scholarly journals GATA-1 binding to the alphaV promoter negatively regulates expression of the integrin alphaV subunit in human leukemic K562 cells.

2002 ◽  
Vol 49 (1) ◽  
pp. 19-28 ◽  
Author(s):  
Małgorzata Czyz ◽  
Marta Stasiak ◽  
Joanna Boncela ◽  
Czesław S Cierniewsk
Keyword(s):  
Vascular Endothelial Cells ◽  
Gene Promoter ◽  
K562 Cells ◽  
Nuclear Extract ◽  
Binding Activity ◽  
Vascular Endothelial ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Mobility Shift Assay ◽  
Lower Expression

Recently we observed that the transcription factors Sp1 and Sp3 bind to the CTCCTCCTC sequence located between positions -194 and -172 of the alphaV promoter region and are directly involved in the regulation of transcriptional activity of the alphaV gene in human umbilical vascular endothelial cells (HUVECs) (Czyz & Cierniewski, 1999, Eur. J. Biochem. 265, 638). In this report we provide evidence that the GATA-1 factor regulates alphaV expression during differentiation of pluripotent K562 cells induced either by phorbol 12-myristate 13-acetate (PMA) or butyric acid (BA) through interaction with the GATA element in the alphaV gene promoter. DNase I footprinting analysis revealed that region -413 to -408, covering the GATA binding site, was protected by nuclear extract from K562 cells. There was no protection of this region by HUVEC nuclear extract. Electrophoretic mobility shift assay (EMSA) analysis of nuclear extract of K562 cells treated with BA revealed an increase in GATA binding activity, which was associated with reduced alphaV mRNA and alphaV protein on the cell surface. Stimulation of K562 cells with PMA resulted in opposite effects: lower expression of GATA-1 was associated with increased levels of alphaV. We conclude that the GATA-1 transcription factor specifically binds to the GATA element in the alphaV gene promoter and negatively regulates alphaV gene expression.

scholarly journals Of the GATA-binding proteins, only GATA-4 selectively regulates the human interleukin-5 gene promoter in interleukin-5-producing cells which express multiple GATA-binding proteins.

1995 ◽  
Vol 15 (7) ◽  
pp. 3830-3839 ◽  
Author(s):  
T Yamagata ◽  
J Nishida ◽  
R Sakai ◽  
T Tanaka ◽  
H Honda ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Regulatory Element ◽  
Gene Promoter ◽  
Nuclear Extract ◽  
Proximal Promoter ◽  
Binding Motif ◽  
Mobility Shift ◽  
Cis Acting ◽  
Interleukin 5 ◽  
Mobility Shift Assay

Interleukin-5 (IL-5) is produced by T lymphocytes and known to support B-cell growth and eosinophilic differentiation of the progenitor cells. Using ATL-16T cells which express IL-5 mRNA, we have identified a region within the human IL-5 gene promoter that regulates IL-5 gene transcription. This cis-acting sequence contains the core binding motif, (A/T)GATA(A/G), for GATA-binding family proteins and thus suggests the involvement of this family members. In this report, we describe the cloning of human GATA-4 (hGATA-4) and show that hGATA-4 selectively interacts with the -70 GATA site within the IL-5 proximal promoter region. By promoter deletion and mutation analyses, we established this region as a positive regulatory element. Cotransfection experiments revealed that both hGATA-4 and phorbol-12-myristate-13-acetate (PMA)-A23187 stimulation are necessary for IL-5 promoter activation. The requirement for another regulatory element called CLE0, which lies downstream of the -70 GATA site, was also demonstrated. ATL-16T cells express mRNAs of three GATA-binding proteins, hGATA-2, hGATA-3, and hGATA-4, and each of them has a potential to bind to the consensus (A/T)GATA(G/A) motif. However, using ATL-16T nuclear extract, we demonstrated that GATA-4 is the only GATA-binding protein that forms a specific DNA-protein complex with the -70 GATA site. An electrophoretic mobility shift assay with extracts of COS cells expressing GATA-binding proteins showed that GATA-4 has the highest binding affinity for the -70 GATA site among the three GATA-binding proteins. When the transactivation abilities were compared among the three, GATA-4 showed the highest activity. These results demonstrate the selective role of GATA-4 in the transcriptional regulation of the IL-5 gene in a circumstance where multiple members of the GATA-binding proteins are expressed.

Mpl Ligand Enhances the Transcription of the Cyclin D3 Gene: A Potential Role for Sp1 Transcription Factor

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4208-4221 ◽  
Author(s):  
Zhengyu Wang ◽  
Ying Zhang ◽  
Jun Lu ◽  
Shinnshin Sun ◽  
Katya Ravid
Keyword(s):  
Potential Role ◽  
Binding Activity ◽  
Cyclin D3 ◽  
Protein Phosphatase 1 ◽  
Mobility Shift ◽  
Sp1 Transcription Factor ◽  
Dnase I Footprinting ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
And Shifts

Cyclin D3 plays a major role in the development of polyploidy in megakaryocytes. The expression of cyclin D3 gene and the level of cyclin D3 protein are increased by the Mpl ligand in the Y10/L8057 megakaryocytic cell line, as indicated by Northern and Western blot analyses, and by nuclear run-on assays and transfection experiments with cyclin D3 promoter constructs. DNase I footprinting of the promoter region showed protected segments, at −75 to −60 bp and at −134 to −92 bp, which display binding sites for the Sp family of transcription factors. Gel mobility shift assay and supershifts with specific antibodies indicate that Sp1 binds to these regions in the cyclin D3 promoter and that Sp1 binding activity is significantly increased by Mpl ligand. Mutation of either Sp1 site both decreases the basal promoter activity and eliminates the induction by Mpl ligand. We find that the nonphosphorylated form of SP1 has greater affinity for the cyclin D3 promoter and that the majority of Sp1 in the cells is nonphosphorylated. Mpl ligand treatment results in increased levels of Sp1 protein, which also appears as nonphosphorylated. Okadaic acid, which inhibits protein phosphatase 1 (PP1) and shifts Sp1 to a phosphorylated form, decreases cyclin D3 gene expression and suppresses Mpl ligand induction. Our data point to the potential of Mpl ligand to activate at once several Sp1-dependent genes during megakaryopoiesis.

Mpl Ligand Enhances the Transcription of the Cyclin D3 Gene: A Potential Role for Sp1 Transcription Factor

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4208-4221 ◽  
Author(s):  
Zhengyu Wang ◽  
Ying Zhang ◽  
Jun Lu ◽  
Shinnshin Sun ◽  
Katya Ravid
Keyword(s):  
Potential Role ◽  
Binding Activity ◽  
Cyclin D3 ◽  
Protein Phosphatase 1 ◽  
Mobility Shift ◽  
Sp1 Transcription Factor ◽  
Dnase I Footprinting ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
And Shifts

Abstract Cyclin D3 plays a major role in the development of polyploidy in megakaryocytes. The expression of cyclin D3 gene and the level of cyclin D3 protein are increased by the Mpl ligand in the Y10/L8057 megakaryocytic cell line, as indicated by Northern and Western blot analyses, and by nuclear run-on assays and transfection experiments with cyclin D3 promoter constructs. DNase I footprinting of the promoter region showed protected segments, at −75 to −60 bp and at −134 to −92 bp, which display binding sites for the Sp family of transcription factors. Gel mobility shift assay and supershifts with specific antibodies indicate that Sp1 binds to these regions in the cyclin D3 promoter and that Sp1 binding activity is significantly increased by Mpl ligand. Mutation of either Sp1 site both decreases the basal promoter activity and eliminates the induction by Mpl ligand. We find that the nonphosphorylated form of SP1 has greater affinity for the cyclin D3 promoter and that the majority of Sp1 in the cells is nonphosphorylated. Mpl ligand treatment results in increased levels of Sp1 protein, which also appears as nonphosphorylated. Okadaic acid, which inhibits protein phosphatase 1 (PP1) and shifts Sp1 to a phosphorylated form, decreases cyclin D3 gene expression and suppresses Mpl ligand induction. Our data point to the potential of Mpl ligand to activate at once several Sp1-dependent genes during megakaryopoiesis.

scholarly journals Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes.

1991 ◽  
Vol 11 (3) ◽  
pp. 1488-1499 ◽  
Author(s):  
H J Roth ◽  
G C Das ◽  
J Piatigorsky
Keyword(s):  
Transcription Factors ◽  
Hela Cell ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Regulatory Elements ◽  
Dnase I ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Dnase I Footprinting

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.

Tissue-specific transcription of the mouse alpha-fetoprotein gene promoter is dependent on HNF-1

1989 ◽  
Vol 9 (10) ◽  
pp. 4204-4212
Author(s):  
M H Feuerman ◽  
R Godbout ◽  
R S Ingram ◽  
S M Tilghman
Keyword(s):  
Specific Binding ◽  
Gene Promoter ◽  
Binding Activity ◽  
Alpha Fetoprotein ◽  
Transient Expression Assay ◽  
Band Shift ◽  
Specific Expression ◽  
Tissue Specific ◽  
Cis Acting ◽  
Dnase I Footprinting

Previous work identified four upstream cis-acting elements required for tissue-specific expression of the alpha-fetoprotein (AFP) gene: three distal enhancers and a promoter. To further define the role of the promoter in regulating AFP gene expression, segments of the region were tested for the ability to direct transcription of a reporter gene in transient expression assay. Experiments showed that the region within 250 base pairs of the start of transcription was sufficient to confer liver-specific transcription. DNase I footprinting and band shift assays indicated that the region between -130 and -100 was recognized by two factors, one of which was highly sequence specific and found only in hepatoma cells. Competition assays suggested that the liver-specific binding activity was HNF-1, previously identified by its binding to other liver-specific promoters. Mutation of the HNF-1 recognition site at -120 resulted in a significant reduction in transcription in transfection assays, suggesting a biological role for HNF-1 in the regulation of AFP expression.

scholarly journals Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver

2005 ◽  
Vol 70 (5) ◽  
pp. 705-712 ◽  
Author(s):  
Miroslava Vujcic ◽  
Natasa Terzic ◽  
Aleksandra Ristic-Fira ◽  
Dusan Kanazir ◽  
Sabera Ruzdijic
Keyword(s):  
Dna Interaction ◽  
Binding Activity ◽  
Regulatory Proteins ◽  
Nuclear Proteins ◽  
Middle Aged ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Supershift Assay ◽  
Mobility Shift Assay ◽  
Oligonucleotide Analogues

Abstract: In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p < 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing. .

DNA-binding factors of B lymphoid cells are susceptible to limited proteolytic cleavage during nuclear extract preparation

1988 ◽  
Vol 8 (4) ◽  
pp. 1812-1815
Author(s):  
E L Mather
Keyword(s):  
Dna Binding ◽  
Nuclear Extract ◽  
Lymphoid Cells ◽  
Chain Gene ◽  
Immunoglobulin Heavy Chain Gene ◽  
Mobility Shift ◽  
Lymphoid Lines ◽  
Dna Binding Factors ◽  
Mobility Shift Assay ◽  
B Lymphoid

DNA-binding proteins that interact with the 3' end of the mouse mu immunoglobulin heavy chain gene were identified by the electrophoretic mobility shift assay. Complexes of distinctly different mobilities were formed by extracts prepared from B lymphoid lines representing different stages of maturation. The apparent stage-specific differences are shown to be due to proteolytic events that occurred during extract preparation.

scholarly journals DNA-binding activity in the excretory–secretory products ofTrichinella pseudospiralis(Nematoda: Trichinelloidea)

Parasitology ◽  
2001 ◽  
Vol 123 (3) ◽  
pp. 301-308 ◽  
Author(s):  
C. H. MAK ◽  
R. C. KO
Keyword(s):  
Dna Binding ◽  
Electrophoretic Mobility ◽  
Protein Complexes ◽  
Binding Activity ◽  
Mobility Shift ◽  
Competition Analysis ◽  
Dna Binding Activity ◽  
Supershift Assay ◽  
Mobility Shift Assay ◽  
Secretory Products

A novel DNA-binding peptide ofMr∼30 kDa was documented for the first time in the excretory–secretory (E–S) products of the infective-stage larvae ofTrichinella pseudospiralis.Larvae recovered from muscles of infected mice were maintained for 48 h in DMEM medium. E–S products of worms extracted from the medium were analysed for DNA-binding activity by the electrophoretic mobility shift assay (EMSA). Multiple DNA-protein complexes were detected. A comparison of theMrof proteins in the complexes indicated that they could bind to the target DNA as a dimer, tetramer or multiples of tetramers. Site selection and competition analysis showed that the binding has a low specificity. A (G/C-rich)-gap-(G/T-rich)-DNA sequence pattern was extracted from a pool of degenerate PCR fragments binding to the E–S products. Results of immunoprecipitation and electrophoretic mobility supershift assay confirmed the authenticity of the DNA-binding protein as an E–S product.

scholarly journals A Sequence of the CIS Gene Promoter Interacts Preferentially with Two Associated STAT5A Dimers: a Distinct Biochemical Difference between STAT5A and STAT5B

1998 ◽  
Vol 18 (10) ◽  
pp. 5852-5860 ◽  
Author(s):  
Frédérique Verdier ◽  
Raquel Rabionet ◽  
Fabrice Gouilleux ◽  
Christian Beisenherz-Huss ◽  
Paule Varlet ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Prolactin Receptor ◽  
Gene Promoter ◽  
Expression Vectors ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Mobility Shift Assay ◽  
Binding Sequence ◽  
Biochemical Difference

ABSTRACT Two distinct genes encode the closely related signal transducer and activator of transcription proteins STAT5A and STAT5B. The molecular mechanisms of gene regulation by STAT5 and, particularly, the requirement for both STAT5 isoforms are still undetermined. Only a few STAT5 target genes, among them the CIS (cytokine-inducible SH2-containing protein) gene, have been identified. We cloned the human CIS gene and studied the human CIS gene promoter. This promoter contains four STAT binding elements organized in two pairs. By electrophoretic mobility shift assay studies using nuclear extracts of UT7 cells stimulated with erythropoietin, we showed that these four sequences bound to STAT5-containing complexes that exhibited different patterns and affinities: the three upstream STAT binding sequences bound to two distinct STAT5-containing complexes (C0 and C1) and the downstream STAT box bound only to the slower-migrating C1 band. Using nuclear extracts from COS-7 cells transfected with expression vectors for the prolactin receptor, STAT5A, and/or STAT5B, we showed that the C1 complex was composed of a STAT5 tetramer and was dependent on the presence of STAT5A. STAT5B lacked this property and bound with a stronger affinity than did STAT5A to the four STAT sequences as a homodimer (C0 complex). This distinct biochemical difference between STAT5A and STAT5B was confirmed with purified activated STAT5 recombinant proteins. Moreover, we showed that the presence on the same side of the DNA helix of a second STAT sequence increased STAT5 binding and that only half of the palindromic STAT binding sequence was sufficient for the formation of a STAT5 tetramer. Again, STAT5A was essential for this cooperative tetrameric association. This property distinguishes STAT5A from STAT5B and could be essential to explain the transcriptional regulation diversity of STAT5.

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.

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