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.

scholarly journals Reversible activation of mouse metal response element-binding transcription factor 1 DNA binding involves zinc interaction with the zinc finger domain.

1997 ◽  
Vol 17 (5) ◽  
pp. 2781-2789 ◽  
Author(s):  
T P Dalton ◽  
D Bittel ◽  
G K Andrews
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Capacity ◽  
Binding Activity ◽  
Response Element ◽  
Dna Binding Activity ◽  
Cell Extracts ◽  
Free Zinc ◽  
Transcription Factor 1

The DNA-binding activity of the Zn finger protein metal response element-binding transcription factor 1 (MTF-1) was rapidly induced both in vivo in mouse Hepa cells, canine MDCK, and human HeLa cells after incubation in medium containing zinc and in vitro in whole-cell extracts to which zinc was added. Acquisition of DNA-binding capacity in the presence of free zinc was temperature and time dependent and did not occur at 4 degrees C. In contrast, activated MTF-1 binding to the metal response element occurred at 4 degrees C. After Zn activation, mouse MTF-1 binding activity was more sensitive to EDTA and was stabilized by DNA binding relative to the Zn finger transcription factor Sp1. After dilution of nuclear or whole-cell extracts from Zn-treated cells and incubation at 37 degrees C, mouse MTF-1 DNA-binding activity was no longer detected but could be completely reconstituted by the subsequent readdition of zinc. In vitro-synthesized, recombinant mouse MTF-1 displayed a similar, reversible temperature- and Zn-dependent activation of DNA-binding activity. Analysis of deletion mutants of recombinant MTF-1 suggests that the Zn finger domain is important for the Zn-dependent activation of DNA-binding capacity. Thus, mouse MTF-1 functions as a reversibly activated sensor of free zinc pools in the cell.

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 The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator.

1994 ◽  
Vol 14 (7) ◽  
pp. 4380-4389 ◽  
Author(s):  
L I Chen ◽  
T Nishinaka ◽  
K Kwan ◽  
I Kitabayashi ◽  
K Yokoyama ◽  
...  
Keyword(s):  
Dna Binding ◽  
Gene Product ◽  
Binding Activity ◽  
Negative Regulator ◽  
Control Element ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Cell Extracts ◽  
Mobility Shift Assay

Studies have demonstrated that the retinoblastoma susceptibility gene product, RB, can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner. The nucleotide sequence of the retinoblastoma control element (RCE) motif, GCCACC or CCACCC, and the Sp1 consensus binding sequence, CCGCCC, can confer equal responsiveness to RB. Here, we report that RB activates transcription of the c-jun gene through the Sp1-binding site within the c-jun promoter. Preincubation of crude nuclear extracts with monoclonal antibodies to RB results in reduction of Sp1 complexes in a mobility shift assay, while addition of recombinant RB in mobility shift assay mixtures with CCL64 cell extracts leads to an enhancement of DNA-binding activity of SP1. These results suggest that RB is directly or indirectly involved in Sp1-DNA binding activity. A mechanism by which RB regulates transactivation is indicated by our detection of a heat-labile and protease-sensitive Sp1 negative regulator(s) (Sp1-I) that specifically inhibits Sp1 binding to a c-jun Sp1 site. This inhibition is reversed by addition of recombinant RB proteins, suggesting that RB stimulates Sp1-mediated transactivation by liberating Sp1 from Sp1-I. Additional evidence for Sp1-I involvement in Sp1-mediated transactivation was demonstrated by cotransfection of RB, GAL4-Sp1, and a GAL4-responsive template into CV-1 cells. Finally, we have identified Sp1-I, a approximately 20-kDa protein(s) that inhibits the Sp1 complexes from binding to DNA and that is also an RB-associated protein. These findings provide evidence for a functional link between two distinct classes of oncoproteins, RB and c-Jun, that are involved in the control of cell growth, and also define a novel mechanism for the regulation of c-jun expression.

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.

scholarly journals cpc-1, the general regulatory gene for genes of amino acid biosynthesis in Neurospora crassa, is differentially expressed during the asexual life cycle

1991 ◽  
Vol 11 (2) ◽  
pp. 928-934 ◽  
Author(s):  
D J Ebbole ◽  
J L Paluh ◽  
M Plamann ◽  
M S Sachs ◽  
C Yanofsky
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Neurospora Crassa ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Recognition Sequence ◽  
Dna Binding Activity ◽  
Cell Extracts

CPCI, the principal regulatory protein required for cross-pathway control of amino acid biosynthetic genes in Neurospora crassa, contains a domain similar to the DNA-binding domain of GCN4, the corresponding general regulator in Saccharomyces cerevisiae. We examined binding by CPC1 synthesized in vitro and by CPC1 present in N. crassa whole-cell extracts. CPCI from both sources was shown to bind to the DNA sequence 5'-ATGACTCAT-3', which is also the preferred recognition sequence of GCN4, CPC1 was confirmed as the source of DNA-binding activity in extracts by immunoblotting. Slightly mobility differences between DNA complexes containing CPCI synthesized in vitro and CPC1 in mycelial extracts were observed. Analyses of N. crassa extracts from different stages of asexual development revealed that CPC1 was abundant immediately following spore germination and through early mycelial growth but was scarce subsequently. CPC1 levels could be increased at any time by imposing amino acid starvation. Copies of the CPC1 response element are located upstream of several genes regulated by cross-pathway control, including cpc-1 itself.

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.

scholarly journals Retinoid-induced apoptosis and Sp1 cleavage occur independently of transcription and require caspase activation.

1997 ◽  
Vol 17 (11) ◽  
pp. 6348-6358 ◽  
Author(s):  
F J Piedrafita ◽  
M Pfahl
Keyword(s):  
T Cells ◽  
Dna Binding ◽  
Cancer Cell ◽  
De Novo ◽  
Binding Activity ◽  
Dependent Manner ◽  
Intact Cells ◽  
Dna Binding Activity ◽  
Induced Apoptosis

Vitamin A and its derivatives, the retinoids, are essential regulators of many important biological functions, including cell growth and differentiation, development, homeostasis, and carcinogenesis. Natural retinoids such as all-trans retinoic acid can induce cell differentiation and inhibit growth of certain cancer cells. We recently identified a novel class of synthetic retinoids with strong anti-cancer cell activities in vitro and in vivo which can induce apoptosis in several cancer cell lines. Using an electrophoretic mobility shift assay, we analyzed the DNA binding activity of several transcription factors in T cells treated with apoptotic retinoids. We found that the DNA binding activity of the general transcription factor Sp1 is lost in retinoid-treated T cells undergoing apoptosis. A truncated Sp1 protein is detected by immunoblot analysis, and cytosolic protein extracts prepared from apoptotic cells contain a protease activity which specifically cleaves purified Sp1 in vitro. This proteolysis of Sp1 can be inhibited by N-ethylmaleimide and iodoacetamide, indicating that a cysteine protease mediates cleavage of Sp1. Furthermore, inhibition of Sp1 cleavage by ZVAD-fmk and ZDEVD-fmk suggests that caspases are directly involved in this event. In fact, caspases 2 and 3 are activated in T cells after treatment with apoptotic retinoids. The peptide inhibitors also blocked retinoid-induced apoptosis, as well as processing of caspases and proteolysis of Sp1 and poly(ADP-ribose) polymerase in intact cells. Degradation of Sp1 occurs early during apoptosis and is therefore likely to have profound effects on the basal transcription status of the cell. Interestingly, retinoid-induced apoptosis does not require de novo mRNA and protein synthesis, suggesting that a novel mechanism of retinoid signaling is involved, triggering cell death in a transcriptional activation-independent, caspase-dependent manner.

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.

scholarly journals Identification of the varR Gene as a Transcriptional Regulator of Virginiamycin S Resistance inStreptomyces virginiae

2001 ◽  
Vol 183 (6) ◽  
pp. 2025-2031 ◽  
Author(s):  
Wises Namwat ◽  
Chang-Kwon Lee ◽  
Hiroshi Kinoshita ◽  
Yasuhiro Yamada ◽  
Takuya Nihira
Keyword(s):  
Dna Binding ◽  
Promoter Region ◽  
Northern Blot Analysis ◽  
Binding Activity ◽  
Binding Motif ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Dna Binding Activity ◽  
Streptomyces Virginiae

ABSTRACT A gene designated varR (for virginiaeantibiotic resistance regulator) was identified in Streptomyces virginiae 89 bp downstream of a varS gene encoding a virginiamycin S (VS)-specific transporter. The deduced varRproduct showed high homology to repressors of the TetR family with a conserved helix-turn-helix DNA binding motif. Purified recombinant VarR protein was present as a dimer in vitro and showed clear DNA binding activity toward the varS promoter region. This binding was abolished by the presence of VS, suggesting that VarR regulates transcription of varS in a VS-dependent manner. Northern blot analysis revealed that varR was cotranscribed with upstream varS as a 2.4-kb transcript and that VS acted as an inducer of bicistronic transcription. Deletion analysis of thevarS promoter region clarified two adjacent VarR binding sites in the varS promoter.

scholarly journals The evaluation of anoxia responsive E2F DNA binding activity in the red eared slider turtle, Trachemys scripta elegans

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4755 ◽  
Author(s):  
Kyle K. Biggar ◽  
Kenneth B. Storey
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Dna Binding ◽  
Complex Dynamics ◽  
Binding Activity ◽  
Trachemys Scripta ◽  
Model Organisms ◽  
Trachemys Scripta Elegans ◽  
Dna Binding Activity

In many cases, the DNA-binding activity of a transcription factor does not change, while its transcriptional activity is greatly influenced by the make-up of bound proteins. In this study, we assessed the protein composition and DNA-binding ability of the E2F transcription factor complex to provide insight into cell cycle control in an anoxia tolerant turtle through the use of a modified ELISA protocol. This modification also permits the use of custom DNA probes that are tailored to a specific DNA binding region, introducing the ability to design capture probes for non-model organisms. Through the use of EMSA and ELISA DNA binding assays, we have successfully determined the in vitro DNA binding activity and complex dynamics of the Rb/E2F cell cycle regulatory mechanisms in an anoxic turtle, Trachemys scripta elegans. Repressive cell cycle proteins (E2F4, Rb, HDAC4 and Suv39H1) were found to significantly increase at E2F DNA-binding sites upon anoxic exposure in anoxic turtle liver. The lack of p130 involvement in the E2F DNA-bound complex indicates that anoxic turtle liver may maintain G1 arrest for the duration of stress survival.

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