspartate), have been found to abolish the activity of the SUP DNA-binding domain. These results strongly suggest that the SUP protein functions in vivo by acting as a DNA-binding protein, likely involved in transcriptional regulation. The association of both an N-terminal and a C-terminal basic region with a single Cys2–His2 zinc finger represents a novel DNA-binding motif suggesting that the mechanism of DNA recognition adopted by the SUP protein is different from that described so far in other zinc finger proteins. PMID:12433998

  17. 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

  18. 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

  19. 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

  20. Identification of zinc finger protein Bcl6 as a novel regulator of early adipose commitment

    PubMed Central

    Hu, Xiaoming; Zhou, Yuanfei; Yang, Yang; Peng, Jie; Song, Tongxing; Xu, Tao; Wei, Hongkui; Jiang, Siwen; Peng, Jian

    2016-01-01

    Adipose tissue is a key determinant of whole-body metabolism and energy homeostasis. Unravelling the transcriptional regulatory process during adipogenesis is therefore highly relevant from a biomedical perspective. In these studies, zinc finger protein B-cell lymphoma 6 (Bcl6) was demonstrated to have a role in early adipogenesis of mesenchymal stem cells. Bcl6 is enriched in preadipose versus non-preadipose fibroblasts and shows upregulated expression in the early stage of adipogenesis. Gain- and loss-of-function studies revealed that Bcl6 acts as a key regulator of adipose commitment and differentiation both in vitro and ex vivo. RNAi-mediated knockdown of Bcl6 in C3H10T1/2 cells greatly inhibited adipogenic potential, whereas Bcl6 overexpression enhanced adipogenic differentiation. This transcription factor also directly or indirectly targets and controls the expression of some early and late adipogenic regulators (i.e. Zfp423, Zfp467, KLF15, C/EBPδ, C/EBPα and PPARγ). We further identified that Bcl6 transactivated the signal transducers and activators of transcription 1 (STAT1), which was determined as a required factor for adipogenesis. Moreover, overexpression of STAT1 rescued the impairment of adipogenic commitment and differentiation induced by Bcl6 knockdown in C3H10T1/2 cells, thereby confirming that STAT1 is a downstream direct target of Bcl6. This study identifies Bcl6 as a positive transcriptional regulator of early adipose commitment. PMID:27251748

  1. 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. PMID:25989233

  2. 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. PMID:23009861

  3. Characterization of Junín virus particles inactivated by a zinc finger-reactive compound.

    PubMed

    García, Cybele C; Ellenberg, Paula C; Artuso, María C; Scolaro, Luis A; Damonte, Elsa B

    2009-07-01

    Our previous studies reported the inhibitory action against arenaviruses of antiretroviral zinc finger-reactive compounds provided by the National Cancer Institute (USA). These compounds were able to inactivate virions as well as to reduce virus yields from infected cells. Here, the inactivation of the arenavirus Junín (JUNV), agent of Argentine hemorrhagic fever, by the aromatic disulfide NSC20625 was analyzed. The treatment of purified JUNV with this compound eliminated infectivity apparently through irreversible modifications in the matrix Z protein detected by: (a) alterations in the electrophoretic migration profile of Z under non-reducing conditions; (b) an electrodense labeling in the internal layer beneath the envelope and around the matrix Z protein, in negatively stained preparations; (c) changes in the subcellular localization of Z in cells transfected with a recombinant fusion protein JUNVZ-eGFP. The infection of Vero cells with JUNV inactivated particles was blocked at the uncoating of viral nucleocapsid from endosomes, providing new evidence for a functional role of Z in this stage of arenavirus cycle. Furthermore, the inactivated JUNV particles retained the immunoreactivity of the surface glycoprotein GP1 suggesting that this disulfide may be useful in the pursuit of an inactivating agent to obtain a vaccine antigen or diagnostic tool. PMID:19463727

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

    PubMed Central

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

    2015-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.06041.001 PMID:25985087

  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. Targeted gene exchange in plant cells mediated by a zinc finger nuclease double cut.

    PubMed

    Schneider, Katja; Schiermeyer, Andreas; Dolls, Anja; Koch, Natalie; Herwartz, Denise; Kirchhoff, Janina; Fischer, Rainer; Russell, Sean M; Cao, Zehui; Corbin, David R; Sastry-Dent, Lakshmi; Ainley, W Michael; Webb, Steven R; Schinkel, Helga; Schillberg, Stefan

    2016-04-01

    Genome modification by homology-directed repair (HDR) is an attractive tool for the controlled genetic manipulation of plants. Here, we report the HDR-mediated gene exchange of expression cassettes in tobacco BY-2 cells using a designed zinc finger nuclease (ZFN). The target contained a 7-kb fragment flanked by two ZFN cutting sites. That fragment was replaced with a 4-kb donor cassette, which integrates gene markers for selection (kanamycin resistance) and for scoring targeting (red fluorescent protein, RFP). Candidates resulting from cassette exchange were identified by molecular analysis of calli generated by transformation via direct DNA delivery. The precision of HDR-mediated donor integration was evaluated by Southern blot analysis, sequencing of the integration locus and analysis of RFP fluorescence by flow cytometry. Screening of 1326 kanamycin-resistant calli yielded 18 HDR events, 16 of which had a perfect cassette exchange at the insert junction and 13 of which produced functional RFP. Our results demonstrate that ZFN-based HDR can be used for high frequency, precise, targeted exchange of fragments of sizes that are commercially relevant in plants. PMID:26426390

  7. 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.

  8. 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

  9. Requirement for the Murine Zinc Finger Protein ZFR in Perigastrulation Growth and Survival

    PubMed Central

    Meagher, Madeleine J.; Braun, Robert E.

    2001-01-01

    The transition from preimplantation to postimplantation development leads to the initiation of complex cellular differentiation and morphogenetic movements, a dramatic decrease in cell cycle length, and a commensurate increase in the size of the embryo. Accompanying these changes is the need for the transfer of nutrients from the mother to the embryo and the elaboration of sophisticated genetic networks that monitor genomic integrity and the homeostatic control of cellular growth, differentiation, and programmed cell death. To determine the function of the murine zinc finger protein ZFR in these events, we generated mice carrying a null mutation in the gene encoding it. Homozygous mutant embryos form normal-appearing blastocysts that implant and initiate the process of gastrulation. Mutant embryos form mesoderm but they are delayed in their development and fail to form normal anterior embryonic structures. Loss of ZFR function leads to both an increase in programmed cell death and a decrease in mitotic index, especially in the region of the distal tip of the embryonic ectoderm. Mutant embryos also have an apparent reduction in apical vacuoles in the columnar visceral endoderm cells in the extraembryonic region. Together, these cellular phenotypes lead to a dramatic development delay and embryonic death by 8 to 9 days of gestation, which are independent of p53 function. PMID:11283266

  10. The Zinc-Finger Antiviral Protein ZAP Inhibits LINE and Alu Retrotransposition

    PubMed Central

    Moldovan, John B.; Moran, John V.

    2015-01-01

    Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome. To investigate the interplay between the L1 retrotransposition machinery and the host cell, we used co-immunoprecipitation in conjunction with liquid chromatography and tandem mass spectrometry to identify cellular proteins that interact with the L1 first open reading frame-encoded protein, ORF1p. We identified 39 ORF1p-interacting candidate proteins including the zinc-finger antiviral protein (ZAP or ZC3HAV1). Here we show that the interaction between ZAP and ORF1p requires RNA and that ZAP overexpression in HeLa cells inhibits the retrotransposition of engineered human L1 and Alu elements, an engineered mouse L1, and an engineered zebrafish LINE-2 element. Consistently, siRNA-mediated depletion of endogenous ZAP in HeLa cells led to a ~2-fold increase in human L1 retrotransposition. Fluorescence microscopy in cultured human cells demonstrated that ZAP co-localizes with L1 RNA, ORF1p, and stress granule associated proteins in cytoplasmic foci. Finally, molecular genetic and biochemical analyses indicate that ZAP reduces the accumulation of full-length L1 RNA and the L1-encoded proteins, yielding mechanistic insight about how ZAP may inhibit L1 retrotransposition. Together, these data suggest that ZAP inhibits the retrotransposition of LINE and Alu elements. PMID:25951186

  11. The zinc finger protein ZNF297B interacts with BDP1, a subunit of TFIIIB.

    PubMed

    Schoenen, Frank; Wirth, Brunhilde

    2006-03-01

    The human gene BDP1, localized on chromosome 5q13 in close proximity to the spinal muscular atrophy determining gene SMN, encodes a large protein consisting of 2254 amino acids (aa). In the first third of the gene, the subunit of the RNA polymerase III (Pol III) transcription factor complex (TFIIIB alpha/beta) is encoded. To further characterize the function of BDP1, we carried out a yeast two-hybrid screen using various parts of BDP1. With the clone BDP1-(1-640) we identified a novel interaction partner, ZNF297B. The ZNF297B gene is localized on chromosome 9q24 and encodes a zinc finger protein of 467 aa possessing the typical structure of a transcription factor. The interaction found in yeast was confirmed by co-immunoprecipitation and refined to the N-terminal region of ZNF297B-(1-127) containing the BTB/POZ domain and the N-terminal end of BDP1-(1-299). The ZNF297B transcript is 5.7 kb in length and ubiquitously expressed, with highest levels found in muscles. Immunofluorescence staining revealed a speckled pattern in the nuclei of HEK293 cells. Due to the essential role of BDP1 in Pol III transcription, we propose that ZNF297B may also regulate these transcriptional pathways. PMID:16542149

  12. Identification of zinc finger protein Bcl6 as a novel regulator of early adipose commitment.

    PubMed

    Hu, Xiaoming; Zhou, Yuanfei; Yang, Yang; Peng, Jie; Song, Tongxing; Xu, Tao; Wei, Hongkui; Jiang, Siwen; Peng, Jian

    2016-06-01

    Adipose tissue is a key determinant of whole-body metabolism and energy homeostasis. Unravelling the transcriptional regulatory process during adipogenesis is therefore highly relevant from a biomedical perspective. In these studies, zinc finger protein B-cell lymphoma 6 (Bcl6) was demonstrated to have a role in early adipogenesis of mesenchymal stem cells. Bcl6 is enriched in preadipose versus non-preadipose fibroblasts and shows upregulated expression in the early stage of adipogenesis. Gain- and loss-of-function studies revealed that Bcl6 acts as a key regulator of adipose commitment and differentiation both in vitro and ex vivo RNAi-mediated knockdown of Bcl6 in C3H10T1/2 cells greatly inhibited adipogenic potential, whereas Bcl6 overexpression enhanced adipogenic differentiation. This transcription factor also directly or indirectly targets and controls the expression of some early and late adipogenic regulators (i.e. Zfp423, Zfp467, KLF15, C/EBPδ, C/EBPα and PPARγ). We further identified that Bcl6 transactivated the signal transducers and activators of transcription 1 (STAT1), which was determined as a required factor for adipogenesis. Moreover, overexpression of STAT1 rescued the impairment of adipogenic commitment and differentiation induced by Bcl6 knockdown in C3H10T1/2 cells, thereby confirming that STAT1 is a downstream direct target of Bcl6. This study identifies Bcl6 as a positive transcriptional regulator of early adipose commitment. PMID:27251748

  13. Transcription Factor Zinc finger and BTB Domain 1 (Zbtb1) Is Essential for Lymphocyte Development*

    PubMed Central

    Punwani, Divya; Simon, Karen; Choi, Youngnim; Dutra, Amalia; Gonzalez-Espinosa, Diana; Pak, Evgenia; Naradikian, Martin; Song, Chang-Hwa; Zhang, Jenny; Bodine, David M.; Puck, Jennifer M.

    2012-01-01

    Absent T lymphocytes were unexpectedly found in homozygotes of a transgenic mouse from an unrelated project. T cell development did not progress beyond double negative stage 1 thymocytes, resulting in a hypocellular, vestigial thymus. B cells were present, but NK cell number and B cell isotype switching were reduced. Transplantation of wild type hematopoietic cells corrected the defect, which was traced to a deletion involving 5 contiguous genes at the transgene insertion site on chromosome 12C3. Complementation using BAC transgenesis implicated zinc finger BTB-POZ domain protein 1 (Zbtb1) in the immunodeficiency, confirming its role in T cell development and suggesting involvement in B and NK cell differentiation. Targeted disruption of Zbtb1 recapitulated the T− B+ NK− severe combined immunodeficiency (SCID) phenotype of the original transgenic animal. Knockouts for Zbtb1 had expanded populations of bone marrow hematopoietic stem cells and also multipotent and early lymphoid lineages, suggesting a differentiation bottleneck for common lymphoid progenitors. Expression of mRNA encoding Zbtb1, a predicted transcription repressor, was greatest in hematopoietic stem cells, thymocytes and pre-B cells, highlighting its essential role in lymphoid development. PMID:22753936

  14. Editing T cell specificity towards leukemia by zinc-finger nucleases and lentiviral gene transfer

    PubMed Central

    Lombardo, Angelo; Magnani, Zulma; Liu, Pei-Qi; Reik, Andreas; Chu, Victoria; Paschon, David E.; Zhang, Lei; Kuball, Jurgen; Camisa, Barbara; Bondanza, Attilio; Casorati, Giulia; Ponzoni, Maurilio; Ciceri, Fabio; Bordignon, Claudio; Greenberg, Philip D.; Holmes, Michael C.; Gregory, Philip D.; Naldini, Luigi; Bonini, Chiara

    2016-01-01

    The transfer of high-avidity T-cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted specificities. We designed zinc-finger nucleases (ZFNs) promoting the disruption of endogenous TCR β and α chain genes. ZFN-treated lymphocytes lacked CD3/TCR surface expression and expanded with IL-7 and IL-15. Upon lentiviral transfer of a TCR for the WT1 tumor antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near-purity, and proved superior in specific antigen recognition to matched TCR-transferred cells. In contrast to TCR-transferred cells, TCR edited lymphocytes did not mediate off-target reactivity while maintaining anti-tumor activity in vivo, thus demonstrating that complete editing of T-cell specificity generate tumor-specific lymphocytes with improved biosafety profile. PMID:22466705

  15. The mesoderm determinant snail collaborates with related zinc-finger proteins to control Drosophila neurogenesis.

    PubMed

    Ashraf, S I; Hu, X; Roote, J; Ip, Y T

    1999-11-15

    The Snail protein functions as a transcriptional regulator to establish early mesodermal cell fate. Later, in germ band-extended embryos, Snail is also expressed in most neuroblasts. Here we present evidence that this expression of Snail is required for central nervous system (CNS) development. The neural function of snail is masked by two closely linked genes, escargot and worniu. Both Escargot and Worniu contain zinc-finger domains that are highly homologous to that of Snail. Although not affecting expression of early neuroblast markers, the deletion of the region containing all three genes correlates with loss of expression of CNS determinants including fushi tarazu, pdm-2 and even-skipped. Transgenic expression of each of the three Snail family proteins can rescue efficiently the fushi tarazu defects, and partially the pdm-2 and even-skipped CNS patterns. These results demonstrate that the Snail family proteins have essential functions during embryonic CNS development, around the time of ganglion mother cell formation. PMID:10562554

  16. Worniu, a Snail family zinc-finger protein, is required for brain development in Drosophila.

    PubMed

    Ashraf, Shovon I; Ganguly, Atish; Roote, John; Ip, Y Tony

    2004-10-01

    The Snail family of zinc-finger transcriptional repressors is essential for morphogenetic cell movements, mesoderm formation, and neurogenesis during embryonic development. These proteins also control cell cycle, cell death, and cancer progression. In Drosophila, three members of this protein family, Snail, Escargot, and Worniu, have essential but redundant functions in asymmetric cell division of neuroblasts. In addition, Snail is critical for early mesoderm formation and Escargot is required for maintaining diploidy in wing imaginal disc cells. In this report, we demonstrate that Worniu plays a role in brain development. We show that alleles of the l(2)35Da complementation group are mutants of worniu. The developing larvae of these mutant alleles fail to shorten their brainstems. The brain phenotype, as well as the lethality, of these mutants can be rescued by worniu transgenes. Moreover, RNAi experiments targeting the worniu transcript show the same nonshortening phenotype in larval brains. worniu is expressed in the neuroblasts of brain hemispheres and ventral ganglions. The results suggest that the loss of Worniu function within the neuroblasts ultimately causes the larval brainstem to fail to go through shortening during development. PMID:15366015

  17. Snail-type zinc finger proteins prevent neurogenesis in Scutoid and transgenic animals of Drosophila.

    PubMed

    Fuse, N; Matakatsu, H; Taniguchi, M; Hayashi, S

    1999-10-01

    Scutoid is a classical dominant gain-of-function mutation of Drosophila, causing a loss of bristles and roughening of the compound eye. Previous genetic and molecular analyses have shown that Scutoid is associated with a chromosomal transposition resulting in a fusion of no-oceli and snail genes. How this gene fusion event leads to the defects in neurogenesis was not known until now. Here have found that snail is ectopically expressed in the eye-antennal and wing imaginal discs in Scutoid larvae, and that this expression is reduced in Scutoid revertants. We have also shown that the expressivity of Scutoid is enhanced by zeste mutations. snail and escargot encode evolutionarily conserved zinc-finger proteins involved in the development of mesoderm and limbs. Snail and Escargot proteins share a common target DNA sequence with the basic helix-loop-helix (bHLH) type proneural gene products. When expressed in the developing external sense organ precursors of the thorax and the eye, these proteins cause a loss of mechanosensory bristles in the thorax and perturbed the development of the compound eye. Such phenotypes resemble those associated with Scutoid. Furthermore, the effect of ectopic Escargot on bristle development is antagonized by coexpression of the bHLH gene asense. Thus, our results suggest that the Scutoid phenotype is due to an ectopic snail expression under the control of no-oceli enhancer, antagonizing neurogenesis through its inhibitory interaction with bHLH proteins. PMID:10552298

  18. The Drosophila gene escargot encodes a zinc finger motif found in snail-related genes.

    PubMed

    Whiteley, M; Noguchi, P D; Sensabaugh, S M; Odenwald, W F; Kassis, J A

    1992-02-01

    Two independent P-element enhancer detection lines were obtained that express lacZ in a pattern of longitudinal stripes early in germband elongation. In this paper, molecular and genetic characterization of a gene located near these transposons is presented. Sequence analysis of a cDNA clone from the region reveals that this gene has a high degree of similarity with the Drosophila snail gene (Boulay et al., 1987). The sequence similarity extends over 400 nucleotides, and includes a region encoding five tandem zinc finger motifs (72% nucleotide identity; 76% amino acid identity). This region is also conserved in the snail homologue from Xenopus laevis (76% nucleotide identity; 83% amino acid identity) (Sargent and Bennett, 1990). We have named the Drosophila snail-related gene escargot (esg), and the region of sequence conservation common to all three genes the 'snailbox'. A number of Drosophila genomic DNA fragments cross-hybridize to a probe from the snailbox region suggesting that snail and escargot are members of a multigene family. The expression pattern of escargot is dynamic and complex. Early in germband elongation, escargot RNA is expressed in a pattern of longitudinal stripes identical to the one observed in the two enhancer detection lines. Later in development, escargot is expressed in cells that will form the larval imaginal tissues, escargot is allelic with l(2)35Ce, an essential gene located near snail in the genome. PMID:1571289

  19. The ken and barbie gene encoding a putative transcription factor with a BTB domain and three zinc finger motifs functions in terminalia development of Drosophila.

    PubMed

    Lukacsovich, Tamas; Yuge, Kazuya; Awano, Wakae; Asztalos, Zoltan; Kondo, Shunzo; Juni, Naoto; Yamamoto, Daisuke

    2003-10-01

    Mutations in the ken and barbie locus are accompanied by the malformation of terminalia in adult Drosophila. Male and female genitalia often remain inside the body, and the same portions of genitalia and analia are missing in a fraction of homozygous flies. Rotated and/or duplicated terminalia are also observed. Terminalia phenotypes are enhanced by mutations in the gap gene tailless, the homeobox gene caudal, and the decapentaplegic gene that encodes a TGFbeta-like morphogen. The ken and barbie gene encodes a protein with three CCHH-type zinc finger motifs that are conserved in several transcription factors such as Krüppel and BCL-6. All defects in ken and barbie mutants are fully rescued by the expression of a wild-type genomic construct, which establishes the causality between phenotypes and the gene. PMID:14518006

  20. Paramagnetic cobalt as a probe of the orientation of an accessory DNA-binding region of the yeast ADR1 zinc-finger protein.

    PubMed

    Schmiedeskamp, M; Klevit, R E

    1997-11-18

    The minimal DNA-binding domain of the yeast ADR1 transcription factor consists of two Cys2-His2 zinc fingers and an additional 20 residues N-terminal and proximal to the fingers. The accessory sequence likely plays a role in contacting DNA. Paramagnetic cobalt was incorporated into the fingers of an ADR1 DNA-binding construct (ADR1z) to serve as a probe of the proximity of the accessory sequence to the zinc fingers. NMR signals from the accessory region are not perturbed by cobalt incorporation. Previous studies showed that this region is random coil in the ADR1z construct in the absence of DNA; it does not adopt a fixed orientation with respect to the cobalt sites. In contrast, many residues of the accessory region are perturbed by cobalt in the DNA-bound form of the protein, suggesting this region becomes constrained. This observation agrees with previous results showing a disorder-to-order transition for the accessory region upon DNA binding. Furthermore, these results indicate that the accessory region lies close to the fingers in the protein-DNA complex. This region thus does not extend along the DNA away from the zinc fingers; it more likely binds the same stretch of DNA contacted by the zinc fingers. Comparison to the behavior of other zinc-finger proteins that utilize an accessory DNA-binding sequence suggested that the region of ADR1 proximal to the zinc fingers might form an alpha-helix. Analysis of sequential NOEs in the accessory region of DNA-bound ADR1z reveals no helical structure. PMID:9369471

  1. The prokaryotic Cys2His2 zinc-finger adopts a novel fold as revealed by the NMR structure of Agrobacterium tumefaciens Ros DNA-binding domain

    PubMed Central

    Malgieri, Gaetano; Russo, Luigi; Esposito, Sabrina; Baglivo, Ilaria; Zaccaro, Laura; Pedone, Emilia M.; Di Blasio, Benedetto; Isernia, Carla; Pedone, Paolo V.; Fattorusso, Roberto

    2007-01-01

    The first putative prokaryotic Cys2His2 zinc-finger domain has been identified in the transcriptional regulator Ros from Agrobacterium tumefaciens, indicating that the Cys2His2 zinc-finger domain, originally thought to be confined to the eukaryotic kingdom, could be widespread throughout the living kingdom from eukaryotic, both animal and plant, to prokaryotic. In this article we report the NMR solution structure of Ros DNA-binding domain (Ros87), providing 79 structural characterization of a prokaryotic Cys2His2 zinc-finger domain. The NMR structure of Ros87 shows that the putative prokaryotic Cys2His2 zinc-finger sequence is indeed part of a significantly larger zinc-binding globular domain that possesses a novel protein fold very different from the classical fold reported for the eukaryotic classical zinc-finger. The Ros87 globular domain consists of 58 aa (residues 9–66), is arranged in a βββαα topology, and is stabilized by an extensive 15-residue hydrophobic core. A backbone dynamics study of Ros87, based on 15N R1, 15N R2, and heteronuclear 15N-{1H}-NOE measurements, has further confirmed that the globular domain is uniformly rigid and flanked by two flexible tails. Mapping of the amino acids necessary for the DNA binding onto Ros87 structure reveals the protein surface involved in the DNA recognition mechanism of this new zinc-binding protein domain. PMID:17956987

  2. High-resolution solution structure of the double Cys2His2 zinc finger from the human enhancer binding protein MBP-1.

    PubMed

    Omichinski, J G; Clore, G M; Robien, M; Sakaguchi, K; Appella, E; Gronenborn, A M

    1992-04-28

    The high-resolution three-dimensional structure of a synthetic 57-residue peptide comprising the double zinc finger of the human enhancer binding protein MBP-1 has been determined in solution by nuclear magnetic resonance spectroscopy on the basis of 1280 experimental restraints. A total of 30 simulated annealing structures were calculated. The backbone atomic root-mean-square distributions about the mean coordinate positions are 0.32 and 0.33 A for the N- and C-terminal fingers, respectively, and the corresponding values for all atoms, excluding disordered surface side chains, are 0.36 and 0.40 A. Each finger comprises an irregular antiparallel sheet and a helix, with the zinc tetrahedrally coordinated to two cysteines and two histidines. The overall structure is nonglobular in nature, and the angle between the long axes of the helices is 47 +/- 5 degrees. The long axis of the antiparallel sheet in the N-terminal finger is approximately parallel to that of the helix in the C-terminal finger. Comparison of this structure with the X-ray structure of the Zif-268 triple finger complexed with DNA indicates that the relative orientation of the individual zinc fingers is clearly distinct in the two cases. This difference can be attributed to the presence of a long Lys side chain in the C-terminal finger of MBP-1 at position 40, instead of a short Ala or Ser side chain at the equivalent position in Zif-268. This finding suggests that different contacts may be involved in the binding of the zinc fingers of MBP-1 and Zif-268 to DNA, consistent with the findings from methylation interference experiments that the two fingers of MBP-1 contact 10 base pairs, while the three fingers of Zif-268 contact only 9 base pairs. PMID:1567844

  3. Spectroscopic Elucidation of the Inhibitory Mechanism of Cys2His2 Zinc Finger Transcription Factors by CobaltIII Schiff Base Complexes

    PubMed Central

    Heffern, Marie C.; Kurutz, Josh

    2014-01-01

    Transcription factors are key regulators in both normal and pathological cell processes. Affecting the activity of these proteins is a promising strategy for understanding gene regulation and developing effective therapeutics. CoIII Schiff base complexes ([Co(acacen)(L)2]+ where L = labile axial ligands) have been shown to be potent inhibitors of a number of zinc metalloproteins including Cys2His2 zinc finger transcription factors. Inhibition by [Co(acacen)(L)2]+ of the target protein is believed to occur through a dissociative exchange of the labile axial ligands for histidine (His) residues essential for function. Here, we report a series of spectroscopic investigations with model peptides of zinc fingers that elucidate the interaction between [Co(acacen)(L)2]+ complexes and zinc finger transcription factors. Observed changes in NMR chemical shifts and 2D 1H-1H NOESY NMR spectra demonstrate the preference of [Co(acacen)(L)2]+ complexes to coordinate His residues over other amino acids. The conformation of [Co(acacen)(L)2]+ upon His-coordination was characterized by 1H NMR, near-UV circular dichroism, and electronic absorption. These studies reveal that the resulting His-coordinated [Co(acacen)(L)2]+ complex possesses an octahedral structure. The effects of [Co(acacen)(L)2]+ complexes on the zinc finger structure were assessed by the degree of hydrogen bonding (probed by 2D NMR) and secondary structure profiles measured by far-UV circular dichroism. These structural studies demonstrate the ability of [Co(acacen)(L)2]+ complexes to disrupt the ββα structure of zinc fingers, resulting in primarily random coil conformations. A mechanism is described wherein [Co(acacen)(L)2]+ complexes inhibit zinc finger transcription factor activity through selectively coordinating His residues in the zinc finger via dissociative ligand exchange and disrupting the ββα structural motif required for gene regulation. PMID:24203451

  4. Recognition of unmodified histone H3 by the first PHD finger of bromodomain-PHD finger protein 2 provides insights into the regulation of histone acetyltransferases monocytic leukemic zinc-finger protein (MOZ) and MOZ-related factor (MORF).

    PubMed

    Qin, Su; Jin, Lei; Zhang, Jiahai; Liu, Lei; Ji, Peng; Wu, Mian; Wu, Jihui; Shi, Yunyu

    2011-10-21

    MOZ (monocytic leukemic zinc-finger protein) and MORF (MOZ-related factor) are histone acetyltransferases important for HOX gene expression as well as embryo and postnatal development. They form complexes with other regulatory subunits through the scaffold proteins BRPF1/2/3 (bromodomain-PHD (plant homeodomain) finger proteins 1, 2, or 3). BRPF proteins have multiple domains, including two PHD fingers, for potential interactions with histones. Here we show that the first PHD finger of BRPF2 specifically recognizes the N-terminal tail of unmodified histone H3 (unH3) and report the solution structures of this PHD finger both free and in complex with the unH3 peptide. Structural analysis revealed that the unH3 peptide forms a third antiparallel β-strand that pairs with the PHD1 two-stranded antiparallel β-sheet. The binding specificity was determined primarily through the recognition of arginine 2 and lysine 4 of the unH3 by conserved aspartic acids of PHD1 and of threonine 6 of the unH3 by a conserved asparagine. Isothermal titration calorimetry and NMR assays showed that post-translational modifications such as H3R2me2as, H3T3ph, H3K4me, H3K4ac, and H3T6ph antagonized the interaction between histone H3 and PHD1. Furthermore, histone binding by PHD1 was important for BRPF2 to localize to the HOXA9 locus in vivo. PHD1 is highly conserved in yeast NuA3 and other histone acetyltransferase complexes, so the results reported here also shed light on the function and regulation of these complexes. PMID:21880731

  5. Generation of Esr1-Knockout Rats Using Zinc Finger Nuclease-Mediated Genome Editing

    PubMed Central

    Dhakal, Pramod; Kubota, Kaiyu; Chakraborty, Damayanti; Lei, Tianhua; Larson, Melissa A.; Wolfe, Michael W.; Roby, Katherine F.; Vivian, Jay L.

    2014-01-01

    Estrogens play pivotal roles in development and function of many organ systems, including the reproductive system. We have generated estrogen receptor 1 (Esr1)-knockout rats using zinc finger nuclease (ZFN) genome targeting. mRNAs encoding ZFNs targeted to exon 3 of Esr1 were microinjected into single-cell rat embryos and transferred to pseudopregnant recipients. Of 17 live births, 5 had biallelic and 1 had monoallelic Esr1 mutations. A founder with monoallelic mutations was backcrossed to a wild-type rat. Offspring possessed only wild-type Esr1 alleles or wild-type alleles and Esr1 alleles containing either 482 bp (Δ482) or 223 bp (Δ223) deletions, indicating mosaicism in the founder. These heterozygous mutants were bred for colony expansion, generation of homozygous mutants, and phenotypic characterization. The Δ482 Esr1 allele yielded altered transcript processing, including the absence of exon 3, aberrant splicing of exon 2 and 4, and a frameshift that generated premature stop codons located immediately after the codon for Thr157. ESR1 protein was not detected in homozygous Δ482 mutant uteri. ESR1 disruption affected sexually dimorphic postnatal growth patterns and serum levels of gonadotropins and sex steroid hormones. Both male and female Esr1-null rats were infertile. Esr1-null males had small testes with distended and dysplastic seminiferous tubules, whereas Esr1-null females possessed large polycystic ovaries, thread-like uteri, and poorly developed mammary glands. In addition, uteri of Esr1-null rats did not effectively respond to 17β-estradiol treatment, further demonstrating that the Δ482 Esr1 mutation created a null allele. This rat model provides a new experimental tool for investigating the pathophysiology of estrogen action. PMID:24506075

  6. Zinc Finger Protein 467 Is a Novel Regulator of Osteoblast and Adipocyte Commitment*

    PubMed Central

    Quach, Julie M.; Walker, Emma C.; Allan, Elizabeth; Solano, Melissa; Yokoyama, Atsushi; Kato, Shigeaki; Sims, Natalie A.; Gillespie, Matthew T.; Martin, T. John

    2011-01-01

    Osteoblasts and adipocytes are derived from common mesenchymal progenitor cells. The bone loss of osteoporosis is associated with altered progenitor differentiation from an osteoblastic to an adipocytic lineage. cDNA microarrays and quantitative real-time PCR (Q-PCR) were carried out in a differentiating mouse stromal osteoblastic cell line, Kusa 4b10, to identify gene targets of factors that stimulate osteoblast differentiation including parathyroid hormone (PTH) and gp130-binding cytokines, oncostatin M (OSM) and cardiotrophin-1 (CT-1). Zinc finger protein 467 (Zfp467) was rapidly down-regulated by PTH, OSM, and CT-1. Retroviral overexpression and RNA interference for Zfp467 in mouse stromal cells showed that this factor stimulated adipocyte formation and inhibited osteoblast commitment compared with controls. Regulation of adipocyte markers, including peroxisome proliferator-activated receptor (PPAR) γ, C/EBPα, adiponectin, and resistin, and late osteoblast/osteocyte markers (osteocalcin and sclerostin) by Zfp467 was confirmed by Q-PCR. Intra-tibial injection of calvarial cells transduced with retroviral Zfp467 doubled the number of marrow adipocytes in C57Bl/6 mice compared with vector control-transduced cells, providing in vivo confirmation of a pro-adipogenic role of Zfp467. Furthermore, Zfp467 transactivated a PPAR-response element reporter construct and recruited a histone deacetylase complex. Thus Zfp467 is a novel co-factor that promotes adipocyte differentiation and suppresses osteoblast differentiation. This has relevance to therapeutic interventions in osteoporosis, including PTH-based therapies currently available, and may be of relevance for the use of adipose-derived stem cells for tissue engineering. PMID:21123171

  7. AAV-Mediated Delivery of Zinc Finger Nucleases Targeting Hepatitis B Virus Inhibits Active Replication

    PubMed Central

    Weber, Nicholas D.; Stone, Daniel; Sedlak, Ruth Hall; De Silva Feelixge, Harshana S.; Roychoudhury, Pavitra; Schiffer, Joshua T.; Aubert, Martine; Jerome, Keith R.

    2014-01-01

    Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy. PMID:24827459

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

    PubMed

    Moehle, Erica A; Moehle, E A; Rock, Jeremy M; Rock, J M; Lee, Ya-Li; Lee, Y L; Jouvenot, Yann; Jouvenot, Y; DeKelver, Russell C; Dekelver, R C; Gregory, Philip D; Gregory, P D; Urnov, Fyodor D; Urnov, F D; Holmes, Michael C; Holmes, M C

    2007-02-27

    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

  9. Zinc-finger nuclease mediated disruption of Rag1 in the LEW/Ztm rat

    PubMed Central

    2012-01-01

    Background Engineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat Rag1 gene essential for the V(D)J recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain. Results After microinjection of Rag1 specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated Rag1 protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the Rag1 protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4+/CD3+/TCRαβ+ as well as CD8+/CD3+/TCRαβ+ positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in Rag1 mutant rats was associated with a hypoplastic thymus that lacked follicular structures. Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient Rag1 mutant rat. Conclusion The Rag1 mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena. PMID:23136839

  10. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

    PubMed

    Weber, Nicholas D; Stone, Daniel; Sedlak, Ruth Hall; De Silva Feelixge, Harshana S; Roychoudhury, Pavitra; Schiffer, Joshua T; Aubert, Martine; Jerome, Keith R

    2014-01-01

    Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy. PMID:24827459

  11. 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

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

    PubMed Central

    Liang, Lingli; Cao, Jing; Lutz, Brianna Marie; Bekker, Alex; Zhang, Wei; Tao, Yuan-Xiang

    2015-01-01

    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 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 naïve 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 antisense 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. PMID:25630025

  13. Efficient Immunoglobulin Gene Disruption and Targeted Replacement in Rabbit Using Zinc Finger Nucleases

    PubMed Central

    Offner, Sonja; Ros, Francesca; Lifke, Valeria; Zeitler, Bryan; Rottmann, Oswald; Vincent, Anna; Zhang, Lei; Jenkins, Shirin; Niersbach, Helmut; Kind, Alexander J.; Gregory, Philip D.; Schnieke, Angelika E.; Platzer, Josef

    2011-01-01

    Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM+ and IgG+ B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields. PMID:21695153

  14. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    SciTech Connect

    Watanabe, Masahito; Umeyama, Kazuhiro; Matsunari, Hitomi; Takayanagi, Shuko; Haruyama, Erika; Nakano, Kazuaki; Fujiwara, Tsukasa; Ikezawa, Yuka; Nakauchi, Hiromitsu; and others

    2010-11-05

    Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  15. 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

  16. Zinc finger protein 131 inhibits estrogen signaling by suppressing estrogen receptor {alpha} homo-dimerization

    SciTech Connect

    Oh, Yohan; Chung, Kwang Chul

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer ZNF131 directly interacts with ER{alpha}. Black-Right-Pointing-Pointer The binding affinity of ZNF131 to ER{alpha} increases upon E2 stimulation. Black-Right-Pointing-Pointer ZNF131 inhibits ER{alpha}-mediated trans-activation by suppressing its homo-dimerization. Black-Right-Pointing-Pointer ZNF131 inhibits ER{alpha}-dimerization and E2-induced breast cancer cell proliferation. Black-Right-Pointing-Pointer ZNF131 inhibits estrogen signaling by acting as an ER{alpha}-co-repressor. -- Abstract: Steroid hormone estrogen elicits various physiological functions, many of which are mediated through two structurally and functionally distinct estrogen receptors, ER{alpha} and ER{beta}. The functional role of zinc finger protein 131 (ZNF131) is poorly understood, but it is assumed to possess transcriptional regulation activity due to the presence of a DNA binding motif. A few recent reports, including ours, revealed that ZNF131 acts as a negative regulator of ER{alpha} and that SUMO modification potentiates the negative effect of ZNF131 on estrogen signaling. However, its molecular mechanism for ER{alpha} inhibition has not been elucidated in detail. Here, we demonstrate that ZNF131 directly interacts with ER{alpha}, which consequently inhibits ER{alpha}-mediated trans-activation by suppressing its homo-dimerization. Moreover, we show that the C-terminal region of ZNF131 containing the SUMOylation site is necessary for its inhibition of estrogen signaling. Taken together, these data suggest that ZNF131 inhibits estrogen signaling by acting as an ER{alpha}-co-repressor.

  17. Zinc Finger Transcription Factors Displaced SREBP Proteins as the Major Sterol Regulators during Saccharomycotina Evolution

    PubMed Central

    Maguire, Sarah L.; Wang, Can; Holland, Linda M.; Brunel, François; Neuvéglise, Cécile; Nicaud, Jean-Marc; Zavrel, Martin; White, Theodore C.; Wolfe, Kenneth H.; Butler, Geraldine

    2014-01-01

    In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs), which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1) and C. albicans (Cph2) have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1) and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina. PMID:24453983

  18. Cysteine 149 in the extracellular finger domain of ASIC1b subunit is critical for zinc-mediated inhibition

    PubMed Central

    JIANG, Q.; INOUE, K; WU, X.; PAPASIAN, C.J.; WANG, J. Q.; XIONG, Z.G.; CHU, X.P.

    2012-01-01

    Acid-sensing ion channel 1b (ASIC1b) is a proton-gated Na+ channel mostly expressed in peripheral sensory neurons. To date, the functional significance of ASIC1b in these cells is unclear due to the lack of a specific inhibitor/blocker. A better understanding of the regulation of ASIC1b may provide a clue for future investigation of its functional importance. One important regulator of acid-sensing ion channels (ASICs) is zinc. In this study, we examined the detailed zinc inhibition of ASIC1b currents and specific amino acid(s) involved in the inhibition. In CHO cells expressing rat ASIC1b subunit, pretreatment with zinc concentration-dependently inhibited the ASIC1b currents triggered by pH dropping from 7.4 to 6.0 with a half-maximum inhibitory concentration of 26 μM. The inhibition of ASIC1b currents by pre-applied zinc was independent of pH, voltage, or extracellular Ca2+. Further, we showed that the effect of zinc is dependent on the extracellular cysteine, but not histidine residue. Mutating cysteine 149, but not cysteine 58 or cysteine 162, located in the extracellular domain of the ASIC1b subunit abolished the zinc inhibition. These findings suggest that cysteine 149 in the extracellular finger domain of ASIC1b subunit is critical for zinc-mediated inhibition and provide the basis for future mechanistic studies addressing the functional significance of zinc inhibition of ASIC1b. PMID:21767613

  19. Remote Laser Welding of Zinc Coated Steel Sheets in an Edge Lap Configuration with Zero Gap

    NASA Astrophysics Data System (ADS)

    Roos, Christian; Schmidt, Michael

    Remote Laser Welding (RLW) of zinc-coated steel sheets is a great challenge for the automotive industry but offers high potentials with respect to flexibility and costs. In state of the art applications, sheets are joined in overlap configuration with a preset gap for a stable zinc degassing. This paper investigates RLW of fillets without a preset gap and conditions for a stable process. The influence of process parameters on weld quality and process stability is shown. Experimental data give evidence, that the degassing of zinc through the capillary and the rear melt pool are the major degassing mechanisms. Furthermore the paper gives experimental validation of the zinc degassing in advance of the process zone to the open side of the fillet. Chemical analysis of the hot-dip galvanized zinc coating proof the iron-zinc-alloys to be the reason for a limited effectiveness of this mechanism in comparison to pure zinc as intermediate.

  20. Clustered organization of homologous KRAB zinc-finger genes with enhanced expression in human T lymphoid cells.

    PubMed Central

    Bellefroid, E J; Marine, J C; Ried, T; Lecocq, P J; Rivière, M; Amemiya, C; Poncelet, D A; Coulie, P G; de Jong, P; Szpirer, C

    1993-01-01

    KRAB zinc-finger proteins (KRAB-ZFPs) constitute a large subfamily of ZFPs of the Krüppel C2H2 type. KRAB (Krüppel-associated box) is an evolutionarily conserved protein domain found N-terminally with respect to the finger repeats. We report here the characterization of a particular subgroup of highly related human KRAB-ZFPs. ZNF91 is one representative of this subgroup and contains 35 contiguous finger repeats at its C-terminus. Three mRNA isoforms with sequence identity to ZNF91 were isolated by the polymerase chain reaction. These encode proteins with a KRAB domain present, partially deleted or absent. Five genomic fragments were characterized, each encoding part of a gene: the ZNF91 gene or one of four distinct, related KRAB-ZFP genes. All exhibit a common exon/intron organization with the variant zinc finger repeats organized in a single exon and the KRAB domain encoded by two separate exons. This positioning of introns supports the hypothesis that the mRNA isoforms encoding polypeptides with variability in the KRAB domain could arise by alternative splicing. By in situ chromosomal mapping studies and by analysis of fragments from a human genomic yeast artificial chromosome library containing KRAB-ZFP genes, we show that these genes occur in clusters; in particular, a gene complex containing over 40 genes has been identified in chromosomal region 19p12-p13.1. These ZNF91-related genes probably arose late during evolution since no homologous genes are detected in the mouse and rat genomes. Although the transcription of members of this KRAB-ZFP gene subgroup is detectable in all human tissues, their expression is significantly higher in human T lymphoid cells. Images PMID:8467795

  1. Expression of a subset of the Arabidopsis Cys(2)/His(2)-type zinc-finger protein gene family under water stress.

    PubMed

    Sakamoto, H; Araki, T; Meshi, T; Iwabuchi, M

    2000-05-01

    The genes encoding Cys(2)/His(2)-type zinc-finger proteins constitute a large family in higher plants. To elucidate the functional roles of these types of protein, four different members of the gene family were cloned from Arabidopsis by PCR-aided methods. One was identical to the already reported gene STZ/ZAT10 and three were as yet unidentified genes, then designated AZF1 (Arabidopsis zinc-finger protein 1), AZF2 and AZF3. The AZF- and STZ-encoded proteins contain two canonical Cys(2)/His(2)-type zinc-finger motifs, separated by a long spacer. Three conserved regions, named B-box, L-box, and DNL-box, were also recognized outside the zinc-finger motifs, as in other members of the two-fingered Cys(2)/His(2)-type zinc-finger protein family. These four genes were positioned on the same branch of a phylogenetic tree constructed based on the zinc-finger motif sequences, suggesting their structural and functional relationship. RNA blot analysis showed that all four genes were mainly expressed in roots and at different levels in other organs. Expression of the four genes responded to water stress. High-salt treatment resulted in elevated levels of expression of all of these genes. Low-temperature treatment increased the expression levels of AZF1, AZF3, and STZ, but not AZF2. Only AZF2 expression was strongly induced by ABA treatment, where the time course of the induction was similar to that caused by high salinity. In situ localization showed that AZF2 mRNA accumulated in the elongation zone of the roots under the salt-stress condition. These results suggest that AZF1, AZF2, AZF3, and STZ are all involved in the water-stress response in an ABA-dependent or -independent pathway to regulate downstream genes. PMID:10806347

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

    PubMed

    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

  3. Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble

    PubMed Central

    Zandarashvili, Levani; Esadze, Alexandre; Vuzman, Dana; Kemme, Catherine A.; Levy, Yaakov; Iwahara, Junji

    2015-01-01

    Although engineering of transcription factors and DNA-modifying enzymes has drawn substantial attention for artificial gene regulation and genome editing, most efforts focus on affinity and specificity of the DNA-binding proteins, typically overlooking the kinetic properties of these proteins. However, a simplistic pursuit of high affinity can lead to kinetically deficient proteins that spend too much time at nonspecific sites before reaching their targets on DNA. We demonstrate that structural dynamic knowledge of the DNA-scanning process allows for kinetically and thermodynamically balanced engineering of DNA-binding proteins. Our current study of the zinc-finger protein Egr-1 (also known as Zif268) and its nuclease derivatives reveals kinetic and thermodynamic roles of the dynamic conformational equilibrium between two modes during the DNA-scanning process: one mode suitable for search and the other for recognition. By mutagenesis, we were able to shift this equilibrium, as confirmed by NMR spectroscopy. Using fluorescence and biochemical assays as well as computational simulations, we analyzed how the shifts of the conformational equilibrium influence binding affinity, target search kinetics, and efficiency in displacing other proteins from the target sites. A shift toward the recognition mode caused an increase in affinity for DNA and a decrease in search efficiency. In contrast, a shift toward the search mode caused a decrease in affinity and an increase in search efficiency. This accelerated site-specific DNA cleavage by the zinc-finger nuclease, without enhancing off-target cleavage. Our study shows that appropriate modulation of the dynamic conformational ensemble can greatly improve zinc-finger technology, which has used Egr-1 (Zif268) as a major scaffold for engineering. PMID:26324943

  4. 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

  5. Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble.

    PubMed

    Zandarashvili, Levani; Esadze, Alexandre; Vuzman, Dana; Kemme, Catherine A; Levy, Yaakov; Iwahara, Junji

    2015-09-15

    Although engineering of transcription factors and DNA-modifying enzymes has drawn substantial attention for artificial gene regulation and genome editing, most efforts focus on affinity and specificity of the DNA-binding proteins, typically overlooking the kinetic properties of these proteins. However, a simplistic pursuit of high affinity can lead to kinetically deficient proteins that spend too much time at nonspecific sites before reaching their targets on DNA. We demonstrate that structural dynamic knowledge of the DNA-scanning process allows for kinetically and thermodynamically balanced engineering of DNA-binding proteins. Our current study of the zinc-finger protein Egr-1 (also known as Zif268) and its nuclease derivatives reveals kinetic and thermodynamic roles of the dynamic conformational equilibrium between two modes during the DNA-scanning process: one mode suitable for search and the other for recognition. By mutagenesis, we were able to shift this equilibrium, as confirmed by NMR spectroscopy. Using fluorescence and biochemical assays as well as computational simulations, we analyzed how the shifts of the conformational equilibrium influence binding affinity, target search kinetics, and efficiency in displacing other proteins from the target sites. A shift toward the recognition mode caused an increase in affinity for DNA and a decrease in search efficiency. In contrast, a shift toward the search mode caused a decrease in affinity and an increase in search efficiency. This accelerated site-specific DNA cleavage by the zinc-finger nuclease, without enhancing off-target cleavage. Our study shows that appropriate modulation of the dynamic conformational ensemble can greatly improve zinc-finger technology, which has used Egr-1 (Zif268) as a major scaffold for engineering. PMID:26324943

  6. 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

  7. 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

    2015-10-01

    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. PMID:26355596

  8. 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. PMID:25119646

  9. Genome-wide identification and expression profiling of the C2H2-type zinc finger protein transcription factor family in tobacco.

    PubMed

    Minglei, Yang; Jiangtao, Chao; Dawei, Wang; Junhua, Hu; Hua, Wu; Daping, Gong; Guanshan, Liu

    2016-04-01

    C2H2 zinc finger protein transcription factor family members have important biological functions in eukaryotes. They not only bind DNA and RNA, but also interact with proteins. In this study, 118 members of the tobacco C2H2 zinc finger protein transcription factor family were identified from the N. tabacum genome database by using Pfam, SMART and Blastp. The analyses of phylogenetic tree, physical and chemical properties, chromosomal mapping, gene structures, protein three-dimensional structures and tissue expression patterns were performed. The results suggested that the peptide length of different subfamily members is significantly different. Phylogenetic and motif analysis revealed that the C2H2 zinc finger protein transcription factor family members can be divided into 5 subfamilies and each member has at least one C2H2 motif. Genes of the family members are distributed across the 22 chromosomes. C2H2 zinc finger protein transcription factor family members are expressed in different tissues although some have higher expression levels in leaves and roots. This study will be helpful for further analysis of the C2H2 zinc finger family proteins in other plants. PMID:27103457

  10. Nucleolin modulates the subcellular localization of GDNF-inducible zinc finger protein 1 and its roles in transcription and cell proliferation

    SciTech Connect

    Dambara, Atsushi; Morinaga, Takatoshi; Fukuda, Naoyuki; Yamakawa, Yoshinori; Kato, Takuya; Enomoto, Atsushi; Asai, Naoya; Murakumo, Yoshiki; Matsuo, Seiichi; Takahashi, Masahide

    2007-10-15

    GZF1 is a zinc finger protein induced by glial cell-line-derived neurotrophic factor (GDNF). It is a sequence-specific transcriptional repressor with a BTB/POZ (Broad complex, Tramtrack, Bric a brac/Poxvirus and zinc finger) domain and ten zinc finger motifs. In the present study, we used immunoprecipitation and mass spectrometry to identify nucleolin as a GZF1-binding protein. Deletion analysis revealed that zinc finger motifs 1-4 of GZF1 mediate its association with nucleolin. When zinc fingers 1-4 were deleted from GZF1 or nucleolin expression was knocked down by short interference RNA (siRNA), nuclear localization of GZF1 was impaired. These results suggest that nucleolin is involved in the proper subcellular distribution of GZF1. In addition, overexpression of nucleolin moderately inhibited the transcriptional repressive activity of GZF1 whereas knockdown of nucleolin expression by siRNA enhanced its activity. Thus, the repressive activity of GZF1 is modulated by the level at which nucleolin is expressed. Finally, we found that knockdown of GZF1 and nucleolin expression markedly impaired cell proliferation. These findings suggest that the physiological functions of GZF1 may be regulated by the protein's association with nucleolin.

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

    PubMed

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

    1996-04-01

    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. PMID:8610125

  12. Identification and characterization of a salt stress-inducible zinc finger protein from Festuca arundinacea

    PubMed Central

    2012-01-01

    Background Increased biotic and abiotic plant stresses due to climate change together with an expected global human population of over 9 billion by 2050 intensifies the demand for agricultural production on marginal lands. Soil salinity is one of the major abiotic stresses responsible for reduced crop productivity worldwide and the salinization of arable land has dramatically increased over the last few decades. Consequently, as land becomes less amenable for conventional agriculture, plants grown on marginal soils will be exposed to higher levels of soil salinity. Forage grasses are a critical component of feed used in livestock production worldwide, with many of these same species of grasses being utilized for lawns, erosion prevention, and recreation. Consequently, it is important to develop a better understanding of salt tolerance in forage and related grass species. Findings A gene encoding a ZnF protein was identified during the analysis of a salt-stress suppression subtractive hybridization (SSH) expression library from the forage grass species Festuca arundinacea. The expression pattern of FaZnF was compared to that of the well characterized gene for delta 1-pyrroline-5-carboxylate synthetase (P5CS), a key enzyme in proline biosynthesis, which was also identified in the salt-stress SSH library. The FaZnF and P5CS genes were both up-regulated in response to salt and drought stresses suggesting a role in dehydration stress. FaZnF was also up-regulated in response to heat and wounding, suggesting that it might have a more general function in multiple abiotic stress responses. Additionally, potential downstream targets of FaZnF (a MAPK [Mitogen-Activated Protein Kinase], GST [Glutathione-S-Transferase] and lipoxygenase L2) were found to be up-regulated in calli overexpressing FaZnF when compared to control cell lines. Conclusions This work provides evidence that FaZnF is an AN1/A20 zinc finger protein that is involved in the regulation of at least two pathways

  13. Zinc finger protein 382 is downregulated by promoter hypermethylation in pediatric acute myeloid leukemia patients

    PubMed Central

    TAO, YAN-FANG; HU, SHAO-YAN; LU, JUN; CAO, LAN; ZHAO, WEN-LI; XIAO, PEI-FANG; XU, LI-XIAO; LI, ZHI-HENG; WANG, NA-NA; DU, XIAO-JUAN; SUN, LI-CHAO; ZHAO, HE; FANG, FANG; SU, GUANG-HAO; LI, YAN-HONG; LI, YI-PING; XU, YUN-YUN; NI, JIAN; WANG, JIAN; FENG, XING; PAN, JIAN

    2014-01-01

    Acute myeloid leukemia (AML) is the second-most common form of leukemia in children. Aberrant DNA methylation patterns are characteristic of AML. Zinc finger protein 382 (ZNF382) has been suggested to be a tumor suppressor gene possibly regulated by promoter hypermethylation in various types of human cancer. However, ZNF382 expression and methylation status in pediatric AML is unknown. In the present study, ZNF382 transcription levels were evaluated by quantitative reverse-transcription PCR. Methylation status was investigated by methylation-specific (MSP) PCR and bisulfate genomic sequencing (BGS). The prognostic significance of ZNF382 expression and promoter methylation was assessed in 105 cases of pediatric AML. The array data suggested that the ZNF382 promoter was hypermethylated in the AML cases examined. MSP PCR and BGS analysis revealed that ZNF382 was hypermethylated in leukemia cell lines. Furthermore, treatment with 5-aza-2′-deoxycytidine (5-Aza) upregulated ZNF382 expression in the selected leukemia cell lines. The aberrant methylation of ZNF382 was observed in 10% (2/20) of the control samples compared with 26.7% (28/105) of the AML samples. ZNF382 expression was significantly decreased in the 105 AML patients compared with the controls. Patients with ZNF382 methylation showed lower ZNF382 transcript levels compared with patients exhibiting no methylation. There were no significant differences in clinical characteristics or cytogenetic analysis between the patients with or without ZNF382 methylation. ZNF382 methylation correlated with minimal residual disease (MRD). Kaplan-Meier survival analysis revealed similar survival times in the samples with ZNF382 methylation, and multivariate analysis revealed that ZNF382 methylation was not an independent prognostic factor in pediatric AML. The epigenetic inactivation of ZNF382 by promoter hypermethylation can be observed in AML cell lines and pediatric AML samples. Therefore, our study suggests that ZNF382

  14. BTB-Zinc Finger Oncogenes Are Required for Ras and Notch-Driven Tumorigenesis in Drosophila

    PubMed Central

    Doggett, Karen; Turkel, Nezaket; Willoughby, Lee F.; Ellul, Jason; Murray, Michael J.; Richardson, Helena E.; Brumby, Anthony M.

    2015-01-01

    During tumorigenesis, pathways that promote the epithelial-to-mesenchymal transition (EMT) can both facilitate metastasis and endow tumor cells with cancer stem cell properties. To gain a greater understanding of how these properties are interlinked in cancers we used Drosophila epithelial tumor models, which are driven by orthologues of human oncogenes (activated alleles of Ras and Notch) in cooperation with the loss of the cell polarity regulator, scribbled (scrib). Within these tumors, both invasive, mesenchymal-like cell morphology and continual tumor overgrowth, are dependent upon Jun N-terminal kinase (JNK) activity. To identify JNK-dependent changes within the tumors we used a comparative microarray analysis to define a JNK gene signature common to both Ras and Notch-driven tumors. Amongst the JNK-dependent changes was a significant enrichment for BTB-Zinc Finger (ZF) domain genes, including chronologically inappropriate morphogenesis (chinmo). chinmo was upregulated by JNK within the tumors, and overexpression of chinmo with either RasV12 or Nintra was sufficient to promote JNK-independent epithelial tumor formation in the eye/antennal disc, and, in cooperation with RasV12, promote tumor formation in the adult midgut epithelium. Chinmo primes cells for oncogene-mediated transformation through blocking differentiation in the eye disc, and promoting an escargot-expressing stem or enteroblast cell state in the adult midgut. BTB-ZF genes are also required for Ras and Notch-driven overgrowth of scrib mutant tissue, since, although loss of chinmo alone did not significantly impede tumor development, when loss of chinmo was combined with loss of a functionally related BTB-ZF gene, abrupt, tumor overgrowth was significantly reduced. abrupt is not a JNK-induced gene, however, Abrupt is present in JNK-positive tumor cells, consistent with a JNK-associated oncogenic role. As some mammalian BTB-ZF proteins are also highly oncogenic, our work suggests that EMT

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

    PubMed

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

    2012-01-01

    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

  16. 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

  17. BTB-Zinc Finger Oncogenes Are Required for Ras and Notch-Driven Tumorigenesis in Drosophila.

    PubMed

    Doggett, Karen; Turkel, Nezaket; Willoughby, Lee F; Ellul, Jason; Murray, Michael J; Richardson, Helena E; Brumby, Anthony M

    2015-01-01

    During tumorigenesis, pathways that promote the epithelial-to-mesenchymal transition (EMT) can both facilitate metastasis and endow tumor cells with cancer stem cell properties. To gain a greater understanding of how these properties are interlinked in cancers we used Drosophila epithelial tumor models, which are driven by orthologues of human oncogenes (activated alleles of Ras and Notch) in cooperation with the loss of the cell polarity regulator, scribbled (scrib). Within these tumors, both invasive, mesenchymal-like cell morphology and continual tumor overgrowth, are dependent upon Jun N-terminal kinase (JNK) activity. To identify JNK-dependent changes within the tumors we used a comparative microarray analysis to define a JNK gene signature common to both Ras and Notch-driven tumors. Amongst the JNK-dependent changes was a significant enrichment for BTB-Zinc Finger (ZF) domain genes, including chronologically inappropriate morphogenesis (chinmo). chinmo was upregulated by JNK within the tumors, and overexpression of chinmo with either RasV12 or Nintra was sufficient to promote JNK-independent epithelial tumor formation in the eye/antennal disc, and, in cooperation with RasV12, promote tumor formation in the adult midgut epithelium. Chinmo primes cells for oncogene-mediated transformation through blocking differentiation in the eye disc, and promoting an escargot-expressing stem or enteroblast cell state in the adult midgut. BTB-ZF genes are also required for Ras and Notch-driven overgrowth of scrib mutant tissue, since, although loss of chinmo alone did not significantly impede tumor development, when loss of chinmo was combined with loss of a functionally related BTB-ZF gene, abrupt, tumor overgrowth was significantly reduced. abrupt is not a JNK-induced gene, however, Abrupt is present in JNK-positive tumor cells, consistent with a JNK-associated oncogenic role. As some mammalian BTB-ZF proteins are also highly oncogenic, our work suggests that EMT

  18. Loss of DNA-binding and new transcriptional trans-activation function in polyomavirus large T-antigen with mutation of zinc finger motif.

    PubMed Central

    Bergqvist, A; Nilsson, M; Bondeson, K; Magnusson, G

    1990-01-01

    A putative zinc finger in polyomavirus large T-antigen was investigated. We were unable to demonstrate unequivocally a requirement for zinc in specific DNA-binding using the chelating agent 1, 10-phenanthroline. An involvement of the putative zinc finger in specific DNA-binding was nevertheless suggested by the properties of a mutant protein with a cys----ser replacement in the finger motif. Probably as a result of the defective DNA-binding, the mutant protein had lost its activity in initiation of viral DNA-replication and in negative regulation of viral early transcription. However, the trans-activation of the viral late promoter was normal. The analysis also revealed a previously unrecognized activity of large T-antigen. The mutant protein trans-activated the viral early promoter. In the wild-type protein this activity is probably concealed by the separate, negative regulatory function. Images PMID:2160069

  19. Zinc finger protein binding to DNA: an energy perspective using molecular dynamics simulation and free energy calculations on mutants of both zinc finger domains and their specific DNA bases.

    PubMed

    Hamed, Mazen Y; Arya, Gaurav

    2016-05-01

    Energy calculations based on MM-GBSA were employed to study various zinc finger protein (ZF) motifs binding to DNA. Mutants of both the DNA bound to their specific amino acids were studied. Calculated energies gave evidence for a relationship between binding energy and affinity of ZF motifs to their sites on DNA. ΔG values were -15.82(12), -3.66(12), and -12.14(11.6) kcal/mol for finger one, finger two, and finger three, respectively. The mutations in the DNA bases reduced the value of the negative energies of binding (maximum value for ΔΔG = 42Kcal/mol for F1 when GCG mutated to GGG, and ΔΔG = 22 kcal/mol for F2, the loss in total energy of binding originated in the loss in electrostatic energies upon mutation (r = .98). The mutations in key amino acids in the ZF motif in positions-1, 2, 3, and 6 showed reduced binding energies to DNA with correlation coefficients between total free energy and electrostatic was .99 and with Van der Waal was .93. Results agree with experimentally found selectivity which showed that Arginine in position-1 is specific to G, while Aspartic acid (D) in position 2 plays a complicated role in binding. There is a correlation between the MD calculated free energies of binding and those obtained experimentally for prepared ZF motifs bound to triplet bases in other reports (), our results may help in the design of ZF motifs based on the established recognition codes based on energies and contributing energies to the total energy. PMID:26196228

  20. An affinity-based scoring scheme for predicting DNA-binding activities of modularly assembled zinc-finger proteins

    PubMed Central

    Sander, Jeffry D.; Zaback, Peter; Joung, J. Keith; Voytas, Daniel F.; Dobbs, Drena

    2009-01-01

    Zinc-finger proteins (ZFPs) have long been recognized for their potential to manipulate genetic information because they can be engineered to bind novel DNA targets. Individual zinc-finger domains (ZFDs) bind specific DNA triplet sequences; their apparent modularity has led some groups to propose methods that allow virtually any desired DNA motif to be targeted in vitro. In practice, however, ZFPs engineered using this ‘modular assembly’ approach do not always function well in vivo. Here we report a modular assembly scoring strategy that both identifies combinations of modules least likely to function efficiently in vivo and provides accurate estimates of their relative binding affinities in vitro. Predicted binding affinities for 53 ‘three-finger’ ZFPs, computed based on energy contributions of the constituent modules, were highly correlated (r = 0.80) with activity levels measured in bacterial two-hybrid assays. Moreover, Kd values for seven modularly assembled ZFPs and their intended targets, measured using fluorescence anisotropy, were also highly correlated with predictions (r = 0.91). We propose that success rates for ZFP modular assembly can be significantly improved by exploiting the score-based strategy described here. PMID:19056825

  1. Crystal structure of the human protein kinase CK2 regulatory subunit reveals its zinc finger-mediated dimerization.

    PubMed Central

    Chantalat, L; Leroy, D; Filhol, O; Nueda, A; Benitez, M J; Chambaz, E M; Cochet, C; Dideberg, O

    1999-01-01

    Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. The structure of a C-terminal truncated form of the human beta subunit has been determined by X-ray crystallography to 1.7 A resolution. One dimer is observed in the asymmetric unit of the crystal. The most striking feature of the structure is the presence of a zinc finger mediating the dimerization. The monomer structure consists of two domains, one entirely alpha-helical and one including the zinc finger. The dimer has a crescent shape holding a highly acidic region at both ends. We propose that this acidic region is involved in the interactions with the polyamines and/or catalytic subunits. Interestingly, conserved amino acid residues among beta subunit sequences are clustered along one linear ridge that wraps around the entire dimer. This feature suggests that protein partners may interact with the dimer through a stretch of residues in an extended conformation. PMID:10357806

  2. The DnaJ-Like Zinc Finger Domain Protein PSA2 Affects Light Acclimation and Chloroplast Development in Arabidopsis thaliana

    PubMed Central

    Wang, Yan-Wen; Chen, Si-Ming; Wang, Wei-Jie; Huang, Xing-Qi; Zhou, Chang-Fang; Zhuang, Zhong; Lu, Shan

    2016-01-01

    The biosynthesis of chlorophylls and carotenoids and the assembly of thylakoid membranes are critical for the photoautotrophic growth of plants. Different factors are involved in these two processes. In recent years, members of the DnaJ-like zinc finger domain proteins have been found to take part in the biogenesis and/or the maintenance of plastids. One member of this family of proteins, PSA2, was recently found to localize to the thylakoid lumen and regulate the accumulation of photosystem I. In this study, we report that the silencing of PSA2 in Arabidopsis thaliana resulted in variegated leaves and retarded growth. Although both chlorophylls and total carotenoids decreased in the psa2 mutant, violaxanthin, and zeaxanthin accumulated in the mutant seedlings grown under growth condition. Lower levels of non-photochemical quenching and electron transport rate were also found in the psa2 mutant seedlings under growth condition compared with those of the wild-type plants, indicating an impaired capability to acclimate to normal light irradiance when PSA2 was silenced. Moreover, we also observed an abnormal assembly of grana thylakoids and poorly developed stroma thylakoids in psa2 chloroplasts. Taken together, our results demonstrate that PSA2 is a member of the DnaJ-like zinc finger domain protein family that affects light acclimation and chloroplast development. PMID:27047527

  3. Engineering HIV-Resistant Human CD4+ T Cells with CXCR4-Specific Zinc-Finger Nucleases

    PubMed Central

    Wilen, Craig B.; Wang, Jianbin; Tilton, John C.; Miller, Jeffrey C.; Kim, Kenneth A.; Rebar, Edward J.; Sherrill-Mix, Scott A.; Patro, Sean C.; Secreto, Anthony J.; Jordan, Andrea P. O.; Lee, Gary; Kahn, Joshua; Aye, Pyone P.; Bunnell, Bruce A.; Lackner, Andrew A.; Hoxie, James A.; Danet-Desnoyers, Gwenn A.; Bushman, Frederic D.; Riley, James L.; Gregory, Philip D.; June, Carl H.; Holmes, Michael C.; Doms, Robert W.

    2011-01-01

    HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5) virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4) in place of or in addition to CCR5 (R5X4) remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals. PMID:21533216

  4. Identification and use of zinc finger transcription factors that increase production of recombinant proteins in yeast and mammalian cells.

    PubMed

    Park, Kyung-Soon; Seol, Wongi; Yang, Hyo-Young; Lee, Seong-Il; Kim, Sung Keun; Kwon, Ryuk Jun; Kim, Eui-Joong; Roh, Young-Hoon; Seong, Baik Lin; Kim, Jin-Soo

    2005-01-01

    Randomized ZFP-TF libraries could induce a specific phenotype without detailed knowledge about the phenotype of interest because, theoretically, the libraries could modulate any gene in the target organism. We have developed a novel method for enhancing the efficiency of recombinant protein production in mammalian and microbial cells using combinatorial libraries of zinc finger protein transcription factors. To this end, we constructed tens of thousands of zinc finger proteins (ZFPs) with distinct DNA-binding specificities and fused these ZFPs to either a transcriptional activation or repression domain to make transcriptional activators or repressors, respectively. Expression vectors that encode these artificial transcription factors were delivered into Saccharomyces cerevisiae or HEK 293 cells along with reporter plasmids that code for human growth hormone (hGH) or SEAP (secreted alkaline phosphatase) (for yeast or HEK, respectively). Expression of the reporter genes was driven by either the cytomegalovirus (CMV) or SV40 virus promoters. After transfection, we screened the cells for increased synthesis of the reporter proteins. From these cells, we then isolated several ZFP-transcription factors (ZFP-TFs) that significantly increased hGH or SEAP synthesis and subjected these regulatory proteins to further characterization. Our results show that randomized ZFP-TF libraries are useful tools for improving the yield of heterologous recombinant protein both in yeast and mammalian cells. PMID:15932240

  5. The DnaJ-Like Zinc Finger Domain Protein PSA2 Affects Light Acclimation and Chloroplast Development in Arabidopsis thaliana.

    PubMed

    Wang, Yan-Wen; Chen, Si-Ming; Wang, Wei-Jie; Huang, Xing-Qi; Zhou, Chang-Fang; Zhuang, Zhong; Lu, Shan

    2016-01-01

    The biosynthesis of chlorophylls and carotenoids and the assembly of thylakoid membranes are critical for the photoautotrophic growth of plants. Different factors are involved in these two processes. In recent years, members of the DnaJ-like zinc finger domain proteins have been found to take part in the biogenesis and/or the maintenance of plastids. One member of this family of proteins, PSA2, was recently found to localize to the thylakoid lumen and regulate the accumulation of photosystem I. In this study, we report that the silencing of PSA2 in Arabidopsis thaliana resulted in variegated leaves and retarded growth. Although both chlorophylls and total carotenoids decreased in the psa2 mutant, violaxanthin, and zeaxanthin accumulated in the mutant seedlings grown under growth condition. Lower levels of non-photochemical quenching and electron transport rate were also found in the psa2 mutant seedlings under growth condition compared with those of the wild-type plants, indicating an impaired capability to acclimate to normal light irradiance when PSA2 was silenced. Moreover, we also observed an abnormal assembly of grana thylakoids and poorly developed stroma thylakoids in psa2 chloroplasts. Taken together, our results demonstrate that PSA2 is a member of the DnaJ-like zinc finger domain protein family that affects light acclimation and chloroplast development. PMID:27047527

  6. New Insights into DNA Recognition by Zinc Fingers Revealed by Structural Analysis of the Oncoprotein ZNF217*

    PubMed Central

    Vandevenne, Marylène; Jacques, David A.; Artuz, Crisbel; Nguyen, Cuong Dinh; Kwan, Ann H. Y.; Segal, David J.; Matthews, Jacqueline M.; Crossley, Merlin; Guss, J. Mitchell; Mackay, Joel P.

    2013-01-01

    Classical zinc fingers (ZFs) are one of the most abundant and best characterized DNA-binding domains. Typically, tandem arrays of three or more ZFs bind DNA target sequences with high affinity and specificity, and the mode of DNA recognition is sufficiently well understood that tailor-made ZF-based DNA-binding proteins can be engineered. We have shown previously that a two-zinc finger unit found in the transcriptional coregulator ZNF217 recognizes DNA but with an affinity and specificity that is lower than other ZF arrays. To investigate the basis for these differences, we determined the structure of a ZNF217-DNA complex. We show that although the overall position of the ZFs on the DNA closely resembles that observed for other ZFs, the side-chain interaction pattern differs substantially from the canonical model. The structure also reveals the presence of two methyl-π interactions, each featuring a tyrosine contacting a thymine methyl group. To our knowledge, interactions of this type have not previously been described in classical ZF-DNA complexes. Finally, we investigated the sequence specificity of this two-ZF unit and discuss how ZNF217 might discriminate its target DNA sites in the cell. PMID:23436653

  7. 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.

  8. Functional analysis of a novel KRAB/C{sub 2}H{sub 2} zinc finger protein Mipu1

    SciTech Connect

    Jiang, Lei; Tang, Daolin; Wang, Kangkai; Zhang, Huali; Yuan, Can; Duan, Dayue; Xiao, Xianzhong . E-mail: Xianzhongxiao@hotmail.com

    2007-05-18

    A novel rat gene, Mipu1, encodes a 608 amino acid protein with an amino-terminal KRAB domain and 14 carboxyl-terminal C{sub 2}H{sub 2} zinc finger motifs. Mipu1 is localized to the nucleus through its KRAB domain or the linker adjacent to its zinc finger region. Using the GST-Mipu1 bound to glutathione-Sepharose beads, a consensus putative DNA binding site (5'-TGTCTTATCGAA-3') was extracted from a random oligonucleotide library. EMSA and target detection assay showed that the probe containing the putative site can bind to purified GST-Mipu1 fusion protein. The oligonucleotide containing the putative site was inserted into the pGL3-promotor vector to produce a reporter construct. The expression of reporter gene was repressed by overexpression of Mipu1 in a dose-dependent manner. Mutation analysis of the consensus sequence indicated that the repression mediated by Mipu1 is sequence-dependent. These results suggest that Mipu1 is a nuclear protein, which functions as a transcriptional repressor.

  9. Structural polymorphism in the major groove of a 5S RNA gene complements the zinc finger domains of transcription factor IIIA.

    PubMed Central

    Huber, P W; Morii, T; Mei, H Y; Barton, J K

    1991-01-01

    Metal complexes that bind to DNA on the basis of shape-selection have been used to map the conformational features of the DNA binding site for transcription factor IIIA. Conformationally distinct segments are detected on the 5S rRNA gene that correspond closely to the binding sites identified for the individual zinc finger domains of the protein. The local conformations are characterized by a major groove opened because of a change in base pair inclination and/or displacement at a central 5'-pyrimidine-purine-3' step, flanked by a widened minor groove, as would arise at the junctions between alternating B- and A-like DNA segments. Docking experiments with a consensus structure of a zinc finger reveal that the mixed A-B binding site accommodates the peptide domain better than either canonical B- or A-DNA helices. The close structural matching of the conformational variations in the 5S rDNA both to the proposed sites of zinc finger binding and to the shape of an individual zinc finger domain points to DNA structural polymorphism as providing an important determinant in recognition. In particular, shape selection in the 5' half of the internal control region may orient the multiple finger domains. Images PMID:1961749

  10. 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

  11. 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

  12. 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. PMID:23975355

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

    PubMed

    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

    2016-04-26

    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

  14. Zfp-37, a new murine zinc finger encoding gene, is expressed in a developmentally regulated pattern in the male germ line.

    PubMed Central

    Burke, P S; Wolgemuth, D J

    1992-01-01

    To begin to examine the function in the mouse testis of genes containing the zinc finger motif, we have screened an adult mouse total testis cDNA library with probes to a conserved region of zinc fingers. We have isolated cDNAs for a new murine zinc finger encoding gene that has been designated Zfp-37. Northern blot hybridization analysis revealed Zfp-37 transcripts at high levels in the testis, the only adult tissue in which Zfp-37 expression was observed. Zfp-37 was also expressed at lower levels in the mid-gestation embryo and placenta. The major testicular transcripts are 2.3 and 2.6 kb. A 4.0 kb transcript was detected at lower levels in the testis as well as in embryo and placenta. Northern blot and in situ hybridization analysis revealed that expression of Zfp-37 was most abundant in germ cells which have completed meiosis and are undergoing the complex morphogenetic changes of spermiogenesis. The pattern of expression of Zfp-37 and the presence of the zinc finger domain suggest that Zfp-37 may have a role in regulating spermiogenesis. Images PMID:1614869

  15. The Role of Cdkn1A-Interacting Zinc Finger Protein 1 (CIZ1) in DNA Replication and Pathophysiology

    PubMed Central

    Liu, Qiang; Niu, Na; Wada, Youichiro; Liu, Ju

    2016-01-01

    Cdkn1A-interacting zinc finger protein 1 (CIZ1) was first identified in a yeast-2-hybrid system searching for interacting proteins of CDK2 inhibitor p21Cip1/Waf1. Ciz1 also binds to CDK2, cyclin A, cyclin E, CDC6, PCNA, TCF4 and estrogen receptor-α. Recent studies reveal numerous biological functions of CIZ1 in DNA replication, cell proliferation, and differentiation. In addition, splicing variants of CIZ1 mRNA is associated with a variety of cancers and Alzheimer’s disease, and mutations of the CIZ1 gene lead to cervical dystonia. CIZ1 expression is increased in cancers and rheumatoid arthritis. In this review, we will summarize the biological functions and molecular mechanisms of CIZ1 in these physiological and pathological processes. PMID:26861296

  16. Generation of plants resistant to tomato yellow leaf curl virus by using artificial zinc-finger proteins.

    PubMed

    Koshino-Kimura, Yoshihiro; Takenaka, Kosuke; Domoto, Fumiya; Aoyama, Yasuhiro; Sera, Takashi

    2008-01-01

    Previously, we designed an artificial zinc-finger protein (AZP) for blocking a replication protein (Rep) of beet severe curly top virus (BSCTV) from binding to its replication origin and demonstrated that transgenic Arabidopsis plants expressing the AZP are completely resistant to the virus infection. Here we applied the AZP technology to tomato yellow leaf curl virus (TYLCV) infective to an important agricultural crop, tomato. We designed and constructed an AZP binding to the direct repeat to block the TYLCV Rep binding. In gel shift assays, we confirmed that the designed AZP has a higher affinity to the replication origin than that of Rep and that the AZP effectively inhibited the Rep binding to its replication origin in vitro. The AZP gene was then introduced into a plant genome with the help of Agrobacterium tumefaciens to generate the transgenic plants. We will discuss properties of the AZP-transgenic plants against TYLCV infection. PMID:18776317

  17. Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System

    PubMed Central

    Koo, Ok Jae; Park, Sol Ji; Lee, Choongil; Kang, Jung Taek; Kim, Sujin; Moon, Joon Ho; Choi, Ji Yei; Kim, Hyojin; Jang, Goo; Kim, Jin-Soo; Kim, Seokjoong; Lee, Byeong-Chun

    2014-01-01

    To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells (RFP+/eGFP+) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system. PMID:25049958

  18. Sensitive and direct electrochemical detection of double-stranded DNA utilizing alkaline phosphatase-labelled zinc finger proteins.

    PubMed

    Noh, Soodong; Ha, Dat Thinh; Yang, Haesik; Kim, Moon-Soo

    2015-06-21

    Direct detection of double-stranded DNA (dsDNA) using zinc finger proteins (ZFPs) is of great importance in biomedical applications such as identifying pathogens and circulating DNAs. However, its sensitivity is still not sufficiently high because limited signalling labels can be conjugated or fused. Herein, we report sensitive and direct detection of dsDNA using (i) alkaline phosphatase (ALP) as a fast catalytic label conjugated to ZFPs along with (ii) electrochemical measurement of an ALP product (l-ascorbic acid) at the indium-tin oxide electrode with a high signal-to-background ratio. ALP is simply conjugated to a ZFP through lysine residues in a ZFP purification tag, a maltose binding protein (MBP). Sandwich-type electrochemical detection of dsDNA allows a detection limit of ca. 100 fM without using DNA amplification. PMID:25969923

  19. Substitution of DNA-Contacting Amino Acids with Functional Variants in the Gata-1 Zinc Finger: A Structurally and Phylogenetically Guided Mutagenesis

    PubMed Central

    Vonderfecht, Tyson R.; Schroyer, Daniel L.; Schenck, Brandy L.; McDonough, Virginia M.; Pikaart, Michael J.

    2008-01-01

    DNA binding functionality among transcription factor proteins is afforded by a number of structural motifs, such as the helix-turn-helix, helix-loop-helix, and zinc finger domains. The common thread among these diverse structures is their sequence-specific binding to essential promoter or other genetic regulatory sequences with high selectivity and affinity. One such motif, present in a wide range of organisms from bacteria to vertebrates, is the Gata-type zinc finger. This family of DNA-binding proteins is characterized by the presence of one or two (Cys)4 metal binding sites which recognize the protein’s eponymous binding site, GATA. Unlike other conserved DNA binding domains, Gata proteins appear to be restricted to binding consensus GATA sequences, or near variations, in DNA. Since the architecture of the Gata finger seems built around recognizing this particular sequence, we set out to define the allowable range of amino acid substitutions along the DNA-binding surface of a Gata finger that could continue to support sequence specific DNA binding activity. Accordingly, we set up a one-hybrid screen in yeast based on the chicken Gata-1 C-terminal zinc finger. Mutant libraries were generated at five amino acids identified in the Gata-DNA structure as likely to mediate sequence-specific contacts between the Gata finger and DNA. These libraries were designed to give as exhaustive amino acid coverage as possible such that almost all alternative amino acids were screened at each of the five probed positions. Screening and characterization of these libraries revealed several functional amino acid substitutions at two leucines which contact the DNA at the 3’ and 5’ flanks of the GATA binding site, but no functional substituents for amino acids near the core of the binding site. This pattern is consistent with amino acid sequences of known DNA-binding Gata fingers. PMID:18328814

  20. Optical response of wurtzite and zinc blende GaP nanowire arrays.

    PubMed

    Aghaeipour, Mahtab; Anttu, Nicklas; Nylund, Gustav; Berg, Alexander; Lehmann, Sebastian; Pistol, Mats-Erik

    2015-11-16

    We compare the optical response of wurtzite and zinc blende GaP nanowire arrays for varying geometry of the nanowires. We measure reflectance spectra of the arrays and extract from these measurements the absorption in the nanowires. To support our experimental findings and to allow for more detailed investigations of the optical response of the nanowire arrays than possible in experiments, we perform electromagnetic modeling. This modeling highlights the validity of the extraction of the absorptance from reflectance spectra, as well as limitations of the extraction due to anti-reflection properties of the nanowires. In our combined experimental and theoretical study, we find for both zinc blende and wurtzite nanowires an absorption resonance that can be tuned into the ultraviolet by decreasing the diameter of the nanowires. This peak stops blue-shifting with decreasing nanowire diameter at a wavelength of approximately 330 nm for zinc blende GaP. In contrast, for the wurtzite GaP nanowires, the resonance continues blue-shifting at 310 nm for the smallest diameters we succeeded in fabricating. We interpret this as a difference in refractive index between wurtzite and zinc blende GaP in this wavelength region. These results open up for optical applications through resonant absorption in the visible and ultraviolet wavelength regions with both zinc blende and wurtzite GaP nanowire arrays. Notably, zinc blende and wurtzite GaP support resonant absorption deeper into the ultraviolet region than previously found for zinc blende and wurtzite InP and InAs. PMID:26698498

  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. Identification of Glis1, a novel Gli-related, Kruppel-like zinc finger protein containing transactivation and repressor functions.

    PubMed

    Kim, Yong-Sik; Lewandoski, Mark; Perantoni, Alan O; Kurebayashi, Shogo; Nakanishi, Gen; Jetten, Anton M

    2002-08-23

    In this study, we describe the identification and characterization of a novel Krüppel-like protein named Gli-similar 1 (Glis1). The Glis1 gene encodes an 84.3-kDa proline-rich protein. Its five tandem zinc finger motifs exhibit highest homology with those of members of the Gli and Zic subfamilies of Krüppel-like proteins. Glis1 was mapped to mouse chromosome 4C6. Northern blot analysis showed that expression of the 3.3-kb Glis1 mRNA is most abundant in placenta and adult kidney and expressed at lower levels in testis. Whole mount in situ hybridization on mouse embryos demonstrated that Glis1 is expressed in a temporal and spatial manner during development; expression was most prominent in several defined structures of mesodermal lineage, including craniofacial regions, branchial arches, somites, vibrissal and hair follicles, limb buds, and myotomes. Confocal microscopic analysis showed that Glis1 is localized to the nucleus. The zinc finger region plays an important role in the nuclear localization of Glis1. Electrophoretic mobility shift assays demonstrated that Glis1 is able to bind oligonucleotides containing the Gli-binding site consensus sequence GACCACCCAC. Although monohybrid analysis showed that in several cell types Glis1 was unable to induce transcription of a reporter, deletion mutant analysis revealed the presence of a strong activation function at the carboxyl terminus of Glis1. The activation through this activation function was totally suppressed by a repressor domain at its amino terminus. Constitutively active Ca(2+)-dependent calmodulin kinase IV enhanced Glis1-mediated transcriptional activation about 4-fold and may be mediated by phosphorylation/activation of a co-activator. Our results suggest that Glis1 may play a critical role in the control of gene expression during specific stages of embryonic development. PMID:12042312

  3. Zinc finger protein 667 expression is upregulated by cerebral ischemic preconditioning and protects cells from oxidative stress

    PubMed Central

    YUAN, DUN; HUANG, JUN; YUAN, XIANRUI; ZHAO, JIE; JIANG, WEIXI

    2013-01-01

    Brain ischemic injury is associated with clinical emergencies such as acute ischemic and hemorrhagic stroke, head trauma, prolonged severe hypotension and cardiac arrest. Ischemic preconditioning (IPC) is the most powerful endogenous mechanism against ischemic injury. However, the majority of IPC treatments are invasive and thus impractical in the clinical setting. Identifying the endogenous neuroprotective mechanism induced by IPC is important for developing new strategies to reduce stroke severity. Zinc finger protein 667 (ZNF667) is a novel zinc finger protein that is upregulated by myocardial IPC. However, its functional role in neuronal ischemia has not been elucidated. In this study, the changes of ZNF667 expression on cerebral IPC and its potential neuroprotective function were investigated. The cerebral ischemia model was established by ameliorated four-vessel occlusion in rats. The northern blot results demonstrated that ZNF667 expression was increased in the hippocampus and cortex at 12 and 24 h after cerebral ischemic pretreatment. To investigate the neuroprotective function of ZNF667, enhanced green fluorescent protein (EGFP)-ZNF667 fusion protein was expressed in C2C12 and brain astrocytoma cells and its subcellular localization was detected by confocal microscopy. EGFP-ZNF667 fusion proteins were localized in the nucleus of C2C12 and brain astrocytoma cells, indicating that ZNF667 may act as a transcription factor in neural cells. To mimic oxidative stress associated with ischemia/reperfusion injury, hydrogen peroxide (H2O2) was used to treat cells. Cell viability was measured by the lactate dehydrogenase (LDH) and WST-1 assays. A decrease in viability was detected in C2C12 and astrocytoma cells following H2O2 treatment, whereas ZNF667 gene overexpression significantly improved cell viability following H2O2 treatment. These results suggested that ZNF667 plays a neuroprotective role by acting as a transcription factor in cerebral IPC. PMID:24648981

  4. The Zinc Finger Transcription Factor ZXDC Activates CCL2 Gene Expression by Opposing BCL6-mediated Repression

    PubMed Central

    Ramsey, Jon E.; Fontes, Joseph D.

    2013-01-01

    The zinc finger X-linked duplicated (ZXD) family of transcription factors has been implicated in regulating transcription of major histocompatibility complex class II genes in antigen presenting cells; roles beyond this function are not yet known. The expression of one gene in this family, ZXD family zinc finger C (ZXDC), is enriched in myeloid lineages and therefore we hypothesized that ZXDC may regulate myeloid-specific gene expression. Here we demonstrate that ZXDC regulates genes involved in myeloid cell differentiation and inflammation. Overexpression of the larger isoform of ZXDC, ZXDC1, activates expression of monocyte-specific markers of differentiation and synergizes with phorbol 12-myristate 13-acetate (which causes differentiation) in the human leukemic monoblast cell line U937. To identify additional gene targets of ZXDC1, we performed gene expression profiling which revealed multiple inflammatory gene clusters regulated by ZXDC1. Using a combination of approaches we show that ZXDC1 activates transcription of a gene within one of the regulated clusters, chemokine (C-C motif) ligand 2 (CCL2; monocyte chemoattractant protein 1; MCP1) via a previously defined distal regulatory element. Further, ZXDC1-dependent up-regulation of the gene involves eviction of the transcriptional repressor B-cell CLL/lymphoma 6 (BCL6), a factor known to be important in resolving inflammatory responses, from this region of the promoter. Collectively, our data show that ZXDC1 is a regulator in the process of myeloid function and that ZXDC1 is responsible for Ccl2 gene de-repression by BCL6. PMID:23954399

  5. Investigating dynamic and energetic determinants of protein nucleic acid recognition: analysis of the zinc finger zif268-DNA complexes

    PubMed Central

    2010-01-01

    Background Protein-DNA recognition underlies fundamental biological processes ranging from transcription to replication and modification. Herein, we present a computational study of the sequence modulation of internal dynamic properties and of intraprotein networks of aminoacid interactions that determine the stability and specificity of protein-DNA complexes. Results To this aim, we apply novel theoretical approaches to analyze the dynamics and energetics of biological systems starting from MD trajectories. As model system, we chose different sequences of Zinc Fingers (ZF) of the Zif268 family bound with different sequences of DNA. The complexes differ for their experimental stability properties, but share the same overall 3 D structure and do not undergo structural modifications during the simulations. The results of our analysis suggest that the energy landscape for DNA binding may be populated by dynamically different states, even in the absence of major conformational changes. Energetic couplings between residues change in response to protein and/or DNA sequence variations thus modulating the selectivity of recognition and the relative importance of different regions for binding. Conclusions The results show differences in the organization of the intra-protein energy-networks responsible for the stabilization of the protein conformations recognizing and binding DNA. These, in turn, are reflected into different modulation of the ZF's internal dynamics. The results also show a correlation between energetic and dynamic properties of the different proteins and their specificity/selectivity for DNA sequences. Finally, a dynamic and energetic model for the recognition of DNA by Zinc Fingers is proposed. PMID:21106075

  6. Identification of a novel human zinc finger protein that specifically interacts with the activation domain of lentiviral Tat proteins.

    PubMed

    Fridell, R A; Harding, L S; Bogerd, H P; Cullen, B R

    1995-06-01

    Transcriptional activation of HIV-1 gene expression by the viral Tat protein requires the interaction of a cellular cofactor with the Tat activation domain. This domain has been shown to consist of the cysteine-rich and core motifs of HIV-1 Tat and is functionally conserved in the distantly related Tat proteins of HIV-2 and EIAV. Using the yeast two-hybrid system, we have identified a novel human gene product, termed HT2A, that specifically and precisely binds to the activation domain of HIV-1 Tat and that can also interact with the HIV-2 and EIAV Tat proteins in vivo. We present data further demonstrating that the interaction between the activation domain of HIV-1 Tat and the HT2A protein can be readily detected in the mammalian cell nucleus. Sequence analysis demonstrates that HT2A is a novel member of the C3HC4 or ring finger family of zinc finger proteins that includes several known oncogenes and transcription factors. Overall, these data suggest that HT2A may play a significant role in mediating the biological activity of the HIV-1 Tat protein in vivo. PMID:7778269

  7. Spatial proximity of the HIV-1 nucleocapsid protein zinc fingers investigated by time-resolved fluorescence and fluorescence resonance energy transfer.

    PubMed

    Mély, Y; Jullian, N; Morellet, N; De Rocquigny, H; Dong, C Z; Piémont, E; Roques, B P; Gérard, D

    1994-10-11

    The three-dimensional structure of peptides encompassing the two zinc-saturated finger motifs of the nucleocapsid protein NCp7 of HIV-1 has been reported by several groups. Whereas the folded structures of the finger motifs were in good agreement, discrepancies existed concerning their spatial relationship since the fingers were found either close to each other [Morellet, N., Jullian, N., De Rocquigny, H., Maigret, B., Darlix, J. L., & Roques, B. P. (1992) Embo J. 11, 3059-3065] or independently folded [Omichinski, J. G., Clore, G. M., Sakaguchi, K., Appella, E., & Gronenborn, A. M. (1991) FEBS Lett. 292, 25-30, Summers, M. F., Henderson, L. E., Chance, M. R., Bess, J. W., Jr., South, T. L., Blake, P. R., Sagi, I., Perez-Alvarado, G., Sowder, R.C., III, Hare, D.R., & Arthur, L. O. (1992) Protein Sci. 1, 563-574]. As in the interacting finger model, Phe16 in the NH2-terminal finger and Trp37 in the COOH-terminal finger were found to be spatially close, the fluorescence properties of the aromatic residues at positions 16 and 37 in the wild-type and two conservatively substituted (12-53) NCp7 peptides were investigated and compared with those of three negative control derivatives where the finger motifs were not in close contact. Direct distance measurements by Tyr-Trp fluorescence resonance energy transfer of the former derivatives yielded a 7-12 A interchromophore distance range which is clearly inconsistent with the 12.5-18 A range measured for the negative controls and thus a random orientation of the zinc finger motifs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7918429

  8. Solution Structure of Ribosomal Protein L40E, a Unique C4 Zinc Finger Protein Encoded by Archaeon Sulfolobus Solfataricus

    SciTech Connect

    Wu, Bin; Lukin, Jonathan A.; Yee, Adelinda; Lemak, Alexander; Semesi, Anthony; Ramelot, Theresa A.; Kennedy, Michael A.; Arrowsmith, Cheryl H.

    2008-01-31

    The ribosomal protein L40E from archaeon Sulfolobus solfataricus is a component of the 50S ribosomal subunit. L40E is a 56-residue, highly basic protein that contains a C4 zinc finger motif, CRKC_X10_CRRC. Homologs are found in both archaea and eukaryotes but are not present in bacteria. Eukaryotic genomes encode L40E as a ubiquitin-fusion protein. L40E was absent from the crystal structure of euryarchaeota 50S ribosomal subunit. Here we report the three-dimensional solution structure of L40E by NMR spectroscopy. The structure of L40E is a three-stranded b-sheet with a simple b2b1b3 topology. There are two unique characteristics revealed by the structure. First, a large and ordered b2–b3 loop twists to pack across the one side of the protein. L40E contains a buried polar cluster comprising Lys19, Lys20, Cys22, Asn29, and Cys36. Second, the surface of L40E is almost entirely positively charged. Ten conserved basic residues are positioned on the two sides of the surface. It is likely that binding of zinc is essential in stabilizing the tertiary structure of L40E to act as a scaffold to create a broad positively charged surface for RNA and/or protein recognition. A portion of this work was performed in the Environmental Molecular Sciences Facility, a DOE national scientific user facility.

  9. The ancient mammalian KRAB zinc finger gene cluster on human chromosome 8q24.3 illustrates principles of C2H2 zinc finger evolution associated with unique expression profiles in human tissues

    PubMed Central

    2010-01-01

    Background Expansion of multi-C2H2 domain zinc finger (ZNF) genes, including the Krüppel-associated box (KRAB) subfamily, paralleled the evolution of tetrapodes, particularly in mammalian lineages. Advances in their cataloging and characterization suggest that the functions of the KRAB-ZNF gene family contributed to mammalian speciation. Results Here, we characterized the human 8q24.3 ZNF cluster on the genomic, the phylogenetic, the structural and the transcriptome level. Six (ZNF7, ZNF34, ZNF250, ZNF251, ZNF252, ZNF517) of the seven locus members contain exons encoding KRAB domains, one (ZNF16) does not. They form a paralog group in which the encoded KRAB and ZNF protein domains generally share more similarities with each other than with other members of the human ZNF superfamily. The closest relatives with respect to their DNA-binding domain were ZNF7 and ZNF251. The analysis of orthologs in therian mammalian species revealed strong conservation and purifying selection of the KRAB-A and zinc finger domains. These findings underscore structural/functional constraints during evolution. Gene losses in the murine lineage (ZNF16, ZNF34, ZNF252, ZNF517) and potential protein truncations in primates (ZNF252) illustrate ongoing speciation processes. Tissue expression profiling by quantitative real-time PCR showed similar but distinct patterns for all tested ZNF genes with the most prominent expression in fetal brain. Based on accompanying expression signatures in twenty-six other human tissues ZNF34 and ZNF250 revealed the closest expression profiles. Together, the 8q24.3 ZNF genes can be assigned to a cerebellum, a testis or a prostate/thyroid subgroup. These results are consistent with potential functions of the ZNF genes in morphogenesis and differentiation. Promoter regions of the seven 8q24.3 ZNF genes display common characteristics like missing TATA-box, CpG island-association and transcription factor binding site (TFBS) modules. Common TFBS modules partly

  10. Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing: identification of novel zinc finger proteins

    PubMed Central

    Hughes, Marcus D.; Zhang, Zhan-Ren; Sutherland, Andrew J.; Santos, Albert F.; Hine, Anna V.

    2005-01-01

    We have successfully linked protein library screening directly with the identification of active proteins, without the need for individual purification, display technologies or physical linkage between the protein and its encoding sequence. By using ‘MAX’ randomization we have rapidly constructed 60 overlapping gene libraries that encode zinc finger proteins, randomized variously at the three principal DNA-contacting residues. Expression and screening of the libraries against five possible target DNA sequences generated data points covering a potential 40 000 individual interactions. Comparative analysis of the resulting data enabled direct identification of active proteins. Accuracy of this library analysis methodology was confirmed by both in vitro and in vivo analyses of identified proteins to yield novel zinc finger proteins that bind to their target sequences with high affinity, as indicated by low nanomolar apparent dissociation constants. PMID:15722478