Single-strand-DNA-binding factors specifically recognize the pyrimidine element in the chick α2(I) collagen gene promoter

A pyrimidine element with mirror repeats centred at position -192 bp of the chick α2(I) collagen promoter interacts with sequence-specific DNA-binding factors. These factors bind to only the pyrimidine strand of this region and have no affinity for the complementary purine strand. Binding activity is also seen with the double-stranded form of this element, but with less affinity than to the single-stranded pyrimidine species. Southwestern blot analyses have shown that proteins of 80 and 134 kDa in chick embryo fibroblast nuclear extracts bind to the pyrimidine strand, whereas only a 134 kDa DNA-binding protein was found in chondrocyte nuclear extracts. The binding mechanism of these nuclear proteins with single-stranded DNA might be based on a non-B-DNA conformation of the pyrimidine element. The position of this binding site in the promoter region, its potential for adopting an unusual secondary structure and the presence of the 80 kDa binding factor in chick embryo fibroblasts, but not in chondrocytes, suggest a possible role for this factor in the expression of the α2(I) collagen gene.

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Only two of the five zinc fingers of the eukaryotic transcriptional repressor PRDI-BF1 are required for sequence-specific DNA binding

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.1940-1949.1992 ◽

1992 ◽

Vol 12 (5) ◽

pp. 1940-1949

Author(s):

A D Keller ◽

T Maniatis

Keyword(s):

Dna Binding ◽

Zinc Finger ◽

Specific Binding ◽

Regulatory Element ◽

Zinc Fingers ◽

Transcriptional Repressor ◽

Gene Promoter ◽

Binding Activity ◽

Necessary And Sufficient ◽

Sequence Requirements

The eukaryotic transcriptional repressor PRDI-BF1 contains five zinc fingers of the C2H2 type, and the protein binds specifically to PRDI, a 14-bp regulatory element of the beta interferon gene promoter. We have investigated the amino acid sequence requirements for specific binding to PRDI and found that the five zinc fingers and a short stretch of amino acids N terminal to the first finger are necessary and sufficient for PRDI-specific binding. The contribution of individual zinc fingers to DNA binding was investigated by inserting them in various combinations into another zinc finger-containing DNA-binding protein whose own fingers had been removed. We found that insertion of PRDI-BF1 zinc fingers 1 and 2 confer PRDI-binding activity on the recipient protein. In contrast, the insertion of PRDI-BF1 zinc fingers 2 through 5, the insertion of zinc finger 1 or 2 alone, and the insertion of zinc fingers 1 and 2 in reverse order did not confer PRDI-binding activity. We conclude that the first two PRDI-BF1 zinc fingers together are sufficient for the sequence-specific recognition of PRDI.

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CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7612-7624.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7612-7624

Author(s):

E M Klenova ◽

R H Nicolas ◽

H F Paterson ◽

A F Carne ◽

C M Heath ◽

...

Keyword(s):

Dna Binding ◽

Cell Lines ◽

Gene Promoter ◽

Differentially Expressed ◽

Ctcf Binding ◽

Ctcf Binding Site ◽

Mobility Shift ◽

Chicken Cell ◽

Nuclear Extracts ◽

Myc Gene

A novel sequence-specific DNA-binding protein, CTCF, which interacts with the chicken c-myc gene promoter, has been identified and partially characterized (V. V. Lobanenkov, R. H. Nicolas, V. V. Adler, H. Paterson, E. M. Klenova, A. V. Polotskaja, and G. H. Goodwin, Oncogene 5:1743-1753, 1990). In order to test directly whether binding of CTCF to one specific DNA region of the c-myc promoter is important for chicken c-myc transcription, we have determined which nucleotides within this GC-rich region are responsible for recognition of overlapping sites by CTCF and Sp1-like proteins. Using missing-contact analysis of all four nucleotides in both DNA strands and homogeneous CTCF protein purified by sequence-specific chromatography, we have identified three sets of nucleotides which contact either CTCF or two Sp1-like proteins binding within the same DNA region. Specific mutations of 3 of 15 purines required for CTCF binding were designed to eliminate binding of CTCF without altering the binding of other proteins. Electrophoretic mobility shift assay of nuclear extracts showed that the mutant DNA sequence did not bind CTCF but did bind two Sp1-like proteins. When introduced into a 3.3-kbp-long 5'-flanking noncoding c-myc sequence fused to a reporter CAT gene, the same mutation of the CTCF binding site resulted in 10- and 3-fold reductions, respectively, of transcription in two different (erythroid and myeloid) stably transfected chicken cell lines. Isolation and analysis of the CTCF cDNA encoding an 82-kDa form of CTCF protein shows that DNA-binding domain of CTCF is composed of 11 Zn fingers: 10 are of C2H2 class, and 1 is of C2HC class. CTCF was found to be abundant and conserved in cells of vertebrate species. We detected six major nuclear forms of CTCF protein differentially expressed in different chicken cell lines and tissues. We conclude that isoforms of 11-Zn-finger factor CTCF which are present in chicken hematopoietic HD3 and BM2 cells can act as a positive regulator of the chicken c-myc gene transcription. Possible functions of other CTCF forms are discussed.

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Repressor Activity of SqrR, a Master Regulator of Persulfide-Responsive Genes, Is Regulated by Heme Coordination

Plant and Cell Physiology ◽

10.1093/pcp/pcaa144 ◽

2020 ◽

Author(s):

Takayuki Shimizu ◽

Yuuki Hayashi ◽

Munehito Arai ◽

Shawn E McGlynn ◽

Tatsuru Masuda ◽

...

Keyword(s):

Dna Binding ◽

Transcriptional Repression ◽

Structural Changes ◽

Gene Promoter ◽

Binding Activity ◽

Heme Binding ◽

Master Regulator ◽

Incremental Change ◽

Dna Binding Activity

Abstract Reactive sulfur species (RSS) are involved in bioactive regulation via persulfidation of proteins. However, how cells regulate RSS-based signaling and RSS metabolism is poorly understood, despite the importance of universal regulation systems in biology. We previously showed that the persulfide-responsive transcriptional factor SqrR acts as a master regulator of sulfide-dependent photosynthesis in proteobacteria. Here, we demonstrated that SqrR also binds heme at a near one-to-one ratio with a binding constant similar to other heme-binding proteins. Heme does not change the DNA-binding pattern of SqrR to the target gene promoter region; however, DNA-binding affinity of SqrR is reduced by the binding of heme, altering its regulatory activity. Circular dichroism spectroscopy clearly showed secondary structural changes in SqrR by the heme binding. Incremental change in the intracellular heme concentration is associated with small, but significant reduction in the transcriptional repression by SqrR. Overall, these results indicate that SqrR has an ability to bind heme to modulate its DNA-binding activity, which may be important for the precise regulation of RSS metabolism in vivo.

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Redox regulation of DNA binding activity of DREF (DNA replication-related element binding factor) in Drosophila

Biochemical Journal ◽

10.1042/bj20031601 ◽

2004 ◽

Vol 378 (3) ◽

pp. 833-838 ◽

Cited By ~ 8

Author(s):

Tae-Yeong CHOI ◽

S. Young PARK ◽

Ho-Sung KANG ◽

Jae-Hun CHEONG ◽

Han-Do KIM ◽

...

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Redox State ◽

Redox Regulation ◽

Binding Activity ◽

Dna Binding Activity ◽

Cell Extracts ◽

Binding Factor ◽

Related Element ◽

Intracellular Redox

DREF [DRE (DNA replication-related element) binding factor] is an 80 kDa polypeptide homodimer which plays an important role in regulating cell proliferation-related genes. Both DNA binding and dimer formation activities are associated with residues 16–115 of the N-terminal region. However, the mechanisms by which DREF dimerization and DNA binding are regulated remain unknown. Here, we report that the DNA binding activity of DREF is regulated by a redox mechanism, and that the cysteine residues are involved in this regulation. Electrophoretic mobility shift analysis using Drosophila Kc cell extracts or recombinant DREF proteins indicated that the DNA binding domain is sufficient for redox regulation. Site-directed mutagenesis and transient transfection assays showed that Cys59 and/or Cys62 are critical both for DNA binding and for redox regulation, whereas Cys91 is dispensable. In addition, experiments using Kc cells indicated that the DNA binding activity and function of DREF are affected by the intracellular redox state. These findings give insight into the exact nature of DREF function in the regulation of target genes by the intracellular redox state.

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E4F and ATF, two transcription factors that recognize the same site, can be distinguished both physically and functionally: a role for E4F in E1A trans activation.

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5138 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5138-5149 ◽

Cited By ~ 17

Author(s):

R J Rooney ◽

P Raychaudhuri ◽

J R Nevins

Keyword(s):

Dna Binding ◽

Binding Activity ◽

Stable Complex ◽

Small Subset ◽

Adenovirus Infection ◽

Binding Assays ◽

Dna Binding Activity ◽

Sequence Recognition ◽

Nuclear Factors ◽

Dna Binding Factors

Previous experiments have identified an element in the adenovirus E4 promoter that is critical for E1A-dependent trans activation and that can confer inducibility to a heterologous promoter. This DNA element is a recognition site for multiple nuclear factors, including ATF, which is likely a family of DNA-binding factors with similar DNA recognition properties. However, ATF activity was found not to be altered in any demonstrable way as a result of adenovirus infection. In contrast, another factor that recognizes this element, termed E4F, was found at only very low levels in uninfected cells but was increased markedly upon adenovirus infection, as measured in DNA-binding assays. Although both the ATF activity and the E4F activity recognized and bound to the same two sites in the E4 promoter, they differed in their sequence recognition of these sites. Furthermore, E4F bound only to a small subset of the ATF recognition sites; for instance, E4F did not recognize the ATF sites in the E2 or E3 promoters. Various E4F and ATF binding sites were inserted into an expression vector and tested by cotransfection assays for responsiveness to E1A. We found that a sequence capable of binding E4F could confer E1A inducibility. In contrast, a sequence that could bind ATF but not E4F did not confer E1A inducibility. We also found that E4F formed a stable complex with the E4 promoter, whereas the ATF DNA complex was unstable and rapidly dissociated. We conclude that the DNA-binding specificity of E4F as well as the alterations in DNA-binding activity of E4F closely correlates with E1A stimulation of the E4 promoter.

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Regulatory Proteins Bind at the Polymorphic NFSE Element of CYP3A4.

Blood ◽

10.1182/blood.v106.11.4453.4453 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4453-4453

Author(s):

Tal David-Kalish ◽

Deborah Rund ◽

Elad Malik ◽

Sara Bar Cohen

Keyword(s):

Dna Binding ◽

Cell Lines ◽

Blast Crisis ◽

Binding Activity ◽

Dna Probe ◽

Luciferase Expression ◽

Cytochrome P450 Enzyme ◽

Mobility Shift ◽

Dna Binding Activity ◽

Nuclear Extracts

Abstract CYP3A4 is the most abundant cytochrome P450 enzyme in the liver and is involved in the metabolism of most clinically used drugs. An A to G substitution in the nifedipine responsive element (NFSE) in the promoter of this gene has been found to be associated with a lower incidence of pediatric therapy-related leukemia (Felix, Proc Natl Acad Sci USA95:13176, 1998) and adult therapy-related leukemia (Rund et al, Leukemia, accepted for publication). To study the effect of this polymorphism on gene expression in hematopoietic cells, we constructed reporter plasmids with the luciferase gene (in pGL3E) under control of the CYP3A4 promoter, using both the polymorphic and normal sequences. These plasmids were transfected into several cell lines of hematopoietic origin and luciferase was quantitated. We used KG1a (myeloid leukemia), K562 (CML blast crisis), and as controls, MelA1, a melanoma line and HepG2, a hepatoma line. Experiments were repeated at least three times for each cell line. The results consistently demonstrated 20–30% lower luciferase activity (in KG1a and K562 respectively) using the polymorphic sequence as compared to the normal sequence while the MelA1 and HepG2 lines showed the opposite effect, a 25% higher luciferase expression with the variant sequence. The results for HepG2 were in agreement with those reported by Rebbeck (Environmental and Molecular Mutagenesis49:299, 2003). To identify the factors binding at NFSE which may influence expression, electrophoretic mobility shift assays were performed using nuclear extracts of both cell lines (K562, KG1a, and HL60) and patient leukemia cells with a DNA probe representing the normal and polymorphic sequences. A gel shift was demonstrated, indicating binding of nuclear extracts to the region of the polymorphism. The database of transacting factors states complete homology of the polymorphic sequence of the NFSE region with the consensus binding site of HSF-1. We therefore performed a series of experiments to determine if HSF-1 is the protein binding at that site. HSF-1 is a multimeric transcription factor which binds to heat shock elements in many promoters which are rapidly transcribed following stress by increases in temperature. We found that recombinant HSF-1 did not bind to the DNA probe alone. However, nuclear extracts of cells which underwent stress by heating to 43°C for one hour (which is known to increase HSF-1 production) demonstrated increased binding to the probe representing the region of the polymorphism and Western blotting demonstrated more HSF-1 in these extracts. Using a Streptavidin-biotin system with a DNA fragment representing the NFSE region, we demonstrated that DNA binding activity to the probe was present in the elution fractions which contained HSF-1, as detected by ECL (enhanced chemoluminescence). Elution fractions which did not show DNA binding activity did not contain detectable HSF-1. We conclude that HSF-1 may be the protein which binds at the NFSE element of the CYP3A promoter but that it binds either as a multimer or as part of a complex of several proteins, which complicates its detection as a DNA binding protein.

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Transcription of the mouse secretory protease inhibitor p12 gene is activated by the developmentally regulated positive transcription factor Sp1

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3796-3806.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3796-3806

Author(s):

S Robidoux ◽

P Gosselin ◽

M Harvey ◽

S Leclerc ◽

S L Guérin

Keyword(s):

Transcription Factor ◽

Mutational Analysis ◽

Dimethyl Sulfate ◽

Gene Promoter ◽

Binding Activity ◽

Regulatory Sequence ◽

Culture Cells ◽

Transcription Factor Sp1 ◽

Nuclear Extracts ◽

Mouse Tissues

We have previously shown that a trans-acting protein produced in some tissue culture cells positively control the transcriptional activity directed by the mouse p12 promoter. This nuclear protein exerts its positive activity by interacting with a regulatory sequence designated p12.A and located between the TATA and CCAAT box elements on the p12 gene promoter. Using DNase I and dimethyl sulfate methylation interference footprinting techniques coupled with gel retardation assays, we found evidence that the protein which binds to the p12.A element is the well-known transcription factor Sp1. Mutational analysis in transient transfection assays confirmed the positive activity exerted by this protein in every cell line tested. In agreement with this observation, we detected a p12.A-Sp1 binding activity in nuclear extracts prepared from all cell lines used. However, a similar binding activity could not be detected in a number of nuclear extracts prepared from normal mouse tissues. In this report, we provide the evidence that the lack of Sp1-binding activity results from the degradation of Sp1 in the kidney, liver, and pancreas of the mouse.

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Down-regulation of nuclear aryl hydrocarbon receptor DNA-binding and transactivation functions: requirement for a labile or inducible factor

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.5653-5660.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 5653-5660

Author(s):

M Reick ◽

R W Robertson ◽

D S Pasco ◽

J B Fagan

Keyword(s):

Dna Binding ◽

Cell Line ◽

Binding Activity ◽

Ah Receptor ◽

Down Regulation ◽

Response Elements ◽

Dna Binding Activity ◽

Nuclear Extracts ◽

Xenobiotic Response

Aryl hydrocarbons (AHs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene activate the sequence-specific DNA-binding activity of the AH receptor. In the rat hepatocyte-derived cell line LCS7, DNA-binding activity peaked after 30 min and was then down-regulated, reaching negligible levels by 2 h. Down-regulation could be blocked, and DNA-binding activity maintained at maximum for many hours by inhibiting protein or RNA synthesis, implying that down-regulation is a mediated process requiring a labile or inducible protein. CYP1A1 transcription and in vivo DNA-protein interactions at xenobiotic response elements were down-regulated in parallel with DNA-binding activity in nuclear extracts, and these changes could also be blocked by inhibitors of protein synthesis. The correlation between AH receptor DNA-binding activity, intensity of in vivo footprints at xenobiotic response elements, and CYP1A1 transcription rate implies that down-regulation of AH receptor DNA-binding activity is important in regulating CYP1A1 transcription and that receptor is required continuously to maintain transcription. This correlation extends to the murine hepatoma cell line Hepa-1c1c7, in which slower kinetics of activation and down-regulation of CYP1A1 transcription paralleled slower activation and down-regulation of AH receptor DNA-binding activity. The difference in kinetics between cell lines also implies that AH receptor DNA-binding activity is modulated by a mechanism that may be influenced by cell-specific regulatory pathways. The above observations in conjunction with mixing experiments and comparisons of cytoplasmic and nuclear extracts indicate that down-regulation of AH receptor DNA-binding activity is probably due either to degradation or to conversion of the receptor to form that is inactive in both DNA binding and transactivation.

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Identification of Proteinase 3 (PR3) Gene Overexpresison and Protein Delocalization in Core Binding Factor Leukemias as a Mechanism Leading to Increased Chemosensitivity.

Blood ◽

10.1182/blood.v112.11.204.204 ◽

2008 ◽

Vol 112 (11) ◽

pp. 204-204

Author(s):

Daniela Cilloni ◽

Ilaria Defilippi ◽

Sonia Carturan ◽

Chiara Maffè ◽

Marisa Pautasso ◽

...

Keyword(s):

Dna Binding ◽

Cell Lines ◽

Mean Value ◽

Binding Activity ◽

Proteinase 3 ◽

Loss Of Function ◽

Core Binding Factor ◽

Secondary Aml ◽

Dna Binding Activity ◽

Binding Factor

Abstract Proteinase 3 (PR3) gene codes for a serine protease with a broad spectrum of proteolytic activity. PR3 is involved in the control of proliferation of myeloid leukemia cells. When abnormally expressed it confers factor-independent growth to hematopoietic cells. The aim of this study was to investigate the role of PR3 gene in leukemic haematopoiesis. We analyzed the expression levels of PR3 by RQ-PCR in 113 BM samples collected from AML patients at diagnosis. The FAB distribution was as follows: M0=5, M1=12, M2=38, M3=12, M4=37, M5=5, M6=4. 19 patients were characterized by t(8;21) and 16 by inv(16). PR3 expression level was also analyzed in 57 BM and 42 PB samples from 88 MDS patients (44 RA, 32 RAEB and 12 secondary-AML) and in 15 BM and 40 PB samples from healthy volunteers. PR3 protein was analyzed by western blot (WB) and its localization determined by immunofluorescence assay using specific antibodies. The transcription factor C/EBPα, which negatively regulates PR3 expression was studied in parallel at the RNA and protein level by RQ-PCR and WB. The DNA binding activity of C/EBPα was investigated by EMSA assay. Gain and loss of function experiments were performed by transfecting COS and 293T cell lines with a plasmid containing the full length PR3 sequence and HL60, Me-1, and Kasumi cell lines with specific shRNA. We found that PR3 is significantly overexpressed in AML samples. The median value of 2−Δ ΔCt is 740, (range 15-5043). Interestingly, patients affected by Core Binding Factor leukemias showed significantly higher PR3 values compared to patients with normal karyotypes (NK) (p<0,0002 for t(8;21), p<0,001 for inv16) and lower C/EPBα levels. EMSA assay demonstrated the absence of C/EBPα DNA binding activity in CBF AML cells but not in NK AML. In addition, PR3 overexpression was detected in 60% of RA patients (mean value: 10, range 3–268), and in all the cases of RAEB (mean value 201: range:128–803) and secondary AML (mean value 589, range 207–7131). WB demonstrated the correlation between the mRNA and protein amount. Interestingly, immunofluorescence demonstrated the de-localization of the protein within the nucleous in CBF AML but it is completely cytoplasmatic in leukemic cells with normal karyotype and in MDS. Transfection experiments with PR3 plasmid demonstrated that PR3 overexpression results into a significantly increased proliferation and reduced apoptosis. By contrast transfection with shRNA triggers apoptosis and cell growth inhibition. In addition, WB demonstrated that nuclear PR3 is able to cleavage the p65subunit of NF-kB into a p56 isoform which lacks any transcriptional activity as confirmed by EMSA. In conclusion, PR3 gene expression and protein are significantly increased in AML and MDS, particularly in CBF leukemias in which the protein is not only increased but also completely delocalized within the nucleous. PR3 overepression My be due to a significant downmodulation of C/EBPα. Ectopic expression of PR3 induces increased proliferation and apoptosis arrest. The abnormal nuclear localization of PR3 in CBF leukemias results into the loss of function of NF-kB thus representing one mechanism of chemo sensitivity in this group of patients.

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Y-box proteins interact with the S1 nuclease-sensitive site in the chicken α 2(I) collagen gene promoter

Biochemical Journal ◽

10.1042/bj3190203 ◽

1996 ◽

Vol 319 (1) ◽

pp. 203-207 ◽

Cited By ~ 11

Author(s):

Dashzeveg BAYARSAIHAN ◽

Badam ENKHMANDAKH ◽

Lewis N LUKENS

Keyword(s):

Type I Collagen ◽

Cold Shock ◽

Gene Promoter ◽

Type I ◽

S1 Nuclease ◽

Expression Screening ◽

Nuclear Extracts ◽

Sensitive Site ◽

Collagen Promoter

The sequence of the chicken α2(I) collagen promoter from -712 to -85, relative to exon 1, has been shown to be important for transcriptional activity. Within this region a pyrimidine/purine asymmetrical element at -200 bp forms an in vitro S1 nuclease-sensitive site. The pyrimidine-rich strand of this element interacts specifically with single-stranded DNA-binding proteins present in fibroblast nuclear extracts [Bayarsaihan and Lukens (1996) Biochem. J. 314, 293–296]. To identify these proteins we performed expression screening of a chick embryo fibroblast cDNA library using a single-stranded polypyrimidine sequence derived from this element. One of the isolated clones was found to encode a member of the cold-shock gene family, either chicken YB-1 or a highly homologous protein. This protein and a known chicken Y-box protein were both found to bind sequence-specifically to the pyrimidine-rich strand of the pyrimidine/purine asymmetrical element in the chicken α2(I) collagen promoter. The binding mechanism of these proteins could be based on the formation of a non-canonical triplex DNA structure (H-DNA). Although members of this widespread and conserved protein family have been reported to modulate the expression of a number of genes, the findings reported here provide the first evidence for a possible role of cold-shock proteins in the regulation of type I collagen genes.

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