Journal articles: 'Transactivation'

1

Wierstra, Inken, and Jürgen Alves. "Despite its strong transactivation domain, transcription factor FOXM1c is kept almost inactive by two different inhibitory domains." Biological Chemistry 387, no. 7 (July 1, 2006): 963–76. http://dx.doi.org/10.1515/bc.2006.120.

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Abstract FOXM1c (MPP2) is an activating transcription factor with several nuclear localization signals, a forkhead domain for DNA binding, and a very strong acidic transactivation domain. Despite its very strong transactivation domain, FOXM1c is kept almost inactive by two different independent inhibitory domains, the N-terminus and the central domain. The N-terminus as a specific negative-regulatory domain directly binds to and thus inhibits the transactivation domain completely. However, it lacks any transrepression potential. In contrast, the central domain functions as a strong RB-independent transrepression domain and as an RB-recruiting negative-regulatory domain. The N-terminus alone is sufficient to eliminate transactivation, while the central domain alone represses the transactivation domain only partially. This hierarchy of the two inhibitory domains offers the possibility to activate the almost inactive wild type in two steps in vitro: deletion of the N-terminus results in a strong transactivator, while additional deletion of the central domain in a very strong transactivator. We suggest that the very high potential of the transactivation domain has to be tightly controlled by these two inhibitory domains because FOXM1 stimulates proliferation by promoting G1/S transition, as well as G2/M transition, and because deregulation of such potent activators of proliferation can result in tumorigenesis.

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2

Boulanger, Marie-Chloé, Chen Liang, Rodney S. Russell, Rongtuan Lin, Mark T. Bedford, Mark A. Wainberg, and Stéphane Richard. "Methylation of Tat by PRMT6 Regulates Human Immunodeficiency Virus Type 1 Gene Expression." Journal of Virology 79, no. 1 (January 1, 2005): 124–31. http://dx.doi.org/10.1128/jvi.79.1.124-131.2005.

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ABSTRACT The human immunodeficiency virus (HIV) transactivator protein, Tat, stimulates transcription from the viral long terminal repeats via an arginine-rich transactivating domain. Since arginines are often known to be methylated, we investigated whether HIV type 1 (HIV-1) Tat was a substrate for known protein arginine methyltransferases (PRMTs). Here we identify Tat as a substrate for the arginine methyltransferase, PRMT6. Tat is specifically associated with and methylated by PRMT6 within cells. Overexpression of wild-type PRMT6, but not a methylase-inactive PRMT6 mutant, decreased Tat transactivation of an HIV-1 long terminal repeat luciferase reporter plasmid in a dose-dependent manner. Knocking down PRMT6 consistently increased HIV-1 production in HEK293T cells and also led to increased viral infectiousness as shown in multinuclear activation of a galactosidase indicator assays. Our study demonstrates that arginine methylation of Tat negatively regulates its transactivation activity and that PRMT6 acts as a restriction factor for HIV replication.

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3

Bull, P., K. L. Morley, M. F. Hoekstra, T. Hunter, and I. M. Verma. "The mouse c-rel protein has an N-terminal regulatory domain and a C-terminal transcriptional transactivation domain." Molecular and Cellular Biology 10, no. 10 (October 1990): 5473–85. http://dx.doi.org/10.1128/mcb.10.10.5473-5485.1990.

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We have shown that the murine c-rel protein can act as a transcriptional transactivator in both yeast and mammalian cells. Fusion proteins generated by linking rel sequences to the DNA-binding domain of the yeast transcriptional activator GAL4 activate transcription from a reporter gene linked in cis to a GAL4 binding site. The full-length mouse c-rel protein (588 amino acids long) is a poor transactivator; however, the C-terminal portion of the protein between amino acid residues 403 to 568 is a potent transcriptional transactivator. Deletion of the N-terminal half of the c-rel protein augments its transactivation function. We propose that c-rel protein has an N-terminal regulatory domain and a C-terminal transactivation domain which together modulate its function as a transcriptional transactivator.

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Bull, P., K. L. Morley, M. F. Hoekstra, T. Hunter, and I. M. Verma. "The mouse c-rel protein has an N-terminal regulatory domain and a C-terminal transcriptional transactivation domain." Molecular and Cellular Biology 10, no. 10 (October 1990): 5473–85. http://dx.doi.org/10.1128/mcb.10.10.5473.

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We have shown that the murine c-rel protein can act as a transcriptional transactivator in both yeast and mammalian cells. Fusion proteins generated by linking rel sequences to the DNA-binding domain of the yeast transcriptional activator GAL4 activate transcription from a reporter gene linked in cis to a GAL4 binding site. The full-length mouse c-rel protein (588 amino acids long) is a poor transactivator; however, the C-terminal portion of the protein between amino acid residues 403 to 568 is a potent transcriptional transactivator. Deletion of the N-terminal half of the c-rel protein augments its transactivation function. We propose that c-rel protein has an N-terminal regulatory domain and a C-terminal transactivation domain which together modulate its function as a transcriptional transactivator.

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5

Pei, D. Q., and C. H. Shih. "An "attenuator domain" is sandwiched by two distinct transactivation domains in the transcription factor C/EBP." Molecular and Cellular Biology 11, no. 3 (March 1991): 1480–87. http://dx.doi.org/10.1128/mcb.11.3.1480-1487.1991.

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C/EBP is a rat liver DNA-binding protein which can act as a transcription factor. Its N-terminal portion contains three distinct domains. The first domain (amino acids 1 to 107) appears to be a highly potent transactivator. The second domain (amino acids 107 to 170) does not appear to exhibit either activation or repression activity. This domain is defined as an "attenuator domain" because its presence under four different sequence contexts reproducibly decreases the effect of transactivation of C/EBP. The third domain (amino acids 171 to 245) is a relatively weaker transactivator with a striking proline-rich motif. Deletional analysis of this third domain has shown that a 45-amino-acid region is sufficient for transactivation. This region (amino acids 171 to 215) contains 12 proline, 6 histidine, and mainly hydrophobic or noncharged amino acids. Further mutational analysis of a highly conserved proline-octamer region within this domain indicates that a specific proline content is not crucial for transactivation.

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6

Pei, D. Q., and C. H. Shih. "An "attenuator domain" is sandwiched by two distinct transactivation domains in the transcription factor C/EBP." Molecular and Cellular Biology 11, no. 3 (March 1991): 1480–87. http://dx.doi.org/10.1128/mcb.11.3.1480.

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C/EBP is a rat liver DNA-binding protein which can act as a transcription factor. Its N-terminal portion contains three distinct domains. The first domain (amino acids 1 to 107) appears to be a highly potent transactivator. The second domain (amino acids 107 to 170) does not appear to exhibit either activation or repression activity. This domain is defined as an "attenuator domain" because its presence under four different sequence contexts reproducibly decreases the effect of transactivation of C/EBP. The third domain (amino acids 171 to 245) is a relatively weaker transactivator with a striking proline-rich motif. Deletional analysis of this third domain has shown that a 45-amino-acid region is sufficient for transactivation. This region (amino acids 171 to 215) contains 12 proline, 6 histidine, and mainly hydrophobic or noncharged amino acids. Further mutational analysis of a highly conserved proline-octamer region within this domain indicates that a specific proline content is not crucial for transactivation.

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7

Omoto, Shinya, Ebiamadon Andi Brisibe, Harumi Okuyama, and Yoichi R. Fujii. "Feline foamy virus Tas protein is a DNA-binding transactivator." Journal of General Virology 85, no. 10 (October 1, 2004): 2931–35. http://dx.doi.org/10.1099/vir.0.80088-0.

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Foamy viruses (FVs) harbour a transcriptional transactivator (Tas) and two Tas-responsive promoter regions, one in the 5′ long terminal repeat (LTR) and the other an internal promoter (IP) in the envelope gene. To analyse the mechanism of transactivation of the FVs, the specificity of feline FV (FFV) Tas protein, which is more distantly related to the respective proteins of non-human primate origin, were investigated. FFV Tas has been shown specifically to activate gene expression from the cognate promoters. No cross-transactivation was noted of the prototype foamy virus and human immunodeficiency virus type 1 LTR. The putative transactivation response element of FFV Tas was mapped to the 5′ LTR U3 region (approximately nt −228 to −195). FFV Tas binds to this element in addition to a previously described sequence (position −66 to −51). It is therefore concluded that FFV Tas is a DNA-binding transactivator that interacts with at least two regions in the virus LTR.

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8

Gutsch, D. E., E. A. Holley-Guthrie, Q. Zhang, B. Stein, M. A. Blanar, A. S. Baldwin, and S. C. Kenney. "The bZIP transactivator of Epstein-Barr virus, BZLF1, functionally and physically interacts with the p65 subunit of NF-kappa B." Molecular and Cellular Biology 14, no. 3 (March 1994): 1939–48. http://dx.doi.org/10.1128/mcb.14.3.1939-1948.1994.

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The Epstein-Barr virus (EBV) BZLF1 (Z) immediate-early transactivator initiates the switch between latent and productive infection in B cells. The Z protein, which has homology to the basic leucine zipper protein c-Fos, transactivates the promoters of several replicative cycle proteins. Transactivation efficiency of the EBV BMRF1 promoter by Z is cell type dependent. In B cells, in which EBV typically exists in a latent form, Z activates the BMRF1 promoter inefficiently. We have discovered that the p65 component of the cellular factor NF-kappa B inhibits transactivation of several EBV promoters by Z. Furthermore, the inhibitor of NF-kappa B, I kappa B alpha, can augment Z-induced transactivation in the B-cell line Raji. Using glutathione S-transferase fusion proteins and coimmunoprecipitation studies, we demonstrate a direct interaction between Z and p65. This physical interaction, which requires the dimerization domain of Z and the Rel homology domain of p65, can be demonstrated both in vitro and in vivo. Inhibition of Z transactivation function by NF-kappa B p65, or possibly by other Rel family proteins, may contribute to the inefficiency of Z transactivator function in B cells and may be a mechanism of maintaining B-cell-specific viral latency.

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9

Gutsch, D. E., E. A. Holley-Guthrie, Q. Zhang, B. Stein, M. A. Blanar, A. S. Baldwin, and S. C. Kenney. "The bZIP transactivator of Epstein-Barr virus, BZLF1, functionally and physically interacts with the p65 subunit of NF-kappa B." Molecular and Cellular Biology 14, no. 3 (March 1994): 1939–48. http://dx.doi.org/10.1128/mcb.14.3.1939.

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The Epstein-Barr virus (EBV) BZLF1 (Z) immediate-early transactivator initiates the switch between latent and productive infection in B cells. The Z protein, which has homology to the basic leucine zipper protein c-Fos, transactivates the promoters of several replicative cycle proteins. Transactivation efficiency of the EBV BMRF1 promoter by Z is cell type dependent. In B cells, in which EBV typically exists in a latent form, Z activates the BMRF1 promoter inefficiently. We have discovered that the p65 component of the cellular factor NF-kappa B inhibits transactivation of several EBV promoters by Z. Furthermore, the inhibitor of NF-kappa B, I kappa B alpha, can augment Z-induced transactivation in the B-cell line Raji. Using glutathione S-transferase fusion proteins and coimmunoprecipitation studies, we demonstrate a direct interaction between Z and p65. This physical interaction, which requires the dimerization domain of Z and the Rel homology domain of p65, can be demonstrated both in vitro and in vivo. Inhibition of Z transactivation function by NF-kappa B p65, or possibly by other Rel family proteins, may contribute to the inefficiency of Z transactivator function in B cells and may be a mechanism of maintaining B-cell-specific viral latency.

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10

Sjöberg, M., and B. Vennström. "Ligand-dependent and -independent transactivation by thyroid hormone receptor beta 2 is determined by the structure of the hormone response element." Molecular and Cellular Biology 15, no. 9 (September 1995): 4718–26. http://dx.doi.org/10.1128/mcb.15.9.4718.

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Chicken thyroid hormone receptor beta 2 (cTR beta 2) is likely to serve specific functions in gene regulation since it possesses a unique N-terminal domain and is expressed in very few tissues. We demonstrate here that TR beta 2 exhibits distinct transactivation properties which are dependent on the availability of ligand and on the structure of the hormone response element. First, a strong ligand-independent transactivation was observed with hormone response elements composed of direct repeats and everted repeats. Second, TR beta 2 was induced by triiodothyronine to transactivate more efficiently than TR beta 0 on palindromic and everted-repeat types of hormone response elements. However, coexpression of the retinoid X receptor reduced the strong transactivation by TR beta 2 but not by TR beta 0 via palindromic response elements, suggesting that TR beta 2 can transactivate as a homodimer. Finally, the N terminus of TR beta 2 contains two distinct transactivation regions rich in tyrosines, which are essential for transactivation. Our results thus show that the activity of the novel transactivating region of TR beta 2 is dependent on the organization of the half-sites in the response element.

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11

Tchórzewski, M., B. Boldyreff, and N. Grankowski. "Extraribosomal function of the acidic ribosomal P1-protein YP1alpha from Saccharomyces cerevisiae." Acta Biochimica Polonica 46, no. 4 (December 31, 1999): 901–10. http://dx.doi.org/10.18388/abp.1999_4112.

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The yeast acidic ribosomal P-proteins YP1alpha, YP1beta, YP2alpha and YP2beta were studied for a possible transactivation potential beside their ribosomal function. The fusions of P-proteins with the GAL4 DNA-binding domain were assayed toward their transcriptional activity with the aid of reporter genes in yeast. Two of the P-proteins, YP1alpha and YP1beta, exhibited transactivation potential, however, only YP1alpha can be regarded as a potent transactivator. This protein was able to transactivate a reporter gene associated with two distinct promoter systems, GAL1 or CYC1. Additionally, truncated proteins of YP1alpha and YP1beta were analyzed. The N-terminal part of YP1alpha fused to GAL4-BD showed transactivation potential but the C-terminal part did not. Our results suggest a putative extraribosomal function for these ribosomal proteins which consequently may be classified as "moonlighting" proteins.

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12

Simcha, Inbal, Michael Shtutman, Daniela Salomon, Jacob Zhurinsky, Einat Sadot, Benjamin Geiger, and Avri Ben-Ze'ev. "Differential Nuclear Translocation and Transactivation Potential of β-Catenin and Plakoglobin." Journal of Cell Biology 141, no. 6 (June 15, 1998): 1433–48. http://dx.doi.org/10.1083/jcb.141.6.1433.

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β-Catenin and plakoglobin are homologous proteins that function in cell adhesion by linking cadherins to the cytoskeleton and in signaling by transactivation together with lymphoid-enhancing binding/T cell (LEF/TCF) transcription factors. Here we compared the nuclear translocation and transactivation abilities of β-catenin and plakoglobin in mammalian cells. Overexpression of each of the two proteins in MDCK cells resulted in nuclear translocation and formation of nuclear aggregates. The β-catenin-containing nuclear structures also contained LEF-1 and vinculin, while plakoglobin was inefficient in recruiting these molecules, suggesting that its interaction with LEF-1 and vinculin is significantly weaker. Moreover, transfection of LEF-1 translocated endogenous β-catenin, but not plakoglobin to the nucleus. Chimeras consisting of Gal4 DNA-binding domain and the transactivation domains of either plakoglobin or β-catenin were equally potent in transactivating a Gal4-responsive reporter, whereas activation of LEF-1– responsive transcription was significantly higher with β-catenin. Overexpression of wild-type plakoglobin or mutant β-catenin lacking the transactivation domain induced accumulation of the endogenous β-catenin in the nucleus and LEF-1–responsive transactivation. It is further shown that the constitutive β-catenin–dependent transactivation in SW480 colon carcinoma cells and its nuclear localization can be inhibited by overexpressing N-cadherin or α-catenin. The results indicate that (a) plakoglobin and β-catenin differ in their nuclear translocation and complexing with LEF-1 and vinculin; (b) LEF-1–dependent transactivation is preferentially driven by β-catenin; and (c) the cytoplasmic partners of β-catenin, cadherin and α-catenin, can sequester it to the cytoplasm and inhibit its transcriptional activity.

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13

Barnabas, Sangeeta, and Ourania M. Andrisani. "Different Regions of Hepatitis B Virus X Protein Are Required for Enhancement of bZip-Mediated Transactivation versus Transrepression." Journal of Virology 74, no. 1 (January 1, 2000): 83–90. http://dx.doi.org/10.1128/jvi.74.1.83-90.2000.

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ABSTRACT The hepatitis B virus X protein (pX) interacts directly with the bZip transactivator CREB and the bZip repressors ICERIIγ and ATF3, increasing their DNA-binding affinity in vitro and their transcriptional efficacy in vivo. However, the mechanism of bZip-pX interaction and of the pX-mediated increase in the bZip transcriptional efficacy remains to be understood. In this study with deletion mutants of pX, we delineated a 67-amino-acid region spanning residues 49 to 115 required for direct CREB, ATF3, and ICER IIγ interaction in vitro and in vivo and increased bZip/CRE binding in vitro. Transient transfections of the pX deletion mutants in AML12 hepatocytes demonstrate that pX49–115 is as effective as the full-length pX in enhancing the ATF3- and ICERIIγ-mediated transrepression. However, this pX region is inactive in increasing the transactivation efficacy of CREB; additional amino acid residues present in pX49–140are required to mediate the increased transactivation efficacy of CREB in vivo. This requirement for different regions of pX in affecting CREB transactivation suggests that amino acid residues 115 to 140 integrate additional events in effecting pX-mediated transactivation, such as concomitant interactions with select components of the basal transcriptional apparatus.

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14

Imamura, Ryu, Esteban S. Masuda, Yoshiyuki Naito, Shin-ichiro Imai, Tadahiro Fujino, Toshiya Takano, Ken-ichi Arai, and Naoko Arai. "Carboxyl-Terminal 15-Amino Acid Sequence of NFATx1 Is Possibly Created by Tissue-Specific Splicing and Is Essential for Transactivation Activity in T Cells." Journal of Immunology 161, no. 7 (October 1, 1998): 3455–63. http://dx.doi.org/10.4049/jimmunol.161.7.3455.

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Abstract NFAT regulates transcription of a number of cytokine and other immunoregulatory genes. We have isolated NFATx, which is one of four members of the NFAT family of transcription factors and is preferentially expressed in the thymus and peripheral blood leukocytes, and an isoform of NFATx, NFATx1. Here we provide evidence showing that 15 amino acids in the carboxyl-terminal end of NFATx1 are required for its maximum transactivation activity in Jurkat T cells. A fusion between these 15 amino acids and the GAL4 DNA binding domain was capable of transactivating reporters driven by the GAL4 DNA binding site. Interestingly, this 15-amino acid transactivation sequence is well conserved in NFAT family proteins, although the sequences contiguous to the carboxyl-terminal regions of the NFAT family are much less conserved. We also report three additional isoforms of NFATx, designated NFATx2, NFATx3, and NFATx4. This transactivation sequence is altered by tissue-specific alternative splicing in newly isolated NFATx isoforms, resulting in lower transactivation activity in Jurkat T cells. NFATx1 is expressed predominantly in the thymus and peripheral blood leukocyte, while the skeletal muscle expressed primarily NFATx2. In Jurkat cells, transcription from the NFAT site of the IL-2 promoter is activated strongly by NFATx1 but only weakly by NFATx2. These data demonstrate that the 15-amino acid sequence of NFATx1 is a major transactivation sequence required for induction of genes by NFATx1 in T cells and possibly regulates NFAT activity through tissue-specific alternative splicing.

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15

Haseeb, Abdul, and Véronique Lefebvre. "The SOXE transcription factors—SOX8, SOX9 and SOX10—share a bi-partite transactivation mechanism." Nucleic Acids Research 47, no. 13 (June 13, 2019): 6917–31. http://dx.doi.org/10.1093/nar/gkz523.

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Abstract SOX8, SOX9 and SOX10 compose the SOXE transcription factor group. They govern cell fate and differentiation in many lineages, and mutations impairing their activity cause severe diseases, including campomelic dysplasia (SOX9), sex determination disorders (SOX8 and SOX9) and Waardenburg-Shah syndrome (SOX10). However, incomplete knowledge of their modes of action limits disease understanding. We here uncover that the proteins share a bipartite transactivation mechanism, whereby a transactivation domain in the middle of the proteins (TAM) synergizes with a C-terminal one (TAC). TAM comprises amphipathic α-helices predicted to form a protein-binding pocket and overlapping with minimal transactivation motifs (9-aa-TAD) described in many transcription factors. One 9-aa-TAD sequence includes an evolutionarily conserved and functionally required EΦ[D/E]QYΦ motif. SOXF proteins (SOX7, SOX17 and SOX18) contain an identical motif, suggesting evolution from a common ancestor already harboring this motif, whereas TAC and other transactivating SOX proteins feature only remotely related motifs. Missense variants in this SOXE/SOXF-specific motif are rare in control individuals, but have been detected in cancers, supporting its importance in development and physiology. By deepening understanding of mechanisms underlying the central transactivation function of SOXE proteins, these findings should help further decipher molecular networks essential for development and health and dysregulated in diseases.

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16

Zheng, Peng-Sheng, Jane Brokaw, and Alison A. McBride. "Conditional Mutations in the Mitotic Chromosome Binding Function of the Bovine Papillomavirus Type 1 E2 Protein." Journal of Virology 79, no. 3 (February 1, 2005): 1500–1509. http://dx.doi.org/10.1128/jvi.79.3.1500-1509.2005.

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ABSTRACT The papillomavirus E2 protein is required for viral transcriptional regulation, DNA replication and genome segregation. We have previously shown that the E2 transactivator protein and BPV1 genomes are associated with mitotic chromosomes; E2 links the genomes to cellular chromosomes to ensure efficient segregation to daughter nuclei. The transactivation domain of the E2 protein is necessary and sufficient for association of the E2 protein with mitotic chromosomes. To determine which residues of this 200-amino-acid domain are important for chromosomal interaction, E2 proteins with amino acid substitutions in each conserved residue of the transactivation domain were tested for their ability to associate with mitotic chromosomes. Chromatin binding was assessed by using immunofluorescence on both spread and directly fixed mitotic chromosomes. E2 proteins defective in the transactivation and replication functions were unable to associate with chromosomes, and those that were competent in these functions were attached to mitotic chromosomes. However, several mutated proteins that were defective for chromosomal interaction could associate with chromosomes after treatment with agents that promote protein folding or when cells were incubated at lower temperatures. These results indicate that precise folding of the E2 transactivation domain is crucial for its interaction with mitotic chromosomes and that this association can be modulated.

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17

Calendar, Richard. "Viral Transactivation." Nature Biotechnology 4, no. 12 (December 1986): 1074–77. http://dx.doi.org/10.1038/nbt1286-1074.

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18

Goodwin, Edward C., Lisa Kay Naeger, David E. Breiding, Elliot J. Androphy, and Daniel DiMaio. "Transactivation-Competent Bovine Papillomavirus E2 Protein Is Specifically Required for Efficient Repression of Human Papillomavirus Oncogene Expression and for Acute Growth Inhibition of Cervical Carcinoma Cell Lines." Journal of Virology 72, no. 5 (May 1, 1998): 3925–34. http://dx.doi.org/10.1128/jvi.72.5.3925-3934.1998.

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ABSTRACT The papillomavirus E2 proteins can function as sequence-specific transactivators or transrepressors of transcription and as cofactors in viral DNA replication. We previously demonstrated that acute expression of the bovine papillomavirus type 1 (BPV1) E2 protein in HeLa and HT-3 cervical carcinoma cell lines greatly reduced cellular proliferation by imposing a specific G1/S phase growth arrest. In this report, we analyzed the effects of a panel of point mutations in the BPV1 E2 protein to identify the functional requirements for acute growth inhibition. Disruption of E2-specific transactivation by mutations within either the transactivation domain or the DNA binding domain severely impaired E2-mediated growth inhibition in HeLa and HT-3 cells, even though these mutants retain various other E2 activities. This result indicates that functional transactivation activity is required for acute E2-mediated growth inhibition. HeLa cells, which contain a wild-type p53 gene, and HT-3 cells, which contain a transactivation-defective p53 gene, exhibited similar responses to the E2 mutants, suggesting that identical functions of the E2 protein were required for growth arrest regardless of p53 status. Replacement of the E2 transactivation domain with that of the herpes simplex virus VP16 generated a chimeric transactivator that efficiently stimulated expression of an E2-responsive reporter plasmid yet was completely defective for growth inhibition, suggesting that an E2-specific transactivation function is required for growth arrest. Surprisingly, the transactivation-defective E2 mutants were also markedly defective in their ability to repress transcription of the native human papillomavirus type 18 (HPV18) E6/E7 oncogenes in HeLa cells and of the HPV18 promoter present in a transfected reporter plasmid. These mutants were also defective in their ability to increase p53 levels. Therefore, efficient repression of the HPV18 promoter in HeLa cells is not merely a consequence of the binding of an E2 protein to appropriately situated binding sites in the promoter.

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Szojka, Zsófia, János András Mótyán, Márió Miczi, Mohamed Mahdi, and József Tőzsér. "Y44A Mutation in the Acidic Domain of HIV-2 Tat Impairs Viral Reverse Transcription and LTR-Transactivation." International Journal of Molecular Sciences 21, no. 16 (August 17, 2020): 5907. http://dx.doi.org/10.3390/ijms21165907.

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HIV transactivator protein (Tat) plays a pivotal role in viral replication through modulation of cellular transcription factors and transactivation of viral genomic transcription. The effect of HIV-1 Tat on reverse transcription has long been described in the literature, however, that of HIV-2 is understudied. Sequence homology between Tat proteins of HIV-1 and 2 is estimated to be less than 30%, and the main difference lies within their N-terminal region. Here, we describe Y44A-inactivating mutation of HIV-2 Tat, studying its effect on capsid production, reverse transcription, and the efficiency of proviral transcription. Investigation of the mutation was performed using sequence- and structure-based in silico analysis and in vitro experiments. Our results indicate that the Y44A mutant HIV-2 Tat inhibited the activity and expression of RT (reverse transcriptase), in addition to diminishing Tat-dependent LTR (long terminal repeat) transactivation. These findings highlight the functional importance of the acidic domain of HIV-2 Tat in the regulation of reverse transcription and transactivation of the integrated provirions.

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Georges, Sara A., Holli A. Giebler, Philip A. Cole, Karolin Luger, Paul J. Laybourn, and Jennifer K. Nyborg. "Tax Recruitment of CBP/p300, via the KIX Domain, Reveals a Potent Requirement for Acetyltransferase Activity That Is Chromatin Dependent and Histone Tail Independent." Molecular and Cellular Biology 23, no. 10 (May 15, 2003): 3392–404. http://dx.doi.org/10.1128/mcb.23.10.3392-3404.2003.

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ABSTRACT Robust transcription of human T-cell leukemia virus type 1 (HTLV-1) genome requires the viral transactivator Tax. Although Tax has been previously shown to interact with the KIX domain of CBP/p300 in vitro, the precise functional relevance of this interaction remains unclear. Using two distinct approaches to interrupt the physical interaction between Tax and KIX, we find that Tax transactivation from chromatin templates is strongly dependent on CBP/p300 recruitment via the KIX domain. Additionally, we find that the primary functional contribution of CBP/p300 to Tax transactivation resides in the intrinsic acetyltransferase activity of the coactivators. These studies unexpectedly uncover a specific requirement for CBP/p300 acetyltransferase activity on chromatin templates assembled with nucleosomes lacking their amino-terminal tails. Together, these data indicate that the KIX domain of CBP/p300 is essential for targeting the acetyltransferase activity of the coactivator to the Tax-CREB (Tax/CREB) complex. Significantly, these observations reveal the presence of one or more CBP/p300 acetyltransferase targets that function specifically on chromatin templates, are independent of the histone tails, and are critical to Tax transactivation.

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Fujinaga, Koh, Dan Irwin, Matthias Geyer, and B. Matija Peterlin. "Optimized Chimeras between Kinase-Inactive Mutant Cdk9 and Truncated Cyclin T1 Proteins Efficiently Inhibit Tat Transactivation and Human Immunodeficiency Virus Gene Expression." Journal of Virology 76, no. 21 (November 1, 2002): 10873–81. http://dx.doi.org/10.1128/jvi.76.21.10873-10881.2002.

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ABSTRACT The human cyclin T1 (hCycT1) protein from the positive transcription elongation factor b (P-TEFb) binds the transactivator Tat and the transactivation response (TAR) RNA stem loop from human immunodeficiency virus type 1 (HIV). This complex activates the elongation of viral transcription. To create effective inhibitors of Tat and thus HIV replication, we constructed mutant hCycT1 proteins that are defective in binding its kinase partner, Cdk9, or TAR. Although these mutant hCycT1 proteins did not increase Tat transactivation in murine cells, their dominant-negative effects were small in human cells. Higher inhibitory effects were obtained when hCycT1 was fused with the mutant Cdk9 protein. Since the autophosphorylation of the C terminus of Cdk9 is required for the formation of the stable complex between P-TEFb, Tat, and TAR, these serines and threonines were changed to glutamate in a kinase-inactive Cdk9 protein. This chimera inhibited Tat transactivation and HIV gene expression in human cells. Therefore, this dominant-negative kinase-inactive mutant Cdk9.hCycT1 chimera could be used for antiviral gene therapy.

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Vijayan, Saptha, Torsten B. Meissner, Kyoung-Hee Lee, Yuen-Joyce Liu, Isaac Downs, Tabasum Sidiq, Chi Zhang, Peter J. van den Elsen, and Koichi S. Kobayashi. "Role of NLRC5 and IRF1 in the induction of MHC class I." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 64.20. http://dx.doi.org/10.4049/jimmunol.202.supp.64.20.

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Abstract MHC class I and MHC class II plays a major role in adaptive immune responses through activation of CD8+ T cells or CD4+ T cells respectively, by presenting intracellular or extracellular antigens. The MHC class I transactivator (CITA), NLRC5 was recently identified as a key transcriptional regulator of MHC class I and related genes. Although promoters of both MHC class I and class II genes share similar cis-regulatory elements, the specificity of NLRC5 to MHC class I gene transactivation remains unclear. To delineate the specificity of NLRC5 in NLRC5-dependent MHC classs I transactivation, we performed co-immunoprecipitation with different deletion mutants of IRF1. The results were further confirmed in mice lacking Irf1 and Nlrc5. We found that the transcription factor IRF1 associates with NLRC5, enabling efficient transactivation of MHC class I and related genes. Double deficiency of Irf1 and Nlrc5 resulted in the severe reduction of MHC class I and related gene expression in vitro and in vivo. Therefore, IRF1 is a key component that determine the specificity of NLRC5-dependent MHC class I gene transactivation, and their involvement in the CITA enhanceosome is critical for MHC class I-dependent immune responses. Further studies on the interaction of IRF1 and NLRC5 would shed light on the potential therapeutic intervention on the MHC class I related diseases such as auto-immune disorders, cancer immunity and in organ transplantation

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Whitelaw, M. L., J. A. Gustafsson, and L. Poellinger. "Identification of transactivation and repression functions of the dioxin receptor and its basic helix-loop-helix/PAS partner factor Arnt: inducible versus constitutive modes of regulation." Molecular and Cellular Biology 14, no. 12 (December 1994): 8343–55. http://dx.doi.org/10.1128/mcb.14.12.8343-8355.1994.

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Gene regulation by dioxins is mediated via the dioxin receptor, a ligand-dependent basic helix-loop-helix (bHLH)/PAS transcription factor. The latent dioxin receptor responds to dioxin signalling by forming an activated heterodimeric complex with a specific bHLH partner, Arnt, an essential process for target DNA recognition. We have analyzed the transactivating potential within this heterodimeric complex by dissecting it into individual subunits, replacing the dimerization and DNA-binding bHLH motifs with heterologous zinc finger DNA-binding domains. The uncoupled Arnt chimera, maintaining 84% of Arnt residues, forms a potent and constitutive transcription factor. Chimeric proteins show that the dioxin receptor also harbors a strong transactivation domain in the C terminus, although this activity was silenced by inclusion of 82 amino acids from the central ligand-binding portion of the dioxin receptor. This central repression region conferred binding of the molecular chaperone hsp90 upon otherwise constitutive chimeras in vitro, indicating that hsp90 has the ability to mediate a cis-repressive function on distant transactivation domains. Importantly, when the ligand-binding domain of the dioxin receptor remained intact, the ability of this hsp90-binding activity to confer repression became conditional rather than irreversible. Our data are consistent with a model in which crucial activities of the dioxin receptor, such as dimerization with Arnt and transactivation, are conditionally repressed by the central ligand- and-hsp90-binding region of the receptor. In contrast, the Arnt protein appears to be free from any repressive activity. Moreover, within the context of the dioxin response element (xenobiotic response element), the C terminus of Arnt conferred a potent, dominating transactivation function onto the native bHLH heterodimeric complex. Finally, the relative transactivation potencies of the individual dioxin receptor and Arnt chimeras varied with cell type and promoter architecture, indicating that the mechanisms for transcriptional activation may differ between these two subunits and that in the native complex the transactivation pathway may be dependent upon cell-specific and promoter contexts.

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Whitelaw, M. L., J. A. Gustafsson, and L. Poellinger. "Identification of transactivation and repression functions of the dioxin receptor and its basic helix-loop-helix/PAS partner factor Arnt: inducible versus constitutive modes of regulation." Molecular and Cellular Biology 14, no. 12 (December 1994): 8343–55. http://dx.doi.org/10.1128/mcb.14.12.8343.

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Gene regulation by dioxins is mediated via the dioxin receptor, a ligand-dependent basic helix-loop-helix (bHLH)/PAS transcription factor. The latent dioxin receptor responds to dioxin signalling by forming an activated heterodimeric complex with a specific bHLH partner, Arnt, an essential process for target DNA recognition. We have analyzed the transactivating potential within this heterodimeric complex by dissecting it into individual subunits, replacing the dimerization and DNA-binding bHLH motifs with heterologous zinc finger DNA-binding domains. The uncoupled Arnt chimera, maintaining 84% of Arnt residues, forms a potent and constitutive transcription factor. Chimeric proteins show that the dioxin receptor also harbors a strong transactivation domain in the C terminus, although this activity was silenced by inclusion of 82 amino acids from the central ligand-binding portion of the dioxin receptor. This central repression region conferred binding of the molecular chaperone hsp90 upon otherwise constitutive chimeras in vitro, indicating that hsp90 has the ability to mediate a cis-repressive function on distant transactivation domains. Importantly, when the ligand-binding domain of the dioxin receptor remained intact, the ability of this hsp90-binding activity to confer repression became conditional rather than irreversible. Our data are consistent with a model in which crucial activities of the dioxin receptor, such as dimerization with Arnt and transactivation, are conditionally repressed by the central ligand- and-hsp90-binding region of the receptor. In contrast, the Arnt protein appears to be free from any repressive activity. Moreover, within the context of the dioxin response element (xenobiotic response element), the C terminus of Arnt conferred a potent, dominating transactivation function onto the native bHLH heterodimeric complex. Finally, the relative transactivation potencies of the individual dioxin receptor and Arnt chimeras varied with cell type and promoter architecture, indicating that the mechanisms for transcriptional activation may differ between these two subunits and that in the native complex the transactivation pathway may be dependent upon cell-specific and promoter contexts.

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Barboric, Matjaz, Fan Zhang, Mojca Besenicar, Ana Plemenitas, and B. Matija Peterlin. "Ubiquitylation of Cdk9 by Skp2 Facilitates Optimal Tat Transactivation." Journal of Virology 79, no. 17 (September 1, 2005): 11135–41. http://dx.doi.org/10.1128/jvi.79.17.11135-11141.2005.

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ABSTRACT By recruiting the positive transcriptional elongation factor b (P-TEFb) to paused RNA polymerase II, the transactivator Tat stimulates transcriptional elongation of the human immunodeficiency virus type 1 (HIV-1) genome. We found that cyclin-dependent kinase 9 (Cdk9), the catalytic subunit of P-TEFb, is ubiquitylated in vivo. This ubiquitylation depended on the Skp1/Cul1/F-box protein E3 ubiquitin ligase Skp2. Likewise, Tat required Skp2 since its transactivation of the HIV-1 long terminal repeat decreased in primary mouse embryonic fibroblasts, which lacked Skp2. The ubiquitylation of Cdk9 by Skp2 facilitated the formation of the ternary complex between P-TEFb, Tat, and transactivation response element. Thus, our findings underscore the requirement of ubiquitylation for the coactivator function in regulating HIV-1 transcriptional elongation.

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Mishra, Rajnikant, Ivan P. Gorlov, Lian Y. Chao, Sanjaya Singh, and Grady F. Saunders. "PAX6, Paired Domain Influences Sequence Recognition by the Homeodomain." Journal of Biological Chemistry 277, no. 51 (October 17, 2002): 49488–94. http://dx.doi.org/10.1074/jbc.m206478200.

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PAX6 functions as a transcription factor and has two DNA-binding domains, a paired domain (PD) and a homeodomain (HD), joined by a glycine-rich linker and followed by a proline-serine-threonine-rich (PST) transactivation region at the C terminus. The mechanism of PAX6 function is not clearly understood, and few target genes in vertebrates have been identified. In this report we described the functional analyses of patient missense mutations from the paired domain region of PAX6 and a paireddomain-less isoform (PD-less) of Pax6 that lacks the paired domain and part of the glycine-rich linker. The PD-less was expressed in the brain, eyes, and pancreas of mouse. The level of expression of this isoform was relatively higher in brain. The mutation sites PAX6-L46R and -C52R were located in the PD of PAX6 on either end of the 5a-polypeptide insert of the alternatively spliced form of PAX6, PAX6-5a. Another PAX6 mutant V53L described in this report was adjacent to C52R. We created corresponding mutations in PAX6 and PAX6-5a, and evaluated their transcriptional activation and DNA binding properties. The PD mutants of PAX6 (L46R, C52R, and V53L) exhibited lower transactivation activities and variable DNA binding ability than wild-type PAX6 with PD DNA-binding consensus sequences. The mutated amino acids containing PAX6-5a isoforms showed unexpected transactivation properties with a reporter containing HD DNA-binding sequences. PAX6-5a-C52R, and -V53L showed lower transactivation activities, but PAX6-5a-L46R had greater transactivation ability than PAX6-5a. The PD-less isoform of Pax6 lost its transactivational ability but could bind to the HD DNA-binding sequences. Functional analysis of the PD-less isoform of Pax6 as well as findings related to missense mutations in the PD suggest that the PD of PAX6 is required for HD function.

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Kohrt, Stephan, Sarah Strobel, Melanie Mann, Heinrich Sticht, Bernhard Fleckenstein, and Andrea Thoma-Kress. "Characterizing the Interaction between the HTLV-1 Transactivator Tax-1 with Transcription Elongation Factor ELL2 and Its Impact on Viral Transactivation." International Journal of Molecular Sciences 22, no. 24 (December 18, 2021): 13597. http://dx.doi.org/10.3390/ijms222413597.

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The human T-cell leukemia virus type 1 (HTLV-1)-encoded transactivator and oncoprotein Tax-1 is essential for HTLV-1 replication. We recently found that Tax-1 interacts with transcription elongation factor for RNA polymerase II 2, ELL2, which enhances Tax-1-mediated transactivation of the HTLV-1 promotor. Here, we characterize the Tax-1:ELL2 interaction and its impact on viral transactivation by confocal imaging, co-immunoprecipitation, and luciferase assays. We found that Tax-1 and ELL2 not only co-precipitate, but also co-localize in dot-like structures in the nucleus. Tax-1:ELL2 complex formation occurred independently of Tax-1 point mutations, which are crucial for post translational modifications (PTMs) of Tax-1, suggesting that these PTMs are irrelevant for Tax-1:ELL2 interaction. In contrast, Tax-1 deletion mutants lacking either N-terminal (aa 1–37) or C-terminal regions (aa 150–353) of Tax-1 were impaired in interacting with ELL2. Contrary to Tax-1, the related, non-oncogenic Tax-2B from HTLV-2B did not interact with ELL2. Finally, we found that ELL2-R1 (aa 1–353), which carries an RNA polymerase II binding domain, and ELL2-R3 (aa 515–640) are sufficient to interact with Tax-1; however, only ELL2-truncations expressing R1 could enhance Tax-1-mediated transactivation of the HTLV-1 promoter. Together, this study identifies domains in Tax-1 and ELL2 being required for Tax-1:ELL2 complex formation and for viral transactivation.

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Campisi, Judith. "Parsing p53 Transactivation." Developmental Cell 20, no. 5 (May 2011): 573–74. http://dx.doi.org/10.1016/j.devcel.2011.04.015.

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Liu, Wang-Jing, Yun-Shiang Chang, Hao-Ching Wang, Jiann-Horng Leu, Guang-Hsiung Kou, and Chu-Fang Lo. "Transactivation, Dimerization, and DNA-Binding Activity of White Spot Syndrome Virus Immediate-Early Protein IE1." Journal of Virology 82, no. 22 (September 3, 2008): 11362–73. http://dx.doi.org/10.1128/jvi.01244-08.

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ABSTRACT Immediate-early proteins from many viruses function as transcriptional regulators and exhibit transactivation activity, DNA binding activity, and dimerization. In this study, we investigated these characteristics in white spot syndrome virus (WSSV) immediate-early protein 1 (IE1) and attempted to map the corresponding functional domains. Transactivation was investigated by transiently expressing a protein consisting of the DNA binding domain of the yeast transactivator GAL4 fused to full-length IE1. This GAL4-IE1 fusion protein successfully activated the Autographa californica multicapsid nucleopolyhedrovirus p35 basal promoter when five copies of the GAL4 DNA binding site were inserted upstream of the TATA box. A deletion series of GAL4-IE1 fusion proteins suggested that the transactivation domain of WSSV IE1 was carried within its first 80 amino acids. A point mutation assay further showed that all 12 of the acidic residues in this highly acidic domain were important for IE1's transactivation activity. DNA binding activity was confirmed by an electrophoresis mobility shift assay using a probe with 32P-labeled random oligonucleotides. The DNA binding region of WSSV IE1 was located in its C-terminal end (amino acids 81 to 224), but mutation of a putative zinc finger motif in this C-terminal region suggested that this motif was not directly involved in the DNA binding activity. A homotypic interaction between IE1 molecules was demonstrated by glutathione S-transferase pull-down assay and a coimmunoprecipitation analysis. A glutaraldehyde cross-linking experiment and gel filtration analysis showed that this self-interaction led to the formation of stable IE1 dimers.

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Sisk, T. J. "Phosphorylation of class II transactivator regulates its interaction ability and transactivation function." International Immunology 15, no. 10 (October 1, 2003): 1195–205. http://dx.doi.org/10.1093/intimm/dxg116.

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LUN, YONG-ZHI, JUN CHENG, QING CHI, XUE-LEI WANG, MENG GAO, and LI-DA SUN. "Transactivation of proto-oncogene c-Myc by hepatitis B virus transactivator MHBst167." Oncology Letters 8, no. 2 (May 28, 2014): 803–8. http://dx.doi.org/10.3892/ol.2014.2190.

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Kim, Eui Tae, Young-Eui Kim, Yong Ho Huh, and Jin-Hyun Ahn. "Role of Noncovalent SUMO Binding by the Human Cytomegalovirus IE2 Transactivator in Lytic Growth." Journal of Virology 84, no. 16 (June 2, 2010): 8111–23. http://dx.doi.org/10.1128/jvi.00459-10.

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ABSTRACT The 86-kDa immediate-early 2 (IE2) protein of human cytomegalovirus (HCMV) is a promiscuous transactivator essential for viral gene expression. IE2 is covalently modified by SUMO at two lysine residues (K175 and K180) and also interacts noncovalently with SUMO. Although SUMOylation of IE2 has been shown to enhance its transactivation activity, the role of SUMO binding is not clear. Here we showed that SUMO binding by IE2 is necessary for its efficient transactivation function and for viral growth. IE2 bound physically to SUMO-1 through a SUMO-interacting motif (SIM). Mutations in SIM (mSIM) or in both SUMOylation sites and SIM (KR/mSIM), significantly reduced IE2 transactivation effects on viral early promoters. The replication of IE2 SIM mutant viruses (mSIM or KR/mSIM) was severely depressed in normal human fibroblasts. Analysis of viral growth curves revealed that the replication defect of the mSIM virus correlated with low-level accumulation of SUMO-modified IE2 and of viral early and late proteins. Importantly, both the formation of viral transcription domains and the association of IE2 with viral promoters in infected cells were significantly reduced in IE2 SIM mutant virus infection. Furthermore, IE2 was found to interact with the SUMO-modified form of TATA-binding protein (TBP)-associated factor 12 (TAF12), a component of the TFIID complex, in a SIM-dependent manner, and this interaction enhanced the transactivation activity of IE2. Our data demonstrate that the interaction of IE2 with SUMO-modified proteins plays an important role for the progression of the HCMV lytic cycle, and they suggest a novel viral mechanism utilizing the cellular SUMO system.

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Cho, Je-Yoel, Yasmin Akbarali, Luiz F. Zerbini, Xuesong Gu, Jay Boltax, Yihong Wang, Peter Oettgen, Dong-Er Zhang, and Towia A. Libermann. "Isoforms of the Ets Transcription Factor NERF/ELF-2 Physically Interact with AML1 and Mediate Opposing Effects on AML1-mediated Transcription of the B Cell-specificblkGene." Journal of Biological Chemistry 279, no. 19 (February 17, 2004): 19512–22. http://dx.doi.org/10.1074/jbc.m309074200.

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We previously isolated different isoforms of a new Ets transcription factor family member, NERF/ELF-2, NERF-2, NERF-1a, and NERF-1b. In contrast to the inhibitory isoforms NERF-1a and NERF-1b, NERF-2 acts as a transactivator of the B cell-specificblkpromoter. We now report that NERF-2 and NERF-1 physically interact with AML1 (RUNX1), a frequent target for chromosomal translocations in leukemia. NERF-2 bound to AML1 via an interaction site located in a basic region upstream of the Ets domain. This is in contrast to most other Ets factors such as Ets-1 that bind to AML1 via the Ets domain, suggesting that different Ets factors utilize different domains for interaction with AML1. The interaction between AML1 and NERF-2 led to cooperative transactivation of theblkpromoter, whereas the interaction between AML1 and NERF-1a led to repression of AML1-mediated transactivation. To delineate the differences in function of the different NERF isoforms, we determined that the transactivation domain of NERF-2 is encoded by the N-terminal 100 amino acids, which have been replaced in NERF-1a by a 19-amino acid transcriptionally inactive sequence. Furthermore, acidic domains A and B, which are conserved in NERF-2 and the related proteins ELF-1 and MEF/ELF-4, but not in NERF-1a, are largely responsible for NERF-2-mediated transactivation. Because translocation of the Ets factor Tel to AML1 is a frequent event in childhood pre-B leukemia, understanding the interaction of Ets factors with AML1 in the context of a B cell-specific promoter might help to determine the function of Ets factors and AML1 in leukemia.

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DeSandro, Angela M., Uma M. Nagarajan, and Jeremy M. Boss. "Associations and Interactions between Bare Lymphocyte Syndrome Factors." Molecular and Cellular Biology 20, no. 17 (September 1, 2000): 6587–99. http://dx.doi.org/10.1128/mcb.20.17.6587-6599.2000.

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ABSTRACT The bare lymphocyte syndrome, a severe combined immunodeficiency due to loss of major histocompatibility complex (MHC) class II gene expression, is caused by inherited mutations in the genes encoding the heterotrimeric transcription factor RFX (RFX-B, RFX5, and RFXAP) and the class II transactivator CIITA. Mutagenesis of the RFX genes was performed, and the properties of the proteins were analyzed with regard to transactivation, DNA binding, and protein-protein interactions. The results identified specific domains within each of the three RFX subunits that were necessary for RFX complex formation, including the ankyrin repeats of RFX-B. DNA binding was dependent on RFX complex formation, and transactivation was dependent on a region of RFX5. RFX5 was found to interact with CIITA, and this interaction was dependent on a proline-rich domain within RFX5. Thus, these studies have defined the protein domains required for the functional regulation of MHC class II genes.

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Cujec, T. P., H. Cho, E. Maldonado, J. Meyer, D. Reinberg, and B. M. Peterlin. "The human immunodeficiency virus transactivator Tat interacts with the RNA polymerase II holoenzyme." Molecular and Cellular Biology 17, no. 4 (April 1997): 1817–23. http://dx.doi.org/10.1128/mcb.17.4.1817.

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The human immunodeficiency virus (HIV) encodes a transcriptional transactivator (Tat), which binds to an RNA hairpin called the transactivation response element (TAR) that is located downstream of the site of initiation of viral transcription. Tat stimulates the production of full-length viral transcripts by RNA polymerase II (pol II). In this study, we demonstrate that Tat coimmunoprecipitates with the pol II holoenzyme in cells and that it binds to the purified holoenzyme in vitro. Furthermore, Tat affinity chromatography purifies a holoenzyme from HeLa nuclear extracts which, upon addition of TBP and TFIIB, supports Tat transactivation in vitro, indicating that it contains all the cellular proteins required for the function of Tat. By demonstrating that Tat interacts with the holoenzyme in the absence of TAR, our data suggest a single-step assembly of Tat and the transcription complex on the long terminal repeat of HIV.

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Sakakibara, Shuhei, Keiji Ueda, Jiguo Chen, Toshiomi Okuno, and Koichi Yamanishi. "Octamer-Binding Sequence Is a Key Element for the Autoregulation of Kaposi's Sarcoma-Associated Herpesvirus ORF50/Lyta Gene Expression." Journal of Virology 75, no. 15 (August 1, 2001): 6894–900. http://dx.doi.org/10.1128/jvi.75.15.6894-6900.2001.

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ABSTRACT The expression of the Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 50 (ORF50) protein, Lyta (lytic transactivator), marks the switch from latent KSHV infection to the lytic phase. ORF50/Lyta upregulates several target KSHV genes, such as K8 (K-bZip), K9 (vIRF1), and ORF57, finally leading to the production of mature viruses. The auto-upregulation of ORF50/Lyta is thought to be an important mechanism for efficient lytic viral replication. In this study, we focused on this autoregulation and identified the promoter element required for it. An electrophoretic mobility shift assay indicated that the octamer-binding protein 1 (Oct-1) bound to this element. Mutations in the octamer-binding motif resulted in refractoriness of the ORF50/Lyta promoter to transactivation by ORF50/Lyta, and Oct-1 expression enhanced this transactivation. These results suggest that the autoregulation of ORF50/Lyta is mediated by Oct-1.

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Yang, Zhilong, Zhangcai Yan, and Charles Wood. "Kaposi's Sarcoma-Associated Herpesvirus Transactivator RTA Promotes Degradation of the Repressors To Regulate Viral Lytic Replication." Journal of Virology 82, no. 7 (January 23, 2008): 3590–603. http://dx.doi.org/10.1128/jvi.02229-07.

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ABSTRACT Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) RTA is an important protein involved in the induction of KSHV lytic replication from latency through activation of the lytic cascade. A number of cellular and viral proteins, including K-RBP, have been found to repress RTA-mediated transactivation and KSHV lytic replication. However, it is unclear as to how RTA overcomes the suppression during lytic reactivation. In this study, we found that RTA can induce K-RBP degradation through the ubiquitin-proteasome pathway and that two regions in RTA are responsible. Moreover, we found that RTA can promote the degradation of several other RTA repressors. RTA mutants that are defective in inducing K-RBP degradation cannot activate RTA responsive promoter as efficiently as wild-type RTA. Interference of the ubiquitin-proteasome pathway affected RTA-mediated transactivation and KSHV reactivation from latency. Our results suggest that KSHV RTA can stimulate the turnover of repressors to modulate viral reactivation. Since herpes simplex virus type 1 transactivator ICP0 and human cytomegalovirus transactivator pp71 also stimulate the degradation of cellular silencers, it is possible that the promotion of silencer degradation by viral transactivators may be a common mechanism for regulating the lytic replication of herpesviruses.

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Li, Liyuan, Chikezie O. Madu, Andrew Lu, and Yi Lu. "HIF-1α Promotes A Hypoxia-Independent Cell Migration." Open Biology Journal 3, no. 1 (January 19, 2010): 8–14. http://dx.doi.org/10.2174/18741967010030100008.

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Hypoxia-inducible factor-1α (HIF-1α) is known as a transactivator for VEGF gene promoter. It can be induced by hypoxia. However, no study has been done so far to dissect HIF-1α-mediated effects from hypoxia or VEGF-mediated effects. By using a HIF-1α knockout (HIF-1α KO) cell system in mouse embryonic fibroblast (MEF) cells, this study analyzes cell migration and HIF-1α, hypoxia and VEGF activation. A hypoxia-mediated HIF-1α induction and VEGF transactivation were observed: both HIF-1α WT lines had significantly increased VEGF transactivation, as an indicator for HIF-1α induction, in hypoxia compared to normoxia; in contrast, HIF-1α KO line had no increased VEGF transactivation under hypoxia. HIF-1α promotes cell migration: HIF-1α-KO cells had a significantly reduced migration compared to that of the HIF-1α WT cells under both normoxia and hypoxia. The significantly reduced cell migration in HIF-1α KO cells can be partially rescued by the restoration of WT HIF-1α expression mediated by adenoviral-mediated gene transfer. Interestingly, hypoxia has no effect on cell migration: the cells had a similar cell migration rate under hypoxic and normoxic conditions for both HIF-1α WT and HIF-1α KO lines, respectively. Collectively, these data suggest that HIF-1α plays a role in MEF cell migration that is independent from hypoxia-mediated effects.

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Ishizaka, Aya, Taketoshi Mizutani, Kazuyoshi Kobayashi, Toshio Tando, Kouhei Sakurai, Toshinobu Fujiwara, and Hideo Iba. "Double Plant Homeodomain (PHD) Finger Proteins DPF3a and -3b Are Required as Transcriptional Co-activators in SWI/SNF Complex-dependent Activation of NF-κB RelA/p50 Heterodimer." Journal of Biological Chemistry 287, no. 15 (February 13, 2012): 11924–33. http://dx.doi.org/10.1074/jbc.m111.322792.

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We have previously shown that DPF2 (requiem/REQ) functions as a linker protein between the SWI/SNF complex and RelB/p52 NF-κB heterodimer and plays important roles in NF-κB transactivation via its noncanonical pathway. Using sensitive 293FT reporter cell clones that had integrated a SWI/SNF-dependent NF-κB reporter gene, we find in this study that the overexpression of DPF1, DPF2, DPF3a, DPF3b, and PHF10 significantly potentiates the transactivating activity of typical NF-κB dimers. Knockdown analysis using 293FT reporter cells that endogenously express these five proteins at low levels clearly showed that DPF3a and DPF3b, which are produced from the DPF3 gene by alternative splicing, are the most critical for the RelA/p50 NF-κB heterodimer transactivation induced by TNF-α stimulation. Our data further show that this transactivation requires the SWI/SNF complex. DPF3a and DPF3b are additionally shown to interact directly with RelA, p50, and several subunits of the SWI/SNF complex in vitro and to be co-immunoprecipitated with RelA/p50 and the SWI/SNF complex from the nuclear fractions of cells treated with TNF-α. In ChIP experiments, we further found that endogenous DPF3a/b and the SWI/SNF complex are continuously present on HIV-1 LTR, whereas the kinetics of RelA/p50 recruitment after TNF-α treatment correlate well with the viral transcriptional activation levels. Additionally, re-ChIP experiments showed DPF3a/b and the SWI/SNF complex associate with RelA on the endogenous IL-6 promoter after TNF-α treatment. In conclusion, our present data indicate that by linking RelA/p50 to the SWI/SNF complex, DPF3a/b induces the transactivation of NF-κB target gene promoters in relatively inactive chromatin contexts.

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Fujinaga, Koh, Thomas P. Cujec, Junmin Peng, Judit Garriga, David H. Price, Xavier Graña, and B. Matija Peterlin. "The Ability of Positive Transcription Elongation Factor b To Transactivate Human Immunodeficiency Virus Transcription Depends on a Functional Kinase Domain, Cyclin T1, and Tat." Journal of Virology 72, no. 9 (September 1, 1998): 7154–59. http://dx.doi.org/10.1128/jvi.72.9.7154-7159.1998.

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ABSTRACT By binding to the transactivation response element (TAR) RNA, the transcriptional transactivator (Tat) from the human immunodeficiency virus increases rates of elongation rather than initiation of viral transcription. Two cyclin-dependent serine/threonine kinases, CDK7 and CDK9, which phosphorylate the C-terminal domain of RNA polymerase II, have been implicated in Tat transactivation in vivo and in vitro. In this report, we demonstrate that CDK9, which is the kinase component of the positive transcription elongation factor b (P-TEFb) complex, can activate viral transcription when tethered to the heterologous Rev response element RNA via the regulator of expression of virion proteins (Rev). The kinase activity of CDK9 and cyclin T1 is essential for these effects. Moreover, P-TEFb binds to TAR only in the presence of Tat. We conclude that Tat–P-TEFb complexes bind to TAR, where CDK9 modifies RNA polymerase II for the efficient copying of the viral genome.

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Chen, Hexin, Graham Wilcox, Gde Kertayadnya, and Charles Wood. "Characterization of the Jembrana Disease Virustat Gene and the cis- andtrans-Regulatory Elements in Its Long Terminal Repeats." Journal of Virology 73, no. 1 (January 1, 1999): 658–66. http://dx.doi.org/10.1128/jvi.73.1.658-666.1999.

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ABSTRACT Jembrana disease virus (JDV) is a newly identified bovine lentivirus that is closely related to the bovine immunodeficiency virus (BIV). JDV contains a tat gene, encoded by two exons, which has potent transactivation activity. Cotransfection of the JDVtat expression plasmid with the JDV promoter chloramphenicol acetyltransferase (CAT) construct pJDV-U3R resulted in a substantial increase in the level of CAT mRNA transcribed from the JDV long terminal repeat (LTR) and a dramatic increase in the CAT protein level. Deletion analysis of the LTR sequences showed that sequences spanning nucleotides −68 to +53, including the TATA box and the predicted first stem-loop structure of the predicted Tat response element (TAR), were required for efficient transactivation. The results, derived from site-directed mutagenesis experiments, suggested that the base pairing in the stem of the first stem-loop structure in the TAR region was important for JDV Tat-mediated transactivation; in contrast, nucleotide substitutions in the loop region of JDV TAR had less effect. For the JDV LTR, upstream sequences, from nucleotide −196 and beyond, as well as the predicted secondary structures in the R region, may have a negative effect on basal JDV promoter activity. Deletion of these regions resulted in a four- to fivefold increase in basal expression. The JDV Tat is also a potent transactivator of other animal and primate lentivirus promoters. It transactivated BIV and human immunodeficiency virus type 1 (HIV-1) LTRs to levels similar to those with their homologous Tat proteins. In contrast, HIV-1 Tat has minimal effects on JDV LTR expression, whereas BIV Tat moderately transactivated the JDV LTR. Our study suggests that JDV may use a mechanism of transactivation similar but not identical to those of other animal and primate lentiviruses.

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Gauthier, Benoit R., Valerie M. Schwitzgebel, Maia Zaiko, Aline Mamin, Beate Ritz-Laser, and Jacques Philippe. "Hepatic Nuclear Factor-3 (HNF-3 or Foxa2) Regulates Glucagon Gene Transcription by Binding to the G1 and G2 Promoter Elements." Molecular Endocrinology 16, no. 1 (January 1, 2002): 170–83. http://dx.doi.org/10.1210/mend.16.1.0752.

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Abstract Glucagon gene expression in the endocrine pancreas is controlled by three islet-specific elements (G3, G2, and G4) and theα -cell-specific element G1. Two proteins interacting with G1 have previously been identified as Pax6 and Cdx2/3. We identify here the third yet uncharacterized complex on G1 as hepatocyte nuclear factor 3 (HNF-3)β, a member of the HNF-3/forkhead transcription family, which plays an important role in the development of endoderm-related organs. HNF-3 has been previously demonstrated to interact with the G2 element and to be crucial for glucagon gene expression; we thus define a second binding site for this transcription on the glucagon gene promoter. We demonstrate that both HNF-3α and -β produced in heterologous cells can interact with similar affinities to either the G1 or G2 element. Pax6, which binds to an overlapping site on G1, exhibited a greater affinity as compared with HNF-3α or -β. We show that both HNF-3β and -α can transactivate glucagon gene transcription through the G2 and G1 elements. However, HNF-3 via its transactivating domains specifically impaired Pax6-mediated transactivation of the glucagon promoter but had no effect on transactivation by Cdx2/3. We suggest that HNF-3 may play a dual role on glucagon gene transcription by 1) inhibiting the transactivation potential of Pax6 on the G1 and G3 elements and 2) direct activation through G1 and G2.

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Wunderlich, Mark, Mithua Ghosh, Karen Weghorst, and Steven J. Berberich. "MdmX Represses E2F1 Transactivation." Cell Cycle 3, no. 4 (April 2, 2004): 470–76. http://dx.doi.org/10.4161/cc.3.4.746.

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Kadakia, Madhavi, Thomas L. Brown, Molly M. McGorry, and Steven J. Berberich. "MdmX inhibits Smad transactivation." Oncogene 21, no. 57 (December 2002): 8776–85. http://dx.doi.org/10.1038/sj.onc.1205993.

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French, Samuel W., Cindy S. Malone, Rhine R. Shen, Mathilde Renard, Sarah E. Henson, Maurine D. Miner, Randolph Wall, and Michael A. Teitell. "Sp1 Transactivation of theTCL1Oncogene." Journal of Biological Chemistry 278, no. 2 (November 5, 2002): 948–55. http://dx.doi.org/10.1074/jbc.m207166200.

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TOLNAY, Mate, Yuang-Taung JUANG, and George C. TSOKOS. "Protein kinase A enhances, whereas glycogen synthase kinase-3β inhibits, the activity of the exon 2-encoded transactivator domain of heterogeneous nuclear ribonucleoprotein D in a hierarchical fashion." Biochemical Journal 363, no. 1 (March 22, 2002): 127–36. http://dx.doi.org/10.1042/bj3630127.

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Heterogeneous nuclear ribonucleoprotein D (hnRNP D) is implicated in transcriptional regulation. Alternative splicing of exons 2 and 7 generates four isoforms of the protein. We report here that only isoforms that contain the product of exon 2 (amino acids 79–97) were able to transactivate. Moreover, the exon 2-encoded protein domain alone was sufficient to drive transcription. TATA-binding protein and p300 interacted with a synthetic peptide corresponding to exon 2, and both proteins co-precipitated with hnRNP D. Stimulation of protein kinase A (PKA) and protein kinase C (PKC) synergistically induced the transactivating ability of hnRNP D, and the exon 2-encoded domain was sufficient for this inducibility. In kinase assays PKA phosphorylated Ser-87 of hnRNP D, whereas glycogen synthase kinase-3β (GSK-3β) phosphorylated Ser-83, but only if Ser-87 had been pre-phosphorylated by PKA. Phosphorylation of Ser-87 enhanced, whereas phosphorylation of Ser-83 repressed, transactivation. Overexpression of GSK-3β inhibited transactivation by hnRNP D, but stimulation of PKC negated the inhibitory effect of GSK-3β. We suggest that a hierarchical phosphorylation pathway regulates the transactivating ability of hnRNP D: PKA activates hnRNP D, but at the same time renders it sensitive to inhibition by GSK-3β; the latter inhibition can be suspended by inactivating GSK-3β with PKC.

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Kretsovali, Androniki, Charalambos Spilianakis, Andreas Dimakopoulos, Takis Makatounakis, and Joseph Papamatheakis. "Self-association of Class II Transactivator Correlates with Its Intracellular Localization and Transactivation." Journal of Biological Chemistry 276, no. 34 (June 18, 2001): 32191–97. http://dx.doi.org/10.1074/jbc.m103164200.

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KISTANOVA, Elena, Helen DELL, Panayota TSANTILI, Eileen FALVEY, Christos CLADARAS, and Margarita HADZOPOULOU-CLADARAS. "The activation function-1 of hepatocyte nuclear factor-4 is an acidic activator that mediates interactions through bulky hydrophobic residues." Biochemical Journal 356, no. 2 (May 24, 2001): 635–42. http://dx.doi.org/10.1042/bj3560635.

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The hepatocyte nuclear factor-4 (HNF-4) contains two transcription activation domains. One domain, activation function-1 (AF-1), consists of the extreme N-terminal 24 amino acids and functions as a constitutive autonomous activator of transcription. This short transactivator belongs to the class of acidic activators, and it is predicted to adopt an amphipathic α-helical structure. Transcriptional analysis of sequential point mutations of the negatively charged residues (Asp and Glu) revealed a stepwise decrease in activity, while mutation of all acidic residues resulted in complete loss of transcriptional activity. Mutations of aromatic and hydrophobic amino acids surrounding the negatively charged residues had a much more profound effect than mutations of acidic amino acids, since even a single mutation of these residues resulted in a dramatic decrease in transactivation, thus demonstrating the importance of hydrophobic residues in AF-1 activity. Like other acidic activators, the AF-1 of HNF-4 binds the transcription factor IIB and the TATA-binding protein directly in vitro. In addition, the cAMP-response-element-binding-protein, a transcriptional adapter involved in the transactivation of a plethora of transcription factors, interacts with the AF-1 of HNF-4 and co-operates in the process of transactivation by HNF-4. The different protein targets of AF-1 suggest that the AF-1 of HNF-4 may be involved in recruiting both general transcription factors and chromatin remodelling proteins during activation of gene expression.

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Liu, Xiangdong, Xiaolin Chen, Vladimir Zachar, Chawnshang Chang, and Peter Ebbesen. "Transcriptional activation of human TR3/nur77 gene expression by human T-lymphotropic virus type I Tax protein through two AP-1-like elements." Journal of General Virology 80, no. 12 (December 1, 1999): 3073–81. http://dx.doi.org/10.1099/0022-1317-80-12-3073.

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The Tax transactivator of human T-lymphotropic virus type I (HTLV-I) is capable of inducing expression of the human immediate-early TR3/nur77 gene. Deletion and mutation analyses of the TR3/nur77 promoter demonstrated that multiple transcription elements in the 121 bp sequence proximal to the transcription start site are required for full Tax transactivation. Mutations of CArG-like, Ets and RCE motifs in this region severely decreased Tax transactivation. Mutation of either of the two identical AP-1-like elements (NAP 1 and 2) immediately upstream of the TATA box caused around 80% reduction of Tax transactivation. Mutation of both NAP elements blocked Tax-mediated activation totally. These two NAP elements could confer Tax-responsiveness on a heterologous basal promoter. Furthermore, the specific NAP-binding complex was only observed in HTLV-I-infected cells. Formation of this specific NAP-binding complex was correlated directly with Tax expression, as demonstrated in JPX-9 cells upon induction of Tax expression. The specific NAP binding could be competed for by consensus AP-1 and CREB elements, indicating that the NAP-binding proteins probably belong to the AP-1 and CREB/ATF transcription factor families. Supershift analysis with antibodies to both the AP-1 and CREB/ATF transcription factor families revealed that only anti-JunD antibody could partially shift this NAP-binding complex, indicating that JunD is a component of the NAP complex. This work suggests that JunD is involved in Tax-regulated TR3/nur77 expression.

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Zhu, Zhengrong, Sean Kim, Taosheng Chen, Jun-Hsiang Lin, Aneka Bell, James Bryson, Yves Dubaquie, et al. "Correlation of High-Throughput Pregnane X Receptor (PXR) Transactivation and Binding Assays." Journal of Biomolecular Screening 9, no. 6 (September 2004): 533–40. http://dx.doi.org/10.1177/1087057104264902.

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Pregnane X receptor (PXR) transactivation and binding assays have been developed into high-throughput assays, which are robust and reproducible (Z′ > 0.5). For most compounds, there was a good correlation between the results of the transactivation and binding assays. EC50 values of compounds in the transactivation assay correlated reasonably well with their IC50 values in the binding assay. However, there were discrepancies with some compounds showing high binding affinity in the binding assay translated into low transactivation. The most likely cause for these discrepancies was an agonist-dependent relationship between binding affinity and transactivation response. In general, compounds that bound to human PXR and transactivated PXR tended to be large hydrophobic molecules.

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Journal articles on the topic 'Transactivation'

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