Cloning and characterization of a novel zinc finger transcriptional repressor. A direct role of the zinc finger motif in repression.
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We have identified a novel transcriptional repressor, AEBP2, that binds to a regulatory sequence (termed AE-1) located in the proximal promoter region of the aP2 gene that encodes the adipose fatty acid-binding protein. Sequence analysis of AEBP2 cDNA revealed that it encodes a protein containing three Gli-Kruppel (Cys2-His2)-type zinc fingers. Northern blot analysis revealed two transcripts (4.5 and 3.5 kilobases) which were ubiquitously expressed in every mouse tissue examined. In co-transfection assays, AEBP2 repressed transcription from the homologous aP2 promoter containing multiple copies of the AE-1 sequence. Moreover, a chimeric construct encoding a fusion AEBP2 protein with the Gal4 DNA-binding domain was able to repress the transcriptional activity of a heterologous promoter containing the Gal4-binding sequence. The transcriptional repression function of AEBP2 was completely abolished when one of the conserved histidine residues and a flanking serine residue in the middle zinc finger were replaced with an arginine residue. The defective transcriptional repression function of the mutant derivative was due neither to lack of expression nor to a failure to localize to the nucleus. Moreover, both the wild-type and mutant derivative of either the histidine-tagged recombinant AEBP2 proteins or the in vitro translated Gal4-AEBP2 fusion proteins were equally able to bind to the target DNA. These results suggest that a portion of the zinc finger structure may play a direct role in transcriptional repression function, but not in DNA binding. (+info)
A human RNA viral cysteine proteinase that depends upon a unique Zn2+-binding finger connecting the two domains of a papain-like fold .
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A cysteine proteinase, papain-like proteinase (PL1pro), of the human coronavirus 229E (HCoV) regulates the expression of the replicase polyproteins, pp1a and ppa1ab, by cleavage between Gly111 and Asn112, far upstream of its own catalytic residue Cys1054. In this report, using bioinformatics tools, we predict that, unlike its distant cellular homologues, HCoV PL1pro and its coronaviral relatives have a poorly conserved Zn2+ finger connecting the left and right hand domains of a papain-like fold. Optical emission spectrometry has been used to confirm the presence of Zn2+ in a purified and proteolytically active form of the HCoV PL1pro fused with the Escherichia coli maltose-binding protein. In denaturation/renaturation experiments using the recombinant protein, its activity was shown to be strongly dependent upon Zn2+, which could be partly substituted by Co2+ during renaturation. The reconstituted, Zn2+-containing PL1pro was not sensitive to 1,10-phenanthroline, and the Zn2+-depleted protein was not reactivated by adding Zn2+ after renaturation. Consistent with the proposed essential structural role of Zn2+, PL1pro was selectively inactivated by mutations in the Zn2+ finger, including replacements of any of four conserved Cys residues predicted to co-ordinate Zn2+. The unique domain organization of HCoV PL1pro provides a potential framework for regulatory processes and may be indicative of a nonproteolytic activity of this enzyme. (+info)
Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA.
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Mice deficient in tristetraprolin (TTP), the prototype of a family of CCCH zinc finger proteins, develop an inflammatory syndrome mediated by excess tumor necrosis factor alpha (TNF-alpha). Macrophages derived from these mice oversecrete TNF-alpha, by a mechanism that involves stabilization of TNF-alpha mRNA, and TTP can bind directly to the AU-rich element (ARE) in TNF-alpha mRNA (E. Carballo, W. S. Lai, and P. J. Blackshear, Science 281:1001-1005, 1998). We show here that TTP binding to the TNF-alpha ARE is dependent upon the integrity of both zinc fingers, since mutation of a single cysteine residue in either zinc finger to arginine severely attenuated the binding of TTP to the TNF-alpha ARE. In intact cells, TTP at low expression levels promoted a decrease in size of the TNF-alpha mRNA as well as a decrease in its amount; at higher expression levels, the shift to a smaller TNF-alpha mRNA size persisted, while the accumulation of this smaller species increased. RNase H experiments indicated that the shift to a smaller size was due to TTP-promoted deadenylation of TNF-alpha mRNA. This CCCH protein is likely to be important in the deadenylation and degradation of TNF-alpha mRNA and perhaps other ARE-containing mRNAs, both in normal physiology and in certain pathological conditions. (+info)
KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins: a potential role for Kruppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing.
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Kruppel-associated box (KRAB) domains are present in approximately one-third of all human zinc finger proteins (ZFPs) and are potent transcriptional repression modules. We have previously cloned a corepressor for the KRAB domain, KAP-1, which is required for KRAB-mediated repression in vivo. To characterize the repression mechanism utilized by KAP-1, we have analyzed the ability of KAP-1 to interact with murine (M31 and M32) and human (HP1alpha and HP1gamma) homologues of the HP1 protein family, a class of nonhistone heterochromatin-associated proteins with a well-established epigenetic gene silencing function in Drosophila. In vitro studies confirmed that KAP-1 is capable of directly interacting with M31 and hHP1alpha, which are normally found in centromeric heterochromatin, as well as M32 and hHP1gamma, both of which are found in euchromatin. Mapping of the region in KAP-1 required for HP1 interaction showed that amino acid substitutions which abolish HP1 binding in vitro reduce KAP-1 mediated repression in vivo. We observed colocalization of KAP-1 with M31 and M32 in interphase nuclei, lending support to the biochemical evidence that M31 and M32 directly interact with KAP-1. The colocalization of KAP-1 with M31 is sometimes found in subnuclear territories of potential pericentromeric heterochromatin, whereas colocalization of KAP-1 and M32 occurs in punctate euchromatic domains throughout the nucleus. This work suggests a mechanism for the recruitment of HP1-like gene products by the KRAB-ZFP-KAP-1 complex to specific loci within the genome through formation of heterochromatin-like complexes that silence gene activity. We speculate that gene-specific repression may be a consequence of the formation of such complexes, ultimately leading to silenced genes in newly formed heterochromatic chromosomal environments. (+info)
FOG-2, a heart- and brain-enriched cofactor for GATA transcription factors.
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Members of the GATA family of zinc finger transcription factors have been shown to play important roles in the control of gene expression in a variety of cell types. GATA-1, -2, and -3 are expressed primarily in hematopoietic cell lineages and are required for proliferation and differentiation of multiple hematopoietic cell types, whereas GATA-4, -5, and -6 are expressed in the heart, where they activate cardiac muscle structural genes. Friend of GATA-1 (FOG) is a multitype zinc finger protein that interacts with GATA-1 and serves as a cofactor for GATA-1-mediated transcription. FOG is coexpressed with GATA-1 in developing erythroid and megakaryocyte cell lineages and cooperates with GATA-1 to control erythropoiesis. We describe a novel FOG-related factor, FOG-2, that is expressed predominantly in the developing and adult heart, brain, and testis. FOG-2 interacts with GATA factors, and interaction of GATA-4 and FOG-2 results in either synergistic activation or repression of GATA-dependent cardiac promoters, depending on the specific promoter and the cell type in which they are tested. The properties of FOG-2 suggest its involvement in the control of cardiac and neural gene expression by GATA transcription factors. (+info)
Distinct leukemia phenotypes in transgenic mice and different corepressor interactions generated by promyelocytic leukemia variant fusion genes PLZF-RARalpha and NPM-RARalpha.
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Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation involving RARalpha and one of four fusion partners: PML, PLZF, NPM, and NuMA genes. To study the leukemogenic potential of the fusion genes in vivo, we generated transgenic mice with PLZF-RARalpha and NPM-RARalpha. PLZF-RARalpha transgenic animals developed chronic myeloid leukemia-like phenotypes at an early stage of life (within 3 months in five of six mice), whereas three NPM-RARalpha transgenic mice showed a spectrum of phenotypes from typical APL to chronic myeloid leukemia relatively late in life (from 12 to 15 months). In contrast to bone marrow cells from PLZF-RARalpha transgenic mice, those from NPM-RARalpha transgenic mice could be induced to differentiate by all-trans-retinoic acid (ATRA). We also studied RARE binding properties and interactions between nuclear corepressor SMRT and various fusion proteins in response to ATRA. Dissociation of SMRT from different receptors was observed at ATRA concentrations of 0.01 microM, 0.1 microM, and 1.0 microM for RARalpha-RXRalpha, NPM-RARalpha, and PML-RARalpha, respectively, but not observed for PLZF-RARalpha even in the presence of 10 microM ATRA. We also determined the expression of the tissue factor gene in transgenic mice, which was detected only in bone marrow cells of mice expressing the fusion genes. These data clearly establish the leukemogenic role of PLZF-RARalpha and NPM-RARalpha and the importance of fusion receptor/corepressor interactions in the pathogenesis as well as in determining different clinical phenotypes of APL. (+info)
MSF (MLL septin-like fusion), a fusion partner gene of MLL, in a therapy-related acute myeloid leukemia with a t(11;17)(q23;q25).
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MLL (ALL1, Htrx, HRX), which is located on chromosome band 11q23, frequently is rearranged in patients with therapy-related acute myeloid leukemia who previously were treated with DNA topoisomerase II inhibitors. In this study, we have identified a fusion partner of MLL in a 10-year-old female who developed therapy-related acute myeloid leukemia 17 months after treatment for Hodgkin's disease. Leukemia cells of this patient had a t(11;17)(q23;q25), which involved MLL as demonstrated by Southern blot analysis. The partner gene was cloned from cDNA of the leukemia cells by use of a combination of adapter reverse transcriptase-PCR, rapid amplification of 5' cDNA ends, and BLAST database analysis to identify expressed sequence tags. The full-length cDNA of 2.8 kb was found to be an additional member of the septin family, therefore it was named MSF (MLL septin-like fusion). Members of the septin family conserve the GTP binding domain, localize in the cytoplasm, and interact with cytoskeletal filaments. A major 4-kb transcript of MSF was expressed ubiquitously; a 1.7-kb transcript was found in most tissues. An additional 3-kb transcript was found only in hematopoietic tissues. By amplification with MLL exon 5 forward primer and reverse primers in MSF, the appropriately sized products were obtained. MSF is highly homologous to hCDCrel-1, which is a partner gene of MLL in leukemias with a t(11;22)(q23;q11.2). Further analysis of MSF may help to delineate the function of MLL partner genes in leukemia, particularly in therapy-related leukemia. (+info)
Free fatty acid-induced insulin resistance is associated with activation of protein kinase C theta and alterations in the insulin signaling cascade.
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To examine the mechanism by which free fatty acids (FFAs) induce insulin resistance in vivo, awake chronically catheterized rats underwent a hyperinsulinemic-euglycemic clamp with or without a 5-h preinfusion of lipid/heparin to raise plasma FFA concentrations. Increased plasma FFAs resulted in insulin resistance as reflected by a approximately 35% reduction in the glucose infusion rate (P < 0.05 vs. control). The insulin resistance was associated with a 40-50% reduction in 13C nuclear magnetic resonance (NMR)-determined rates of muscle glycogen synthesis (P < 0.01 vs. control) and muscle glucose oxidation (P < 0.01 vs. control), which in turn could be attributed to a approximately 25% reduction in glucose transport activity as assessed by 2-[1,2-3H]deoxyglucose uptake in vivo (P < 0.05 vs. control). This lipid-induced decrease in insulin-stimulated muscle glucose metabolism was associated with 1) a approximately 50% reduction in insulin-stimulated insulin receptor substrate (IRS)-1-associated phosphatidylinositol (PI) 3-kinase activity (P < 0.05 vs. control), 2) a blunting in insulin-stimulated IRS-1 tyrosine phosphorylation (P < 0.05, lipid-infused versus glycerol-infused), and 3) a four-fold increase in membrane-bound, or active, protein kinase C (PKC) theta (P < 0.05 vs. control). We conclude that acute elevations of plasma FFA levels for 5 h induce skeletal muscle insulin resistance in vivo via a reduction in insulin-stimulated muscle glycogen synthesis and glucose oxidation that can be attributed to reduced glucose transport activity. These changes are associated with abnormalities in the insulin signaling cascade and may be mediated by FFA activation of PKC theta. (+info)