Mapping Protein-binding Sites on DNA by DNase I Footprinting

2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.prot3947
Author(s):  
Joseph Sambrook ◽  
David W. Russell
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Dnase I ◽  
Protein Binding Sites ◽  
Dnase I Footprinting

DNase I Footprinting to Identify Protein Binding Sites

BIO-PROTOCOL ◽  
2013 ◽  
Vol 3 (14) ◽  
Author(s):  
Isabelle Gaugué ◽  
Dominique Bréchemier-Baey ◽  
Jacqueline Plumbridge
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Dnase I ◽  
Protein Binding Sites ◽  
Dnase I Footprinting

scholarly journals Protein-binding sites within the 5' DNase I-hypersensitive region of the chicken alpha D-globin gene.

1987 ◽  
Vol 7 (6) ◽  
pp. 2059-2069 ◽  
Author(s):  
B Kemper ◽  
P D Jackson ◽  
G Felsenfeld
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Dnase I ◽  
Specific Factor ◽  
Mobility Shift ◽  
Protein Binding Sites ◽  
Dnase I Footprinting ◽  
Flanking Region

We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared.

Protein-binding sites within the 5' DNase I-hypersensitive region of the chicken alpha D-globin gene

1987 ◽  
Vol 7 (6) ◽  
pp. 2059-2069
Author(s):  
B Kemper ◽  
P D Jackson ◽  
G Felsenfeld
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Dnase I ◽  
Specific Factor ◽  
Mobility Shift ◽  
Protein Binding Sites ◽  
Dnase I Footprinting ◽  
Flanking Region

We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared.

scholarly journals Identification of regulatory sequences and protein-binding sites in the liver-type promoter of a gene encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

1991 ◽  
Vol 11 (2) ◽  
pp. 1099-1106 ◽  
Author(s):  
F P Lemaigre ◽  
S M Durviaux ◽  
G G Rousseau
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Specific Protein ◽  
Regulatory Sequences ◽  
Alternative Promoters ◽  
Gene Encoding ◽  
Protein Binding Sites ◽  
Cis Acting ◽  
Dnase I Footprinting ◽  
Nuclear Extracts

The liver-type and muscle-type isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase are encoded by one gene that uses two alternative promoters. We have identified cis-acting sequences and protein-binding sites on the liver-type promoter. Transfection assays with deleted promoters showed that maximal promoter activity is contained within 360 bp upstream of the cap site. DNase I footprinting experiments with liver and spleen nuclear extracts and with purified proteins revealed several protein-binding sites in this region. These included four binding sites for nuclear factor I, one site that contains an octamer consensus but showed a liver-specific footprint pattern, two liver-specific protein-binding sites, and one poly(dG)-containing binding site. Transfection of cells of hepatic origin suggested that all these sites except one are involved in transcriptional regulation. The region between -360 and -2663 contained an element that functioned as a silencer in a nonhepatic cell line. We conclude that in liver transcription from the liver-type promoter of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene is controlled by ubiquitous and tissue-specific factors and involves activating and derepressing mechanisms.

Identification and characterization of a T-cell-specific enhancer adjacent to the murine CD4 gene

1991 ◽  
Vol 11 (11) ◽  
pp. 5506-5515
Author(s):  
S Sawada ◽  
D R Littman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Binding ◽  
Binding Sites ◽  
Dnase I ◽  
Minor Effect ◽  
Enhancer Activity ◽  
Enhancer Element ◽  
Protein Binding Sites ◽  
Factor Binding

Expression of the CD4 and CD8 glycoproteins is a tightly regulated process tied to the maturation of functionally distinct classes of thymocytes. Therefore, understanding of the mechanism of expression of the genes encoding CD4 and CD8 is likely to yield important insight into regulation of the differentiated functions of T cells. Here, we report the identification of a T-cell-specific enhancer in a DNase I-hypersensitive region about 13 kb 5' of the transcription initiation site of the murine CD4 gene. Within the minimal enhancer element, at least three nuclear protein binding sites were identified by DNase I footprint analysis. One site contains the consensus motif for TCF-1 alpha/LEF-1, a recently identified HMG box transcription factor primarily expressed in pre-B and T cells. By Southwestern (DNA-protein) blotting and binding competition analyses, the protein binding to this site was found to be indistinguishable from TCF-1 alpha/LEF-1. Mutagenesis of this site resulted in loss of factor binding but had a relatively minor effect on enhancer activity. In contrast, mutations in another site, containing two consensus binding motifs for basic helix-loop-helix proteins, abolished factor binding and dramatically reduced enhancer activity. None of the protein binding sites had activity on its own, suggesting that the CD4 enhancer requires the interaction of multiple regulatory sites.

scholarly journals Identification and characterization of a T-cell-specific enhancer adjacent to the murine CD4 gene.

1991 ◽  
Vol 11 (11) ◽  
pp. 5506-5515 ◽  
Author(s):  
S Sawada ◽  
D R Littman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Binding ◽  
Binding Sites ◽  
Dnase I ◽  
Minor Effect ◽  
Enhancer Activity ◽  
Enhancer Element ◽  
Protein Binding Sites ◽  
Factor Binding

Expression of the CD4 and CD8 glycoproteins is a tightly regulated process tied to the maturation of functionally distinct classes of thymocytes. Therefore, understanding of the mechanism of expression of the genes encoding CD4 and CD8 is likely to yield important insight into regulation of the differentiated functions of T cells. Here, we report the identification of a T-cell-specific enhancer in a DNase I-hypersensitive region about 13 kb 5' of the transcription initiation site of the murine CD4 gene. Within the minimal enhancer element, at least three nuclear protein binding sites were identified by DNase I footprint analysis. One site contains the consensus motif for TCF-1 alpha/LEF-1, a recently identified HMG box transcription factor primarily expressed in pre-B and T cells. By Southwestern (DNA-protein) blotting and binding competition analyses, the protein binding to this site was found to be indistinguishable from TCF-1 alpha/LEF-1. Mutagenesis of this site resulted in loss of factor binding but had a relatively minor effect on enhancer activity. In contrast, mutations in another site, containing two consensus binding motifs for basic helix-loop-helix proteins, abolished factor binding and dramatically reduced enhancer activity. None of the protein binding sites had activity on its own, suggesting that the CD4 enhancer requires the interaction of multiple regulatory sites.

Identification of regulatory sequences and protein-binding sites in the liver-type promoter of a gene encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

1991 ◽  
Vol 11 (2) ◽  
pp. 1099-1106
Author(s):  
F P Lemaigre ◽  
S M Durviaux ◽  
G G Rousseau
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Specific Protein ◽  
Regulatory Sequences ◽  
Alternative Promoters ◽  
Gene Encoding ◽  
Protein Binding Sites ◽  
Cis Acting ◽  
Dnase I Footprinting ◽  
Nuclear Extracts

The liver-type and muscle-type isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase are encoded by one gene that uses two alternative promoters. We have identified cis-acting sequences and protein-binding sites on the liver-type promoter. Transfection assays with deleted promoters showed that maximal promoter activity is contained within 360 bp upstream of the cap site. DNase I footprinting experiments with liver and spleen nuclear extracts and with purified proteins revealed several protein-binding sites in this region. These included four binding sites for nuclear factor I, one site that contains an octamer consensus but showed a liver-specific footprint pattern, two liver-specific protein-binding sites, and one poly(dG)-containing binding site. Transfection of cells of hepatic origin suggested that all these sites except one are involved in transcriptional regulation. The region between -360 and -2663 contained an element that functioned as a silencer in a nonhepatic cell line. We conclude that in liver transcription from the liver-type promoter of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene is controlled by ubiquitous and tissue-specific factors and involves activating and derepressing mechanisms.

Cloning and characterization of the 5′-flanking region of the rat glutamate-cysteine ligase catalytic subunit

2001 ◽  
Vol 357 (2) ◽  
pp. 447-455 ◽  
Author(s):  
Heping YANG ◽  
Jiaohong WANG ◽  
Zong-Zhi HUANG ◽  
Xiaopeng OU ◽  
Shelly C. LU
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Dnase I ◽  
Luciferase Expression ◽  
Mrna Levels ◽  
Start Site ◽  
Glutamate Cysteine Ligase ◽  
Dnase I Footprinting ◽  
H4iie Cells ◽  
Flanking Region

Glutamate-cysteine ligase (GCL), the rate-limiting enzyme in glutathione synthesis, is made up of two subunits, a catalytic (heavy) subunit (GCLC) and a modifier (light) subunit (GCLM), which are differentially regulated. Increased hepatic GCLC expression occurs during rapid growth, oxidative stress and after ethanol treatment. To facilitate studies of GCLC transcriptional regulation, we have cloned and characterized a 1.8kb 5′-flanking region of the rat GCLC (GenBank accession number AF218362). A consensus TATA box and one transcriptional start site are located at 302 and 197 nucleotides upstream of the translational start site, respectively. The promoter contains consensus binding sites for many transcription factors including nuclear factor κB (NF-κB) and activator protein 1 (AP-1). The rat GCLC promoter was able to efficiently drive luciferase expression in H4IIE cells. Sequential deletion analysis revealed that three DNA regions, −595 to −111, −1108 to −705 and −705 to −595, are involved in positive (the first two regions) and negative (the latter region) gene regulation. Specific protein binding to these regions was confirmed by DNase I footprinting and electrophoretic mobility-shift assays (EMSAs). Ethanol-fed livers exhibit increased protein binding to region −416 to −336 on DNase I footprinting analysis, which was found to be NF-κB and AP-1 on EMSA and supershift analysis. Acetaldehyde treatment of H4IIE cells led to a time- and dose-dependent increase in GCLC mRNA levels, binding of NF-κB and AP-1 to the GCLC promoter, and luciferase activity driven by the GCLC promoter fragment containing these binding sites.

EVALUATION OF TISSUE STEROID BINDING IN VITRO

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S223-S246 ◽  
Author(s):  
C. R. Wira ◽  
H. Rochefort ◽  
E. E. Baulieu
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Experimental System ◽  
Specific Protein ◽  
Coupling Mechanism ◽  
Target Tissue ◽  
Protein Binding Sites ◽  
Definition Of ◽  
Hormone Specificity

ABSTRACT The definition of a RECEPTOR* in terms of a receptive site, an executive site and a coupling mechanism, is followed by a general consideration of four binding criteria, which include hormone specificity, tissue specificity, high affinity and saturation, essential for distinguishing between specific and nonspecific binding. Experimental approaches are proposed for choosing an experimental system (either organized or soluble) and detecting the presence of protein binding sites. Techniques are then presented for evaluating the specific protein binding sites (receptors) in terms of the four criteria. This is followed by a brief consideration of how receptors may be located in cells and characterized when extracted. Finally various examples of oestrogen, androgen, progestagen, glucocorticoid and mineralocorticoid binding to their respective target tissues are presented, to illustrate how researchers have identified specific corticoid and mineralocorticoid binding in their respective target tissue receptors.

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