scholarly journals Functional interference between the ubiquitous and constitutive octamer transcription factor 1 (OTF-1) and the glucocorticoid receptor by direct protein-protein interaction involving the homeo subdomain of OTF-1.

1992 ◽  
Vol 12 (11) ◽  
pp. 4960-4969 ◽  
Author(s):  
E Kutoh ◽  
P E Strömstedt ◽  
L Poellinger
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Protein Interaction ◽  
Binding Activity ◽  
Dependent Manner ◽  
Protein Protein Interaction ◽  
Dna Binding Activity ◽  
Functional Interference

The ubiquitous and constitutive octamer transcription factor OTF-1 (Oct 1) is the target of positive regulation by the potent herpes simplex virus trans-activator VP16, which forms a complex with the homeodomain of OTF-1. Here we present evidence that the glucocorticoid receptor can negatively regulate OTF-1 function by a mechanism that is independent of DNA binding. In vivo-expressed glucocorticoid receptor inhibited in a hormone-dependent manner activation of a minimal promoter construct carrying a functional octamer site. Moreover, expression of the receptor in vivo resulted in hormone-dependent repression of OTF-1-dependent DNA-binding activity in nuclear extract. In vitro, the DNA-binding activity of partially purified OTF-1 was repressed following incubation with purified glucocorticoid receptor. Cross-linking and immunoprecipitation experiments indicated that the functional interference may be due to a strong association between these two proteins in solution. Finally, preliminary evidence indicates that the homeo subdomain of OTF-1 that directs formation of a complex with VP16 may also be critical for interaction with the glucocorticoid receptor. Thus, OTF-1 is a target for both positive and negative regulation by protein-protein interaction. Moreover, the functional interference between OTF-1 and the glucocorticoid receptor represents a novel regulatory mechanism in the cross-coupling of signal transduction pathways of nuclear receptors and constitutive transcription factors.

Functional interference between the ubiquitous and constitutive octamer transcription factor 1 (OTF-1) and the glucocorticoid receptor by direct protein-protein interaction involving the homeo subdomain of OTF-1

1992 ◽  
Vol 12 (11) ◽  
pp. 4960-4969
Author(s):  
E Kutoh ◽  
P E Strömstedt ◽  
L Poellinger
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Protein Interaction ◽  
Binding Activity ◽  
Dependent Manner ◽  
Protein Protein Interaction ◽  
Dna Binding Activity ◽  
Functional Interference

The ubiquitous and constitutive octamer transcription factor OTF-1 (Oct 1) is the target of positive regulation by the potent herpes simplex virus trans-activator VP16, which forms a complex with the homeodomain of OTF-1. Here we present evidence that the glucocorticoid receptor can negatively regulate OTF-1 function by a mechanism that is independent of DNA binding. In vivo-expressed glucocorticoid receptor inhibited in a hormone-dependent manner activation of a minimal promoter construct carrying a functional octamer site. Moreover, expression of the receptor in vivo resulted in hormone-dependent repression of OTF-1-dependent DNA-binding activity in nuclear extract. In vitro, the DNA-binding activity of partially purified OTF-1 was repressed following incubation with purified glucocorticoid receptor. Cross-linking and immunoprecipitation experiments indicated that the functional interference may be due to a strong association between these two proteins in solution. Finally, preliminary evidence indicates that the homeo subdomain of OTF-1 that directs formation of a complex with VP16 may also be critical for interaction with the glucocorticoid receptor. Thus, OTF-1 is a target for both positive and negative regulation by protein-protein interaction. Moreover, the functional interference between OTF-1 and the glucocorticoid receptor represents a novel regulatory mechanism in the cross-coupling of signal transduction pathways of nuclear receptors and constitutive transcription factors.

A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor

1986 ◽  
Vol 6 (12) ◽  
pp. 4723-4733
Author(s):  
L A Chodosh ◽  
R W Carthew ◽  
P A Sharp
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Site ◽  
Rna Polymerase Ii ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Affinity Column ◽  
Dna Binding Activity ◽  
Dna Fragment

A simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44- to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

scholarly journals Constitutive nuclear NFκB/rel DNA-binding activity of rat thymocytes is increased by stimuli that promote apoptosis, but not inhibited by pyrrolidine dithiocarbamate

1995 ◽  
Vol 312 (3) ◽  
pp. 833-838 ◽  
Author(s):  
A F G Slater ◽  
M Kimland ◽  
S A Jiang ◽  
S Orrenius
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Gradient Centrifugation ◽  
Binding Activity ◽  
Pyrrolidine Dithiocarbamate ◽  
Nf Kappa B ◽  
Dna Binding Activity ◽  
Apoptotic Activity

Rat thymocytes spontaneously undergo apoptotic death in cell culture, and are also sensitive to the induction of apoptosis by various stimuli. We show that unstimulated thymocytes constitutively express a p50-containing nuclear factor kappa B (NF kappa B)/rel DNA-binding activity in their nuclei. When the cells were fractionated by density-gradient centrifugation this activity was found to be most pronounced in immature CD4+8+ thymocytes, the cell population that undergoes selection by apoptosis in vivo and that is most sensitive to external inducers of apoptosis in vitro. The intensity of the NF kappa B/rel protein-DNA complex was significantly enhanced 30 min after exposing thymocytes to methylprednisolone or etoposide, two agents well known to induce apoptosis in these cells. Expression of this DNA-binding activity therefore correlates with the subsequent occurrence of apoptosis. By analogy to other systems, it has been suggested that antioxidants such as pyrrolidine dithiocarbamate (PDTC) inhibit thymocyte apoptosis by preventing the activation of an NF kappa B/rel transcription factor. However, we have found that etoposide induces a very similar enhancement of the NF kappa B/rel DNA-binding activity in the presence or absence of PDTC, despite a pronounced inhibition of apoptotic DNA fragmentation in the former situation. Dithiocarbamates therefore do not exert their anti-apoptotic activity in thymocytes by inhibiting the activation of this transcription factor.

One exon of the human LSF gene includes conserved regions involved in novel DNA-binding and dimerization motifs

1994 ◽  
Vol 14 (8) ◽  
pp. 5076-5087
Author(s):  
M K Shirra ◽  
Q Zhu ◽  
H C Huang ◽  
D Pallas ◽  
U Hansen
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Binding Activity ◽  
Protein Protein Interaction ◽  
Dna Binding Activity ◽  
Dna Binding Sites ◽  
Alternative Mrna Splicing ◽  
Alternatively Spliced

The transcription factor LSF, identified as a HeLa protein that binds the simian virus 40 late promoter, recognizes direct repeats with a center-to-center spacing of 10 bp. The characterization of two human cDNAs, representing alternatively spliced mRNAs, provides insight into the unusual DNA-binding and oligomerization properties of LSF. The sequence of the full-length LSF is identical to that of the transcription factors alpha CP2 and LBP-1c and has similarity to the Drosophila transcription factor Elf-1/NTF-1. Using an epitope-counting method, we show that LSF binds DNA as a homodimer. LSF-ID, which is identical to LBP-1d, contains an in-frame internal deletion of 51 amino acids resulting from alternative mRNA splicing. Unlike LSF, LSF-ID did not bind LSF DNA-binding sites. Furthermore, LSF-ID did not affect the binding of LSF to DNA, suggesting that the two proteins do not interact. Of three short regions with a high degree of homology between LSF and Elf-1/NTF-1, LSF-ID lacks two, which are predicted to form beta-strands. Double amino acid substitutions in each of these regions eliminated specific DNA-binding activity, similarly to the LSF-ID deletion. The dimerization potential of these mutants was measured both by the ability to inhibit the binding of LSF to DNA and by direct protein-protein interaction studies. Mutations in one homology region, but not the other, functionally eliminated dimerization.

scholarly journals M-phase-specific phosphorylation of the POU transcription factor GHF-1 by a cell cycle-regulated protein kinase inhibits DNA binding.

1995 ◽  
Vol 15 (12) ◽  
pp. 6694-6701 ◽  
Author(s):  
C Caelles ◽  
H Hennemann ◽  
M Karin
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Protein Kinase ◽  
Dna Binding ◽  
Binding Activity ◽  
Dependent Manner ◽  
Mitotic Kinase ◽  
Dna Binding Activity ◽  
M Phase ◽  
A Cell

GHF-1 is a member of the POU family of homeodomain proteins. It is a cell-type-specific transcription factor responsible for determination and expansion of growth hormone (GH)- and prolactin-expressing cells in the anterior pituitary. It was previously suggested that cyclic AMP (cAMP)-responsive protein kinase A (PKA) phosphorylates GHF-1 at a site within the N-terminal arm of its homeodomain, thereby inhibiting its binding to the GH promoter. These results, however, are inconsistent with the physiological stimulation of GH production by the cAMP pathway. As reported here, cAMP agonists and PKA do not inhibit GHF-1 activity in living cells and although they stimulate the phosphorylation of GHF-1, the inhibitory phosphoacceptor site within the homeodomain is not affected. Instead, this site, Thr-220, is subject to M-phase-specific phosphorylation. As a result, GHF-1 DNA binding activity is transiently inhibited during the M phase. This activity is regained once cells enter G1, a phase during which GHF-1 phosphorylation is minimal. Thr-220 of GHF-1 is the homolog of the mitotic phosphoacceptor site responsible for the M-phase-specific inhibition of Oct-1 DNA binding Ser-382. As this site is conserved in all POU proteins, it appears that all members of this group are similarly regulated. A specific kinase activity distinct in its substrate specificity and susceptibility to inhibitors from the Cdc2 mitotic kinase or PKA was identified in extracts of mitotic cells. This novel activity could be involved in regulating the DNA binding activity of all POU proteins in a cell cycle-dependent manner.

Characterization of the mouse metal-regulatory-element-binding proteins, metal element protein-1 and metal regulatory transcription factor-1

2001 ◽  
Vol 353 (3) ◽  
pp. 591-601 ◽  
Author(s):  
Olivier LAROCHELLE ◽  
Gale STEWART ◽  
Pierre MOFFATT ◽  
Véronique TREMBLAY ◽  
Carl SÉGUIN
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Activity ◽  
Dependent Manner ◽  
Rnase Protection ◽  
Dna Binding Activity ◽  
Cell Extracts ◽  
Metal Element ◽  
Transcription Factor 1

Metal activation of metallothionein gene transcription depends mainly on the presence of regulatory DNA sequences termed metal-regulatory elements (MREs) and involves MRE-binding transcription factor-1 (MTF-1) interacting with the MREs in a Zn2+-dependent manner. We previously identified and characterized a nuclear protein, termed metal element protein-1 (MEP-1), specifically binding with high affinity to MRE elements. The precise relationship between MTF-1 and MEP-1 was unclear, and to determine whether MEP-1 and MTF-1 were distinct protein species, we performed DNA binding analyses to characterize the binding properties of both proteins. Electrophoretic mobility-shift assays showed that MTF-1, produced in COS cells, produces a slower-migrating band compared with that obtained with purified MEP-1. Using an anti-MTF-1 antibody, we showed that both the MTF-1–MRE and the MEP-1–MRE complexes are supershifted by an anti-MTF-1 antibody, thus demonstrating that MEP-1 is antigenically related to MTF-1. RNase protection analyses carried out with RNA prepared from different tissues and cell lines failed to reveal the presence of MTF-1 splicing variants. This indicates that MEP-1 may be a proteolytic fragment of MTF-1. MTF-1 DNA-binding activity was rapidly activated in vivo by Zn2+ ions but not by Cd2+, UV irradiation or PMA, and occurred on ice as well as at 21°C. In control and Zn2+-treated cell extracts, DNA-binding activity was not enhanced in vitro following the addition of exogenous Zn2+ or a preincubation at 37°C. However, recombinant MTF-1 produced in vitro required Zn2+ activation for DNA binding. Interestingly, treatment of nuclear extracts with calf intestine phosphatase completely abrogated MTF-1 DNA-binding activity, thus suggesting that phosphorylation is involved in the regulation of MTF-1 activity.

Estradiol upregulates mesangial cell MMP-2 activity via the transcription factor AP-2

2002 ◽  
Vol 282 (1) ◽  
pp. F164-F169 ◽  
Author(s):  
Michael Guccione ◽  
Sharon Silbiger ◽  
Jun Lei ◽  
Joel Neugarten
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Protein Expression ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Binding Activity ◽  
Dependent Manner ◽  
Protein Levels ◽  
Dna Binding Activity ◽  
Metalloproteinase Activity

The accumulation of extracellular matrix in the glomerular mesangium reflects the net balance between the synthesis and degradation of matrix components. We have shown that estradiol suppresses the synthesis of types I and IV collagen by cultured mesangial cells (Kwan G, Neugarten J, Sherman M, Ding Q, Fotadar U, Lei J, and Silbiger S. Kidney Int 50: 1173–1179, 1996; Neugarten J, Acharya A, Lei J, and Silbiger S. Am J Physiol Renal Physiol 279: F309–F318, 2000; Neugarten J, Medve I, Lei J, and Silbiger SR. Am J Physiol Renal Physiol 277: F1–F8, 1999; Neugarten J and Silbiger S. Am J Kidney Dis 26: 147–151, 1995; Silbiger S, Lei J, and Neugarten J. Kidney Int 55: 1268–1276, 1998; Silbiger S, Lei J, Ziyadeh FN, and Neugarten J. Am J Physiol Renal Physiol 274: F1113–F1118, 1998). In the present study, we evaluated the effects of sex hormones on the activity of matrix metalloproteinase-2 (MMP-2) in murine mesangial cells, the synthesis of which is regulated by the transcription factor activator protein-2 (AP-2). Estradiol stimulated MMP-2 activity by increasing MMP-2 protein levels in a dose-dependent manner. These effects occurred at physiological concentrations of estradiol and were receptor mediated. Estradiol also increased AP-2 protein levels and increased binding of mesangial cell nuclear extracts to an AP-2 consensus binding sequence oligonucleotide. The ability of estradiol to increase AP-2 protein expression, AP-2/DNA binding activity, MMP-2 protein expression, and metalloproteinase activity was reversed by PD-98059, a selective inhibitor of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling cascade. We conclude that estradiol upregulates the MAPK cascade, which in turn stimulates the synthesis of AP-2 protein. The resultant increased AP-2/DNA binding activity leads to increased synthesis of MMP-2 and increased metalloproteinase activity. Stimulation of metalloproteinase activity by estradiol may contribute to the protective effect of female gender on renal disease progression.

scholarly journals I kappa B/MAD-3 masks the nuclear localization signal of NF-kappa B p65 and requires the transactivation domain to inhibit NF-kappa B p65 DNA binding.

1992 ◽  
Vol 3 (12) ◽  
pp. 1339-1352 ◽  
Author(s):  
P A Ganchi ◽  
S C Sun ◽  
W C Greene ◽  
D W Ballard
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Binding Activity ◽  
Transactivation Domain ◽  
Nf Kappa B ◽  
Dna Binding Activity ◽  
Localization Signal

The active nuclear form of the NF-kappa B transcription factor complex is composed of two DNA binding subunits, NF-kappa B p65 and NF-kappa B p50, both of which share extensive N-terminal sequence homology with the v-rel oncogene product. The NF-kappa B p65 subunit provides the transactivation activity in this complex and serves as an intracellular receptor for a cytoplasmic inhibitor of NF-kappa B, termed I kappa B. In contrast, NF-kappa B p50 alone fails to stimulate kappa B-directed transcription, and based on prior in vitro studies, is not directly regulated by I kappa B. To investigate the molecular basis for the critical regulatory interaction between NF-kappa B and I kappa B/MAD-3, a series of human NF-kappa B p65 mutants was identified that functionally segregated DNA binding, I kappa B-mediated inhibition, and I kappa B-induced nuclear exclusion of this transcription factor. Results from in vivo expression studies performed with these NF-kappa B p65 mutants revealed the following: 1) I kappa B/MAD-3 completely inhibits NF-kappa B p65-dependent transcriptional activation mediated through the human immunodeficiency virus type 1 kappa B enhancer in human T lymphocytes, 2) the binding of I kappa B/MAD-3 to NF-kappa B p65 is sufficient to retarget NF-kappa B p65 from the nucleus to the cytoplasm, 3) selective deletion of the functional nuclear localization signal present in the Rel homology domain of NF-kappa B p65 disrupts its ability to engage I kappa B/MAD-3, and 4) the unique C-terminus of NF-kappa B p65 attenuates its own nuclear localization and contains sequences that are required for I kappa B-mediated inhibition of NF-kappa B p65 DNA binding activity. Together, these findings suggest that the nuclear localization signal and transactivation domain of NF-kappa B p65 constitute a bipartite system that is critically involved in the inhibitory function of I kappa B/MAD-3. Unexpectedly, our in vivo studies also demonstrate that I kappa B/MAD-3 binds directly to NF-kappa B p50. This interaction is functional as it leads to retargeting of NF-kappa B p50 from the nucleus to the cytoplasm. However, no loss of DNA binding activity is observed, presumably reflecting the unique C-terminal domain that is distinct from that present in NF-kappa B p65.

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