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.

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.

E-box- and MEF-2-independent muscle-specific expression, positive autoregulation, and cross-activation of the chicken MyoD (CMD1) promoter reveal an indirect regulatory pathway

1994 ◽  
Vol 14 (8) ◽  
pp. 5474-5486
Author(s):  
C A Dechesne ◽  
Q Wei ◽  
J Eldridge ◽  
L Gannoun-Zaki ◽  
P Millasseau ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
The Other ◽  
Indirect Pathway ◽  
Specific Expression ◽  
Fibroblast Cultures ◽  
E Box ◽  
Myogenic Factors

Members of the MyoD family of gene-regulatory proteins (MyoD, myogenin, myf5, and MRF4) have all been shown not only to regulate the transcription of numerous muscle-specific genes but also to positively autoregulate and cross activate each other's transcription. In the case of muscle-specific genes, this transcriptional regulation can often be correlated with the presence of a DNA consensus in the regulatory region CANNTG, known as an E box. Little is known about the regulatory interactions of the myogenic factors themselves; however, these interactions are thought to be important for the activation and maintenance of the muscle phenotype. We have identified the minimal region in the chicken MyoD (CMD1) promoter necessary for muscle-specific transcription in primary cultures of embryonic chicken skeletal muscle. The CMD1 promoter is silent in primary chick fibroblast cultures and in muscle cell cultures treated with the thymidine analog bromodeoxyuridine. However, CMD1 and chicken myogenin, as well as, to a lesser degree, chicken Myf5 and MRF4, expressed in trans can activate transcription from the minimal CMD1 promoter in these primary fibroblast cultures. Here we show that the CMD1 promoter contains numerous E-box binding sites for CMD1 and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific expression, autoregulation, or cross activation depends upon the presence of of these E-box or MEF-2 binding sites in the CMD1 promoter. These results demonstrate that the autoregulation and cross activation of the chicken MyoD promoter through the putative direct binding of the myogenic basic helix-loop-helix regulatory factors is mediated through an indirect pathway that involves unidentified regulatory elements and/or ancillary factors.

scholarly journals Hybrid Dynamic Pharmacophore Models as Effective Tools to Identify Novel Chemotypes for Anti-TB Inhibitor Design: A Case Study With Mtb-DapB

2020 ◽  
Vol 8 ◽  
Author(s):  
Chinmayee Choudhury ◽  
Anshu Bhardwaj
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Chemical Space ◽  
Pharmacophore Model ◽  
Model Systems ◽  
Receptor Interaction ◽  
Hybrid Molecules ◽  
Wide Range ◽  
Binding Pockets ◽  
Pharmacophore Models

Antimicrobial resistance (AMR) is one of the most serious global public health threats as it compromises the successful treatment of deadly infectious diseases like tuberculosis. New therapeutics are constantly needed but it takes a long time and is expensive to explore new biochemical space. One way to address this issue is to repurpose the validated targets and identify novel chemotypes that can simultaneously bind to multiple binding pockets of these targets as a new lead generation strategy. This study reports such a strategy, dynamic hybrid pharmacophore model (DHPM), which represents the combined interaction features of different binding pockets contrary to the conventional approaches, where pharmacophore models are generated from single binding sites. We have considered Mtb-DapB, a validated mycobacterial drug target, as our model system to explore the effectiveness of DHPMs to screen novel unexplored compounds. Mtb-DapB has a cofactor binding site (CBS) and an adjacent substrate binding site (SBS). Four different model systems of Mtb-DapB were designed where, either NADPH/NADH occupies CBS in presence/absence of an inhibitor 2, 6-PDC in the adjacent SBS. Two more model systems were designed, where 2, 6-PDC was linked to NADPH and NADH to form hybrid molecules. The six model systems were subjected to 200 ns molecular dynamics simulations and trajectories were analyzed to identify stable ligand-receptor interaction features. Based on these interactions, conventional pharmacophore models (CPM) were generated from the individual binding sites while DHPMs were created from hybrid-molecules occupying both binding sites. A huge library of 1,563,764 publicly available molecules were screened by CPMs and DHPMs. The screened hits obtained from both types of models were compared based on their Hashed binary molecular fingerprints and 4-point pharmacophore fingerprints using Tanimoto, Cosine, Dice and Tversky similarity matrices. Molecules screened by DHPM exhibited significant structural diversity, better binding strength and drug like properties as compared to the compounds screened by CPMs indicating the efficiency of DHPM to explore new chemical space for anti-TB drug discovery. The idea of DHPM can be applied for a wide range of mycobacterial or other pathogen targets to venture into unexplored chemical space.

scholarly journals A novel cis-acting element required for lipopolysaccharide-induced transcription of the murine interleukin-1 beta gene.

1995 ◽  
Vol 15 (1) ◽  
pp. 112-119 ◽  
Author(s):  
S A Godambe ◽  
D D Chaplin ◽  
T Takova ◽  
L M Read ◽  
C J Bellone
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Competitive Inhibition ◽  
Interleukin 1 ◽  
Sodium Dodecyl ◽  
Regulatory Elements ◽  
Nuclear Factor ◽  
Interleukin 1 Beta ◽  
Factor Binding ◽  
Beta Gene

Regulatory elements important for transcription of the murine interleukin-1 beta (IL-1 beta) gene lie within a DNase I-hypersensitive region located > 2,000 bp upstream from the transcription start site. We have identified within this region a novel positive regulatory element that is required for activation of an IL-1 beta promoter-chloramphenicol acetyltransferase (CAT) fusion gene in the murine macrophage line RAW264.7. Electrophoretic mobility shift analysis of the 3' portion (-2315 to -2106) of the hypersensitive region revealed at least two nuclear factor binding sites, one of which is located between positions -2285 and -2256. Competitive inhibition studies localized the binding site to a 15-bp sequence between -2285 and -2271. Nuclear factor binding was lost by mutation of the 6-bp sequence from -2280 to -2275. The specific retarded complex formed with RAW264.7 nuclear extract was not detected under similar conditions with nuclear extracts from RLM-11, a murine T-cell line which does not express IL-1 beta RNA. Mutation of the 6-bp sequence (-2280 to -2275) in the chimeric IL-1 beta promoter -4093 +I CAT plasmid virtually eliminated the activation of this reporter gene by lipopolysaccharide (LPS) in transfected RAW264.7 cells. Multimerization of the 15-bp sequence containing the core wild-type 6-bp sequence 5' of minimal homologous or heterologous promoters in CAT reporter plasmids resulted in significant enhancement of CAT expression compared with parallel constructs containing the mutant 6-bp core sequence. This element was LPS independent and position and orientation dependent. The multimerized 15-bp sequence did not enhance expression in RLM-11 cells. Methylation interference revealed contact residues from -2281 to -2271, CCAAAAAGGAA. Because a search of the NIH TFD data bank with the 11-bp binding site sequence found no homology to known nuclear factor binding sites, we have designated this sequence the IL1 beta -upstream nuclear factor 1 (IL1 beta -UNF1) target. UV cross-linking and sodium dodecyl sulfate-polyacrylamide electrophoresis identified an IL1 beta -UNF1-specific binding factor approximately 85 to 90 kDa in size.

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.

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.

scholarly journals Inactivating Mutation of the Mouse Tissue Inhibitor of Metalloproteinases-2(Timp-2) Gene Alters ProMMP-2 Activation

2000 ◽  
Vol 275 (34) ◽  
pp. 26416-26422 ◽  
Author(s):  
John J. Caterina ◽  
Susan Yamada ◽  
Nancy C. M. Caterina ◽  
Glenn Longenecker ◽  
Kenn Holmbäck ◽  
...  
Keyword(s):  
Mouse Tissue ◽  
Tissue Inhibitor Of Metalloproteinases ◽  
Tissue Inhibitor ◽  
Inactivating Mutation

scholarly journals Structure-aided prediction of mammalian transcription factor complexes in conserved non-coding elements

2013 ◽  
Vol 368 (1632) ◽  
pp. 20130029 ◽  
Author(s):  
Harendra Guturu ◽  
Andrew C. Doxey ◽  
Aaron M. Wenger ◽  
Gill Bejerano
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Protein Interactions ◽  
Binding Sites ◽  
Three Dimensional ◽  
Structural Data ◽  
Regulatory Elements ◽  
Computational Method ◽  
Web Resource ◽  
Three Dimensional Models

Mapping the DNA-binding preferences of transcription factor (TF) complexes is critical for deciphering the functions of cis -regulatory elements. Here, we developed a computational method that compares co-occurring motif spacings in conserved versus unconserved regions of the human genome to detect evolutionarily constrained binding sites of rigid TF complexes. Structural data were used to estimate TF complex physical plausibility, explore overlapping motif arrangements seldom tackled by non-structure-aware methods, and generate and analyse three-dimensional models of the predicted complexes bound to DNA. Using this approach, we predicted 422 physically realistic TF complex motifs at 18% false discovery rate, the majority of which (326, 77%) contain some sequence overlap between binding sites. The set of mostly novel complexes is enriched in known composite motifs, predictive of binding site configurations in TF–TF–DNA crystal structures, and supported by ChIP-seq datasets. Structural modelling revealed three cooperativity mechanisms: direct protein–protein interactions, potentially indirect interactions and ‘through-DNA’ interactions. Indeed, 38% of the predicted complexes were found to contain four or more bases in which TF pairs appear to synergize through overlapping binding to the same DNA base pairs in opposite grooves or strands. Our TF complex and associated binding site predictions are available as a web resource at http://bejerano.stanford.edu/complex .

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