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

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.

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DNase I Footprinting to Identify Protein Binding Sites

BIO-PROTOCOL ◽

10.21769/bioprotoc.824 ◽

2013 ◽

Vol 3 (14) ◽

Cited By ~ 1

Author(s):

Isabelle Gaugué ◽

Dominique Bréchemier-Baey ◽

Jacqueline Plumbridge

Keyword(s):

Protein Binding ◽

Binding Sites ◽

Dnase I ◽

Protein Binding Sites ◽

Dnase I Footprinting

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Mapping Protein-binding Sites on DNA by DNase I Footprinting

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot3947 ◽

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

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

Biochemical Journal ◽

10.1042/bj3570447 ◽

2001 ◽

Vol 357 (2) ◽

pp. 447-455 ◽

Cited By ~ 14

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.

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Identification and characterization of a chicken alpha-globin enhancer.

Molecular and Cellular Biology ◽

10.1128/mcb.9.3.893 ◽

1989 ◽

Vol 9 (3) ◽

pp. 893-901 ◽

Cited By ~ 28

Author(s):

J A Knezetic ◽

G Felsenfeld

Keyword(s):

Globin Gene ◽

Regulatory Elements ◽

Dnase I ◽

Specific Factor ◽

Gene Promoters ◽

Globin Genes ◽

Mobility Shift ◽

Enhancer Element ◽

Alpha Globin

We identify and describe the properties of an enhancer within the chicken alpha-globin gene cluster. This cluster consists of one gene (pi) expressed only in primitive erythrocytes and two (alpha A and alpha D) expressed in both primitive and definitive cell lineages. The genes are linked together in the order 5'-pi-alpha D-alpha A-3' and occupy a region about 10 kilobase pairs long. The enhancer is located at the 3' end of the cluster, about 750 base pairs 3' to the alpha A translation stop site. When assayed by transfection into either primitive or definitive primary chicken erythrocytes, this element stimulated expression from plasmids containing the alpha D- or alpha A-globulin gene promoters. Except for sites in the alpha-globin promoters, no other stimulatory activity was observed in DNA taken from other regions of the alpha-globin locus. Moderate resolution DNase I hypersensitivity studies as well as DNase I footprinting revealed three regions of protein binding, each containing a similar core DNA sequence within the enhancer element. Gel mobility shift studies demonstrated that all three regions bind the recently identified erythrocyte-specific factor, EryfI, which has binding sites in the regulatory regions of all chicken globin genes. Our data suggest that the enhancer we have identified may act in vivo only on the alpha A gene; expression of the alpha D gene is affected by another EryfI site located in the alpha D promoter. Such a mechanism would be consistent with the observed relative abundances of alpha A- and alpha D-globin in vivo. The simplicity of these regulatory elements may reflect the limited repertoire of expression of these genes during development.

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Identification and characterization of a chicken alpha-globin enhancer

Molecular and Cellular Biology ◽

10.1128/mcb.9.3.893-901.1989 ◽

1989 ◽

Vol 9 (3) ◽

pp. 893-901

Author(s):

J A Knezetic ◽

G Felsenfeld

Keyword(s):

Globin Gene ◽

Regulatory Elements ◽

Dnase I ◽

Specific Factor ◽

Gene Promoters ◽

Globin Genes ◽

Mobility Shift ◽

Enhancer Element ◽

Alpha Globin

We identify and describe the properties of an enhancer within the chicken alpha-globin gene cluster. This cluster consists of one gene (pi) expressed only in primitive erythrocytes and two (alpha A and alpha D) expressed in both primitive and definitive cell lineages. The genes are linked together in the order 5'-pi-alpha D-alpha A-3' and occupy a region about 10 kilobase pairs long. The enhancer is located at the 3' end of the cluster, about 750 base pairs 3' to the alpha A translation stop site. When assayed by transfection into either primitive or definitive primary chicken erythrocytes, this element stimulated expression from plasmids containing the alpha D- or alpha A-globulin gene promoters. Except for sites in the alpha-globin promoters, no other stimulatory activity was observed in DNA taken from other regions of the alpha-globin locus. Moderate resolution DNase I hypersensitivity studies as well as DNase I footprinting revealed three regions of protein binding, each containing a similar core DNA sequence within the enhancer element. Gel mobility shift studies demonstrated that all three regions bind the recently identified erythrocyte-specific factor, EryfI, which has binding sites in the regulatory regions of all chicken globin genes. Our data suggest that the enhancer we have identified may act in vivo only on the alpha A gene; expression of the alpha D gene is affected by another EryfI site located in the alpha D promoter. Such a mechanism would be consistent with the observed relative abundances of alpha A- and alpha D-globin in vivo. The simplicity of these regulatory elements may reflect the limited repertoire of expression of these genes during development.

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1488 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1488-1499 ◽

Cited By ~ 34

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.

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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.

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.1099 ◽

1991 ◽

Vol 11 (2) ◽

pp. 1099-1106 ◽

Cited By ~ 15

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.

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