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Query: UNIPROT:P06889 (Mol)
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The inactivating effect of sequence-specific promoter methylations was extensively studied by using the late E2A promoter of adenovirus type 2 (Ad2) DNA. The modification of the three 5' CCGG 3' sequences at nucleotides +24, +6 and -215, relative to the cap site in this promoter, sufficed to silence the gene in transient expression either in Xenopus laevis oocytes or in mammalian cells, and after the fixation of the E2A promoter-chloramphenicol-acetyltransferase (CAT) gene construct in the genome of hamster cells. It will now be demonstrated that the inactivation of the late promoter of Ad2 DNA can be reversed by transactivating functions that are encoded in the 13S messenger RNA of the E1A region of Ad2 DNA. The reactivation of a methylation-inactivated eukaryotic promoter by transactivating functions has general significance in that the value of a regulatory signal can be fully realized only by its controlled reversibility. It was demonstrated in transient expression experiments that the 5' CCGG 3'-methylated late E2A promoter was at least partly reactivated in cell lines constitutively expressing the E1 region of Ad2 or of adenovirus type 5 (Ad5) DNA. The reactivation led to transcriptional initiation at the authentic cap sites of the late E2A promoter and was not associated with promoter demethylation, at least not in both DNA complements. Reactivation of the methylation-inactivated E2A promoter could also be demonstrated in two BHK21 cell lines (mc14 and mc20), which carried the late E2A promoter-CAT gene assembly in an integrated form. In these cell lines the late E2A promoter was methylated and the CAT gene was not expressed. By transfection of cell lines mc14 and mc20, the reactivating functions were shown to reside in the pAd2E1A-13 S cDNA clone of Ad2 DNA. The pAd2E1A-12 S cDNA clone or the pAd2E1B clone showed no reactivating function. These findings implicated the E1A 289 amino acid residue protein of Ad2, a well-known transactivator, as the reactivating function of the endogenous, previously dormant, late E2A promoter-CAT gene assembly. The methylated promoter was not demethylated, at least not in both complements, and it was shown that reactivation of the methylated promoter entailed transcriptional initiation at the authentic late E2A cap site. Since E1A and E1B jointly had a more pronounced effect, it was conceivable that genes in both regions acted together in the abrogation of the inhibitory effect of promoter methylations in the late E2A promoter.
J Mol Biol 1988 Jul 20
PMID:Reactivation of the methylation-inactivated late E2A promoter of adenovirus type 2 by E1A (13 S) functions. 297 12

At early times after infection with herpes simplex virus, transcription from beta-promoters is initiated only in the presence of a functional 174,000 Mr phosphoprotein (ICP4), encoded by an immediate early (alpha) gene (IE4). A transient expression assay was used to analyze the requirement for two (ICP4 and ICP0) of the five alpha-gene products in the transcriptional regulation of model alpha and beta-gene promoters. These studies reveal that cells cotransfected with plasmids containing the alpha-gene sequences for infected cell proteins (ICPs) 4 and 0 and a thymidine kinase (TK, a beta-gene) gene or the thymidine kinase promoter fused to a chloramphenicol acetyltransferase (CAT) cassette accumulate 10 to 20-fold more RNA or exhibit 10 to 20-fold more CAT activity than cells cotransfected with a plasmid encoding either alpha-gene protein and a thymidine kinase indicator gene. Functional ICP4 is required for enhanced transcriptional activation in the transient expression assay system. It is also required for the uniform dispersal of ICP0 throughout the nucleus as shown by immunofluorescence staining analysis of transfected cells. Two alpha-promoter-CAT fusions were used as targets to study what effects ICP4, ICP0 and Vmw65 (the virion-associated alpha-gene transactivator) have on expression from alpha-promoters that contain all of the sequences that confer alpha-gene regulation, or only the core sequence governing basal level expression. We conclude that ICP4 can activate alpha-gene expression from the core sequence and, depending on its abundance, activate or repress expression from a promoter containing the sequences required for alpha-gene regulation. Independent of these alpha-regulatory sequences cotransfection with low levels of sequences encoding both ICP0 and ICP4 activate expression. At higher ratios of effector (both ICP4 and ICP0) the target accumulation of CAT activity decreases. Although a ts allele of IE4 (cloned from the mutant virus tsK) does not activate alpha-gene expression it can enhance the ability of ICP0 to activate a target containing alpha-regulatory sequences. Virus studies involving tsK support the conclusion that functional ICP4 is required to activate beta-promoters and to repress expression from alpha-promoters and help to explain the pleiotropic effects of the tsK mutation. These analyses have also revealed the presence of a novel RNA species that overlaps the sequences encoding ICP0. Our results suggest that co-ordinate regulation of HSV gene expression is mediated by the functional interaction of at least two alpha-gene products, ICP0 and ICP4.
J Mol Biol 1986 Oct 05
PMID:Co-ordinate regulation of herpes simplex virus gene expression is mediated by the functional interaction of two immediate early gene products. 302 83

Thyroid hormone receptor acts as a hormone-dependent transcriptional transactivator and as a transcriptional repressor in the absence of thyroid hormone. Specifically, thyroid hormone receptor can repress retinoic acid-induced gene expression through interactions with retinoic acid receptor. (Retinoic acid is a potent teratogen in the frog Xenopus laevis, acting at early embryonic stages to interfere with the formation of anterior structures. Endogenous retinoic acid is thought to act in normal anterior-posterior axis formation.) We have previously shown that thyroid hormone receptor RNA (alpha isotype) is expressed and polysome-associated during Xenopus embryogenesis preceding thyroid gland maturation and endogenous thyroid hormone production (D. E. Banker, J. Bigler, and R. N. Eisenman, Mol. Cell. Biol. 11:5079-5089, 1991). To determine whether thyroid hormone receptor might influence the effects of retinoic acid in early frog development, we have examined the results of ectopic thyroid hormone receptor expression on retinoic acid teratogenesis. We demonstrate that microinjections of full-length thyroid hormone receptor RNA protect injected embryos from retinoic acid teratogenesis. DNA binding is apparently essential to this protective function, as truncated thyroid hormone receptors, lacking DNA-binding domains but including hormone-binding and dimerization domains, do not protect from retinoic acid. We have shown that microinjections of these dominant-interfering thyroid hormone receptors, as well as anti-thyroid hormone receptor antibodies, increase retinoic acid teratogenesis in injected embryos, presumably by inactivating endogenous thyroid hormone receptor. This finding suggests that endogenous thyroid hormone receptors may act to limit retinoic acid sensitivity. On the other hand, after thyroid hormone treatment, ectopic thyroid hormone receptor mediates teratogenesis that is indistinguishable from the dorsoanterior deficiencies produced in retinoic acid teratogenesis. The previously characterized retinoic acid-responsive gene, Xhox.lab2, can be induced by thyroid hormone in embryos ectopically expressing thyroid hormone receptor and is less responsive to retinoic acid in such embryos. The fact that both thyroid hormone and retinoic acid can affect overlapping gene expression pathways to produce abnormal embryonic axes and can regulate the same early-expressed gene suggests a model in which thyroid hormone receptor blocks retinoic acid receptor-mediated teratogenesis by directly repressing retinoic acid-responsive genes.
Mol Cell Biol 1993 Dec
PMID:Thyroid hormone receptor can modulate retinoic acid-mediated axis formation in frog embryogenesis. 750 77

The hepatitis B virus (HBV) X protein (pX) is capable of activating transcription regulated by viral and cellular promoters containing binding sites for different transcription factors, including AP1. In this study we have analyzed the mechanisms of AP1 induction by pX. The hepatitis B virus transactivator was able to activate TRE (12-O-tetradecanoylphorbol-13-acetate response element)-directed transcription in different cell lines, including HepG2, HeLa, CV1, and PLC/PRF/5 cells. pX-induced AP1 activation in HepG2 cells was associated with an increase in the DNA-binding activity of c-Jun/c-Fos heterodimers, which was not dependent either on an increase in the overall amount of c-Fos and c-Jun proteins in the cells or on formation of dimers between pX and the two proteins, thus suggesting the involvement of posttranslational modifications of the transcription factor. The observation that the overexpression of c-Jun and c-Fos in the cells results in a strong augmentation of the effect of pX on TRE-directed transcription is additional evidence indicating the involvement of posttranscriptional modifications of c-Jun/c-Fos heterodimers. The increased AP1 binding observed in the presence of pX was unaffected by the protein kinase C inhibitors calphostin C and sphingosine and by the protein kinase A inhibitor HA1004, while it was almost completely blocked by staurosporine, a potent and nonspecific protein kinase inhibitor, suggesting that protein kinase C- and A-independent phosphorylation events might play a role in the phenomenon. The ability of pX also to increase TRE-directed transcription in cell lines in which AP1-binding activity is not increased (i.e., HeLa, CV1, and PLC/PRF/5 cells) suggests that pX can activate canonical TRE sites by different mechanisms as well.
Mol Cell Biol 1994 Feb
PMID:Induction of the DNA-binding activity of c-jun/c-fos heterodimers by the hepatitis B virus transactivator pX. 750 9

The regulation of transcriptional elongation plays a central role in the expression of a number of cellular and viral genes. For example, levels of c-myc RNA change during cellular proliferation and differentiation via alterations in transcriptional attenuation near the 5' end of the c-myc gene. The protein that regulates transcription through attenuation sites in c-myc has not been identified. However, a candidate protein of equivalent function exists in the human immunodeficiency virus (HIV) genome, where the transactivator Tat increases transcriptional elongation through the HIV LTR and coding sequences by interacting with the trans-acting-response (TAR) RNA stem-loop that is found at the 5' end of all viral transcripts. By placing TAR 3' to the P2 promoter of the mouse c-myc gene, we demonstrate that Tat can also direct read-through transcription in mouse c-myc in transfected HeLa cells. Thus we identified a viral transactivator whose cellular counterpart regulates transcriptional attenuation within c-myc and other proto-oncogenes.
J Mol Biol 1994 Nov 04
PMID:Human immunodeficiency virus type 1 tat directs transcription through attenuation sites within the mouse c-myc gene. 752 69

We have investigated HGF-induced signal transduction in two normal mouse epithelial cell lines (M23 and MM55). Both cell lines display HGF-induced mitogenesis and high level HGF-induced autophosphorylation of MET/HGFR. In both M23 and MM55 cells, HGF induces association with MET/HGFR and increased tyrosine phosphorylation of the SH2-domain containing proteins PI3K, GAP and NCK. PLC-gamma exhibited neither HGF-induced increases in tyrosine phosphorylation nor an association with MET/HGFR in these cell lines. Additionally, HGF induced increased transcription of c-fos, c-jun, junB, junD, and c-myc early response genes in both cell lines. We therefore suggest that the second messenger proteins PI3K, GAP and NCK, and possibly the protein products of the c-fos, c-jun, junB, junD and c-myc genes, are important elements in the HGF-induced mitogenic pathway in the normal mouse epithelial cell lines M23 and MM55.
Biochem Mol Biol Int 1995 Jul
PMID:Hepatocyte growth factor-induced signal transduction in two normal mouse epithelial cell lines. 754 43

Tat strongly activates transcription of the HIV-1 provirus by stimulating both initiation and elongation. This transactivator binds to the TAR RNA element, but can also associate with cellular transcription factors, interacting with upstream promoter sequences. To achieve a better understanding of the role of Tat in the assembly of the transcriptional initiation complex in the living cell, we have examined how the activity of this protein is modified when the general transcription factor involved in the first step of this process, TBP, is overexpressed. The activity of Tat, either wild-type or fused to the DNA binding domain of GAL4 (GBTat), was tested using reporter constructs containing GAL4 binding sites upstream of a minimal promoter corresponding to the HIV-1 TATA box, with or without the TAR element. We found that overexpression of TBP led to a dramatic increase in the activity of the GBTat protein. In order to activate GBTat, TBP must be able to interact with the TATA box. Analysis of several Tat mutants indicated that both the cysteine-rich and the core domains of this transactivator are necessary and sufficient to activate transcription when TBP is overexpressed. In vitro experiments showed that Tat binds specifically to TBP. There was a correlation between the ability of different Tat mutants to bind TBP and their capacity to activate transcription in vivo. With the natural HIV-1 promoter, overexpression of TBP first stimulated and then suppressed the Tat-induced activity. This inhibition was abrogated by an increase in the intracellular levels of Tat. These experimental data indicate that Tat stimulates initiation of transcription by interacting with TBP in vivo.
J Mol Biol 1995 Jul 07
PMID:Evidence for functional interaction between the HIV-1 Tat transactivator and the TATA box binding protein in vivo. 760 68

Immediate early gene (IEG) products, such as FOS and JUN, may partially mediate the long-term transcriptional response of CNS cells to specific changes in their environment. To determine whether IEG products might be involved in the immature brain's response to hypoxia-ischemia (H-I), 7-day-old rat pups were subjected to unilateral common carotid artery ligation followed by 3 h of hypoxia (8% O2/92% N2) at 37 degrees C, which results in pathological changes only in specific regions of the hemisphere ipsilateral to ligation. Time course experiments were performed, in which animals were sacrificed between 1 and 24 h after H-I. RNAs from several brain regions were analyzed by Northern blot hybridization for their relative concentrations of nine IEG mRNAs (c-fos, c-jun, junB, TIS 1 (nur77), TIS7, TIS8 (zif268), TIS10, TIS11, and TIS21). Induction of all IEGs, except TIS7 and TIS10, was observed in ipsilateral forebrain, and, less frequently, in contralateral forebrain, at 1, 2, and 3 h post-hypoxia. In some animals, lower levels of expression were also detected at 4, 18 and 24 h. With minor exceptions, co-induction of all seven IEGs was observed in a given RNA sample. Induction of two other mRNAs, representing the heat shock and astrocytic responses, were also observed. Hsp70 mRNA levels were increased only in the brains of animals exhibiting IEG induction. However, hsp70 induction was confined to the ipsilateral forebrain, implying a more direct relationship between its expression and permanent morphological damage. GFAP mRNA induction occurred predominantly in ipsilateral forebrain samples at 18 and 24 h post-hypoxia. Levels of B-actin and ubiquitin mRNAs were relatively constant in the same RNA samples. In control experiments c-fos mRNA induction was not detected after sham ligation with hypoxia, ligation with sham hypoxia, or hypoxia alone. These results suggest that the immature brain is highly responsive to H-I at the level of gene expression, involving at least three different rapid response systems.
Brain Res Mol Brain Res 1993 May
PMID:Immediate early gene induction after neonatal hypoxia-ischemia. 768 83

The distinct effects of cytokines on cellular growth and differentiation suggest that specific signaling pathways mediate these diverse biological activities. Fibroblast growth factors (FGFs) are well-established inhibitors of skeletal muscle differentiation and may operate via activation of specific signaling pathways distinct from recently identified mitogen signaling pathways. We examined whether platelet-derived growth factor (PDGF)-activated signaling pathways are sufficient to mediate FGF-dependent repression of myogenesis by introducing the PDGF beta receptor into a mouse skeletal muscle cell line. Addition of PDGF-BB to cells expressing the PDGF beta receptor activated the PDGF beta receptor tyrosine kinase, stimulated mitogen-activated protein (MAP) kinase, and increased the steady-state levels of junB and c-fos mRNAs. Despite the activation of these intracellular signaling molecules, PDGF beta receptor activation elicited no detectable effect on cell proliferation or differentiation. In contrast to PDGF-BB, addition of FGF-2 to myoblasts activated signaling pathways that resulted in DNA synthesis and repression of differentiation. Because of the low number of endogenous FGF receptors expressed, FGF-stimulated signaling events, including tyrosine phosphorylation and activation of MAP kinase, could be detected only in cells expressing higher levels of a transfected FGF receptor cDNA. As the PDGF beta receptor- and FGF receptor-stimulated signaling pathways yield different biological responses in these skeletal muscle cells, we hypothesize that FGF-mediated repression of skeletal muscle differentiation activates signaling pathways distinct from those activated by the PDGF beta receptor. Activation of PDGF beta receptor tyrosine kinase activity, stimulation of MAP kinase, and upregulation of immediate-early gene expression are not sufficient to repress skeletal muscle differentiation.
Mol Cell Biol 1995 Jun
PMID:A requirement for fibroblast growth factor in regulation of skeletal muscle growth and differentiation cannot be replaced by activation of platelet-derived growth factor signaling pathways. 776 Aug 19

We have engineered GH3 cells with reporter genes under control of the growth hormone and prolactin promoters and measured protein production. The results indicate very low level production of reporter proteins from the cells regardless of the promoter used to drive expression. This was surprising in light of the observation that the cells still produced high levels of endogenous growth hormone and prolactin. Chinese hamster ovary (CHO) cells were engineered to express the Pit-1 transactivator. Transfection of reporter genes under control of the prolactin promoter demonstrated a clear enhancement of expression levels compared to the same promoter in parental CHO cells. Pit-1 expression is not sufficient, however, for high level, stable expression from the growth hormone promoter. These results indicate that the growth hormone and prolactin promoters are not sufficient for high level, stable expression even in normally permissive cells and suggest that Pit-1 alone is not sufficient for strong promoter activity from the integrated plasmids.
Mol Cell Endocrinol 1995 Feb
PMID:Endogenous and exogenous pituitary-specific promoters are differentially controlled. 776 27


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