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 .

Molecular cloning of a zinc finger protein which binds to the heptamer of the signal sequence for V ( D … (1994 Jan)
molecular cloning of a zinc finger protein which binds to the heptamer of the signal sequence for V ( D ) J recombination . The somatic V ( D ) J recombination for the assembly of the Ig and TCR genes is mediated by the recombination signal sequences ( Rss ) and the V ( D ) J recombinase . A cDNA clone was isolated from a lambda gt11 expression library made from mouse thymocyte poly ( A ) RNA , using the Rss as a ligand . The deduced amino acid sequence of the putative protein , designated recognition component ( Rc ) , reveals a pair of Cys2 His2 zinc fingers followed by a Glu and Asp rich acidic domain . In addition , there are five copies of the Ser / Thr Pro X Arg / Lys sequence , which are putative DNA binding units . The zinc finger acidic domain structures present in Rc are also found in several enhancer binding proteins , such as those for the kappa B motif of the Ig kappa light chain enhancer or related sequences . bacterial fusion proteins for Rc bind preferentially to the Rss heptamer and to the kappa B motif . The dual affinities of Rc for the Rss heptamer and the kappa B motif suggest a possible link between Ig transcription and somatic recombination . The formation of multiple gel shifted DNA protein complexes for Rc and its DNA ligand suggests that these complexes tend to …

Genomic organisation and characterisation of the neural sex determination gene fruitless ( fru ) in the hawaiian species drosophila heteroneura. (2000 May)
genomic organisation and characterisation of the neural sex determination gene fruitless ( fru ) in the hawaiian species drosophila heteroneura . there are several mechanisms for the determination of sex . sexual behaviour is part of the sex determination cascade , and in drosophila melanogaster male courtship is controlled in part by the fruitless gene . As part of a study of sexual behaviour in hawaiian drosophila , we have cloned the neural sex determination gene fru from the hawaiian picture wing species drosophila heteroneura . The fru gene has at least seven exons covering a region of 18kb and encodes three transcripts , fruA , fruB and fruC . Each transcript encodes a single ORF of 841 , 678 and 691aa , respectively . The FRUA and FRUB proteins have a BTB protein protein binding domain and two zinc finger like domains and are well conserved with the D . melanogaster proteins . The FRUC protein has a BTB domain but no zinc finger like domains . The fru gene is expressed in 1 7 day old adult males as a 5 . 1kb transcript . This transcript is not seen in adult females , so the fru gene has a different pattern of sex differential expression in the hawaiian drosophila compared with D . melanogaster .

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 .

Specific interactions of the autoantigen L7 with multi zinc finger protein ZNF7 and ribosomal protein S7. (1997 Oct)
specific interactions of the autoantigen L7 with multi zinc finger protein ZNF7 and ribosomal protein S7 . The eucaryotic protein L7 , which associates with the large subunit of ribosomes , has been shown to be a major autoantigen in systemic autoimmune arthritis . The N terminus carries a sequence motif that is similar to the leucine zipper domain of eucaryotic transcription factors . This domain promotes the homodimerization of protein L7 through alpha helical coiled coil formation and binds to distinct mrnas , thereby inhibiting their cell free translation . using a yeast two hybrid selection , we have identified from a jurkat T lymphoma cDNA library ribosomal protein S7 and the multi zinc finger protein ZNF7 as proteins that interact with protein L7 . A fragment of L7 carrying the leucine zipper like domain is fully sufficient to mediate these interactions . their potential biological significance is indicated by low apparent dissociation constants of S7 L7 ( 15 x 10 ( 9 ) M ) and , respectively , ZNF7 L7 ( 2 x 10 ( 9 ) M ) complexes and co immunoprecipitation of proteins S7 , ZNF7 , and L7 from a cell lysate with an anti L7 antibody . We also show that ZNF7 like L7 and S7 can exist in a ribosome bound form . This study provides further evidence suggesting that L7 is involved in translational regulation through interactions with components of the translational apparatus .

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 .

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 …

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 zinc finger like domain in the 54kda protein of several pestiviruses. (1997 Jul)
A zinc finger like domain in the 54kda protein of several pestiviruses .

Variations of the C2H2 zinc finger motif in the yeast genome and classification of yeast zinc finger proteins. (1997 Jul)
variations of the C2H2 zinc finger motif in the yeast genome and classification of yeast zinc finger proteins . The prosite pattern Zinc finger C2H2 was extended to permit the detection of all C2H2 zinc fingers and their parent proteins in the recently completed sequence of the yeast genome . additionally , a new computer program was written that extracts other zinc binding motifs ( non C2H2 fingers ) , overlapping with the classical zinc finger pattern , from the found set of yeast C2H2 fingers . The complete and correct detection of all fingers is a prerequisite for the classification of the yeast zinc finger proteins in functional terms . The detected 53 yeast C2H2 zinc finger proteins do not contain finger clusters with 10 or more repeats , as is frequently found in higher eukaryotes . Only three proteins contain four or more fingers in a cluster . moreover , nearly all 27 yeast proteins with tandem arrays of two or three finger domains can be classified into nine subgroups with high sequence conservation in their finger clusters , in particular of their DNA recognition helices . these results and application of the recently elaborated finger / DNA recognition rules suggest that the yeast proteins belonging to the same subgroup may recognize identical or very similar DNA sites .

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 .

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 .

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 .

From repression domains to designer zinc finger proteins : a novel strategy of intracellular immunization against HIV. (1996 Oct)
From repression domains to designer zinc finger proteins : a novel strategy of intracellular immunization against HIV . tissue specific gene regulation of eukaryotic organisms is to a large extent mediated by transcription factors that interact with genomic DNA sequences in a sequence specific manner . The purpose of this synopsis is to put forward the potential of designer zinc finger proteins in treating infections of human immunodeficiency virus ( HIV ) . artificial transcription factors containing designer zinc finger structures fused to activator or repressor domains have been designated transcription response modifiers ( TRMs ) . The principle of engineering TRMs has been derived from the analysis of human krüppel type zinc finger genes and their products . Our research efforts encompass two fascinating features that are displayed by the human krüppel type zinc finger protein KOX1 : 1 ) the krüppel type zinc finger domains display rules of sequence specific DNA recognition , and 2 ) the evolutionarily conserved krüppel associated box ( KRAB ) presents one of the strongest transcriptional repressors identified so far in mammalian organisms . The KRAB repressor activity is postulated to be mediated through co repressor molecules , such as silencing mediating protein 1 ( SMP 1 ) . Thus , the structural organization and functional analysis of zinc finger proteins revealed principles of zinc finger transcription factors that are applicable for reducing the viral load in individuals infected with HIV . In this article , a novel concept of generating therapeutic proteins …

Identification of a zinc finger encoding gene in onchocerca volvulus. (1994 Jan)
identification of a zinc finger encoding gene in onchocerca volvulus . eukaryotic transcription factors can be identified and classified according to conserved DNA binding motifs and conserved regulatory domains . The functional and structural analysis , and the conservation of these genes among metazoans emphasizes the importance of these DNA binding proteins in development and differentiation . In order to identify genes with common DNA binding motifs in the genome of onchocerca volvulus we have cloned a zinc finger encoding gene of the structure C2 H2 . using the caenorhabditis elegans sex determining tra 1 gene as a probe we have identified two tra 1 related genes in the genome of O . volvulus . The zinc finger region of one of these genes ( ovzf1 ) was subcloned and sequenced . The predicted protein has at least eight consecutive zinc fingers and each finger possesses the characteristic paired cysteine and histidine residues and the proper spacing of the amino acids between the conserved residues .