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T Hai

Publications and source records attributed to T Hai.

32 records · Page 2Linked to original sources
PubMed1997

The hepatitis B virus X protein enhances the DNA binding potential and transcription efficacy of bZip transcription factors.

The hepatitis B virus X protein interacts with the basic-region, leucine zipper protein (bZip) domain of cAMP response element-binding protein increasing its affinity for the cAMP response element site in vitro and its transcriptional efficacy in vivo (Williams, J. S., and Andrisani, O. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3819-3823). Here we examine pX interactions with bZip transcription factors ATF3, gadd153/Chop10, ICER IIgamma, and NF-IL6. We demonstrate direct interactions in vitro between pX and the bZip proteins tested. In contrast MyoD and Gal4(1-147) fail to interact with pX. We also demonstrate by the mammalian two-hybrid assay the direct interaction of pX with cAMP response element- binding protein, ICER IIgamma, ATF3, and NF-IL6 in hepatocytes. In addition, pX increases the DNA binding potential of bZip proteins for their cognate DNA-binding site in vitro. In transient transfections in hepatocytes (AML12 cell line), pX increases the transcriptional efficacy of the bZip transcription factors. NF-IL6-mediated transcriptional activation is enhanced 3-fold by pX. Most interestingly, pX augments the repression mediated by bZip repressors ATF3 and ICER IIgamma, by 6- and 7-fold, respectively, demonstrating for the first time the involvement of pX in gene repression. We conclude that pX is an enhancer of the DNA binding potential of bZip transcription factors, thereby increasing the transactivation or repression efficacy of bZip-responsive genes.

Animals↗
PubMed1997

Tissue-specific pattern of stress kinase activation in ischemic/reperfused heart and kidney.

In this report we investigate the molecular mechanisms that contribute to tissue damage following ischemia and ischemia coupled with reperfusion (ischemia/reperfusion) in the rat heart and kidney. We observe the activation of three stress-inducible mitogen-activated protein (MAP) kinases in these tissues: p38 MAP kinase and the 46- and 55-kDa isoforms of Jun N-terminal kinase (JNK46 and JNK55). The heart and kidney show distinct time courses in the activation of p38 MAP kinase during ischemia but no activation of either JNK46 or JNK55. These two tissues also respond differently to ischemia/reperfusion. In the heart we observe activation of JNK55 and p38 MAP kinase, whereas in the kidney all three kinases are active. We also examined the expression pattern of two stress-responsive genes, c-Jun and ATF3. Our results indicate that in the heart both genes are induced by ischemia and ischemia/reperfusion. However, in the kidney c-Jun and ATF3 expression is induced only by ischemia/reperfusion. To correlate these molecular events with tissue damage we examined DNA laddering, a common marker of apoptosis. A significant increase in DNA laddering was evident in both heart and kidney following ischemia/reperfusion and correlated with the pattern of kinase activation, supporting a link between stress kinase activation and apoptotic cell death in these tissues.

Activating Transcription Factor 2↗
PubMed1997

gadd153/Chop10, a potential target gene of the transcriptional repressor ATF3.

Recently, we demonstrated that the function of ATF3, a stress-inducible transcriptional repressor, is negatively regulated by a bZip protein, gadd153/Chop10. In this report, we present evidence that ATF3 can repress the expression of its own inhibitor, gadd153/Chop10. First, ATF3 represses a chloramphenicol acetyltransferase reporter gene driven by the gadd153/Chop10 promoter when assayed by a transfection assay in vivo and a transcription assay in vitro. Second, the gadd153/Chop10 promoter contains two functionally important binding sites for ATF3: an AP-1 site and a C/EBP-ATF composite site, a previously unidentified binding site for ATF3. The absence of either site reduces the ability of ATF3 to repress the promoter. Third, overexpression of ATF3 by transient transfection results in a reduction of the endogenous gadd153/Chop10 mRNA level. Fourth, as described previously, ATF3 is induced in the liver upon CCl4 treatment. Intriguingly, we show in this report that gadd153/Chop10 mRNA is not present in areas where ATF3 is induced. Taken together, these results strongly suggest that ATF3 represses the expression of gadd153/Chop10. The mutual negative regulation between ATF3 and gadd153/Chop10 is discussed.

Activating Transcription Factor 3↗
PubMed1996

ATF3 gene. Genomic organization, promoter, and regulation.

ATF3 gene, which encodes a member of the activating transcription factor/cAMP responsive element binding protein (ATF/CREB) family of transcription factors, is induced by many physiological stresses. As a step toward understanding the induction mechanisms, we isolated the human ATF3 gene and analyzed its genome organization and 5'-flanking region. We found that the human ATF3 mRNA is derived from four exons distributed over 15 kilobases. Sequence analysis of the 5'-flanking region revealed a consensus TATA box and a number of transcription factor binding sites including the AP-1, ATF/CRE, NF-kappa B, E2F, and Myc/Max binding sites. As another approach to understanding the mechanisms by which the ATF3 gene is induced by stress signals, we studied the regulation of the ATF3 gene in tissue culture cells by anisomycin, an approach that has been used to study the stress responses in tissue culture cells. We showed that anisomycin at a low concentration activates the ATF3 promoter and stabilizes the ATF3 mRNA. Significantly, co-transfection of DNAs expressing ATF2 and c-Jun activates the ATF3 promoter. A possible mechanism implicating the C-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) stress-inducible signaling pathway in the induction of the ATF3 gene is discussed.

Activating Transcription Factor 3↗
PubMed1996

Analysis of ATF3, a transcription factor induced by physiological stresses and modulated by gadd153/Chop10.

We demonstrate that ATF3, a member of the ATF/CREB family of transcription factors, is induced in a variety of stressed tissues: mechanically injured liver, toxin-injured liver, blood-deprived heart, and postseizure brain. We also demonstrate that an ATF3-interacting protein, gadd153/Chop10, forms a nonfunctional heterodimer with ATF3: the heterodimer, in contrast to the ATF3 homodimer, does not bind to the ATF/cyclic AMP response element consensus site and does not repress transcription. Interestingly, ATF3 and gadd153/Chop10 are expressed in inverse but overlapping manners during the liver's response to carbon tetrachloride (CCl4): the level of gadd153/Chop10 mRNA is high in the normal liver and greatly decreases upon CCl4 treatment; the level of ATF3 mRNA, on the other hand, is low in the normal liver and greatly increases upon CCl4 treatment. We hypothesize that in nonstressed liver, gadd153/Chop10 inhibits the limited amount of ATF3 by forming an inactive heterodimer with it, whereas in CCl4-injured liver, the synthesis of gadd153/Chop10 is repressed, allowing the induced ATF3 to function.

Activating Transcription Factor 3↗
PubMed1994

ATF3 and ATF3 delta Zip. Transcriptional repression versus activation by alternatively spliced isoforms.

ATF3 is a member of the mammalian activating transcription factor/cAMP responsive element binding protein (ATF/CREB) family of transcription factors. In this report, we demonstrate that, contrary to the implication of its name, ATF3 represses rather than activates transcription from promoters with ATF sites. We also present evidence suggesting that one possible mechanism by which ATF3 represses transcription is to stabilize inhibitory co-factors at the promoter. In addition, we describe a naturally occurring, alternatively spliced, form of ATF3: ATF3 delta Zip. ATF3 delta Zip lacks the leucine zipper domain and does not bind to DNA. In contrast to ATF3, ATF3 delta Zip stimulates transcription, presumably by sequestering inhibitory co-factors away from the promoter. It is possible that ATF3 delta Zip is a physiologically important regulator and that it, together with ATF3, regulates the expression of specific target genes.

Activating Transcription Factor 3↗
PubMed1994

Expression vectors for affinity purification and radiolabeling of proteins using Escherichia coli as host.

We have constructed two convenient vectors to produce foreign proteins in Escherichia coli. The first vector was developed to produce histidine (His)-tagged fusion proteins. In addition to encoding six contiguous His residues, it contains three unique restriction sites that allow cloning of a blunt-ended DNA fragment in three different reading frames. Therefore, one can clone any gene of interest in this vector to make a fusion protein tagged with six His at the N terminus. The His-tag allows purification of the fusion protein to almost homogeneity by a nickel-chelating column in a single step. The second vector is a derivative of the first vector; it encodes two tandem phosphorylation sites for heart muscle kinase (HMK) immediately downstream from the His residues. Therefore, the resulting fusion protein can be radiolabeled using [gamma-32P]ATP and HMK in vitro. The labeled protein can then be used as a probe to detect protein-protein interaction.

Adenosine Triphosphate↗
PubMed1993

Dimerization specificity of the leucine zipper-containing bZIP motif on DNA binding: prediction and rational design.

We propose an interhelical salt bridge rule to explain the dimerization specificity between the two amphipathic alpha-helices in the leucine zipper structure. Using the bZIP class of DNA-binding proteins as a model system, we predicted and designed novel dimerization partners. We predicted that ATF4, a member of the ATF/CREB family of transcription factors, would preferentially form heterodimers with IGEBP1, a member of the C/EBP superfamily. These predictions were verified using a gel mobility-shift assay. To further test the value of this interhelical salt bridge rule, we modified the bZIP protein C/EBP attempting to design molecules that would form preferentially heterodimers with C/EBP or molecules that would not interact with C/EBP. These designed molecules behaved as predicted. Therefore, we conclude that this interhelical salt bridge rule is useful in understanding the dimerization specificity of bZIP proteins. In addition, we suggest that this rule could be used to design novel "dominant-negative" molecules to specifically inhibit the function of target leucine zipper proteins in vivo.

Activating Transcription Factor 4↗
PubMed1993

Cyclic AMP-independent ATF family members interact with NF-kappa B and function in the activation of the E-selectin promoter in response to cytokines.

We previously reported that NF-kappa B and a complex we referred to as NF-ELAM1 play a central role in cytokine-induced expression of the E-selectin gene. In this study we identify cyclic AMP (cAMP)-independent members of the ATF family binding specifically to the NF-ELAM1 promoter element. The NF-ELAM1 element (TGACATCA) differs by a single nucleotide substitution from the cAMP-responsive element consensus sequence. We demonstrate that this sequence operates in a cAMP-independent manner to induce transcription and thus define it as a non-cAMP-responsive element (NCRE). We show that ATFa is a component of the NF-ELAM1 complex and its overexpression activates the E-selectin promoter. In addition, ATFa, ATF2, and ATF3 interact directly with NF-kappa B in vitro, linking two unrelated families of transcription factors in a novel protein-protein interaction. Furthermore, we demonstrate that the ability of overexpressed NF-kappa B to transactivate the E-selectin promoter in vivo is dependent on the NF-ELAM1 complex. Our results suggest that a direct interaction between ATFs and NF-kappa B is, at least in part, the mechanism by which these factors specifically regulate E-selectin promoter activity.

Activating Transcription Factors↗
PubMed1992

Synthesis and properties of polymerized, diaspirin cross-linked hemoglobins.

During the course of our studies it became clear that there were therapeutic applications for which a polymeric hemoglobin having an extended half-life in circulation would be appropriate. Therefore, a process for the glutaraldehyde-polymerization of diaspirin cross-linked hemoglobin (DCLHb) was developed and used to prepare glutaraldehyde-polymerized DCLHb (GP-DCLHb) in lactated Ringer's solution in sufficient quantities for biological testing. Both isovolemic exchange-transfusion and "top-load" studies (rats; primates and swine, respectively) were completed in which a broad spectrum of physiologic, histopathologic and analytical parameters were monitored and assessed. In general, GP-DCLHb in lactated Ringer's solution was well-tolerated physiologically. When compared to DCLHb, GP-DCLHb offers the advantages of reduced renal clearance of hemoglobin and an extended half-life in circulation. GP-DCLHb has the disadvantages that (1) glutaraldehyde is an ineffective virucidal agent under the conditions of the polymerization reaction and a separate virus inactivation step is required; (2) low-endotoxin (LAL-negative) GP-DCLHb solutions are pyrogenic (rabbits); and (3) unusual deposition of hemoglobin-containing material in the small arterioles of the liver and kidney (rats) was sometimes seen even after a period of time (2 weeks) during which treatment-related organ pathologies are usually resolved, a finding peculiar to GP-DCLHb among the various hemoglobin derivatives we have tested.

Animals↗
PubMed1992

Trace element analyses of diaspirin cross-linked hemoglobin solutions.

A sensitive assay using inductively coupled plasma atomic emission spectroscopy (ICP-AES) has been applied to the measurement of trace elements in diaspirin cross-linked hemoglobin (DCLHb) solutions. Calcium, magnesium, zinc and iron were the only elements detected at greater than background levels. Ag, Al, As, B, Ba, Bi, Cd, Co, Cr, Cu, Mn, Mo, Pb, Sb, Se, Si, Sr, Ti, and V were present at concentrations either equal to the acid blanks or were not detected. Detection of non-heme iron (not incorporated into the hemoglobin porphyrin ring) in a 10 g/dL hemoglobin solution required the development of a special protocol. In this protocol a chelator, DTPA, was added to hemoglobin solutions to complex with both free and non-specifically bound non-heme iron. The resulting iron:DTPA complexes were separated from the hemoglobin molecules by ultrafiltration and the ultra-filtrate analyzed by ICP-AES. A modification of this assay in which the DTPA was omitted was used to measure the free non-heme iron in solution. Typical concentrations of chelatable (free and non-specifically bound) and solution (free) non-heme iron in DCLHb production lots at the completion of manufacture were 0.5-1.0 ppm and 0.1-0.3 ppm, respectively.

Aspirin↗
PubMed1991

Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity.

The Fos/Jun and ATF/CREB families of transcription factors function in coupling extracellular signals to alterations in expression of specific target genes. Like many eukaryotic transcription factors, these proteins bind to DNA as dimers. Dimerization is mediated by a structure known as the "leucine-zipper" motif. Although Fos/Jun and ATF/CREB were previously thought to interact preferentially with different DNA regulatory elements (the AP-1/TRE and ATF/CRE sites, respectively), we find that members of these two families form selective cross-family heterodimers. The resulting heterodimers display distinguishable DNA binding specificities from each other and from their parental homodimers. These findings indicate that the Fos/Jun and ATF/CREB families of transcription factors are not as distinct as was previously thought. We suggest that they can be grouped into a superfamily of transcription factors.

Activating Transcription Factor 2↗
PubMed1988

Transcription factor ATF interacts with the TATA factor to facilitate establishment of a preinitiation complex.

The mammalian activator protein ATF stimulates transcription from the adenovirus E4 promoter by binding to multiple upstream promoter and enhancer elements. DNAase footprint analyses have revealed that there are cooperative interactions between ATF and TFIID (the mammalian TATA factor) when both are bound simultaneously to the promoter and that these interactions in turn facilitate promoter recognition by RNA polymerase II and the general initiation factors TFIIB and TFIIE. However, the complex of TFIID and the other general factors is stable following oligonucleotide-mediated dissociation of ATF from the complete preinitiation complex. These results indicate that TFIID is a direct target for ATF, that these interactions facilitate assembly of a complete preinitiation complex, and that the role of ATF might be transient.

Adenoviridae↗