Zinc finger transcription factors designed for bispecific coregulation of erbb2 and erbb3 receptors : insights into ErbB receptor biology. (2005 Oct)
Zinc finger transcription factors designed for bispecific coregulation of erbb2 and erbb3 receptors : insights into ErbB receptor biology . signaling through the ErbB family of tyrosine kinase receptors in normal and cancer derived cell lines contributes to cell growth and differentiation . In this work , we altered the levels of erbb2 and erbb3 receptors , individually and in combination , by using 6 finger and 12 finger synthetic zinc finger protein artificial transcription factors ( ATFs ) in an epidermoid squamous cell carcinoma line , A431 . We successfully designed 12 finger ATFs capable of coregulating erbb3 and ICAM 1 or erbb2 and erbb3 . With ATFs , the effects of changes in erbb2 and erbb3 receptor levels were evaluated by using cell proliferation , cell migration , and cell signaling assays . Cell proliferation was increased when erbb2 and erbb3 were both overexpressed . Cell migration on collagen was decreased when erbb2 was down regulated , yet migration on laminin was significantly increased with erbb3 overexpression . erbb2 and erbb3 overexpression also stimulated the phosphatidylinositol 3 kinase and mitogen activated protein kinase pathways . Our ATF approach has elucidated differences in ErbB receptor mediated proliferation , migration , and intracellular signaling that cannot be explained merely by the presence or absence of particular ErbB receptors and emphasizes the dynamic nature of the ErbB signaling system . The transcription factor approach developed here provides a gene economical route to the regulation of multiple genes and may be important …

Identification of a novel krueppel related zinc finger gene ( znf184 ) mapping to 6p21. (1997 May)
identification of a novel krueppel related zinc finger gene ( znf184 ) mapping to 6p21 . 3 . cDNA selection and exon trapping were performed on cosmids mapping to a region 3 Mb distal to HLA A . analysis of resulting fragments indicated the presence of two zinc finger transcripts , and one of these was used to isolate a partial cDNA ( znf184 ) from a placental library . The second transcript contained additional sequence of the 5 end of the gene , extending the sequence to 2678 bp . sequence analysis indicates that znf184 is a classical krueppel zinc finger with 19 highly conserved zinc finger motifs at the C terminus and a krueppel associated box at the N terminus of the protein . This gene encodes a 3 . 2 kb transcript that is highly expressed in testis and expressed at a moderate to low level in all other tissues tested . This zinc finger gene maps to a region approximately 200 kb distal to the microsatellite marker d6s105 and approximately 300 kb proximal to d6s1260 .

Cloning , genomic organization and promoter activity of the mouse zinc finger protein gene ZF 12 The human zinc finger … (2003 Jun)
cloning , genomic organization and promoter activity of the mouse zinc finger protein gene ZF 12 The human zinc finger protein znf191 is a krüppel like transcription factor , which may be relevant to many diseases such as neuropsychiatric , cardiovascular and liver caner diseases . To elucidate the function of znf191 by gene targeting , it is necessary to clone and characterize of the homologous gene in model organisms ( mice ) . The mouse homologous gene ( ZF 12 ) was cloned and sequenced for the first time , the genbank accession number is ay052495 . It contains four exons and three introns ; all intronic splice sites exhibited consensus GT / AG sequences . The single nucleotide polymorphisms ( SNPs ) in exon 2 and the alternative length of 3 untranslated region ( 3 UTR ) have been found . The linkage of the ZF 12 gene and the zinc finger protein gene Zfp 35 has been found , so the ZF 12 gene can be localized to B3 to C or beside of chromosome 18 . We assessed approximately 1 . 2 kb of 5 flanking region of the ZF 12 gene for basal promoter activity . A series of deletion mutants of 5 flanking region linked to the luciferase gene was constructed . basal level expression of these constructs was tested in COS 7 cells , nih3t3 cells and HeLa cells . By measuring luciferase activity , which was transiently expressed in the transfected cells …

Isolation and characterisation of a diverse family of arabidopsis two and three fingered C2H2 zinc finger protein genes and cdnas. (1997 Apr)
isolation and characterisation of a diverse family of arabidopsis two and three fingered C2H2 zinc finger protein genes and cdnas . In animal systems the C2H2 zinc finger protein ( ZFP ) gene family is the largest group of regulatory proteins and its members have a wide and important role in growth and development . It is likely that this family of ZFP transcription factors will also be important in plants . We have used a PCR approach employing highly degenerate oligonucleotide primers to isolate several arabidopsis genomic and cDNA clones encoding potential ZFPs . In addition we have used the sequence information from these clones to identify two ESTs as members of this family . Five two fingered and one three fingered ZFPs have been identified . outside of the zinc finger regions , there is considerable sequence diversity , including the sequence and length of the interfinger region ; the expression pattern of each gene is different . This is the first report of the isolation of a three fingered plant C2H2 ZFP gene and we discuss its possible evolutionary origin from two fingered ZFP genes .

Synthetic zinc finger peptides : old and novel applications. (2004 Jul)
synthetic zinc finger peptides : old and novel applications . In the last decade , the efforts in clarifying the interaction between zinc finger proteins and DNA targets strongly stimulated the creativity of scientists in the field of protein engineering . In particular , the versatility and the modularity of zinc finger ( ZF ) motives make these domains optimal building blocks for generating artificial zinc finger peptides ( ZFPs ) . ZFPs can act as transcription modulators potentially able to control the expression of any desired gene , when fused to an appropriate effector domain . artificial ZFPs open the possibility to re program the expression of specific genes at will and can represent a powerful tool in basic science , biotechnology and gene therapy . In this review we will focus on old , novel and possible future applications of artificial ZFPs .

DNA target selectivity by the vitamin D3 receptor : mechanism of dimer binding to an asymmetric repeat element. (1993 Aug)
DNA target selectivity by the vitamin D3 receptor : mechanism of dimer binding to an asymmetric repeat element . The 1 , 25 dihydroxyvitamin D3 receptor , like other members of the nuclear receptor superfamily , forms dimers in solution that are probably stabilized by a dyad symmetrical interface formed by the ligand binding domain . This receptor , however , recognizes DNA targets that are not dyad symmetric but rather are organized as direct repeats of a hexameric sequence with a characteristic 3 bp spacing . using molecular modeling and site directed mutagenesis , we have identified regions within the vitamin D3 receptor zinc finger region that confer selectivity for direct repeats with appropriate spacing . reflecting the organization of the DNA target , these regions , mapping to the tip of the first zinc finger module and the N and C termini of the second finger module , direct asymmetrical protein protein contacts . A stereochemical model is proposed for these interactions .

Mizip , a highly conserved , vertebrate specific melanin concentrating hormone receptor 1 interacting zinc finger protein. (2002 Sep)
mizip , a highly conserved , vertebrate specific melanin concentrating hormone receptor 1 interacting zinc finger protein . using the yeast two hybrid system a novel protein was identified from human brain that interacts with the C terminus of melanin concentrating hormone receptor 1 ( MCH R1 ) . This protein , characterized by a myeloid translocation protein 8 , nervy , deaf1 proteins ( MYND ) zinc finger domain , is termed MCH R1 interacting zinc finger protein , mizip . It is fully conserved in man , rat , mouse and highly conserved in xenopus and zebrafish , but not detectable in invertebrates . mizip gene organization in human ( six exons on chromosome 9q34 . 3 ) and mouse is highly conserved , yet in rodents an additional exon is generated giving rise to alternatively spliced mrnas . mizip is expressed in brain , testis and stomach , where expression of MCH and MCH R1 was previously reported . mizip interaction with MCH R1 was verified by overlay and pull down assays as well as by co transfection experiments in human embryonic kidney 293 cells . mizip is cytoplasmically localized but gets recruited to the plasma membrane when cells are co transfected with MCH R1 supporting the notion that mizip is involved in the function of MCH R1 .

Early hematopoietic zinc finger protein zinc finger protein 521 : a candidate regulator of diverse immature cells. (2008 Mar)
early hematopoietic zinc finger protein zinc finger protein 521 : a candidate regulator of diverse immature cells . The early hematopoietic zinc finger protein / zinc finger protein 521 ( EHZF / znf521 ) is a recently identified , 1131 amino acid long nuclear factor that contains 30 zinc fingers distributed in clusters throughout its sequence . A 13 AA motif , that binds to components of the nuclear remodelling and histone deacetylation ( NuRD ) complex and is conserved in several trascriptional co repressors , is located at the amino terminal end of the molecule . EHZF / znf521 expression is high in the most immature cells of the haematopoietic system and declines with differentiation . Its transcript is also abundant in brain , particularly in the cerebellum . Its murine counterpart , Evi3 / zfp521 , is enriched in haematopoietic and neural stem cells , in cerebellar granule neuron precursors and in the developing striatum . enforced expression of EHZF / znf521 in haematopoietic progenitors results in their expansion and in inhibition of differentiation . EHZF / znf521 is a member of the BMP signalling pathway and an inhibitor of the transcription factor OLF1 / EBF1 , implicated in the differentiation of neural progenitors and in the specification of the B cell lineage . EHZF expression is observed in most acute myelogenous leukaemias and is particularly high in those with rearrangements of the MLL gene , where EHZF may contribute to the leukaemic phenotype . EHZF / znf521 …

Gene regulation in planta by plant derived engineered zinc finger protein transcription factors. (2005 Apr)
Gene regulation in planta by plant derived engineered zinc finger protein transcription factors . The ability to modify plant traits is of great commercial potential in agricultural biotechnology . To this end we have engineered plant based zinc finger protein transcription factors ( ZFP TFs ) that minimize the use of non plant DNA sequences . This novel architecture supports the use of tandem arrays of zinc finger DNA recognition domains such that the ZFP TF binds a contiguous DNA target site thus emulating the design of ZFP TFs described previously for mammalian gene regulation . We show that this plant based ZFP TF architecture supports high affinity DNA binding while allowing the specificity of the DNA protein interaction to be determined by the amino acid sequences of the recognition helices . This plant based backbone thus supports the use of previously characterized DNA recognition helices originally identified in a mammalian ZFP context without using mammalian DNA sequences . moreover , we show that plant based ZFP TFs employing this new architecture can up regulate endogenous ADH activity by 20 fold in transgenic arabidopsis . Thus plant based ZFP TFs are shown to be potent regulators of gene expression in vivo .

CDNA cloning , DNA binding , and evolution of mammalian transcription factor IIIA. (2002 Jan)
cDNA cloning , DNA binding , and evolution of mammalian transcription factor IIIA . cDNA for rat transcription factor IIIA ( tfiiia ) was cloned by degenerate PCR and rapid amplification of cDNA ends . This cDNA coded for a protein with nine Cys ( 2 ) His ( 2 ) zinc fingers and a non finger C terminal tail ; 63 amino acid ( aa ) sequence identity was observed with the xenopus tfiiia zinc finger region . recombinant rat protein containing only the nine fingers afforded dnase I protection of the identical nucleotides protected by xenopus laevis native tfiiia on the xenopus 5S RNA gene internal control region . A putative mouse tfiiia clone was identified in an expressed sequence tag database by sequence similarity to rat tfiiia . recombinant nine finger protein from this clone afforded dnase I protection of the xenopus 5S rRNA gene like the native frog protein as did a recombinant nine finger form of a putative human tfiiia clone . these DNA binding results demonstrate that these clones code for the respective mammalian tfiiias . rodent and human tfiiias share about 87 aa sequence identity in their zinc finger regions and have evolved to about the same extent as X . laevis and xenopus borealis tfiiias . A monoclonal antibody against human p53 tumor suppressor bound to rat and mouse tfiiia but not to human tfiiia in western blots . The N terminal regions of rodent and human tfiiia do not contain the …

Identification of DNA recognition sequences and protein interaction domains of the multiple Zn finger protein Roaz. (1998 Nov)
identification of DNA recognition sequences and protein interaction domains of the multiple Zn finger protein Roaz . Roaz , a rat C2H2 zinc finger protein , plays a role in the regulation of olfactory neuronal differentiation through its interaction with the Olf 1 / EBF transcription factor family . An additional role for the Roaz / Olf 1 / EBF heterodimeric protein is suggested by its ability to regulate gene activation at a distinct promoter lacking Olf 1 / EBF binding sites . using an in vitro binding site selection assay ( selex ) , we demonstrate that Roaz protein binds to novel inverted perfect or imperfect repeats of gcaccc separated by 2 bp . We show that Roaz is capable of binding to a canonical consensus recognition sequence with high affinity ( Kd 3 nM ) . analysis of the structural requirement for protein dimerization and DNA binding by Roaz reveals the role of specific zinc finger motifs in the Roaz protein for homodimerization and heterodimerization with the Olf 1 / EBF transcription factor . The DNA binding domain of Roaz is mapped to the N terminal 277 amino acids , containing the first seven zinc finger motifs , which confers weak monomeric binding to a single half site and a stronger dimeric binding to the inverted repeat in a binding site dependent manner . Full length protein can form dimers on both the inverted repeat and direct repeat but not on a single half site . these findings …

Metal and DNA binding properties and mutational analysis of the transcription activating factor , B , of coliphage 186 : … (1997 Aug)
metal and DNA binding properties and mutational analysis of the transcription activating factor , B , of coliphage 186 : a prokaryotic C4 zinc finger protein . coliphage 186 B is a 72 amino acid protein belonging to the Ogr family of analogous transcription factors present in P2 like phage , which contain a Cys X2 Cys X22 Cys X4 Cys presumptive zinc finger motif . The molecular characterization of these proteins has been hampered by their insolubility , a difficulty overcome in the present study by obtaining B as a soluble cadmium containing derivative ( CdB ) . atomic absorption spectroscopy showed the presence of one atom of cadmium per molecule of purified CdB . The UV absorption spectrum revealed a shoulder at 250 nm , characteristic of CysS Cd ( II ) ligand to metal charge transfer transitions , and the difference absorption coefficient after acidification ( delta epsilon 248 , 24 mM 1 cm 1 ) indicated the presence of a Cd ( Cys S ) 4 center . Gel mobility shift analysis of CdB with a 186 late promoter demonstrated specific DNA binding ( KD , app 3 4 microm ) and the protein was shown to activate transcription in vitro from a promoter reporter plasmid construct . The B DNA binding site was mapped by gel shift and dnaase I cleavage protection experiments to an area between 70 and 43 relative to the transcription start site , coincident with the consensus sequence , gttgt N8 …

The transactivation domain within cysteine / histidine rich region 1 of CBP comprises two novel zinc binding modules. (2000 Jun)
The transactivation domain within cysteine / histidine rich region 1 of CBP comprises two novel zinc binding modules . cAMP response element binding protein binding protein ( CBP ) is a transcriptional coactivator that interacts with a number of DNA binding proteins and cofactor proteins involved in the regulation of transcription . relatively little is known about the structure of CBP , but it has been noted that it contains three domains that are rich in cysteine and histidine ( CH1 , CH2 , and CH3 ) . The sequence of CH2 conforms to that of a leukemia associated protein domain ( PHD finger ) , and it has been postulated that this and both CH1 and CH3 may be zinc finger domains . This has not , however , been demonstrated experimentally . We have studied CH1 and show that it is composed of two novel zinc binding modules , which we term zinc bundles . Each bundle contains the sequence Cys X ( 4 ) Cys X ( 8 ) His X ( 3 ) Cys , and we show that a synthetic peptide comprising one zinc bundle from CH1 can fold in a zinc dependent manner . CH3 also appears to contain two zinc bundles , one with the variant sequence Cys X ( 2 ) Cys X ( 9 ) His X ( 3 ) Cys , and we demonstrate that this variant motif also undergoes Zn ( II ) induced folding . CH1 acts as …

A binding shift assay for the zinc bound and zinc free HIV 1 nucleocapsid protein by capillary electrophoresis. (1998 Aug)
A binding shift assay for the zinc bound and zinc free HIV 1 nucleocapsid protein by capillary electrophoresis . affinity capillary electrophoresis was used to detect a shift in mobility when a zinc ion binds to the highly basic nucleocapsid protein ( NCp7 ) of HIV 1 . NCp7 contains two Cys X2 Cys X4 His X4 Cys zinc fingers . With constant concentrations of NCp7 as a receptor and various concentrations of zinc as a ligand in the sample buffer and the electrophoresis buffer , we observed changes in electrophoretic mobilities of NCp7 protein when complexes were formed with zinc . scatchard analysis of the mobility indicates the presence of at least two types of binding sites for zinc . At pH 6 . 0 , one site is shown to bind zinc strongly with a binding constant Kb 3 . 25 x 10 ( 5 ) M 1 and the second site has a Kb 1 . 8 x 10 ( 5 ) M 1 . The binding of zinc to the first zinc finger decreased the affinity of zinc for the second zinc finger approximately twofold . The Hill coefficient for this negative cooperativity is 0 . 9 . A series of NCp7 mutants were also examined in the assay to determine their ability to bind zinc . This assay affords a quick method to observe a zinc ion binding to NCp7 and to calculate binding constants . copyright

A novel human striated muscle RING zinc finger protein , SMRZ , interacts with smt3b via its RING domain. (2001 Jun)
A novel human striated muscle RING zinc finger protein , SMRZ , interacts with smt3b via its RING domain . The RING domain is a conserved zinc finger motif , which serves as a protein protein interaction interface . searches of a human heart expressed sequence tag data base for genes encoding the RING domain identified a novel cDNA , named striated muscle RING zinc finger protein ( SMRZ ) . The SMRZ cDNA is 1 . 9 kilobase pairs in length and encodes a polypeptide of 288 amino acid residues ; analysis of the peptide sequence demonstrated an N terminal RING domain . fluorescence in situ hybridization localized SMRZ to chromosome 1p33 34 . northern blots demonstrated that SMRZ is expressed exclusively in striated muscle . In the cardiovascular system , SMRZ is more highly expressed in the fetal heart than in the adult heart ( slightly higher expression in the ventricle than in the atrium ) , suggesting that SMRZ is developmentally regulated . SMRZ was found to interact with smt3b , a ubiquitin like protein , through the SMRZ RING domain . This interaction was abolished by mutagenesis of conserved RING domain residues . transient transfection of SMRZ into c2c12 myoblasts showed localization of SMRZ to the nucleus . these data suggest that SMRZ may play an important role in striated muscle cell embryonic development and perhaps in cell cycle regulation .

Znf268 , a novel kruppel like zinc finger protein , is implicated in early human liver development. (2004 Nov)
znf268 , a novel kruppel like zinc finger protein , is implicated in early human liver development . The advancement in gene knockout and transgenesis have brought about enormous improvement in our understanding of mouse embryogenesis in the past decade or so . On the other hand , relatively little is known about human embryogenesis due largely to the lack of easy access to human embryos and tissues for biomedical studies . We have previously isolated a novel zinc finger gene , znf268 , from a 3 week old human embryo cDNA library in an effort to identify genes important for human embryonic development . To investigate the potential involvement of znf268 in human embryogenesis , we report here the spatial and temporal regulation of its expression during development . northern blot and western blot analyses revealed that znf268 is expressed in early embryos , predominantly , if not exclusively , in fetal liver with little detectable expression in other fetal organs . interestingly , unlike most zinc finger proteins , znf268 protein was found to be localized mainly in the cytoplasm of embryonic hepatocytes . This subcellular localization was substantiated by the localization of EGFP znf268 fusion protein overexpressed in the transfected COS7 cells . these results suggest that znf268 plays a role in early human liver development most likely by functioning through a cytoplasmic mechanism .

Characterization of the thermosensitive ts453 reovirus mutant : increased dsrna binding of sigma 3 protein correlates with interferon resistance. (1998 Aug)
characterization of the thermosensitive ts453 reovirus mutant : increased dsrna binding of sigma 3 protein correlates with interferon resistance . The mutation harbored by the reovirus ts453 thermosensitive mutant has been assigned to the S4 gene encoding the major outer capsid protein sigma 3 . previous gene sequencing has identified a nonconservative amino acid substitution located near the zinc finger of sigma 3 protein in the mutant . coexpression in COS cells of the sigma 3 protein presenting this amino acid substitution ( N16K ) , together with the other major capsid protein mu 1 , has also revealed an altered interaction between the two proteins ; this altered interaction prevents the sigma 3 dependent cleavage of mu 1 to mu 1C . This could explain the lack of outer capsid assembly observed during ts453 virus infection at nonpermissive temperature . In the present study , we pursued the characterization of this mutant sigma 3 protein . although the N16K mutation is located close to the zinc finger region , it did not affect the ability of the protein to bind zinc . In contrast , this mutation , as well as mutations within the zinc finger motif itself , can increase the binding of the protein to double stranded RNA ( dsrna ) . It also appears that the N16K mutant protein is more efficiently transported to the nucleus than the wild type protein , an observation consistent with the postulated role of dsrna binding in sigma 3 nuclear …

Nucleolin modulates the subcellular localization of GDNF inducible zinc finger protein 1 and its roles in transcription and cell proliferation. (2007 Oct)
nucleolin modulates the subcellular localization of GDNF inducible zinc finger protein 1 and its roles in transcription and cell proliferation . 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 .

Isolation , tissue expression , and chromosomal assignment of a novel human gene which encodes a protein with RING finger … (1999 Jan)
isolation , tissue expression , and chromosomal assignment of a novel human gene which encodes a protein with RING finger motif . We identified a novel gene encoding a RING finger ( c3hc4 type zinc finger ) protein from a human neuroblastoma full length enriched cDNA library . This cDNA clone consists of 1919 nucleotides with an open reading frame of a 485 amino acid protein . From reverse transcription ( RT ) polymerase chain reaction ( PCR ) analysis , the messenger RNA was ubiquitously expressed in various human adult tissues . The chromosomal location of the gene was determined on the chromosome 6p21 . 3 region by PCR based analyses with both a human / rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel .

FYVE finger proteins as effectors of phosphatidylinositol 3 phosphate. (1999 Jun)
FYVE finger proteins as effectors of phosphatidylinositol 3 phosphate . phosphatidylinositol 3 phosphate ( ptdins ( 3 ) P ) , generated via the phosphorylation of phosphatidylinositol by phosphatidylinositol 3 kinase ( PI 3 kinase ) , plays an essential role in intracellular membrane traffic . The underlying mechanism is still not understood in detail , but the recent identification of the FYVE finger as a protein domain that binds specifically to ptdins ( 3 ) P provides a number of potential effectors for ptdins ( 3 ) P . The FYVE finger ( named after the first letter of the four proteins containing it ; fab1p , YOTB , vac1p and EEA1 ) is a double zinc binding domain that is conserved in more than 30 proteins from yeast to mammals . It is found in several proteins involved in intracellular traffic , and FYVE finger mutations that affect zinc binding are associated with the loss of function of several of these proteins . The interaction of FYVE fingers with ptdins ( 3 ) P may serve three alternative functions : first , to recruit cytosolic FYVE finger proteins to ptdins ( 3 ) P containing membranes ( in concert with accessory molecules ) ; second , to enrich for membrane bound FYVE finger proteins into ptdins ( 3 ) P containing microdomains within the membrane ; and third , to modulate the activity of membrane bound FYVE finger proteins .