Overexpression of CCAAT Displacement Protein Represses the Promiscuously Active Proximal gp91phox Promoter

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3151-3160 ◽  
Author(s):  
Diana Catt ◽  
Shannon Hawkins ◽  
Ann Roman ◽  
Wen Luo ◽  
David G. Skalnik
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Transcriptional Activity ◽  
Binding Activity ◽  
Proximal Promoter ◽  
Modular Organization ◽  
Transcriptional Activators ◽  
Cis Element ◽  
Dna Binding Activity ◽  
Ccaat Displacement Protein

CCAAT displacement protein (CDP) is a transcriptional repressor that restricts expression of the gp91phox gene to mature myeloid cells. CDP interacts with multiple sites within the −450 to +12 bp human gp91phox promoter, and down-regulation of CDP DNA-binding activity is required for induction of gp91phox transcription in mature phagocytes. Truncation of the gp91phox promoter to −102 to +12 bp removes 4 CDP-binding sites and reveals a promiscuous promoter activity that is active in some nonphagocytic cells. A cis-element at −90 bp is required for derepressed transcription and serves as a binding site for multiple transcriptional activators. We now report that this element also serves as a binding site for CDP. The affinity of CDP for this element is relatively weak compared with upstream CDP-binding sites within the promoter, consistent with the promiscuous transcriptional activity exhibited by the −102 to +12 bp gp91phox promoter fragment. Further analysis of the proximal promoter reveals an additional weak-affinity CDP-binding site centered at approximately −20 bp. Overexpression of cloned CDP represses the −102 to +12 bp gp91phox promoter, indicating that these proximal CDP-binding sites are functionally significant. The constellation of transcriptional activators and a repressor that interacts with the −90 bp cis-element is identical to that observed for a promoter element at −220 bp, reflecting the highly modular organization of the gp91phoxpromoter. These studies illustrate the complex interplay between transcriptional activators and a repressor that contribute to the myeloid-restricted expression of the gp91phox gene.

Overexpression of CCAAT Displacement Protein Represses the Promiscuously Active Proximal gp91phox Promoter

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3151-3160 ◽  
Author(s):  
Diana Catt ◽  
Shannon Hawkins ◽  
Ann Roman ◽  
Wen Luo ◽  
David G. Skalnik
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Transcriptional Activity ◽  
Binding Activity ◽  
Proximal Promoter ◽  
Modular Organization ◽  
Transcriptional Activators ◽  
Cis Element ◽  
Dna Binding Activity ◽  
Ccaat Displacement Protein

Abstract CCAAT displacement protein (CDP) is a transcriptional repressor that restricts expression of the gp91phox gene to mature myeloid cells. CDP interacts with multiple sites within the −450 to +12 bp human gp91phox promoter, and down-regulation of CDP DNA-binding activity is required for induction of gp91phox transcription in mature phagocytes. Truncation of the gp91phox promoter to −102 to +12 bp removes 4 CDP-binding sites and reveals a promiscuous promoter activity that is active in some nonphagocytic cells. A cis-element at −90 bp is required for derepressed transcription and serves as a binding site for multiple transcriptional activators. We now report that this element also serves as a binding site for CDP. The affinity of CDP for this element is relatively weak compared with upstream CDP-binding sites within the promoter, consistent with the promiscuous transcriptional activity exhibited by the −102 to +12 bp gp91phox promoter fragment. Further analysis of the proximal promoter reveals an additional weak-affinity CDP-binding site centered at approximately −20 bp. Overexpression of cloned CDP represses the −102 to +12 bp gp91phox promoter, indicating that these proximal CDP-binding sites are functionally significant. The constellation of transcriptional activators and a repressor that interacts with the −90 bp cis-element is identical to that observed for a promoter element at −220 bp, reflecting the highly modular organization of the gp91phoxpromoter. These studies illustrate the complex interplay between transcriptional activators and a repressor that contribute to the myeloid-restricted expression of the gp91phox gene.

The rat albumin promoter: cooperation with upstream elements is required when binding of APF/HNF1 to the proximal element is partially impaired by mutation or bacterial methylation

1989 ◽  
Vol 9 (11) ◽  
pp. 4759-4766
Author(s):  
F Tronche ◽  
A Rollier ◽  
I Bach ◽  
M C Weiss ◽  
M Yaniv
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Transcriptional Activity ◽  
Transient Transfection ◽  
Tata Box ◽  
Specific Factor ◽  
Target Sequence ◽  
Functional Interaction ◽  
Dna Methylase ◽  
Ccaat Box

We have characterized in the accompanying paper (P. Herbomel, A. Rollier, F. Tronche, M.-O. Ott, M. Yaniv, and M. C. Weiss, Mol. Cell. Biol. 9:4750-4758, 1989) six different elements in the albumin promoter. One of them, the proximal element (PE), is the binding site for a strictly liver specific factor, APF/HNF1. This binding site contains a bacterial DAM DNA methylase methylation target sequence which, when methylated, decreases the affinity of the protein for this element. When the different albumin promoter constructions were prepared in an Escherichia coli deoxyadenosine methylase-negative strain, the respective contributions of the elements to the overall promoter activity were strikingly different. An intact proximal element plus the TATA box gave almost full transcriptional activity in transient transfection experiments and only in differentiated hepatoma cells of line H4II, whereas the distal elements (distal element III [DEIII], the NF1-binding site DEII, and the E/CBP-binding site DEI) had become essentially dispensable. Mutations affecting the CCAAT box showed only a two- to threefold decrease. When PE was methylated, mutated, or replaced by the homologous element from the alpha-fetoprotein gene, activity in the context of the short promoter (PE plus the TATA box) was abolished. However, activity was restored in the presence of the upstream elements, showing that cooperation with factors binding to the CCAAT box and distal elements favors the functional interaction of the liver-specific APF/HNF1 factor with lower-affinity binding sites.

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.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

Androgen-dependent and DNA binding-independent association of androgen receptor with chromatic regions coding androgen-induced non-coding RNAs

2021 ◽  
Author(s):  
Takahiro Sawada ◽  
Koichi Nishimura ◽  
Jinichi Mori ◽  
Yoshiaki Kanemoto ◽  
Alexander Kouzmenko ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Specific Antigen ◽  
Binding Activity ◽  
Lncap Cells ◽  
Coding Regions ◽  
Dna Binding Activity ◽  
Genome Wide ◽  
Non Coding Rnas ◽  
Independent Association

Abstract Androgen induces the binding of its receptor (AR) to androgen-responsive elements (AREs), while genome-wide studies showed that most androgen-induced AR binding sites on chromatin were unrelated to AREs. Enhancer RNAs (eRNAs), a class of non-coding RNAs(ncRNAs), are transcribed from super-enhancers (SEs), and trigger the formation of large ribonucleoprotein (RNP) condensates of transcription factors. By in silico search, an SE is found to be located on the locus of KLK3 that encodes prostate specific antigen (PSA). On the KLK3 SE, androgen-induced expression of ncRNAs was detected and designated as KLK3eRNAs in LNCaP cells, and androgen-induced association of AR and FOXA1 on the KLK3eRNA coding regions was detected. Such androgen-induced association of an AR mutant lacking DNA binding activity on the KLK3eRNA coding regions was undetectable on an exogenous ARE. Thus, the present findings suggest a molecular basis of androgen-induced association of AR with chromatin on ARE-unrelated sequences.

scholarly journals Differential functional significance of AP-1 binding sites in the promoter of the gene encoding mouse tissue inhibitor of metalloproteinases-3

1997 ◽  
Vol 324 (2) ◽  
pp. 547-553 ◽  
Author(s):  
Hyungtae KIM ◽  
William D. PENNIE ◽  
Yi SUN ◽  
Nancy H. COLBURN
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Functional Significance ◽  
Regulatory Elements ◽  
Binding Activity ◽  
Model Systems ◽  
Mouse Tissue ◽  
Significant Activity ◽  
Tissue Inhibitor Of Metalloproteinases ◽  
Tissue Inhibitor

Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular-matrix-associated protein that suppresses tumorigenicity or invasion in several model systems. We have identified, by in vitro footprinting, six AP-1 (activator protein-1) or AP-1-like binding sites in the mouse TIMP-3 promoter that bind purified c-Jun homodimers. Electrophoretic mobility shift assays revealed that the non-consensus fifth AP-1 binding site (AP-720; nt -720 to -714) had the strongest binding activity for recombinant c-Jun protein, and that the fourth binding site (AP-763; nt -763 to -754) and AP-720 showed strong binding activity for cellular nuclear proteins. Antibody supershift and blocking experiments suggest that AP-720, but not AP-763, binds authentic AP-1 components. Transient transfection reporter assays of deletion constructs showed that the region spanning AP-720 has the highest transcriptional activity, and that sequences 5′ to this region (nt -2846 to -747) may contain negative regulatory elements. The deletion construct containing about 500 nt 5′ to the transcriptional start, but no AP-1 sites, showed lower but significant activity, suggesting both AP-1-dependent and -independent regulation of the mouse TIMP-3 promoter. Mutational inactivation of AP-720 abolished the activity increment that distinguished the reporter construct containing both AP-720 and sixth AP-1 binding site (AP-617; nt -617 to -611) from that containing only AP-617. In summary, we report here that both AP-1 and non-AP-1 elements contribute to activity, with the non-consensus AP-1 site at -720 showing the greatest functional significance among the AP-1 sites.

scholarly journals Generation of three different fragments of bound C3 with purified factor I or serum. II. Location of binding sites in the C3 Fragments for Factors B and H, complement receptors , and bovine conglutinin

1983 ◽  
Vol 158 (2) ◽  
pp. 334-352 ◽  
Author(s):  
GD Ross ◽  
SL Newman ◽  
JD Lambris ◽  
JE Devery-Pocius ◽  
JA Cain ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Complement Receptors ◽  
Phagocytic Cells ◽  
Factor I ◽  
D Region ◽  
The Many ◽  
Low Ionic Strength ◽  
Inhibition Studies

The many different recognized functions of C3 are dependent upon the ability of the activated C3 molecule both to bind covalently to protein and carbohydrate surfaces and to provide binding sites for as many as eleven different proteins. The location of the binding sites for six of these different proteins (factors B and H, complement receptors CR(1), CR(2) and CR(3) and conglutinin) was examined in the naturally occurring C3-fragments generated by C3 activation (C3b) and degradation by Factor I (iC3b, C3c, C3d,g) and trypsin (C3d). Evidence was obtained for at least four distinct binding sites in C3 for these six different C3 ligands. One binding site for B was detectable only in C3b, whereas a second binding site for H and CR(1) was detectable in both C3b and iC3b. The affinity of the binding site for H and CR(1) was charge dependent and considerably reduced in iC3b as compared to C3b. H binding to iC3b-coated sheep erythrocytes (EC3bi) was measurable only in low ionic strength buffer (4 mS). The finding that C3c-coated microspheres bound to CR(1), indicated that this second binding site was still intact in the C3c fragment. However, H binding to C3c was not examined. A third binding site in C3 for CR(2) was exposed in the d region by factor I cleavage of C3b into iC3b, and the activity of this site was unaffected by the further I cleavage of iC3b into C3d,g. Removal of the 8,000-dalton C3g fragment from C3d,g with trypsin forming C3d, resulted in reduced CR2 activity. However, because saturating amounts of monoclonal anti-C3g did not block the CR(2)-binding activity of EC3d,g, it appears unlikely that the g region of C3d,g or iC3b forms a part of the CR(2)-binding site. In addition, detergent-solubilized EC3d (C3d-OR) inhibited the CR(2)-binding activity of EC3d,g. Monocytes and neutrophils, that had been previously thought to lack CR(2) because of their inability to form EC3d rosettes, did bind EC3d,g containing greater than 5 × 10(4) C3d,g molecules per E. The finding that monocyte and neutrophil rosettes with EC3d,g were inhibited by C3d-OR, suggested that these phagocytic cells might indeed express very low numbers of CR(2), and that these CR(2) were detectable with EC3d,g and not with EC3d because C3d,g had a higher affinity for CR2 than did C3d. A fourth C3 binding site for CR(3) and conglutinin (K) was restricted to the iC3b fragment. Because of simultaneous attachment of iC3b to phagocyte CR3 and CR(3), the characteristics of iC3b binding to CR3 could only be examined with phagocytes on which the CR(1) had been blocked with anti-CR(1). Inhibition studies with EDTA and N-acetyl-D-glucosamine demonstrated a requirement for both calcium cations and carbohydrate in the binding of EC3bi to CR3 and to K. However, CR(3) differed from K in that magnesium cations were required in addition to calcium for maximum CR(3) binding activity, and NADG produced less inhibition of CR(3) activity than of K activity.

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