Changes in pH and NADPH Regulate the DNA Binding Activity of Neuronal PAS Domain Protein 2, a Mammalian Circadian Transcription Factor

Biochemistry ◽  
2015 ◽  
Vol 54 (2) ◽  
pp. 250-259 ◽  
Author(s):  
Katsuhiro Yoshii ◽  
Fumihisa Tajima ◽  
Sumio Ishijima ◽  
Ikuko Sagami
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Activity ◽  
Pas Domain ◽  
Dna Binding Activity ◽  
Domain Protein ◽  
Circadian Transcription

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.

Cloning and characterization of E2F-2, a novel protein with the biochemical properties of transcription factor E2F

1993 ◽  
Vol 13 (12) ◽  
pp. 7802-7812
Author(s):  
M Ivey-Hoyle ◽  
R Conroy ◽  
H E Huber ◽  
P J Goodhart ◽  
A Oliff ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Hela Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Identity ◽  
Biochemical Properties ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Novel Protein

E2F is a mammalian transcription factor that appears to play an important role in cell cycle regulation. While at least two proteins (E2F-1 and DP-1) with E2F-like activity have been cloned, studies from several laboratories suggest that additional homologs may exist. A novel protein with E2F-like properties, designated E2F-2, was cloned by screening a HeLa cDNA library with a DNA probe derived from the DNA binding domain of E2F-1 (K. Helin, J. A. Lees, M. Vidal, N. Dyson, E. Harlow, and A. Fattaey, Cell 70:337-350, 1992). E2F-2 exhibits overall 46% amino acid identity to E2F-1. Both the sequence and the function of the DNA and retinoblastoma gene product binding domains of E2F-1 are conserved in E2F-2. The DNA binding activity of E2F-2 is dramatically enhanced by complementation with particular sodium dodecyl sulfate-polyacrylamide gel electrophoresis-purified components of HeLa cell E2F, and anti-E2F-2 antibodies cross-react with components of purified HeLa cell E2F. These observations are consistent with a model in which E2F binds DNA as a heterodimer of two distinct proteins, and E2F-2 is functionally and immunologically related to one of these proteins.

Treatment of cultured myotubes with the proteasome inhibitor β-lactone increases the expression of the transcription factor C/EBPβ

2007 ◽  
Vol 292 (1) ◽  
pp. C216-C226 ◽  
Author(s):  
Wei Wei ◽  
Hongmei Yang ◽  
Michael Menconi ◽  
Peirang Cao ◽  
Chester E. Chamberlain ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Transcription Factor ◽  
Dna Binding ◽  
Proteasome Inhibitor ◽  
Binding Activity ◽  
Dominant Negative ◽  
Dna Binding Activity ◽  
Cultured Myotubes ◽  
Nuclear Levels ◽  
Factor C

The role of the proteasome in the regulation of cellular levels of the transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) is poorly understood. We tested the hypothesis that C/EBPβ levels in cultured myotubes are regulated, at least in part, by proteasome activity. Treatment of cultured L6 myotubes, a rat skeletal muscle cell line, with the specific proteasome inhibitor β-lactone resulted in increased nuclear levels of C/EBPβ as determined by Western blotting and immunofluorescent detection. This effect of β-lactone reflected inhibited degradation of C/EBPβ. Surprisingly, the increased C/EBPβ levels in β-lactone-treated myotubes did not result in increased DNA-binding activity. In additional experiments, treatment of the myotubes with β-lactone resulted in increased nuclear levels of growth arrest DNA damage/C/EBP homologous protein (Gadd153/CHOP), a dominant-negative member of the C/EBP family that can form heterodimers with other members of the C/EBP family and block DNA binding. Coimmunoprecipitation and immunofluorescent detection provided evidence that C/EBPβ and Gadd153/CHOP interacted and colocalized in the nuclei of the β-lactone-treated myotubes. When Gadd153/CHOP expression was downregulated by transfection of myotubes with siRNA targeting Gadd153/CHOP, C/EBPβ DNA-binding activity was restored in β-lactone-treated myotubes. The results suggest that C/EBPβ is degraded by a proteasome-dependent mechanism in skeletal muscle cells and that Gadd153/CHOP can interact with C/EBPβ and block its DNA-binding activity. The observations are important because they increase the understanding of the complex regulation of the expression and activity of C/EBPβ in skeletal muscle.

scholarly journals Two monomers of yeast transcription factor ADR1 bind a palindromic sequence symmetrically to activate ADH2 expression.

1991 ◽  
Vol 11 (3) ◽  
pp. 1566-1577 ◽  
Author(s):  
S K Thukral ◽  
A Eisen ◽  
E T Young
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Dimethyl Sulfate ◽  
Binding Activity ◽  
Single Amino Acid ◽  
Minor Effect ◽  
Palindromic Sequence ◽  
Amino Terminal ◽  
Dna Binding Activity

ADR1 is a transcription factor from Saccharomyces cerevisiae that regulates ADH2 expression through a 22-bp palindromic sequence (UAS1). Size fractionation studies revealed that full-length ADR1 and a truncated ADR1 protein containing the first 229 amino acids, which has the complete DNA-binding domain, ADR1:17-229, exist as monomers in solution. However, two complexes were formed with target DNA-binding sites. UV-cross-linking studies suggested that these two complexes represent one and two molecules of ADR1 bound to DNA. Studies of ADR1 complexes formed with wild-type UAS1, asymmetrically altered UAS1, and one half of UAS1 showed that ADR1 can bind to one half of UAS1 and gives rise to a complex containing one molecule of ADR1. Dimethyl sulfate interference studies were consistent with this interpretation and in addition indicated that purine contact sites in each half of UAS1 were identical. Increasing the distance between the two halves of UAS1 had at most a minor effect of the thermodynamics of formation of the two complexes. These data are more consistent with ADR1 binding as two independent monomers, one to each half of UAS1. However, binding of two ADR1 monomers at UAS1 is apparently essential for transactivation in vivo. Further, we have identified a stretch of 18 amino acid residues amino terminal to the zinc two-finger domains of ADR1 which is essential for DNA-binding activity. Single amino acid substitutions of residues in this region resulted in severely reduced DNA-binding activity.

scholarly journals A Highly Organized Structure Mediating Nuclear Localization of a Myb2 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

2011 ◽  
Vol 10 (12) ◽  
pp. 1607-1617 ◽  
Author(s):  
Chien-Hsin Chu ◽  
Lung-Chun Chang ◽  
Hong-Ming Hsu ◽  
Shu-Yi Wei ◽  
Hsing-Wei Liu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Trichomonas Vaginalis ◽  
Simian Virus 40 ◽  
Binding Activity ◽  
T Antigen ◽  
Structural Domain ◽  
Dna Binding Activity

ABSTRACT Nuclear proteins usually contain specific peptide sequences, referred to as nuclear localization signals (NLSs), for nuclear import. These signals remain unexplored in the protozoan pathogen, Trichomonas vaginalis . The nuclear import of a Myb2 transcription factor was studied here using immunodetection of a hemagglutinin-tagged Myb2 overexpressed in the parasite. The tagged Myb2 was localized to the nucleus as punctate signals. With mutations of its polybasic sequences, 48KKQK51 and 61KR62, Myb2 was localized to the nucleus, but the signal was diffusive. When fused to a C-terminal non-nuclear protein, the Myb2 sequence spanning amino acid (aa) residues 48 to 143, which is embedded within the R2R3 DNA-binding domain (aa 40 to 156), was essential and sufficient for efficient nuclear import of a bacterial tetracycline repressor (TetR), and yet the transport efficiency was reduced with an additional fusion of a firefly luciferase to TetR, while classical NLSs from the simian virus 40 T-antigen had no function in this assay system. Myb2 nuclear import and DNA-binding activity were substantially perturbed with mutation of a conserved isoleucine (I74) in helix 2 to proline that altered secondary structure and ternary folding of the R2R3 domain. Disruption of DNA-binding activity alone by point mutation of a lysine residue, K51, preceding the structural domain had little effect on Myb2 nuclear localization, suggesting that nuclear translocation of Myb2, which requires an ordered structural domain, is independent of its DNA binding activity. These findings provide useful information for testing whether myriad Mybs in the parasite use a common module to regulate nuclear import.

Identification of Cyclin D3 as a Direct Transcriptional Target of E2A Using Damid.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1618-1618
Author(s):  
John K. Choi ◽  
Siyuan Song ◽  
Jonathan Cooperman ◽  
Danielle L. Letting ◽  
Gerd A. Blobel
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
B Cell ◽  
Target Genes ◽  
Cell Development ◽  
Binding Activity ◽  
B Cell Development ◽  
Cyclin D3 ◽  
Dna Binding Activity ◽  
E Boxes

Abstract The transcription factor E2A is required for very early B cell development. The exact mechanism by which E2A promotes B cell development is unclear and cannot be explained by the known E2A targets, components of the pre-B cell receptor and cyclin dependent kinase inhibitors, indicating additional pathways and targets remain to be identified. We had previously reported that E2A can promote precursor B cell expansion, promote G1 cell cycle progression, and induce the expressions of multiple G1 phase cyclins including cyclin D3, suggesting that E2A induction of these genes may contribute to early B cell development. To better understand the mechanism by which E2A induces these cyclins, we characterized the relationship between E2A and the cyclin D3 gene promoter. E2A transactivated a luciferase reporter plasmid containing the 1kb promoter of cyclin D3 that contains two consensus E2A binding sites (E-boxes); however, deletion of the E-boxes did not disrupt the transactivation by E2A. We hypothesized three possible mechanisms: 1) indirect activation of cyclin D3 via another transcription factor, 2) binding of E2A to cryptic non-E-boxes, or 3) recruitment of E2A to the promoter via interaction with other DNA binding factor. To test the first possibility, promoter occupancy was examined using the DamID approach. In this approach, a fusion protein consisting of E. coli DNA adenosine methyltransferase (DAM) and a transcription factor of interest is expressed at low levels, resulting in specific methylation of adenosine residues within 2–5 kb of the transcription factor target sites. A fusion construct composed of E2A and DAM (E47Dam), was subcloned in lentiviral vectors, and used to transduce precursor B cell lines. The methylated adenosine residues were detected using a sensitive ligation-mediated PCR (LM-PCR) assay that required only 1 ug of genomic DNA and can detect methylation even if only 3% of the cells express E47Dam; no methylated adenosines were detected in control cells, indicating that all methylated residues resulted from E47Dam. Specific adenosine methylation was identified at the IgH intronic enhancer, a known E2A target site, but not at the non-target sites, CD19, HPRT, and GAPDH promoters. Specific methylation was detected at the cyclin D3 promoter but not 10 kb down-stream, despite similar concentrations of E-boxes at both sites. Chromatin immunoprecipitation analysis confirmed the DamID findings and further localized the binding to within 1 kb of the two E-boxes in the cyclin D3 promoter. To distinguish between the two remaining mechanisms (cryptic non-E-boxes versus recruitment via other DNA binding factors), two point mutations were introduced into E47Dam that disrupted its DNA binding activity. The mutated E47Dam continued to methylate at the cyclin D3 promoter. We conclude that E2A can be recruited to the cyclin D3 promoter, independent of E-boxes or E2A DNA binding activity. Our findings raise the possibility that some direct E2A target genes may lack functional E-boxes. Furthermore, mutated E2A, lacking an E2A DNA binding domain, that is seen in 6% of pediatric ALLs may still activate a subset of E2A target genes. Finally, our application of lentiviral vectors and LM-PCR to the DamID approach should permit analysis of primary human precursor B cells, despite the limitations in cell number and transduction efficiency.

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