scholarly journals The immunosuppressive fungal metabolite gliotoxin specifically inhibits transcription factor NF-kappaB.

1996 ◽  
Vol 183 (4) ◽  
pp. 1829-1840 ◽  
Author(s):  
H L Pahl ◽  
B Krauss ◽  
K Schulze-Osthoff ◽  
T Decker ◽  
E B Traenckner ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Tyrosine Kinases ◽  
Opportunistic Infections ◽  
Pathogenic Fungi ◽  
Intact Cells ◽  
High Concentrations ◽  
Nf Kappab

Opportunistic infections, such as aspergillosis, are among the most serious complications suffered by immunocompromised patients. Aspergillus fumigatus and other pathogenic fungi synthesize a toxic epipolythiodioxopiperazine metabolite called gliotoxin. Gliotoxin exhibits profound immunosuppressive activity in vivo. It induces apoptosis in thymocytes, splenocytes, and mesenteric lymph node cells and can selectively deplete bone marrow of mature lymphocytes. The molecular mechanism by which gliotoxin exerts these effects remains unknown. Here, we report that nanomolar concentrations of gliotoxin inhibited the activation of transcription factor NF-kappaB in response to a variety of stimuli in T and B cells. The effect of gliotoxin was specific because, at the same concentrations, the toxin did not affect activation of the transcription factor NF-AT or of interferon-responsive signal transducers and activators of transcription. Likewise, the activity of the constitutively DNA-binding transcription factors Oct-1 and cyclic AMP response element binding protein (CREB), as well as the activation of protein tyrosine kinases p56lck and p59fyn, was not altered by gliotoxin. Very high concentrations of gliotoxin prevented NF-kappaB DNA binding in vitro. However, in intact cells, inhibition of NF-kappaB did not occur at the level of DNA binding; rather, the toxin appeared to prevent degradation of IkappaB-alpha, NF-kappaB's inhibitory subunit. Our data raise the possibility that the immunosuppression observed during aspergillosis results in part from gliotoxin-mediated NF-kappaB inhibition.

scholarly journals Degradation of Myogenic Transcription Factor MyoD by the Ubiquitin Pathway In Vivo and In Vitro: Regulation by Specific DNA Binding

1998 ◽  
Vol 18 (10) ◽  
pp. 5670-5677 ◽  
Author(s):  
Ossama Abu Hatoum ◽  
Shlomit Gross-Mesilaty ◽  
Kristin Breitschopf ◽  
Aviad Hoffman ◽  
Hedva Gonen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
26S Proteasome ◽  
Intact Cells ◽  
Free System ◽  
Ubiquitin System

ABSTRACT MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

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.

The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP

1992 ◽  
Vol 12 (6) ◽  
pp. 2662-2672
Author(s):  
Z Kozmik ◽  
S Wang ◽  
P Dörfler ◽  
B Adams ◽  
M Busslinger
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
B Cell ◽  
Lymphoid Cells ◽  
Binding Studies ◽  
Specific Transcription Factor ◽  
High Affinity ◽  
B Lymphoid

The CD19 protein is expressed on the surface of all B-lymphoid cells with the exception of terminally differentiated plasma cells and has been implicated as a signal-transducing receptor in the control of proliferation and differentiation. Here we demonstrate complete correlation between the expression pattern of the CD19 gene and the B-cell-specific transcription factor BSAP in a large panel of B-lymphoid cell lines. The human CD19 gene has been cloned, and several BSAP-binding sites have been mapped by in vitro protein-DNA binding studies. In particular, a high-affinity BSAP-binding site instead of a TATA sequence is located in the -30 promoter region upstream of a cluster of heterogeneous transcription start sites. Moreover, this site is occupied by BSAP in vivo in a CD19-expressing B-cell line but not in plasma or HeLa cells. This high-affinity site has been conserved in the promoters of both human and mouse CD19 genes and was furthermore shown to confer B-cell specificity to a beta-globin reporter gene in transient transfection experiments. In addition, BSAP was found to be the only abundant DNA-binding activity of B-cell nuclear extracts that interacts with the CD19 promoter. Together, this evidence strongly implicates BSAP in the regulation of the CD19 gene.

scholarly journals The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP.

1992 ◽  
Vol 12 (6) ◽  
pp. 2662-2672 ◽  
Author(s):  
Z Kozmik ◽  
S Wang ◽  
P Dörfler ◽  
B Adams ◽  
M Busslinger
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
B Cell ◽  
Lymphoid Cells ◽  
Binding Studies ◽  
Specific Transcription Factor ◽  
High Affinity ◽  
B Lymphoid

The CD19 protein is expressed on the surface of all B-lymphoid cells with the exception of terminally differentiated plasma cells and has been implicated as a signal-transducing receptor in the control of proliferation and differentiation. Here we demonstrate complete correlation between the expression pattern of the CD19 gene and the B-cell-specific transcription factor BSAP in a large panel of B-lymphoid cell lines. The human CD19 gene has been cloned, and several BSAP-binding sites have been mapped by in vitro protein-DNA binding studies. In particular, a high-affinity BSAP-binding site instead of a TATA sequence is located in the -30 promoter region upstream of a cluster of heterogeneous transcription start sites. Moreover, this site is occupied by BSAP in vivo in a CD19-expressing B-cell line but not in plasma or HeLa cells. This high-affinity site has been conserved in the promoters of both human and mouse CD19 genes and was furthermore shown to confer B-cell specificity to a beta-globin reporter gene in transient transfection experiments. In addition, BSAP was found to be the only abundant DNA-binding activity of B-cell nuclear extracts that interacts with the CD19 promoter. Together, this evidence strongly implicates BSAP in the regulation of the CD19 gene.

scholarly journals Intact RNA-binding Domains Are Necessary for Structure-specific DNA Binding and Transcription Control by CBTF122duringXenopusDevelopment

2004 ◽  
Vol 279 (50) ◽  
pp. 52447-52455 ◽  
Author(s):  
Garry P. Scarlett ◽  
Stuart J. Elgar ◽  
Peter D. Cary ◽  
Anna M. Noble ◽  
Robert L. Orford ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Rna Binding ◽  
Transcription Activator ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
Rna Binding Domains ◽  
Ccaat Box

CBTF122is a subunit of theXenopusCCAAT box transcription factor complex and a member of a family of double-stranded RNA-binding proteins that function in both transcriptional and post-transcriptional control. Here we identify a region of CBTF122containing the double-stranded RNA-binding domains that is capable of binding either RNA or DNA. We show that these domains bind A-form DNA in preference to B-form DNA and that the -59 to -31 region of the GATA-2 promoter (anin vivotarget of CCAAT box transcription factor) adopts a partial A-form structure. Mutations in the RNA-binding domains that inhibit RNA binding also affect DNA bindingin vitro. In addition, these mutations alter the ability of CBTF122fusions with engrailed transcription repressor and VP16 transcription activator domains to regulate transcription of the GATA-2 genein vivo. These data support the hypothesis that the double-stranded RNA-binding domains of this family of proteins are important for their DNA binding bothin vitroandin vivo.

scholarly journals τ91, an Essential Subunit of Yeast Transcription Factor IIIC, Cooperates with τ138 in DNA Binding

1998 ◽  
Vol 18 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Rosalía Arrebola ◽  
Nathalie Manaud ◽  
Sophie Rozenfeld ◽  
Marie-Claude Marsolier ◽  
Olivier Lefebvre ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Essential Gene ◽  
Open Reading Frames ◽  
Class Iii ◽  
Gene Encoding ◽  
Promoter Recognition

ABSTRACT Transcription factor IIIC (TFIIIC) (or τ) is a large multisubunit and multifunctional factor required for transcription of all class III genes in Saccharomyces cerevisiae. It is responsible for promoter recognition and TFIIIB assembly. We report here the cloning and characterization of TFC6, an essential gene encoding the 91-kDa polypeptide, τ91, present in affinity-purified TFIIIC. τ91 has a predicted molecular mass of 74 kDa. It harbors a central cluster of His and Cys residues and has basic and acidic amino acid regions, but it shows no specific similarity to known proteins or predicted open reading frames. The TFIIIC subunit status of τ91 was established by the following biochemical and genetic evidence. Antibodies to τ91 bound TFIIIC-DNA complexes in gel shift assays; in vivo, a B block-deficient U6 RNA gene (SNR6) harboring GAL4 binding sites was reactivated by fusing the GAL4 DNA binding domain to τ91; and a point mutation in TFC6 (τ91-E330K) was found to suppress the thermosensitive phenotype of a tfc3-G349Emutant affected in the B block binding subunit (τ138). The suppressor mutation alleviated the DNA binding and transcription defects of mutant TFIIIC in vitro. These results indicated that τ91 cooperates with τ138 for DNA binding. Recombinant τ91 by itself did not interact with a tRNA gene, although it showed a strong affinity for single-stranded DNA.

scholarly journals Id Helix-Loop-Helix Proteins Antagonize Pax Transcription Factor Activity by Inhibiting DNA Binding

2001 ◽  
Vol 21 (2) ◽  
pp. 524-533 ◽  
Author(s):  
E. Claire Roberts ◽  
Richard W. Deed ◽  
Toshiaki Inoue ◽  
John D. Norton ◽  
Andrew D. Sharrocks
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Cellular Proliferation ◽  
Regulatory Function ◽  
Transcription Factor Family ◽  
Helix Loop Helix ◽  
Id Proteins ◽  
Proliferation And Differentiation

ABSTRACT The Id subfamily of helix-loop-helix (HLH) proteins plays a fundamental role in the regulation of cellular proliferation and differentiation. The major mechanism by which Id proteins are thought to inhibit differentiation is through interaction with other HLH proteins and inhibition of their DNA-binding activity. However, Id proteins have also been shown to interact with other proteins involved in regulating cellular proliferation and differentiation, suggesting a more widespread regulatory function. In this study we demonstrate functional interactions between Id proteins and members of the Pax-2/-5/-8 subfamily of paired-domain transcription factors. Members of the Pax transcription factor family have key functions in regulating several developmental processes exemplified by B lymphopoiesis, in which Pax-5 plays an essential role. Id proteins bind to Pax proteins in vitro and in vivo. Binding occurs through the paired DNA-binding domain of the Pax proteins and results in the disruption of DNA-bound complexes containing Pax-2, Pax-5, and Pax-8. In vivo, Id proteins modulate the transcriptional activity mediated by Pax-5 complexes on the B-cell-specific mb-1 promoter. Our results therefore demonstrate a novel facet of Id function in regulating cellular differentiation by functionally antagonizing the action of members of the Pax transcription factor family.

scholarly journals Crystal structure of the DNA binding domain of the transcription factor T-bet suggests simultaneous recognition of distant genome sites

2016 ◽  
Vol 113 (43) ◽  
pp. E6572-E6581 ◽  
Author(s):  
Ce Feng Liu ◽  
Gabriel S. Brandt ◽  
Quyen Q. Hoang ◽  
Natalia Naumova ◽  
Vanja Lazarevic ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Transcription Factor ◽  
Dna Binding ◽  
Quaternary Structure ◽  
Regulatory Element ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Chromosome Conformation

The transcription factor T-bet (Tbox protein expressed in T cells) is one of the master regulators of both the innate and adaptive immune responses. It plays a central role in T-cell lineage commitment, where it controls the TH1 response, and in gene regulation in plasma B-cells and dendritic cells. T-bet is a member of the Tbox family of transcription factors; however, T-bet coordinately regulates the expression of many more genes than other Tbox proteins. A central unresolved question is how T-bet is able to simultaneously recognize distant Tbox binding sites, which may be located thousands of base pairs away. We have determined the crystal structure of the Tbox DNA binding domain (DBD) of T-bet in complex with a palindromic DNA. The structure shows a quaternary structure in which the T-bet dimer has its DNA binding regions splayed far apart, making it impossible for a single dimer to bind both sites of the DNA palindrome. In contrast to most other Tbox proteins, a single T-bet DBD dimer binds simultaneously to identical half-sites on two independent DNA. A fluorescence-based assay confirms that T-bet dimers are able to bring two independent DNA molecules into close juxtaposition. Furthermore, chromosome conformation capture assays confirm that T-bet functions in the direct formation of chromatin loops in vitro and in vivo. The data are consistent with a looping/synapsing model for transcriptional regulation by T-bet in which a single dimer of the transcription factor can recognize and coalesce distinct genetic elements, either a promoter plus a distant regulatory element, or promoters on two different genes.

scholarly journals Cooperative binding of the Xenopus RNA polymerase I transcription factor xUBF to repetitive ribosomal gene enhancers

1992 ◽  
Vol 12 (11) ◽  
pp. 4970-4980
Author(s):  
C D Putnam ◽  
C S Pikaard
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Consensus Sequence ◽  
Dnase I ◽  
Ribosomal Gene ◽  
Minimum Requirement ◽  
Dna Binding Domains ◽  
Binding Domains

Upstream binding factor (UBF) is a DNA-binding transcription factor implicated in ribosomal gene promoter and enhancer function in vertebrates. UBF is unusual in that it has multiple DNA-binding domains with homology to high-mobility-group (HMG) nonhistone chromosomal proteins 1 and 2. However, a recognizable DNA consensus sequence for UBF binding is lacking. In this study, we have used gel retardation and DNase I footprinting to examine Xenopus UBF (xUBF) binding to Xenopus laevis ribosomal gene enhancers. We show that UBF has a minimum requirement for about 60 bp of DNA, the size of the short enhancer variant in X. laevis. Stronger UBF binding occurs on the longer enhancer variant (81 bp) and on multiple enhancers linked head to tail. In vivo, Xenopus ribosomal gene enhancers exist in blocks of 10 alternating 60- and 81-bp repeats within the intergenic spacer. In vitro, UBF binds cooperatively to probes with 10 enhancers, with five intermediate complexes observed in titration experiments. This suggests that, on average, one UBF dimer binds every two enhancers. A single UBF dimer can produce a DNase I footprint ranging in size from approximately 30 to about 115 bp on enhancer probes of different lengths. This observation is consistent with the hypothesis that multiple DNA-binding domains or subdomains within UBF bind independently, forming more-stable interactions on longer probes.

scholarly journals Multiple Roles of the τ131 Subunit of Yeast Transcription Factor IIIC (TFIIIC) in TFIIIB Assembly

2002 ◽  
Vol 22 (1) ◽  
pp. 298-308 ◽  
Author(s):  
Hélène Dumay-Odelot ◽  
Joël Acker ◽  
Rosalia Arrebola ◽  
André Sentenac ◽  
Christian Marck
Keyword(s):  
Transcription Factor ◽  
Transcription Initiation ◽  
In Vitro Transcription ◽  
Initiation Factor ◽  
Physical Interaction ◽  
Restrictive Temperature ◽  
Class Iii ◽  
High Concentrations

ABSTRACT Yeast transcription factor IIIC (TFIIIC) plays a key role in assembling the transcription initiation factor TFIIIB on class III genes after TFIIIC-DNA binding. The second largest subunit of TFIIIC, τ131, is thought to initiate TFIIIB assembly by interacting with Brf1/TFIIIB70. In this work, we have analyzed a TFIIIC mutant (τ131-ΔTPR2) harboring a deletion in τ131 removing the second of its 11 tetratricopeptide repeats. Remarkably, this thermosensitive mutation was selectively suppressed in vivo by overexpression of B”/TFIIIB90, but not Brf1 or TATA-binding protein. In vitro, the mutant factor preincubated at restrictive temperature bound DNA efficiently but lost transcription factor activity. The in vitro transcription defect was abolished at high concentrations of B” but not Brf1. Copurification experiments of baculovirus-expressed proteins confirmed a direct physical interaction between τ131 and B”. τ131, therefore, appears to be involved in the recruitment of both Brf1 and B”.

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