Conserved region of the rat alpha-lactalbumin promoter is a target site for protein binding in vitro.
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ABSTRACT:
SUBMITTER: Lubon H
PROVIDER: S-EPMC1135422 | biostudies-other | 1988 Dec
REPOSITORIES: biostudies-other
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Project description:BackgroundThe dystrophin glycoprotein complex is disrupted in Duchenne muscular dystrophy and many other neuromuscular diseases. The principal heterodimeric partner of dystrophin at the heart of the dystrophin glycoprotein complex in the main clinically affected tissues (skeletal muscle, heart and brain) is its distant relative, alpha-dystrobrevin. The alpha-dystrobrevin gene is subject to complex transcriptional and post-transcriptional regulation, generating a substantial range of isoforms by alternative promoter use, alternative polyadenylation and alternative splicing. The choice of isoform is understood, amongst other things, to determine the stoichiometry of syntrophins (and their ligands) in the dystrophin glycoprotein complex.ResultsWe show here that, contrary to the literature, most alpha-dystrobrevin genes, including that of humans, encode three distinct syntrophin-binding sites, rather than two, resulting in a greatly enhanced isoform repertoire. We compare in detail the quantitative tissue-specific expression pattern of human and mouse alpha-dystrobrevin isoforms, and show that two major gene features (the novel syntrophin-binding site-encoding exon and the internal promoter and first exon of brain-specific isoforms alpha-dystrobrevin-4 and -5) are present in most mammals but specifically ablated in mouse and rat.ConclusionLineage-specific mutations in the murids mean that the mouse brain has fewer than half of the alpha-dystrobrevin isoforms found in the human brain. Our finding that there are likely to be fundamental functional differences between the alpha-dystrobrevins (and therefore the dystrophin glycoprotein complexes) of mice and humans raises questions about the current use of the mouse as the principal model animal for studying Duchenne muscular dystrophy and other related disorders, especially the neurological aspects thereof.
| S-EPMC2796648 | biostudies-literature
Project description:Three subtypes of alpha 2-adrenergic receptors (alpha 2A, alpha 2B and alpha 2C) have been described that differ in their primary sequence and tissue-specific expression and are encoded by three distinct genes. Previous work has shown that the human alpha 2A-adrenergic receptor gene promoter consists of a TATA-box (TATAAA), palindromic sequence (CCCACGTGGG) and GC-box (GGGGCGG) motif. Sequence analysis of the putative promoter region of the rat alpha 2A-adrenergic receptor gene showed that these promoter regions are conserved in their sequence and relative location. We analysed the transcriptional activity of these regions using RINm5F, a rat insulinoma cell line that expresses the endogenous alpha 2A-adrenergic receptor gene. These results showed that the region from -484 to -92 has a negative effect on transcription, as deletion of this region in alpha 2A-adrenergic receptor gene-chloramphenicol acetyltransferase reporter constructs increased reporter gene activity. This region included the GC-box sequence which is a consensus binding site for the nuclear factor SP1, which is a positive activator of transcription. Gel-mobility-shift assays and supershift assays with an antibody that recognizes SP1 showed binding of the SP1 nuclear factor as well as other nuclear factors to this GC-box region. Additional nuclear factors bind to the downstream palindromic region. We suggest that positive- and negative-acting nuclear factors contribute to the activity of the alpha 2-adrenergic receptor promoter.
| S-EPMC1136033 | biostudies-other
Project description:To study the function of Paf1C in mouse ESCs, we generated an ES cell line stably expressing a location and affinity purification (LAP)-tagged Ctr9 fusiuon protein using the bacterial astificial chromosome (BAC)-based TransgeneOmics approach. To investigate whether pluripotency and lineage control genes differentially regulated upon Paf1C depletion are direct targets of the Paf1C, we analyzed the binding of the Ctr9-LAP fusion protein by ChIP-chip and identified 2175 promoter regions that were bound by Ctr9. The supplementary bed file contains promoter regions bound by Ctr9. Keywords: ChIP-chip
2009-01-31 | GSE14654 | GEO
Project description:BackgroundGranulosa cell tumors (GCT) of the ovary often express aromatase and synthesize estrogen, which in turn may influence their progression. Recently a specific point mutation (C134W) in the FOXL2 protein was identified in >94% of adult-type GCT and it is likely to contribute to their development. A number of genes are known to be regulated by FOXL2, including aromatase/CYP19A1, but it is unclear which are direct targets and whether the C134W mutation alters their regulation. Recently, it has been reported that FOXL2 forms a complex with steroidogenic factor 1 (SF-1) which is a known regulator of aromatase in granulosa cells.Methodology/principal findingsIn this work, the human GCT-derived cell lines KGN and COV434 were heterozygous and wildtype for the FOXL2:C134W mutation, respectively. KGN had abundant FOXL2 mRNA expression but it was not expressed in COV434. Expression of exogenous FOXL2:C134W in COV434 cells induced higher expression of a luciferase reporter for the ovarian specific aromatase promoter, promoter II (PII) (-516bp) than expression of wildtype FOXL2, but did not alter induction of a similar reporter for the steroidogenic acute regulatory protein (StAR) promoter (-1300bp). Co-immunoprecipitation confirmed that FOXL2 bound SF-1 and that it also bound its homologue, liver receptor homologue 1 (LRH-1), however, the C134W mutation did not alter these interactions or induce a selective binding of the proteins. A highly conserved putative binding site for FOXL2 was identified in PII. FOXL2 was demonstrated to bind the site by electrophoretic mobility shift assays (EMSA) and site-directed mutagenesis of this element blocked its differential induction by wildtype FOXL2 and FOXL2:C134W.Conclusions/significanceThese findings suggest that aromatase is a direct target of FOXL2:C134W in adult-type GCT via a single distinctive and highly conserved binding site in PII and therefore provide insight into the pathogenic mechanism of this mutation.
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Project description:Transcription of the serum albumin gene occurs almost exclusively in the liver and is controlled in part by a strong liver-specific promoter. The upstream region of the serum albumin gene promoter is highly conserved among species and is footprinted in vitro by a number of nuclear proteins. However, the role of the upstream promoter region in regulating transcription and the identity of the transcription factors that bind to this region have not been established. In the present study, deletion analysis of the rat serum albumin promoter in transiently transfected HepG2 cells demonstrated that elimination of the region between -207 and -153 bp caused a two-fold decrease in promoter activity (P<0.05). Additional analysis of the -207 to -124 bp promoter interval led to the identification of two potential binding sites for hepatocyte nuclear factor-3 (HNF-3) located at -168 to -157 bp (site X) and -145 to -134 bp (site Y). Electrophoretic mobility-shift assays performed with the HNF-3 X and Y sites demonstrated that both sites are capable of binding HNF-3alpha and HNF-3beta. Placement of a single copy of the HNF-3 X site upstream from a minimal promoter increased promoter activity by about four-fold in HepG2 cells, and the reporter construct containing this site could be transactivated if co-transfected with an HNF-3 expression construct. Furthermore, inactivation of the HNF-3 X site by site-directed mutagenesis within the context of the -261 bp albumin promoter construct resulted in a 40% decrease in transcription (P<0.05). These results indicate that the positive effect of the -207 to -153 bp promoter interval is attributable to the presence of the HNF-3 X site within this interval. Additional results obtained with transfected HepG2 cells suggest that the HNF-3 Y site plays a lesser role in activation of transcription than the X site.
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Project description:?-Lactalbumin (?-LA) is a small (Mr 14,200), acidic (pI 4-5), Ca2+-binding protein. ?-LA is a regulatory component of lactose synthase enzyme system functioning in the lactating mammary gland. The protein possesses a single strong Ca2+-binding site, which can also bind Mg2+, Mn2+, Na+, K+, and some other metal cations. It contains several distinct Zn2+-binding sites. Physical properties of ?-LA strongly depend on the occupation of its metal binding sites by metal ions. In the absence of bound metal ions, ?-LA is in the molten globule-like state. The binding of metal ions, and especially of Ca2+, increases stability of ?-LA against the action of heat, various denaturing agents and proteases, while the binding of Zn2+ to the Ca2+-loaded protein decreases its stability and causes its aggregation. At pH 2, the protein is in the classical molten globule state. ?-LA can associate with membranes at neutral or slightly acidic pH at physiological temperatures. Depending on external conditions, ?-LA can form amyloid fibrils, amorphous aggregates, nanoparticles, and nanotubes. Some of these aggregated states of ?-LA can be used in practical applications such as drug delivery to tissues and organs. ?-LA and some of its fragments possess bactericidal and antiviral activities. Complexes of partially unfolded ?-LA with oleic acid are cytotoxic to various tumor and bacterial cells. ?-LA in the cytotoxic complexes plays a role of a delivery carrier of cytotoxic fatty acid molecules into tumor and bacterial cells across the cell membrane. Perhaps in the future the complexes of ?-LA with oleic acid will be used for development of new anti-cancer drugs.
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Project description:Ebola virus (EBOV) in humans causes a severe illness with high mortality rates. Several strategies have been developed in the past to treat EBOV infection, including the antibody cocktail ZMapp, which has been shown to be effective in nonhuman primate models of infection 1 and has been used under compassionate-treatment protocols in humans 2 . ZMapp is a mixture of three chimerized murine monoclonal antibodies (mAbs)3-6 that target EBOV-specific epitopes on the surface glycoprotein7,8. However, ZMapp mAbs do not neutralize other species from the genus Ebolavirus, such as Bundibugyo virus (BDBV), Reston virus (RESTV) or Sudan virus (SUDV). Here, we describe three naturally occurring human cross-neutralizing mAbs, from BDBV survivors, that target an antigenic site in the canonical heptad repeat 2 (HR2) region near the membrane-proximal external region (MPER) of the glycoprotein. The identification of a conserved neutralizing antigenic site in the glycoprotein suggests that these mAbs could be used to design universal antibody therapeutics against diverse ebolavirus species. Furthermore, we found that immunization with a peptide comprising the HR2-MPER antigenic site elicits neutralizing antibodies in rabbits. Structural features determined by conserved residues in the antigenic site described here could inform an epitope-based vaccine design against infection caused by diverse ebolavirus species.
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Project description:Differential HLA-C levels influence several human diseases, but the mechanisms responsible are incompletely characterized. Using a validated prediction algorithm, we imputed HLA-C cell surface levels in 228 individuals from the 1000 Genomes dataset. We tested 68,726 SNPs within the MHC for association with HLA-C level. The HLA-C promoter region variant, rs2395471, 800 bp upstream of the transcription start site, gave the most significant association with HLA-C levels (p = 4.2 × 10-66). This imputed expression quantitative trait locus, termed impeQTL, was also shown to associate with HLA-C expression in a genome-wide association study of 273 donors in which HLA-C mRNA expression levels were determined by quantitative PCR (qPCR) (p = 1.8 × 10-20) and in two cohorts where HLA-C cell surface levels were determined directly by flow cytometry (n = 369 combined, p < 10-15). rs2395471 is located in an Oct1 transcription factor consensus binding site motif where the A allele is predicted to have higher affinity for Oct1 than the G allele. Mobility shift electrophoresis demonstrated that Oct1 binds to both alleles in vitro, but decreased HLA-C promoter activity was observed in a luciferase reporter assay for rs2395471_G relative to rs2395471_A on a fixed promoter background. The rs2395471 variant accounts for up to 36% of the explained variation of HLA-C level. These data strengthen our understanding of HLA-C transcriptional regulation and provide a basis for understanding the potential consequences of manipulating HLA-C levels therapeutically.
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Project description:Pub1p, a highly abundant poly(A)+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3) shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion. This structural trait is conserved in Pub1p metazoan homologues, the TIA-1 family, defining a new class of RRM-type domains that we propose to name TRRM (TIA-1 C-terminal domain-like RRM). Pub1p TRRM and the N-terminal RRM1-RRM2 tandem bind RNA with high selectivity for U-rich sequences, with TRRM showing additional preference for UA-rich ones. RNA-mediated chemical shift changes map to β-sheet and protein loops in the three RRMs. Additionally, NMR titration and biochemical in vitro cross-linking experiments determined that Pub1p TRRM interacts specifically with the N-terminal region (1-402) of yeast eIF4G1 (Tif4631p), very likely through the conserved Box1, a short sequence motif neighbouring the Pab1p binding site in Tif4631p. The interaction involves conserved residues of Pub1p TRRM, which define a protein interface that mirrors the Pab1p-Tif4631p binding mode. Neither protein nor RNA recognition involves the novel N-terminal helix, whose functional role remains unclear. By integrating these new results with the current knowledge about Pub1p, we proposed different mechanisms of Pub1p recruitment to the mRNPs and Pub1p-mediated mRNA stabilization in which the Pub1p/Tif4631p interaction would play an important role.
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Project description:An opportunistic pathogen, Klebsiella pneumoniae is known to cause life-threating nosocomial infection with a high rate of morbidity and mortality. Evolutions of multi-drug-resistant and hyper-virulent strains of K. pneumoniae make the situation worse. Currently, there is no incisive drug molecule available for drug-resistant hyper-virulent K. pneumoniae infection that emphasizes the need for identification of novel and more promising drug targets in K. pneumoniae. Recently, various non-canonical structures of nucleic acids especially G-quadruplex (G4) motifs have been identified as potential therapeutic targets against several human pathogenic bacteria and viruses including Mycobacterium tuberculosis, Streptococcus pneumoniae, human immunodeficiency virus (HIV), Ebola, and Nipah. Therefore, in present study we screened the K. pneumoniae genomes for identification of evolutionary conserved G4 structure-forming motifs as promising anti-bacterial drug targets. Bioinformatics analysis revealed the presence of six highly conserved G4 motifs in the promoter region of five essential genes that play a critical role in nutrient transport and metabolism. Biophysical studies showed the formation of G4 structure by these conserved motifs. Circular Dichroism melting analysis showed the stabilization of these G4 motifs by a well-known G4-stabilizing agent, BRACO-19. The stabilization of these motifs by BRACO-19 was also able to stop the primer extension process, which is an essential phenomenon for expression of the G4-harboring gene. The addition of G4-specific ligand at low micromolar range was observed to be lethal for the growth of this bacteria and negatively controlled the expression of the G4-harboring genes via G4 structure stabilization. These observations strengthen the formation of G4 structures by the predicted G4 motif in vivo, which can be stabilized by G4 ligands like BRACO-19. This stabilization of G4 structures can attenuate the expression of G4-harboring essential genes and thus play a critical role in the regulation of gene expression. Thus, taking all given result in consideration, for the first time, this study showed the new therapeutic avenue for combating K. pneumoniae infection by characterizing the conserved G4 motifs as promising therapeutic targets.
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