scholarly journals Both N- and C-terminal domains of RelB are required for full transactivation: role of the N-terminal leucine zipper-like motif.

1993 ◽  
Vol 13 (3) ◽  
pp. 1572-1582 ◽  
Author(s):  
P Dobrzanski ◽  
R P Ryseck ◽  
R Bravo
Keyword(s):  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Leucine Zipper ◽  
Wild Type ◽  
Nf Kappa B ◽  
Hybrid Molecules ◽  
In Cis ◽  
Rel Homology Domain

RelB, a member of the Rel family of transcription factors, can stimulate promoter activity in the presence of p50-NF-kappa B or p50B/p49-NF-kappa B in mammalian cells. Transcriptional activation analysis reveals that the N and C termini of RelB are required for full transactivation in the presence of p50-NF-kappa B. RelB/p50-NF-kappa B hybrid molecules containing the Rel homology domain of p50-NF-kappa B and the N and C termini of RelB have high transcriptional activity compared with wild-type p50-NF-kappa B. The N and C termini of RelB cooperate in transactivation in cis or trans configuration. Alterations in the structure of the leucine zipper-like motif present in the N terminus of RelB significantly decrease the transcriptional capacity of RelB and of different RelB/p50-NF-kappa B hybrid molecules.

Both N- and C-terminal domains of RelB are required for full transactivation: role of the N-terminal leucine zipper-like motif

1993 ◽  
Vol 13 (3) ◽  
pp. 1572-1582
Author(s):  
P Dobrzanski ◽  
R P Ryseck ◽  
R Bravo
Keyword(s):  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Leucine Zipper ◽  
Wild Type ◽  
Nf Kappa B ◽  
Hybrid Molecules ◽  
In Cis ◽  
Rel Homology Domain

RelB, a member of the Rel family of transcription factors, can stimulate promoter activity in the presence of p50-NF-kappa B or p50B/p49-NF-kappa B in mammalian cells. Transcriptional activation analysis reveals that the N and C termini of RelB are required for full transactivation in the presence of p50-NF-kappa B. RelB/p50-NF-kappa B hybrid molecules containing the Rel homology domain of p50-NF-kappa B and the N and C termini of RelB have high transcriptional activity compared with wild-type p50-NF-kappa B. The N and C termini of RelB cooperate in transactivation in cis or trans configuration. Alterations in the structure of the leucine zipper-like motif present in the N terminus of RelB significantly decrease the transcriptional capacity of RelB and of different RelB/p50-NF-kappa B hybrid molecules.

scholarly journals Conservation of transcriptional activation functions of the NF-kappa B p50 and p65 subunits in mammalian cells and Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (3) ◽  
pp. 1666-1674 ◽  
Author(s):  
P A Moore ◽  
S M Ruben ◽  
C A Rosen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Transcriptional Activator ◽  
Genetic System ◽  
Nf Kappa B ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activation Domains ◽  
Inhibitory Molecule

The NF-kappa B transcription factor complex is composed of a 50-kDa (p50) and a 65-kDa (p65) subunit. Both subunits bind to similar DNA motifs and elicit transcriptional activation as either homo- or heterodimers. By using chimeric proteins that contain the DNA binding domain of the yeast transcriptional activator GAL4 and subdomains of p65, three distinct transcriptional activation domains were identified. One domain was localized to a region of 42 amino acids containing a potential leucin zipper structure, consistent with earlier reports. Two other domains, both acidic and rich in prolines, were also identified. Of perhaps more significance, the same minimal activation domains that were functional in mammalian cells were also functional in the yeast Saccharomyces cerevisiae. Coexpression of the NF-kappa B inhibitory molecule, I kappa B, reduced the transcriptional activity of p65 significantly, suggesting the ability of I kappa B to function in a similar manner in S. cerevisiae. Surprisingly, while the conserved rel homology domain of p65 demonstrated no transcriptional activity in either mammalian cells or S. cerevisiae, the corresponding domain in p50 was a strong transcriptional activator in S. cerevisiae. The observation that similar domains elicit transcriptional activation in mammalian cells and S. cerevisiae demonstrates strong conservation of the transcriptional machinery required for NF-kappa B function and provides a powerful genetic system to study the transcriptional mechanisms of these proteins.

scholarly journals Role of aspartate 351 in transactivation and active conformation of estrogen receptor α

2005 ◽  
Vol 35 (3) ◽  
pp. 449-464 ◽  
Author(s):  
Jeong Hoon Kim ◽  
Mee Hyun Lee ◽  
Byoung Jin Kim ◽  
Jun Hyun Kim ◽  
Seong Jun Han ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Steroid Receptors ◽  
Estrogen Receptor Α ◽  
Orphan Nuclear Receptor ◽  
Active Conformation ◽  
Wild Type

Estrogen-dependent transcriptional activation by estrogen receptor α (ERα) depends on the conformation of helices 3 and 12 in the ligand-binding domain. To better understand the function of helix 3 in ERα, we examined the role of charged residues, which are conserved in most steroid receptors in helix 3, in estrogen-dependent transactivation. The replacement of Asp-351 with lysine (D351K) or leucine (D351 L) completely abolished estrogen-dependent transactivation without affecting estrogen-binding, DNA-binding and homodimerization activities in ERα. The mutations dramatically reduced the ligand-dependent activation function 2 activity and impaired the ability of ERα to bind p160 coactivators. In addition, the D351K mutant effectively inhibited the transcriptional activation activity of wild-type ERα. Furthermore Asp-351 was required not only for the estrogen-dependent conformational change of wild-type ERα but also for the constitutive transcriptional activity and ligand-independent active conformation of ERα mutant Y537N. Similarly, in the orphan nuclear receptor called estrogen-related receptor 3 (ERR3), the replacement of Asp-273 (the corresponding amino acid to Asp-351 in ERα) with lysine abolished constitutive transcriptional activity of ERR3 without affecting DNA-binding activity and impaired the ability of the receptor to interact with p160 coactivators. These data suggest a role of Asp-351 in inducing and stabilizing the active conformation of ERα, and our results experimentally confirm the concept that Asp-351 in helix 3 interacts with the amide hydrogen of L540 in helix 12 to form a transcriptionally competent surface for binding p160 coactivators.

Conservation of transcriptional activation functions of the NF-kappa B p50 and p65 subunits in mammalian cells and Saccharomyces cerevisiae

1993 ◽  
Vol 13 (3) ◽  
pp. 1666-1674
Author(s):  
P A Moore ◽  
S M Ruben ◽  
C A Rosen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Transcriptional Activator ◽  
Genetic System ◽  
Nf Kappa B ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activation Domains ◽  
Inhibitory Molecule

The NF-kappa B transcription factor complex is composed of a 50-kDa (p50) and a 65-kDa (p65) subunit. Both subunits bind to similar DNA motifs and elicit transcriptional activation as either homo- or heterodimers. By using chimeric proteins that contain the DNA binding domain of the yeast transcriptional activator GAL4 and subdomains of p65, three distinct transcriptional activation domains were identified. One domain was localized to a region of 42 amino acids containing a potential leucin zipper structure, consistent with earlier reports. Two other domains, both acidic and rich in prolines, were also identified. Of perhaps more significance, the same minimal activation domains that were functional in mammalian cells were also functional in the yeast Saccharomyces cerevisiae. Coexpression of the NF-kappa B inhibitory molecule, I kappa B, reduced the transcriptional activity of p65 significantly, suggesting the ability of I kappa B to function in a similar manner in S. cerevisiae. Surprisingly, while the conserved rel homology domain of p65 demonstrated no transcriptional activity in either mammalian cells or S. cerevisiae, the corresponding domain in p50 was a strong transcriptional activator in S. cerevisiae. The observation that similar domains elicit transcriptional activation in mammalian cells and S. cerevisiae demonstrates strong conservation of the transcriptional machinery required for NF-kappa B function and provides a powerful genetic system to study the transcriptional mechanisms of these proteins.

scholarly journals SUMO-1 Modification of PIASy, an E3 Ligase, Is Necessary for PIASy-Dependent Activation of Tcf-4

2005 ◽  
Vol 25 (9) ◽  
pp. 3506-3518 ◽  
Author(s):  
Motomasa Ihara ◽  
Hideki Yamamoto ◽  
Akira Kikuchi
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Wnt Pathway ◽  
E3 Ligase ◽  
Promyelocytic Leukemia ◽  
Wild Type ◽  
Sumoylation Site ◽  
Promyelocytic Leukemia Protein ◽  
Distinct Distribution

ABSTRACT We have previously shown that modification of Tcf-4, a transcription factor in the Wnt pathway, with SUMO by PIASy, a SUMO E3 ligase, enhances its transcriptional activity. Since PIASy itself was also modified with SUMO-1, we studied the role of sumoylation of PIASy in the regulation of Tcf-4. Lys35 was found to be a sumoylation site of PIASy. PIASyK35R, in which Lys35 was mutated to Arg, did not enhance sumoylation of Tcf-4, although this PIASy mutant did not lose the ligase activity of sumoylation for other proteins. Wild-type PIASy and PIASyK35R showed a distinct distribution in the nucleus, although both were colocalized with Tcf-4. Promyelocytic leukemia protein, which is involved in transcriptional regulation, was associated with PIASyK35R more frequently than wild-type PIASy in the nucleus. PIASyK35R could not stimulate the transcriptional activity of Tcf-4 under the conditions in which wild-type PIASy enhanced it. Conjugation of SUMO-1 to the amino terminus of PIASyK35R neither enhanced sumoylation of Tcf-4 nor stimulated the transcriptional activity of Tcf-4. These results suggest that sumoylation of Lys35 in PIASy determines the nuclear localization of PIASy and that it is necessary for PIASy-dependent sumoylation and transcriptional activation of Tcf-4.

Transcriptional activities of p73 splicing variants are regulated by inter-variant association

2001 ◽  
Vol 356 (3) ◽  
pp. 859-866 ◽  
Author(s):  
Yoshihide UEDA ◽  
Makoto HIJIKATA ◽  
Shinji TAKAGI ◽  
Tsutomu CHIBA ◽  
Kunitada SHIMOTOHNO
Keyword(s):  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Structural Difference ◽  
Terminal Region ◽  
Wild Type ◽  
Splicing Variants ◽  
Terminal Structure ◽  
Tumour Suppressor P53 ◽  
Two Hybrid

p73 has been identified as a gene that encodes a protein with significant identity with the tumour suppressor p53. The main structural difference between p73 and p53 is the additional C-terminal region of p73. Six isoforms of p73 with differing C-terminal structures, α, β, γ, δ, ∊ and ξ, have been reported. These variants differ in transcriptional activity on p53-responsive promoters. Here we report a possible mechanism of transcriptional activation by p73 splicing variants. C-terminal deletion mutants of p73α showed a significantly higher level of transcriptional activity than wild-type p73α, suggesting that the C-terminal structure of p73α functions to repress the transcriptional activity of p73α. The results of immunoprecipitation assays and two-hybrid assays in mammalian cells showed that the p73 variants interacted with each other, but not with p53. The transcriptional activity of p73β was reduced by co-expression with either p73α or p73∊, which bears an identical C-terminal structure to p73α. Co-expression of the C-terminal portion of p73α or p73∊ with p73β also resulted in reduced transcriptional activity. Moreover, we observed that the level of endogenous p21 protein induced by p73β was decreased by co-expression of full-length p73∊ or the C-terminal region of p73α or p73∊. These observations suggest that p73-mediated gene expression is regulated by the interactions of p73 splicing variants in the cell.

scholarly journals Glucocorticoid Receptor Phosphorylation Differentially Affects Target Gene Expression

2008 ◽  
Vol 22 (8) ◽  
pp. 1754-1766 ◽  
Author(s):  
Weiwei Chen ◽  
Thoa Dang ◽  
Raymond D. Blind ◽  
Zhen Wang ◽  
Claudio N. Cavasotto ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Target Genes ◽  
Divalent Cations ◽  
Phosphorylation Site ◽  
Transcriptional Response ◽  
Receptor Occupancy ◽  
Wild Type ◽  
Interacting Protein

Abstract The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

scholarly journals GAGA Factor Isoforms Have Distinct but Overlapping Functions In Vivo

2001 ◽  
Vol 21 (24) ◽  
pp. 8565-8574 ◽  
Author(s):  
Anthony J. Greenberg ◽  
Paul Schedl
Keyword(s):  
Fusion Protein ◽  
Transcriptional Activation ◽  
Functional Difference ◽  
Wild Type ◽  
Gaga Factor ◽  
Phenotypic Analysis ◽  
Alternatively Spliced

ABSTRACT The Drosophila melanogaster GAGA factor (encoded by the Trithorax-like [Trl] gene) is required for correct chromatin architecture at diverse chromosomal sites. The Trl gene encodes two alternatively spliced isoforms of the GAGA factor (GAGA-519 and GAGA-581) that are identical except for the length and sequence of the C-terminal glutamine-rich (Q) domain. In vitro and tissue culture experiments failed to find any functional difference between the two isoforms. We made a set of transgenes that constitutively express cDNAs coding for either of the isoforms with the goal of elucidating their roles in vivo. Phenotypic analysis of the transgenes in Trl mutant background led us to the conclusion that GAGA-519 and GAGA-581 perform different, albeit largely overlapping, functions. We also expressed a fusion protein with LacZ disrupting the Q domain of GAGA-519. This LacZ fusion protein compensated for the loss of wild-type GAGA factor to a surprisingly large extent. This suggests that the Q domain either is not required for the essential functions performed by the GAGA protein or is exclusively used for tetramer formation. These results are inconsistent with a major role of the Q domain in chromatin remodeling or transcriptional activation. We also found that GAGA-LacZ was able to associate with sites not normally occupied by the GAGA factor, pointing to a role of the Q domain in binding site choice in vivo.

Retrovirus-induced interference with collagen I gene expression in Mov13 fibroblasts is maintained in the absence of DNA methylation

1991 ◽  
Vol 11 (1) ◽  
pp. 47-54
Author(s):  
H Chan ◽  
S Hartung ◽  
M Breindl
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Chromatin Structure ◽  
Transcriptional Activity ◽  
Xenopus Laevis Oocytes ◽  
Regulatory Sequences ◽  
Type I ◽  
Wild Type ◽  
Col1a1 Gene

We have studied the role of DNA methylation in repression of the murine alpha 1 type I collagen (COL1A1) gene in Mov13 fibroblasts. In Mov13 mice, a retroviral provirus has inserted into the first intron of the COL1A1 gene and blocks its expression at the level of transcriptional initiation. We found that regulatory sequences in the COL1A1 promoter region that are involved in the tissue-specific regulation of the gene are unmethylated in collagen-expressing wild-type fibroblasts and methylated in Mov13 fibroblasts, confirming and extending earlier observations. To directly assess the role of DNA methylation in the repression of COL1A1 gene transcription, we treated Mov13 fibroblasts with the demethylating agent 5-azacytidine. This treatment resulted in a demethylation of the COL1A1 regulatory sequences but failed to activate transcription of the COL1A1 gene. Moreover, the 5-azacytidine treatment induced a transcription-competent chromatin structure in the retroviral sequences but not in the COL1A1 promoter. In DNA transfection and microinjection experiments, we found that the provirus interfered with transcriptional activity of the COL1A1 promoter in Mov13 fibroblasts but not in Xenopus laevis oocytes. In contrast, the wild-type COL1A1 promoter was transcriptionally active in Mov13 fibroblasts. These experiments showed that the COL1A1 promoter is potentially transcriptionally active in the presence of proviral sequences and that Mov13 fibroblasts contain the trans-acting factors required for efficient COL1A1 gene expression. Our results indicate that the provirus insertion in Mov13 can inactivate COL1A1 gene expression at several levels. It prevents the developmentally regulated establishment of a transcription-competent methylation pattern and chromatin structure of the COL1A1 domain and, in the absence of DNA methylation, appears to suppress the COL1A1 promoter in a cell-specific manner, presumably by assuming a dominant chromatin structure that may be incompatible with transcriptional activity of flanking cellular sequences.

scholarly journals The role of the 5′-flanking sequence of a human tRNAGlu gene in modulation of its transcriptional activity in vitro

1990 ◽  
Vol 272 (3) ◽  
pp. 797-803 ◽  
Author(s):  
E S Gonos ◽  
J P Goddard
Keyword(s):  
Transcriptional Activity ◽  
Potential Model ◽  
Trna Gene ◽  
Deletion Mutants ◽  
Wild Type ◽  
Flanking Sequence ◽  
Cell Extracts ◽  
Transcription In Vitro

The role of a tRNA-like structure within the 5′-flanking sequence of a human tRNA(Glu) gene in the modulation of its transcription in vitro by HeLa cell extracts has been investigated using several deletion mutants of a recombinant of the gene which lacked part or all of the tRNA-like structure. The transcriptional efficiency of four mutants was the same as that of the wild-type recombinant, two mutants had decreased transcriptional efficiency, one was more efficient, and one, lacking part of the 5′ intragenic control region, was inactive. Correlation of the transcriptional efficiencies with the position and the size of the 5′-flanking sequence that was deleted indicated that the tRNA-like structure may be deleted without loss of transcriptional efficiency. Current models for the modulation of tRNA gene transcription by the 5′-flanking sequence are assessed in the light of the results obtained, and a potential model is presented.

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