Putative Zinc Finger Protein Research Articles

Characteristics of the Intron Involvement in the Mitogen-induced Expression of Zfp-36

Zfp-36, the gene encoding the putative zinc finger protein tristetraprolin (TTP), is rapidly induced in fibroblasts by a variety of growth factors. Recent gene knockout experiments have shown that TTP-deficient mice developed arthritis, cachexia, and autoimmunity, all apparently mediated by an excess of tumor necrosis factor alpha. We recently showed that full serum inducibility of Zfp-36 requires elements in the promoter; in addition, removal of the single intron strikingly inhibited serum-induced TTP expression. We show here that replacement of the intron with unrelated sequences, or removal of 95% of the intron but retention of the splice sites, each resulted in the maintenance of approximately 45 and 19%, respectively, of full serum-induced expression. In addition, deletion of intron sequences base pairs 601-655 decreased the serum-induced expression of TTP by 65%. Sequence base pairs 618-626 bound specifically to the transcription factor Sp1; mutation of this binding motif decreased TTP expression by 70%, suggesting that Sp1 binding to this motif contributes to serum induction of Zfp-36. We conclude that full serum-induced expression of Zfp-36 depends on the activation of conventional promoter elements as well as elements in the single intron, and that the presence per se of the intron in its natural location also contributes significantly to the regulated expression of this gene.

Isolation and mapping of a family of putative zinc-finger protein cDNAs from rice.

To understand the functions of rice homologues of the Arabidopsis flowering-time gene CONSTANS (CO) and salt-tolerance gene STO, we performed a similarity search of the single-run sequence data of cDNA clones accumulated by the Rice Genome Research Program, and isolated seven rice cDNA clones (S3574, C60910, S12569, R2931, R1479, R1577, and E10707) coding for proteins containing one of two zinc-finger-like motifs. Comparison of the deduced amino acid sequences between these cDNAs and the CO gene revealed significant similarities (46%-61%) in the region of zinc-finger motifs. A domain having a high content of basic amino acids at the C-terminus of the CO protein was found in the corresponding region of proteins predicted by from cDNAs S3574, C60910, and S12569. Two amino acid sequences, "CCADEAAL" and "FCV(L)EDRA," which were present inside each zinc-finger in the Arabidposis regulatory protein STO, were also found in each of the two zinc-finger regions of proteins predicted from cDNAs R2931, R1479, R1577, and E10707. Using restriction fragment length polymorphism (RFLP) linkage analysis, we determined the chromosomal location of the seven cDNA clones. The position of R2931 on the RFLP linkage map was closely linked to Hd-3, one of the putative quantitative trait loci (QTL) controlling heading date in rice.

Noc2, a Putative Zinc Finger Protein Involved in Exocytosis in Endocrine Cells

We have cloned a cDNA encoding a novel protein of 302 amino acids (designated Noc2, no C2 domain) that has 40.7% amino acid identity with and 77.9% similarity to the N-terminal region of rabphilin-3A, a target molecule of Rab3A. However, unlike rabphilin-3A, Noc2 lacks two C2 domains that are thought to interact with Ca2+ and phospholipids. Noc2 is expressed predominantly in endocrine tissues and hormone-secreting cell lines and at very low levels in brain. Immunoblot analysis of subcellular fractions of the insulin-secreting cell line MIN6 and immunocytochemistry reveal that Noc2 is a 38-kDa protein present in the cytoplasm. Overexpression of Noc2 in PC12 cells cotransfected with growth hormone enhances high K+-induced growth hormone secretion. Screening a mouse embryonic cDNA library with the yeast two-hybrid system shows that Noc2 interacts with the LIM domain-containing protein zyxin, a component of the cytoskeleton, and this interaction is further confirmed by the coimmunoprecipitation experiment. Accordingly, Noc2 is probably involved in regulated exocytosis in endocrine cells by interacting with the cytoskeleton.

Identification of Glucose-Regulated Genes in Human Mesangial Cells by mRNA Differential Display

Diabetic nephropathy is characterised by an accumulation of extracellular matrix proteins in the glomerular mesangium. Hyperglycaemia is a major factor promoting this progressive expansion of the mesangial matrix. We have used the technique of mRNA differential display to investigate changes in gene expression in cultured human mesangial cells following long-term (21 days) exposure to either physiologic (4 mM) or pathologic (30 mM)d-glucose concentrations. Approximately 12,000 mRNA species were screened for evidence of altered expression and several hundred candidate cDNA fragments were obtained. Northern blot and RT-PCR analysis of ten randomly chosen candidate cDNA fragments revealed three exhibiting increased mRNA expression under elevatedd-glucose levels. Nucleotide sequence analysis identified two of the cDNA fragments as encoding prolyl 4-hydroxylase α-subunit and thrombospondin-1. The third cDNA fragment represents a novel glucose-regulated gene, encoding a putative zinc finger protein. Upregulated expression of these genes in response to high levels ofd-glucose may contribute significantly to the disease process. mRNA differential display is a useful tool to investigate the mechanism of diabetic nephropathy.

A novel genetic system to isolate a dominant negative effector on DNA- binding activity of Oct-2

Recent studies have revealed that interactions between transcription factors play an important role in regulation of gene expression in eukaryotic cells. To isolate cDNA clones that dominantly inhibit the DNA-binding activity of Oct-2, chosen as a representative factor, we have developed a novel screening system. This employs an Escherichia coli tester strain carrying a modified lac operon as a reporter gene, with the lac operator sequence replaced by an octamer sequence. Oct-2 expressed in this tester strain represses the expression of the reporter gene and changes the phenotype of the cell from Lac+to Lac-. Introduction of a cDNA expression library prepared from a human T-cell line into the Oct-2-harboring tester strain allowed selection of three Lac+clones out of 1 x 10(5) transformants. One of them, hT86, encoding a putative zinc finger protein was found to derepress beta-galactosidase activity in the Oct-2-harboring tester strain at the transcriptional level. In gel mobility shift assays, hT86 attenuated the intensity of the retarded band composed of the octamer probe and Oct-2, suggesting a dominant negative effect on the DNA-binding activity of Oct-2. The strategy described here provides a new approach for studying protein-protein interactions that govern the complex regulation of gene expression.

Thyroid Hormone-Responsive Genes in Developing Cerebellum Include a Novel Synaptotagmin and ahairlessHomolog

Proper development of the mammalian CNS requires sufficient thyroid hormone; thyroid hormone deficiency during a brief perinatal period produces severe neurological defects in humans and experimental animals. Thyroid hormone exerts its effects through nuclear receptors, which modulate the transcription of downstream genes in response to hormone binding. Surprisingly, few genes that are regulated by thyroid hormone receptors in the CNS have been described. Here, I report the isolation and characterization of genes that are expressed in response to thyroid hormone in developing rat brain. One such gene (Srg1) encodes a novel protein related to synaptotagmin, a protein involved in regulating neurotransmitter release; another (hr) encodes a putative zinc finger protein related to the product of a recently identified mouse gene, hairless. Both Srg1 and hr are induced rapidly (<4 hr), suggesting that they are regulated directly by thyroid hormone. The temporal and spatial expression of both Srg1 and hr is characteristic of genes important to nervous system development. Srg1 and hr are likely part of a cascade of gene activation induced by thyroid hormone that is critical for CNS organization and development.

BCL-6 expression in reactive lymphoid tissue and in B-cell non-Hodgkin's lymphomas.

Chromosomal abnormalities involving 3q27 have recently been associated with diffuse large B-cell lymphomas and, less frequently, with follicular lymphomas. Molecular studies have led to the identification of the BCL-6/LAZ-3 gene, located at 3q27 and coding for a putative zinc-finger protein that might act as a transcriptional regulator during cell differentiation and development. Rearrangement of BCL-6 results in truncation of the gene in its 5' portion, leaving the protein intact; a resultant deregulation of its expression has been hypothesized. In order to test this hypothesis, the expression of BCL-6 protein was investigated in human reactive lymphoid tissue and compared with a group of non-Hodgkin's lymphomas (NHLs) with or without 3q27 anomalies and/or BCL-6 gene rearrangement. BCL-6 protein is consistently expressed in reactive lymphoid tissues, where it is restricted to the follicle centre. The protein is also widely expressed in NHL: all follicular lymphomas tested showed a pattern of expression similar to the reactive B follicle, independently of the presence of BCL-6 gene rearrangement and/or 3q27 anomalies. In the diffuse large B-cell lymphomas, there was more variation in BCL-6 expression, but a correlation with 3q27 anomalies and/or BCL-6 rearrangement was not found. Deregulation of the BCL-6 gene did not result in an aberrant tissue expression as detected by immunohistochemistry.

The Drosophila Polycomb group gene Sex comb on midleg (Scm) encodes a zinc finger protein with similarity to polyhomeotic protein.

The Sex comb on midleg (Scm) gene is a member of the Polycomb group (PcG) of genes in Drosophila melanogaster. The PcG genes encode transcriptional repressors required for proper spatial expression of homeotic genes. We report the isolation of new Scm mutations and the molecular characterization of the Scm gene. Scm mRNA is expressed maternally, at peak levels in early embryos and then at lower levels throughout the remainder of development. Scm encodes a putative zinc finger protein of 877 amino acids. Scm protein is similar to polyhomeotic, another member of the PcG, both in the zinc finger region and in a separate C-terminal domain of 60 amino acids, which we term the SPM domain. Sequence analysis of an Scm mutant allele suggests a functional requirement for the SPM domain. Scm protein also bears homology in multiple domains to a mouse protein, Rae-28 (Nomura, M., Takihara, Y. and Shimada, K. (1994) Differentiation 57,39-50) and to a fly tumor suppressor protein, the product of the lethal(3)malignant brain tumor gene (Wismar, J. et al., (1995) Mech. Dev. 53, 141-154). Possible functional relationships among these proteins and potential biochemical roles for Scm protein in PcG repression are discussed.

A zinc finger protein that represses transcription of the human MHC class II gene, DPA.

The proximal promoters of all MHC class II genes contain a sequence element, the 19-bp X box, that is conserved in both sequence and position. Extensive analysis using a wide variety of approaches has demonstrated that the integrity of the X box is essential for transcription initiation from all class II genes studied. However, the X box is now recognized to contain two subregions, termed X1 and X2. Radiolabeled oligonucleotides corresponding to the X2 box of the MHC class II genes DPA and DQB were used to screen B cell and T cell expression libraries. A novel cDNA, termed XBR (X box repressor), encoding a putative zinc finger protein that binds specifically to the DPA X2 box was isolated from a human T cell line. The XBR gene encodes a 7-kb message that is ubiquitously transcribed, although at higher levels in tissues of the lymphocytic compartment. Southern blots indicate that this gene is single copy in primates and contains regions that are highly divergent in other species. Overexpression of XBR in a B cell line resulted in a dramatic reduction of transcription from a reporter gene construct driven by the DPA promoter, but not from similar constructs with mutations in the X2 box. Similarly, overexpression of XBR reduced induction of reporter gene activity driven from the DPA promoter in HeLa cells treated with IFN-gamma. XBR may, therefore, mediate transcriptional repression, thus preventing inappropriate MHC class II expression. XBR function may in part explain the dominant trans-acting repression of MHC class II expression reported in cell fusion experiments.

Huckebein specifies aspects of CNS precursor identity required for motoneuron axon pathfinding

huckebein encodes a putative zinc finger protein expressed in a subset of Drosophila CNS precursors, including the NB 4-2/GMC 4-2a/RP2 cell lineage. In huckebein mutant embryos, GMC 4-2a does not express the cell fate marker EVEN-SKIPPED; conversely, huckebein overexpression produces a duplicate EVEN-SKIPPED-positive GMC 4-2a. We use Dil to trace the entire NB 4-2 lineage in wild-type and huckebein mutant embryos. Loss of huckebein does not affect the number, position, or type of neurons in the NB 4-2 lineage; however, all motoneurons show axon pathfinding defects and never terminate at the correct muscle. Thus, huckebein regulates aspects of GMC and neuronal identity required for proper motoneuron axon pathfinding in the NB 4-2 lineage.

Promoter Analysis of Zfp-36 the Mitogen-inducible Gene Encoding the Zinc Finger Protein Tristetraprolin

The gene encoding the putative zinc finger protein tristetraprolin (TTP), Zfp-36, is rapidly induced by a variety of mitogens and growth factors. We show here that 77 base pairs 5' of the transcription start site are sufficient for full serum inducibility of the mouse Zfp-36 promoter. This region of the promoter includes consensus sequences for the binding of the transcription factors EGR-1, AP2, and Sp1. In addition, we have identified a previously undescribed element, TTP promoter element 1 (TPE1); this 10-base pair sequence includes a palindrome and is identical in the human, bovine, and mouse genes. Each of the three binding elements, EGR-1, AP2, and TPE1, contribute to the serum induction of Zfp-36 and can confer serum-inducible expression on a heterologous minimal promoter. Gel mobility shift assays demonstrated the formation of complexes consisting of this region of the promoter and cellular nuclear proteins and demonstrated that the extent of complex formation could be altered by treatment of the cells with serum or insulin. These results suggest that the response of Zfp-36 to serum and other mitogens is mediated by a series of cis-acting elements acting in concert to confer full inducible transcription of this gene.

Analysis of cubitus interruptus regulation in Drosophila embryos and imaginal disks.

The cubitus interruptus (ci) gene of Drosophila is expressed in all anterior compartment cells in both embryos and imaginal disks where it encodes a putative zinc-finger protein related to the vertebrate Gli and C. elegans Tra-1 proteins. Using ci/lacZ fusions, we located regulatory sequences responsible for the normal pattern of ci expression, and obtained evidence that separate elements regulate its expression in embryos and imaginal disks. Mutants that delete a portion of this regulatory region express ci ectopically in the posterior compartments of their wing imaginal disks and have wings with malformed posterior compartments. Similar deletions of ci/lacZ fusion constructs also result in ectopic posterior compartment expression. Evidence that the engrailed protein normally represses ci in posterior compartments includes the expansion of ci expression into posterior compartment cells that lack engrailed function, diminution of ci expression upon overexpression of engrailed protein in anterior compartment cells, and the ability of engrailed protein to bind to the ci regulatory region in vivo and in vitro. We suggest that engrailed protein directly represses ci expression in posterior compartment cells.

Chromosome mapping of human (ZNF147) and mouse genes for estrogen-responsive finger protein (efp), a member of the RING finger family

We have previously identified an estrogen-responsive gene, efp (estrogen-responsive finger protein), that encodes a putative zinc finger protein ( Proc. Natl. Acad. Sci. USA 90: 11117–11121, 1993). The efp protein has a RING finger, a variant type of zinc finger motif, B1 box, and B2 box, each having a pair of zinc fingers, present in a family of apparent DNA-binding proteins. Some members of this family have transformation capabilities when found in chromosomal translocations. Chromosome mapping of the efp gene by fluorescence in situ hybridization reveals that human EFP (ZNF147) is located at 17q23.1 and that mouse Efp is located at 11C. These results provide additional evidence that the mouse 11C region displays conserved synteny with the 17q23.1 region of the human genome.

Identification of a novel TGF-beta-regulated gene encoding a putative zinc finger protein in human osteoblasts.

The TGF-beta family of growth factors has been extensively studied and found to play major roles in bone physiology and disease. A novel, TGF-beta-inducible early gene (TIEG) in normal human fetal osteoblasts (hFOB) has been identified using differential-display PCR. Using this differentially expressed cDNA fragment of TIEG to screen a hOB cDNA library, a near full-length cDNA for this gene was isolated. Northern analyses indicated that the steady-state levels of the 3.5 kb TIEG mRNA increased within 30 min of TGF-beta treatment of human osteoblasts and reached a maximum of 10-fold above control levels at 120 min post-treatment. This regulation was independent of new protein synthesis. Computer sequence analyses indicates that TIEG mRNA encodes for a 480 amino-acid protein. The TIEG protein contains three zinc finger motifs, several proline-rich src homology-3 (SH3) binding domains at the C-terminal end, and is homologous in this region to the zinc finger-containing transcription factor family of genes. A growth factor/cytokine-specific induction of TIEG has been shown. TIEG expression in hFOB cells was highly induced by TGF-beta and bone morphogenetic protein-2 (BMP-2), with a moderate induction by epidermal growth factor (EGF), but no induction by other growth factors/cytokines was observed. In addition to osteoblastic cells, high levels of TIEG expression were detected in skeletal muscle tissue, while low or no detectable levels were found in brain, lung, liver or kidney. Because TIEG is an early induced putative transcription factor gene, and shows a growth factor induction and tissue specificity, its protein product might play an important role as a signalling molecule in osteoblastic cells.

Characterization of the TGF beta 1-inducible hic-5 gene that encodes a putative novel zinc finger protein and its possible involvement in cellular senescence.

Transforming growth factor (TGF) beta 1 is a potent cytokine that inhibits the growth of several types of cells. Our earlier study suggested that the mouse osteoblastic cell line, MC3T3-E1, was sensitive to growth inhibition by TGF beta 1 and that this effect was partly mediated by H2O2. To identify the molecules that participate in the negative regulation of growth by these stimuli, we carried out differential screening of cDNA libraries and isolated a set of genes induced by TGF beta 1. Among the clones isolated, one originally named tsc-5 was found to be induced by H2O2 as well as TGF beta 1. Analysis of this cDNA renamed hic (hydrogen peroxide-inducible clone)-5 suggested that Hic-5 protein has four LIM motifs, each of which contained two (or one) putative zinc fingers. The expression of hic-5 mRNA was repressed in Ki-ras-transformed mouse fibroblasts and in several cell lines established from human tumor. On the other hand, its expression was augmented in the in vitro senescent process of human diploid fibroblasts. Among the mouse organs examined, hic-5 was highly expressed in the lung and spleen. Finally, a colony formation assay using an hic-5 expression vector driven by the cytomegalovirus promoter suggested that hic-5 overexpression had a cytostatic effect on cellular growth, depending upon the cell type. Although the relationship between hic-5 function and the signal transduction pathway of TGF beta 1 remains unresolved, these results implied that hic-5 has some role in the growth-inhibitory pathway associated with in vitro senescence, and that down-regulation of hic-5 contributes to tumorigenesis.

The yeast MOT2 gene encodes a putative zinc finger protein that serves as a global negative regulator affecting expression of several categories of genes, including mating-pheromone-responsive genes.

The STE4 gene encodes the beta subunit of a heterotrimeric G protein that is an essential component of the pheromone signal transduction pathway. To identify downstream component(s) of Ste4, we sought pseudo-revertants that restored mating competence to ste4 mutants. The suppressor mot2 was isolated as a recessive mutation that restored conjugational competence to a temperature-sensitive ste4 mutant and simultaneously conferred a temperature-sensitive growth phenotype. The MOT2 gene encodes a putative zinc finger protein, the deletion of which resulted in temperature-sensitive growth, increased expression of FUS1 in the absence of pheromones, and suppression of a deletion of the alpha-factor receptor. On the other hand, sterility resulting from deletion of STE4 was not suppressed by the mot2 deletion. These phenotypes are similar to those associated with temperature-sensitive mutations in CDC36 and CDC39, which are proposed to encode general negative regulators of transcription rather than factors involved in the pheromone response pathway. Deletion of MOT2 also caused increased transcription of unrelated genes such as GAL7 and PHO84. Overexpression of MOT2 suppresses the growth defect of temperature-sensitive mutations in CDC36 and CDC39. These observations suggest that Mot2 functions as a general negative regulator of transcription in the same processes as Cdc36 and Cdc39.

The yeast MOT2 gene encodes a putative zinc finger protein that serves as a global negative regulator affecting expression of several categories of genes, including mating-pheromone-responsive genes.

The STE4 gene encodes the beta subunit of a heterotrimeric G protein that is an essential component of the pheromone signal transduction pathway. To identify downstream component(s) of Ste4, we sought pseudo-revertants that restored mating competence to ste4 mutants. The suppressor mot2 was isolated as a recessive mutation that restored conjugational competence to a temperature-sensitive ste4 mutant and simultaneously conferred a temperature-sensitive growth phenotype. The MOT2 gene encodes a putative zinc finger protein, the deletion of which resulted in temperature-sensitive growth, increased expression of FUS1 in the absence of pheromones, and suppression of a deletion of the alpha-factor receptor. On the other hand, sterility resulting from deletion of STE4 was not suppressed by the mot2 deletion. These phenotypes are similar to those associated with temperature-sensitive mutations in CDC36 and CDC39, which are proposed to encode general negative regulators of transcription rather than factors involved in the pheromone response pathway. Deletion of MOT2 also caused increased transcription of unrelated genes such as GAL7 and PHO84. Overexpression of MOT2 suppresses the growth defect of temperature-sensitive mutations in CDC36 and CDC39. These observations suggest that Mot2 functions as a general negative regulator of transcription in the same processes as Cdc36 and Cdc39.

Identification of genes encoding zinc finger proteins, non-histone chromosomal HMG protein homologue, and a putative GTP phosphohydrolase in the genome of Chilo iridescent virus.

Five RNA transcripts of about 1.2 to 1.7 kilobases were mapped to a part of the genome of insect iridescent virus type 6 (Chilo iridescent virus; CIV) between genome coordinates 0.832 and 0.856 within the EcoRI DNA fragment F. The nucleotide sequence of this particular region (5702 base pairs) of the CIV genome was determined. The DNA sequence contains a number of perfect direct, inverted, and palindromic repeats including three clusters of tandemly organized repetitive DNA elements located between the nucleotide positions 1534 to 1566, 3720 to 3780, and 4350 to 4450. Eight long open reading frames (ORFs; EF1 to 8) were detected in the sequenced region of the CIV genome. ORF EF1 encodes a putative protein of 221 amino acid residues (aa) that is closely related to eukaryotic nonhistone chromosomal proteins of the high mobility group (HMG) superfamily. Virus encoded homologues of HMG proteins have not been reported so far. The EF2 gene product (145 aa) contains a specific zinc finger motif and belongs to a distinct group of identified and putative zinc finger proteins including a second putative protein (239 aa) of CIV encoded in the EcoRI DNA fragment Y (1984 bp; 0.381 to 0.391 viral map units). The product of EF6 (127 aa) is related to D250 ORF product of African swine fever virus (ASFV) and belongs to the recently described protein family sharing a highly conserved sequence motif with bacterial antimutator GTP phosphohydrolase MutT. Thus the sequenced region of the CIV genome encodes three putative proteins which may be directly involved in the replication and/or transcription of the viral DNA.

The putative zinc‐finger protein WZF1 interacts with a cis‐acting element of wheat histone genes

A nonamer motif (CATCCAACG) that is one of the cis-acting elements identified in the proximal promoter region of some wheat histone genes is included in the region that interacts with the wheat DNA-binding protein, HBP (histone gene-binding protein)-2. To obtain structural and functional information about this DNA-binding protein, we attempted to isolate a cDNA clone encoding HBP-2 on the basis of its ability to bind to a nonamer-containing 38-bp DNA fragment. Southwestern screening of a wheat cDNA library with concatenated 38-residue oligonucleotides as the probe produced one candidate clone. Nucleotide sequence analyses of this cDNA clone and the corresponding genomic clone showed that the protein deduced from the nucleotide sequence consisted of 261 amino acids and contained a set of zinc-finger motifs similar to those found in many eukaryotic transcription factors. The protein, named WZF1 (wheat zinc-finger protein 1), which was expressed from the cDNA in Escherichia coli cells, bound specifically and metal-ion-dependently to the nonamer-containing oligonucleotide. The WZF1 mRNA was highly expressed in the root apexes of wheat seedlings, but less so in the proximal portion of young leaves; whereas, histone H3 mRNA was highly expressed in both tissues. The expression patterns of the WZF1 and histone H3 genes in the early stages of germination differed, expression of the WZF1 gene being almost constant but not that of the H3 gene. The relationship of WZF1 and HBP-2 and the possible role of WZF1 in the histone gene expression were discussed.

Identification of a gene encoding a novel zinc finger protein in Saccharomyces cerevisiae

In the course of a comprehensive genomic screen for cell cycle regulated genes in the yeast Saccharomyces cerevisiae (Price et al., 1991) we identified and characterized a transcriptional unit encoding a putative zinc finger protein named FZF1 (EMBL accession number: X67787). This gene encodes a protein containing five zinc fingers of the Cys2His2 class, three of which are positioned in tandem at the N-terminus. The fourth and fifth finger follow after an interruption of 61 and 66 amino acids, respectively. While FZF1 is constitutively expressed at a very low level, its deletion is not essential for growth. Its similarity with known transcription factors, however, suggests that the FZF1 gene product may serve as a transcription factor in yeast.