scholarly journals Autoactivation by a Candida glabrata copper metalloregulatory transcription factor requires critical minor groove interactions.

1996 ◽  
Vol 16 (2) ◽  
pp. 724-734 ◽  
Author(s):  
K A Koch ◽  
D J Thiele
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Candida Glabrata ◽  
Regulatory Element ◽  
Critical Role ◽  
Minor Groove ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Binding Studies

Rapid transcriptional autoactivation of the Candida glabrata AMT1 copper metalloregulatory transcription factor gene is essential for survival in the presence of high extracellular copper concentrations. Analysis of the interactions between purified recombinant AMT1 protein and the AMT1 promoter metal regulatory element was carried out by a combination of missing-nucleoside analysis, ethylation interference, site-directed mutagenesis, and quantitative in vitro DNA binding studies. The results of these experiments demonstrate that monomeric AMT1 binds the metal regulatory element with very high affinity and utilizes critical contacts in both the major and minor grooves. A single adenosine residue in the minor groove, conserved in all known yeast Cu metalloregulatory transcription factor DNA binding sites, plays a critical role in both AMT1 DNA binding in vitro and Cu-responsive AMT1 gene transcription in vivo. Furthermore, a mutation in the AMT1 Cu-activated DNA binding domain which converts a single arginine, found in a conserved minor groove binding domain, to lysine markedly reduces AMT1 DNA binding affinity in vitro and results in a severe defect in the ability of C. glabrata cells to mount a protective response against Cu toxicity.

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.

Methylation of RUNX1 by the Nuclear PRMT5/COPR5 Complex Regulates Its Cellular Location in Leukemia Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3403-3403
Author(s):  
Xinyang Zhao ◽  
Ly P. Vu ◽  
Fabiana Perna ◽  
Fan Liu ◽  
Hao Xu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Arginine Methylation ◽  
Binding Domain ◽  
Dependent Manner ◽  
Dna Binding Domain ◽  
Sm Proteins ◽  
Differentiation Potential ◽  
Hematopoietic Stem

Abstract Abstract 3403 RUNX1 is a transcription factor that is required for definitive hematopoietic development, and helps regulate long term hematopoietic stem cell self-renewal, platelet production, and lymphocyte development during adult hematopoiesis. RUNX1 is known to be modified via phosphorylation, acetylation, ubiquitination and methylation, for example on R208 and R210 by PRMT1, which activates its activating function. We continue to investigate how the methylation of RUNX1 by other protein arginine methyl transferases (PRMTs) regulates its function. Loop 9 of the DNA binding domain (the Runt domain) of RUNX1 contains an SGRGK sequence that is also present on the tails of histone H2A and H4. The histone tails of H4 and H2A can be methylated by a purified PRMT5 complex in vitro. An enzymatically active in vitro PRMT5 complex capable of methylating histones and SM proteins requires two subunits: both PRMT5 and MEP50, a WD 40 repeat domain protein. Nevertheless, this purified PRMT5/MEP50 complex cannot methylate the DNA binding domain of the RUNX1 protein in vitro. We show that RUNX1 also can be symmetrically methylated at R142 within the SGRGK motif in vitro by a nuclear PRMT5/MEP50 complex which also contains COPR5. We show after RUNX1 is methylated on R142 within the nucleus of HEL cells, RUNX1 is exported to the cytoplasm in a CRM1 dependent manner, as the export of methylated RUNX1 is blocked by lemptomycin B. CRM1 interacts with PRMT5, supporting that PRMT5 mediated arginine methylation tags protein for nuclear export. Therefore, PRMT5 not only involves in epigenetic regulation by methylation of histones but also it can directly controls the level of transcription factor proteins within the nucleus. Polycytocemia Vera patients who express the Jak2V617F mutation have low PRMT5 activity due to JAK2V617F mediated PRMT5 phosphorylation (Liu et al 2011). How Jak2 signaling affects RUNX1 methylation and RUNX1 localization within the nucleus is still under investigation. By controlling the amount of RUNX1 available within the cell nucleus, PRMT5 may regulate lineage differentiation potential and growth potential of hematopoietic stem and progenitor cells. The nuclear localization of RUNX1 can be changed through post translational modification such as arginine methylation in addition to point mutations and translocations involving RUNX1 found patients with leukemia and pre-leukemic diseases. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Functional and conserved domains of the Drosophila transcription factor encoded by the segmentation gene knirps

1994 ◽  
Vol 14 (12) ◽  
pp. 7899-7908
Author(s):  
N Gerwin ◽  
A La Rosée ◽  
F Sauer ◽  
H P Halbritter ◽  
M Neumann ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Broad Band ◽  
Binding Domain ◽  
Orphan Receptor ◽  
Dna Binding Domain ◽  
Coding Region ◽  
Amino Acid Region

The Drosophila gap gene knirps (kni) is required for abdominal segmentation. It encodes a steroid/thyroid orphan receptor-type transcription factor which is distributed in a broad band of nuclei in the posterior region of the blastoderm. To identify essential domains of the kni protein (KNI), we cloned and sequenced the DNA encompassing the coding region of nine kni mutant alleles of different strength and kni-homologous genes of related insect species. We also examined in vitro-modified versions of KNI in various assay systems both in vitro and in tissue culture. The results show that KNI contains several functional domains which are arranged in a modular fashion. The N-terminal 185-amino-acid region which includes the DNA-binding domain and a functional nuclear location signal fails to provide kni activity to the embryo. However, a truncated KNI protein that contains additional 47 amino acids exerts rather strong kni activity which is functionally defined by a weak kni mutant phenotype of the embryo. The additional 47-amino-acid stretch includes a transcriptional repressor domain which acts in the context of a heterologous DNA-binding domain of the yeast transcriptional activator GAL4. The different domains of KNI as defined by functional studies are conserved during insect evolution.

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 Prebending the estrogen response element destabilizes binding of the estrogen receptor DNA binding domain.

1997 ◽  
Vol 17 (6) ◽  
pp. 3173-3180 ◽  
Author(s):  
J Kim ◽  
G de Haan ◽  
A M Nardulli ◽  
D J Shapiro
Keyword(s):  
Estrogen Receptor ◽  
Transcription Factor ◽  
Dna Binding ◽  
Minor Groove ◽  
Dna Topology ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Eukaryotic Transcription ◽  
Estrogen Response ◽  
Direct Demonstration

Binding of many eukaryotic transcription regulatory proteins to their DNA recognition sequences results in conformational changes in DNA. To test the effect of altering DNA topology by prebending a transcription factor binding site, we examined the interaction of the estrogen receptor (ER) DNA binding domain (DBD) with prebent estrogen response elements (EREs). When the ERE in minicircle DNA was prebent toward the major groove, which is in the same direction as the ER-induced DNA bend, there was no significant effect on ER DBD binding relative to the linear counterparts. However, when the ERE was bent toward the minor groove, in a direction that opposes the ER-induced DNA bend, there was a four- to eightfold reduction in ER DBD binding. Since reduced binding was also observed with the ERE in nicked circles, the reduction in binding was not due to torsional force induced by binding of ER DBD to the prebent ERE in covalently closed minicircles. To determine the mechanism responsible for reduced binding to the prebent ERE, we examined the effect of prebending the ERE on the association and dissociation of the ER DBD. Binding of the ER DBD to ERE-containing minicircles was rapid when the EREs were prebent toward either the major or minor groove of the DNA (k(on) of 9.9 x 10(6) to 1.7 x 10(7) M(-1) s(-1)). Prebending the ERE toward the minor groove resulted in an increase in k(off) of four- to fivefold. Increased dissociation of the ER DBD from the ERE is, therefore, the major factor responsible for reduced binding of the ER DBD to an ERE prebent toward the minor groove. These data provide the first direct demonstration that the interaction of a eukaryotic transcription factor with its recognition sequence can be strongly influenced by altering DNA topology through prebending the DNA.

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 The Yeast Heat Shock Transcription Factor Changes Conformation in Response to Superoxide and Temperature

2000 ◽  
Vol 11 (5) ◽  
pp. 1753-1764 ◽  
Author(s):  
Sengyong Lee ◽  
Tage Carlson ◽  
Noah Christian ◽  
Kristi Lea ◽  
Jennifer Kedzie ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Dna Binding ◽  
Conformational Change ◽  
Mutational Analysis ◽  
Binding Domain ◽  
Heat Shock Transcription Factor ◽  
Dna Binding Domain ◽  
Yeast Saccharomyces Cerevisiae

In vitro DNA-binding assays demonstrate that the heat shock transcription factor (HSF) from the yeast Saccharomyces cerevisiae can adopt an altered conformation when stressed. This conformation, reflected in a change in electrophoretic mobility, requires that two HSF trimers be bound to DNA. Single trimers do not show this change, which appears to represent an alteration in the cooperative interactions between trimers. HSF isolated from stressed cells displays a higher propensity to adopt this altered conformation. Purified HSF can be stimulated in vitro to undergo the conformational change by elevating the temperature or by exposing HSF to superoxide anion. Mutational analysis maps a region critical for this conformational change to the flexible loop between the minimal DNA-binding domain and the flexible linker that joins the DNA-binding domain to the trimerization domain. The significance of these findings is discussed in the context of the induction of the heat shock response by ischemic stroke, hypoxia, and recovery from anoxia, all known to stimulate the production of superoxide.

scholarly journals Assembly of a Functional Beta Interferon Enhanceosome Is Dependent on ATF-2–c-jun Heterodimer Orientation

2000 ◽  
Vol 20 (13) ◽  
pp. 4814-4825 ◽  
Author(s):  
James V. Falvo ◽  
Bhavin S. Parekh ◽  
Charles H. Lin ◽  
Ernest Fraenkel ◽  
Tom Maniatis
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Site ◽  
Leucine Zipper ◽  
Critical Role ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Beta Interferon

ABSTRACT Heterodimeric transcription factors, including the basic region-leucine zipper (bZIP) protein ATF-2–c-jun, are well-characterized components of an enhanceosome that mediates virus induction of the human beta interferon (IFN-β) gene. Here we report that within the IFN-β enhanceosome the ATF-2–c-jun heterodimer binds in a specific orientation, which is required for assembly of a complex between ATF-2–c-jun and interferon regulatory factor 3 (IRF-3). We demonstrate that correct orientation of the ATF-2–c-jun binding site is required for virus induction of the IFN-β gene and for IRF-3-dependent activation of a composite ATF-2– c-jun–IRF site in the IFN-β promoter. We also show that in vitro the DNA-bound ATF-2–c-jun heterodimer adopts a fixed orientation upon the binding of IRF-3 at an adjacent site in the IFN-β enhancer and that the DNA-binding domain of IRF-3 is sufficient to mediate this effect. In addition, we show that the DNA-binding domain of ATF-2 is necessary and sufficient for selective protein-protein interactions with IRF-3. Strikingly, in vivo chromatin immunoprecipitation experiments with IFN-β reporter constructs reveal that recruitment of IRF-3 to the IFN-β promoter upon virus infection is dependent on the orientation of the ATF-2–c-jun heterodimer binding site. These observations demonstrate functional and physical cooperativity between the bZIP and IRF transcription factor families and illustrate the critical role of heterodimeric transcription factors in formation of the IFN-β enhanceosome.

scholarly journals Functional and conserved domains of the Drosophila transcription factor encoded by the segmentation gene knirps.

1994 ◽  
Vol 14 (12) ◽  
pp. 7899-7908 ◽  
Author(s):  
N Gerwin ◽  
A La Rosée ◽  
F Sauer ◽  
H P Halbritter ◽  
M Neumann ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Broad Band ◽  
Binding Domain ◽  
Orphan Receptor ◽  
Dna Binding Domain ◽  
Coding Region ◽  
Amino Acid Region

The Drosophila gap gene knirps (kni) is required for abdominal segmentation. It encodes a steroid/thyroid orphan receptor-type transcription factor which is distributed in a broad band of nuclei in the posterior region of the blastoderm. To identify essential domains of the kni protein (KNI), we cloned and sequenced the DNA encompassing the coding region of nine kni mutant alleles of different strength and kni-homologous genes of related insect species. We also examined in vitro-modified versions of KNI in various assay systems both in vitro and in tissue culture. The results show that KNI contains several functional domains which are arranged in a modular fashion. The N-terminal 185-amino-acid region which includes the DNA-binding domain and a functional nuclear location signal fails to provide kni activity to the embryo. However, a truncated KNI protein that contains additional 47 amino acids exerts rather strong kni activity which is functionally defined by a weak kni mutant phenotype of the embryo. The additional 47-amino-acid stretch includes a transcriptional repressor domain which acts in the context of a heterologous DNA-binding domain of the yeast transcriptional activator GAL4. The different domains of KNI as defined by functional studies are conserved during insect evolution.

Mixed Cu+ and Zn2+ Coordination in the DNA-Binding Domain of the AMT1 Transcription Factor from Candida glabrata

Biochemistry ◽  
1994 ◽  
Vol 33 (32) ◽  
pp. 9566-9577 ◽  
Author(s):  
Joanne L. Thorvaldsen ◽  
Andrew K. Sewell ◽  
Amie M. Tanner ◽  
John M. Peltier ◽  
Ingrid J. Pickering ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Candida Glabrata ◽  
Binding Domain ◽  
Dna Binding Domain
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