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Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response.

In response to accumulation of unfolded proteins in the endoplasmic reticulum (ER), a homoeostatic response, termed the unfolded protein response (UPR), is activated in all eukaryotic cells. The UPR involves only transcriptional regulation in yeast, and approx. 6% of all yeast genes, encoding not only proteins to augment the folding capacity in the ER, but also proteins working at various stages of secretion, are induced by ER stress [Travers, Patil, Wodicka, Lockhart, Weissman and Walter (2000) Cell (Cambridge, Mass.) 101, 249-258]. In the present study, we conducted microarray analysis of HeLa cells, although our analysis covered only a small fraction of the human genome. A great majority of human ER stress-inducible genes (approx. 1% of 1800 genes examined) were classified into two groups. One group consisted of genes encoding ER-resident molecular chaperones and folding enzymes, and these genes were directly regulated by the ER-membrane-bound transcription factor activating transcription factor (ATF) 6. The ER-membrane-bound protein kinase double-stranded RNA-activated protein kinase-like ER kinase (PERK)-mediated signalling pathway appeared to be responsible for induction of the remaining genes, which are not involved in secretion, but may be important after cellular recovery from ER stress. In higher eukaryotes, the PERK-mediated translational-attenuation system is known to operate in concert with the transcriptional-induction system. Thus we propose that mammalian cells have evolved a strategy to cope with ER stress different from that of yeast cells.

Activating Transcription Factor 6↗

Clustering of the YNA1 gene encoding a Zn(II)2Cys6 transcriptional factor in the yeast Hansenula polymorpha with the nitrate assimilation genes YNT1, YNI1 and YNR1, and its involvement in their transcriptional activation.

The genes encoding the nitrate transporter (YNT1), nitrite reductase (YNI1) and nitrate reductase (YNR1) are clustered in the yeast Hansenula polymorpha. In addition, DNA sequencing of the region containing these genes demonstrated that a new open reading frame called YNA1 (yeast nitrate assimilation) was located between YNR1 and YNI1. The YNA1 gene encodes a protein of 529 residues belonging to the family of Zn(II)2Cys6 fungal transcriptional factors, and has the highest similarity to the transcriptional factors encoded by nirA, and to a smaller extent to nit-4, involved in the nitrate induction of the gene involved in the assimilation of this compound in filamentous fungi. Northern blot analysis showed the presence of the YNA1 transcript in cells incubated in nitrate, nitrate plus ammonium, ammonium, and nitrogen-free media, with a decrease in its levels in those cells incubated in ammonium. In nitrate the strain Deltayna1::URA3, with a disrupted YNA1 gene, neither grew nor expressed the genes YNT1, YNI1 and YNR1. In the gene cluster YNT1-YNI1-YNA1-YNR1, the four genes were transcribed independently in the YNT1-->YNR1 direction and the transcription start sites were determined by primer extension.

Amino Acid Sequence↗

DNase I footprinting, DNA bending and in vitro transcription analyses of ClcR and CatR interactions with the clcABD promoter: evidence of a conserved transcriptional activation mechanism.

In Pseudomonas putida, benzoate and 3-chlorobenzoate are converted to catechol and 3-chlorocatechol, respectively, which are then catabolized to tricarboxylic acid cycle intermediates via the catBCA and clcABD pathways. The catBCA and clcABD operons are regulated by homologous transcriptional activators CatR and ClcR. Previous studies have demonstrated that in addition to sequence similarities, CatR and ClcR share functional similarities which allow catR to complement clcR. In this study, we demonstrate that CatR activates the clcABD promoter in vitro without inducer, but more transcript is produced when inducer is added. DNase I footprinting and DNA-bending analyses demonstrate that CatR binds to and bends the clcABD promoter to the same angle as does ClcR plus its inducer, 2-chloromuconate. This implies that CatR binds to the clc promoter in its active conformation. Transcription of the clcABD promoter by the alpha-subunit truncation mutant (alpha-235) of RNA polymerase was sharply reduced, indicating that the alpha-subunit C-terminal domain is important. However, a small amount of transcript was produced under these conditions, indicating that other contact sites on the RNA polymerase may play a role in activation.

Bacterial Proteins↗

Two essential components of the Saccharomyces cerevisiae transcription factor TFIIIB: transcription and DNA-binding properties.

RNA polymerase III transcription factor TFIIIB from Saccharomyces cerevisiae contains at least two polypeptides, with apparent masses of 90 and 70 kDa, that were previously identified by photocrosslinking to DNA. It is shown here that TFIIIB can be chromatographically separated into two components, each of which is required for efficient tRNA gene transcription. DNA-protein photocrosslinking experiments show these two components separately contain the 90- and 70-kDa TFIIIB-specific polypeptides. The 70-kDa component forms a heparin-sensitive complex with transcription factor TFIIIC and DNA, stabilizes TFIIIC interaction with the tRNA gene promoter elements, and protects against DNase I digestion in the 3' portion of the upstream DNA sequence that is occupied by TFIIIB. The 90-kDa component of TFIIIB, which only detectably interacts with the TFIIIC-DNA complex when the 70-kDa component is also present, generates the complete DNase I protection pattern of TFIIIB and bestows heparin-insensitivity on the TFIIIB-DNA complex. The resolution of TFIIIB into two functional components further defines the probable steps and interactions involved in the formation of stable transcription complexes.

Chromatography, Ion Exchange↗

Tax protein of human T-cell leukemia virus type I binds to the ankyrin motifs of inhibitory factor kappa B and induces nuclear translocation of transcription factor NF-kappa B proteins for transcriptional activation.

Human T-cell leukemia virus type I causes adult T-cell leukemia and tropical spastic paraparesis, and its regulator protein Tax has been implicated in the pathogenic activity of human T-cell leukemia virus type I. Tax activates transcription of viral and cellular genes through specific enhancers: the 21-bp enhancer of human T-cell leukemia virus type I, the nuclear factor kappa B (NF-kappa B)-binding site of the interleukin 2 receptor alpha gene, and the serum-responsive element of c-fos. Tax binds to enhancer-binding proteins including cAMP-responsive element-binding protein, cAMP-responsive element modulator, transcription factor NF-kappa B p50 and p67SRF, and associates with each enhancer DNA indirectly. In addition to this mechanism, we report here that Tax binds to inhibitory factor kappa B gamma (I-kappa B) gamma, which forms a complex with NF-kappa B protein heterodimer p50-p65 or homodimer p50-p50 and retains them in the cytoplasm. Tax binding to I-kappa B gamma induces nuclear translocation of NF-kappa B p65. In association with this nuclear translocation of p65, transcription directed by the kappa B enhancer is strongly activated. Tax binds to the ankyrin motifs of I-kappa B gamma, suggesting its possible interaction with many other proteins carrying ankyrin motifs contributing to various regulatory processes. This is a different mechanism of transcriptional activation by the oncoprotein Tax and seems to be independent from the trans-activation through indirect binding to enhancer DNAs.

Animals↗

Human general transcription factor TFIIA: characterization of a cDNA encoding the small subunit and requirement for basal and activated transcription.

The human general transcription factor TFIIA is one of several factors involved in specific transcription by RNA polymerase II, possibly by regulating the activity of the TATA-binding subunit (TBP) of TFIID. TFIIA purified from HeLa extracts consists of 35-, 19-, and 12-kDa subunits. Here we describe the isolation of a cDNA clone (hTFIIA gamma) encoding the 12-kDa subunit. Using expression constructs derived from hTFIIA gamma and TFIIA alpha/beta (which encodes a 55-kDa precursor to the alpha and beta subunits of natural TFIIA), we have constructed a synthetic TFIIA with a polypeptide composition similar to that of natural TFIIA. The recombinant complex supports the formation of a DNA-TBP-TFIIA complex and mediates both basal and Gal4-VP16-activated transcription by RNA polymerase II in TFIIA-depleted nuclear extracts. In contrast, TFIIA has no effect on tRNA and 5S RNA transcription by RNA polymerase III in this system. We also present evidence that both the p55 and p12 recombinant subunits interact with TBP and that the basic region of TBP is critical for the TFIIA-dependent function of TBP in nuclear extracts.

Amino Acid Sequence↗

Synergistic transcriptional activation of the tissue inhibitor of metalloproteinases-1 promoter via functional interaction of AP-1 and Ets-1 transcription factors.

The tissue inhibitor of metalloproteinases-1 (TIMP-1) is an inhibitor of the extracellular matrix-degrading metalloproteinases. We characterized response elements that control TIMP-1 gene expression. One contains a binding site that selectively binds c-Fos and c-Jun in vitro and confers a response to multiple AP-1 family members in vivo. Adjacent to this is a binding site for Ets domain proteins. Although c-Ets-1 alone did not activate transcription from this element, it enhanced transcription synergistically with AP-1 either in the context of the natural promoter or when the sequence was linked upstream of a heterologous promoter. Furthermore, a complex of c-Jun and c-Fos interacted with c-Ets-1 in vitro. These results suggest that AP-1 tethers c-Ets-1 to the TIMP-1 promoter via protein-protein interaction to achieve Ets-dependent transcriptional regulation. Collectively, our results indicate that TIMP-1 expression is controlled by several DNA response elements that respond to variations in the level and activity of AP-1 and Ets transcriptional regulatory proteins.

Animals↗

Mapping of the transcriptional repression domain of the lymphoid-specific transcription factor oct-2A.

The lymphoid-specific transcription factor Oct-2a is implicated in B cell-specific transcriptional activity via the octamer motif. Structure/function analysis of various Oct-2a effector regions in the context of the GAL4 DNA-binding domain revealed that Oct-2a contains two functionally different activation domains at the N and the C termini. The transcriptional activity of both domains is strongly potentiated by interactions with distinct B cell-specific coactivators. Recently, we have identified a repression domain located within the N terminus of Oct-2a (amino acids 2-99). When this domain was transferred to a potent activator, transcription was strongly inhibited. In this study we present a deletion analysis of the N-terminal region of Oct-2a to determine the minimal repression domain. We identified a stretch of 23 amino acids, rich in serine and threonine residues, which was responsible for most of the repression activity. We show that repression is strongly dependent on the type of enhancer present in the reporter plasmid as well as on the cell line tested. The possibility that Oct-2a can act as an activator and/or a repressor may have important consequences for the function of Oct-2a in B cell differentiation and other developmental processes.

Amino Acid Sequence↗

Chicken ovalbumin upstream promoter transcription factor, a transcriptional activator of HIV-1 gene expression in human brain cells.

Viral infection of the central nervous system by the human immunodeficiency virus type 1 leads to a wide range of neuropathological disorders. However, the molecular mechanisms governing transcription of the human immunodeficiency virus type 1 genome in brain remain unclear. We have recently established that in brain cells, proteins belonging to the steroid/thyroid/retinoic acid receptor family bind to the -352 to -320 region of the long terminal repeat (LTR). Here, by supershift experiments, we have identified chicken ovalbumin upstream promoter transcription factor (COUP-TF), an orphan member of this nuclear receptor family, as one of the major proteins interacting with this LTR site. Cotransfection studies revealed that COUP-TF is able to dramatically activate LTR-directed gene transcription in human oligodendroglioma but not in astrocytoma cells. This activation occurs through two mechanisms, depending on the LTR sequence. Moreover, in neuronal cells COUP-TF and dopamine, a catecholamine neurotransmitter, enhance LTR-directed transcription by acting on the proximal LTR region. These results reveal the importance of COUP-TF and the dopamine signaling pathway as activators of human immunodeficiency virus type 1 gene expression in brain.

Brain↗

cDNA cloning and characterization of murine transcriptional enhancer factor-1-related protein 1, a transcription factor that binds to the M-CAT motif.

The M-CAT motif is a cis-regulatory DNA sequence that is essential for muscle-specific transcription of several genes. Previously, we had shown that both muscle-specific (A1) and ubiquitous (A2) factors bind to an essential M-CAT motif in the myosin heavy chain beta gene and that the ubiquitous factor is transcriptional enhancer factor (TEF)-1. Here we report the isolation of mouse cDNAs encoding two forms (a and b) of a TEF-1-related protein, TEFR1. The TEFR1a cDNA encodes a 427-amino acid protein. The coding region of TEFR1b is identical to 1a in both nucleotide and predicted amino acid sequence except for the absence of 43 amino acids downstream of the TEA DNA-binding domain. Three TEFR1 transcripts (approximately 7, approximately 3.5, and approximately 2 kilobase pairs) are enriched in differentiated skeletal muscle (myotubes) relative to undifferentiated skeletal muscle (myoblasts) and non-muscle cells in culture. In situ hybridization analysis indicated that TEFR1 transcripts are enriched in the skeletal muscle lineage during mouse embryogenesis. Transient expression of fusion proteins of TEFR1 and the yeast GAL4 DNA-binding domain in cell lines activated the expression of chloramphenicol acetyltransferase (CAT) reporter constructs containing GAL4 binding sites, indicating that TEFR1 contains an activation domain. An anti-TEFR1 polyclonal antibody supershifted the muscle-specific M-CAT.A1 factor complex in gel mobility shift assays, suggesting that TEFR1 is a major component of this complex. Our results suggest that TEFR1 might play a role in the embryonic development of skeletal muscle in the mouse.

Amino Acid Sequence↗

Transcriptional activation domain of the herpesvirus protein VP16 becomes conformationally constrained upon interaction with basal transcription factors.

The transcriptional activation domain of the herpesvirus protein VP16 resides in the carboxyl-terminal 78 amino acids (residues 413-490). Fluorescence analyses of this domain indicated that critical amino acids are solvent-exposed in highly mobile segments. To examine interactions between VP16 and components of the basal transcriptional machinery, we incorporated (at position 442 or 473 of VP16) tryptophan analogs that can be selectively excited in complexes with other Trp-containing proteins. TATA-box binding protein (TBP) (but not transcription factor B (TFIIB)) caused concentration-dependent changes in the steady-state anisotropy of VP16, from which equilibrium binding constants were calculated. Quenching of the fluorescence from either position (442 or 473) was significantly affected by TBP, whereas TFIIB affected quenching only at position 473. 7-aza-Trp residues at either position showed a emission spectral shift in the presence of TBP (but not TFIIB), indicating a change to a more hydrophobic environment. In anisotropy decay experiments, TBP reduced the segmental motion at either position; in contrast, TFIIB induced a slight change only at position 473. Our results support models of TBP as a target protein for transcriptional activators and suggest that ordered structure in the VP16 activation domain is induced upon interaction with target proteins.

Binding Sites↗

Bcl-2 transcription from the proximal P2 promoter is activated in neuronal cells by the Brn-3a POU family transcription factor.

The BCL-2 protein is able to protect neuronal and other cell types from apoptotic programmed cell death and plays a key role in regulating the rate of apoptosis during development of the nervous system. We have previously demonstrated that the Brn-3a POU domain transcription factor protects sensory neurons from apoptotic programmed cell death induced by nerve growth factor withdrawal. We report here that Bcl-2 transcription is predominantly initiated from the Bcl-2 P2 promoter in both the ND7 neuronal cell line and primary dorsal root ganglion neurons, in contrast to the predominant use of the Bcl-2 P1 promoter in other cell types. Moreover, Bcl-2 transcription initiated from the P2 region increases in ND7 cells stably overexpressing Brn-3a, resulting in enhanced BCL-2 protein levels. Similarly, the Bcl-2 P2 promoter is directly activated by Brn-3a in co-transfection assays in both ND7 cells and dorsal root ganglion neurons. Analysis of the Bcl-2 regulatory sequence revealed a binding site for Brn-3a that is required for maximal activation by Brn-3a both in transfected cells and during differentiation of ND7 cells. Together these data identify Brn-3a as the first transcription factor regulating Bcl-2 activity specifically in neuronal cells and indicate that the anti-apoptotic effect of Brn-3a is likely to be mediated, at least in part, via the up-regulation of Bcl-2 expression.

Base Sequence↗

Inhibition of activating transcription factor 1- and cAMP-responsive element-binding protein-activated transcription by an intracellular single chain Fv fragment.

Activating transcription factor 1 (ATF1) and cAMP-responsive element (CRE)-binding protein (CREB) activate transcription through CREs located in the promoters of cellular and viral genes. We previously described a monoclonal antibody (mAb41.4) that prevents ATF1 binding to DNA and reduces CRE-driven promoter activity in vitro (Orten, D. J., Strawhecker, J. M., Sanderson, S. D., Huang, D., Prytowsky, M. B. , and Hinrichs, S. H. (1994) J. Biol. Chem. 269, 32254-32263). A single chain Fv (scFv) fragment from the mAb41.4-expressing hybridoma was generated to provide a means to investigate transcription factor function via intracellular expression of the scFv fragment. The affinity of scFv4 (subgroup: VL kappa-III, VH miscellaneous) for ATF1 was similar to that of the parental mAb and the Fab fragment, but it demonstrated greater inhibitory activity and reacted with CREB. scFv4 disrupted the binding of both ATF1 and CREB in electrophoretic mobility shift assays and reduced expression of CRE-driven expression in vitro. Transient expression of scFv had no effect on the non-CRE-containing adenovirus major late promoter. The proliferating cell nuclear antigen promoter, containing two CREs, was significantly more sensitive to inhibition by scFv than the cytomegalovirus immediate-early promoter, containing five CREs. Cotransfection of either ATF1 or CREB in the presence of scFv restored basal levels of expression. The intracellular expression of scFv provides a unique means to investigate the roles of the transcription factors ATF1 and CREB.

Activating Transcription Factor 1↗

Functional interaction between the p160 coactivator proteins and the transcriptional enhancer factor family of transcription factors.

SRC1, initially identified as a nuclear receptor coactivator, was found to interact with a member of the transcriptional enhancer factor (TEF) family of transcription factors, TEF-4. The interaction, which occurs in both intact cells and in a cell-free system, is mediated by the highly conserved basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) domain present in the N-terminal region of SRC1. Moreover, all three members of the p160 family of nuclear receptor coactivators, SRC1, TIF2, and RAC3, are able to potentiate transcription from a TEF response element in transient transfection experiments, and this activation requires the presence of the bHLH-PAS domain. These results suggest that the p160 proteins could be bona fide coactivators of the TEF family of transcription factors.

Animals↗

Transforming growth factor-beta induction of smooth muscle cell phenotpye requires transcriptional and post-transcriptional control of serum response factor.

Transforming growth factor-beta induces a smooth muscle cell phenotype in undifferentiated mesenchymal cells. To elucidate the mechanism(s) of this phenotypic induction, we focused on the molecular regulation of smooth muscle-gamma-actin, whose expression is induced at late stages of smooth muscle differentiation and developmentally restricted to this lineage. Transforming growth factor-beta induced smooth muscle-gamma-actin protein, cytoskeletal localization, and mRNA expression in mesenchymal cells. Smooth muscle-gamma-actin promoter-luciferase reporter activity was enhanced by transforming growth factor-beta, and deletion analysis revealed that CArG box 2 in the promoter was necessary for this transcriptional activation. CArG motifs bind transcriptional activator serum response factor; gel shift analyses revealed increased binding of serum response factor-containing complexes to this site in response to transforming growth factor-beta, paralleled by increased serum response factor protein expression. Serum response factor expression was found to be up-regulated by transforming growth factor-beta via transcriptional activation of the gene and post-transcriptional regulation. Using mesenchymal cells stably transfected with wild type or dominant-negative serum response factor, we demonstrated that its expression is sufficient for induction of a smooth muscle phenotype in mesenchymal cells and is necessary for transforming growth factor-beta-mediated smooth muscle induction.

Actins↗

Interaction and cooperation of mi transcription factor (MITF) and myc-associated zinc-finger protein-related factor (MAZR) for transcription of mouse mast cell protease 6 gene.

The mi transcription factor (MITF) is a basic-helix-loop-helix leucine zipper (bHLH-Zip) transcription factor that is important for the normal phenotypic expression of mast cells. Most transcription factors function in cooperation with other factors by protein-protein interactions. To search proteins interacting with MITF, we carried out a yeast two-hybrid screen and isolated Myc-associated zinc-finger protein related factor (MAZR) as a partner of MITF. When expressed with MITF in NIH/3T3 cells, MAZR was colocalized with MITF. The association of MAZR with MITF was further confirmed by a co-immunoprecipitation study and in vitro binding assay. The zinc-finger domain of MAZR and the Zip domain of MITF were essential for the interaction. MAZR was expressed in cultured mast cells and MST mastocytoma cells containing mouse mast cell protease (mMCP)-6 transcript abundantly. The overexpression of dominant negative MAZR in MST mastocytoma cells reduced the amount of mMCP-6 mRNA. The simultaneous transfection of MAZR and MITF significantly increased the promoter activity of the mMCP-6 gene, indicating that the MAZR and MITF synergistically transactivated the mMCP-6 gene. MAZR appeared to play important roles in the normal phenotypic expression of mast cells in association with MITF.

3T3 Cells↗

Transcriptional regulation of mouse delta-opioid receptor gene. Role of Ikaros in the stimulated transcription of mouse delta-opioid receptor gene in activated T cells.

Delta-opioid receptors (DOR) present on T cells have been shown to mediate the immunomodulatory effects of endogenous and synthetic DOR agonists on T cells. Considerable evidence indicates that there is stimulated transcription of DOR gene in activated T cells, which is correlated with augmented expression of DOR and enhanced capacity of DOR agonists to affect the T-cell's functions. However, the molecular mechanism underlying the stimulated transcription of the DOR gene in activated T cells is still unclear. In the present study, we analyzed a 1.3-kb DNA fragment immediately upstream of the translation start site (-1300 to +1 bp, with the translation start site designated as +1) of the mouse DOR gene in EL-4 cells, a mouse lymphoma T cell line that exhibits enhanced expression of DOR transcripts when activated by phytohemagglutinin. Through both in vivo and in vitro experiments, we have demonstrated that increased binding activity of Ikaros at the Ikaros-binding site (-378 to -374) in the DOR promoter is required for the stimulated transcription of DOR gene in phytohemagglutinin-activated T cells.

Animals↗

Transcriptional regulation of mouse delta-opioid receptor gene by CpG methylation: involvement of Sp3 and a methyl-CpG-binding protein, MBD2, in transcriptional repression of mouse delta-opioid receptor gene in Neuro2A cells.

Opioid receptors are expressed in a cell type-specific manner. Here we show that the mouse delta-opioid receptor (mDOR) gene is regulated by promoter region CpG methylation. The mDOR promoter containing a putative CpG island is highly methylated in Neuro2A cells, correlating with the repression of this gene in these cells. This is in contrast with the unmethylated state of the mDOR promoter in NS20Y cells, which express a high level of mDOR. Repression of mDOR transcription in Neuro2A cells could be partially relieved by chemically induced demethylation with 5-aza-2'-deoxycytidine. In addition, in vitro methylation of the luciferase reporter gene driven by the mDOR promoter resulted in an inhibition of transcription in NS20Y cells. Methyl-CpG-binding protein complex 1 (MeCP1) has been implicated in methylation-mediated transcriptional repression of several genes. Electrophoretic mobility shift assays showed that fully methylated, but not unmethylated, mDOR promoter fragment formed a MeCP1-like protein complex that contained methyl-CpG-binding domain protein 2 (MBD2) and Sp3. Furthermore, the expression level of Sp3 was decreased when Neuro2A cells were demethylated with 5-aza-2'-deoxycytidine, and increasing Sp3 levels in Schneider's Drosophila line 2 cells led to the repression of mDOR promoter activity when the promoter was methylated. These results demonstrate that Sp3 and MBD2 are involved in the transcriptional repression of mDOR in Neuro2A cells through binding to the methylated CpG sites in the promoter region and may play a role in the cell type-specific expression of mDOR.

Animals↗