Nuclear Protein Binding Sites Research Articles

Novel Insights into the Molecular Interaction of a Panduratin A Derivative with the Non Structural Protein (NS3) of Dengue Serotypes: A Molecular Dynamics Study.

The ligand PKP10 having substitution of Cl- at R2 and R3 positions of ring A of Panduratin A i.e., ((1R,2S,5S)-5-(2,3-dichlorophenyl)-3-methyl-2-(3-methylbut-2-nyl)cyclohex-3- enyl)(2,6-dihydroxy-4-methylphenyl)methanone hydrate) has been observed to block the Nuclear Receptor Binding Protein binding site of Non Structural protein 3 in all dengue serotypes. In continuation with our earlier study, we have reported sixty novel Panduratin A derivatives compounds where substitution was done in positions 2 and 3 position of the benzyl ring A of Panduratin A with various substituents. We selected ((1R,2S,5S)-5-(2,3-dichlorophenyl)-3-methyl-2-(3-methylbut-2-nyl)cyclohex-3- nyl) (2,6-dihydroxy-4-methylphenyl) methanone hydrate) (PKP10) for molecular dynamics (MD) simulations as it constantly produced lowest CDocker interaction energy of among all the sixty five derivatives. The CDocker interaction energy was predicted to be -140.804, -79.807, -78.217 and -84.073 Kcalmol-1 respectively against NS3 protein of dengue serotypes (DENV1-4). To understand the dynamics of the PKP10 with NS3 protein, each complex was subjected to molecular dynamics simulations of 50 ns in aqueous solution. MD (Molecular Dynamics) simulation study revealed that the binding of ligand PKP10 at the active site of NS3 induces a conformational change in all serotypes which was well supported by principal component analysis. To the best of our knowledge, this is first ever study which provided atomistic insights into the interaction of PKP10 with NS3 protein of dengue serotypes. The result from our study along with in vitro studies is expected to open up better avenues to develop inhibitors for dengue virus in the near future.

Increased glycolipid storage produced by the inheritance of a complex intronic haplotype in the α-galactosidase A (GLA) gene.

BackgroundAccumulation of galactosphingolipids is a general characteristic of Fabry disease, a lysosomal storage disorder caused by the deficient activity of α-galactosidase A encoded by the GLA gene. Although many polymorphic GLA haplotypes have been described, it is still unclear whether some of these variants are causative of disease symptoms. We report the study of an inheritance of a complex intronic haplotype (CIH) (c.-10C > T, c.369 + 990C > A, c.370-81_370-77delCAGCC, c.640-16A > G, c.1000-22C > T) within the GLA gene associated with Fabry-like symptoms and galactosphingolipid accumulation.We analysed α-Gal A activity in plasma, leukocytes and skin fibroblasts in patients, and measured accumulation of galactosphingolipids by enzymatic methods and immunofluorescence techniques. Additionally, we evaluated GLA expression using quantitative PCR, EMSA, and cDNA cloning.ResultsCIH carriers had an altered GLA expression pattern, although most of the carriers had high residual enzyme activity in plasma, leukocytes and in skin fibroblasts. Nonetheless, CIH carriers had significant galactosphingolipid accumulation in fibroblasts in comparison with controls, and also glycolipid deposits in renal tubules and glomeruli. EMSA assays indicated that the c.-10C > T variant in the promoter affected a nuclear protein binding site.ConclusionsThus, inheritance of the CIH caused an mRNA deregulation altering the GLA expression pattern, producing a tissue glycolipid storage.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0267-z) contains supplementary material, which is available to authorized users.

The –928 G/C and –362 G/C Single-Nucleotide Polymorphisms in the Promoter of MCP-1: Increased Transcriptional Activity and Novel Binding Sites

Background: The –928 guanine (G)/cytosine (C) and –362 G/C single-nucleotide polymorphisms (SNPs) in the proximal promoter region of the monocyte chemoattractant protein 1 gene have been associated with an increased risk for intimal medial thickness and carotid atherosclerosis, respectively. We characterized the transcriptional activity of these two SNPs in vitro and identified transcription factors that bind to them. Methods: The proximal promoter region spanning bases –2746 to +440 was sequenced in subjects with carotid atherosclerosis. Two SNPs consisting of C-for-G substitution at bases –928 and –362 were characterized. Each observed haplotype was inserted into a luciferase reporter and transfected into mammalian cells. Results: Stimulation with 12-O-tetradecanoylphorbol 13-acetate increased transcriptional activity of the –928 C plasmid (p = 0.005). The basal transcription activities of the plasmids containing –928 C and –362 C were also increased (p < 0.001 and p < 0.0001, respectively). Electrophoretic mobility shift assay (EMSA) and DNA footprinting identified an Ah receptor nuclear translocator protein (ARNT) binding site at the –928 C SNP. EMSA data indicated signal transducers and activators of transcription (STAT) binding at the –362 G SNP. A nuclear binding protein, poly(ADP-ribose) polymerase (PARP) 1, was purified from the –928 C SNP site and identified by mass spectroscopy. Conclusion: The –928 C SNP and the –362 C SNP are associated with increased transcriptional activity in vitro, but the –362 G site is not. The –928 C SNP is associated with PARP-1 and ARNT binding, and the –362 G is associated with a STAT binding site.

A common variant associated with dyslexia reduces expression of the KIAA0319 gene.

Numerous genetic association studies have implicated the KIAA0319 gene on human chromosome 6p22 in dyslexia susceptibility. The causative variant(s) remains unknown but may modulate gene expression, given that (1) a dyslexia-associated haplotype has been implicated in the reduced expression of KIAA0319, and (2) the strongest association has been found for the region spanning exon 1 of KIAA0319. Here, we test the hypothesis that variant(s) responsible for reduced KIAA0319 expression resides on the risk haplotype close to the gene's transcription start site. We identified seven single-nucleotide polymorphisms on the risk haplotype immediately upstream of KIAA0319 and determined that three of these are strongly associated with multiple reading-related traits. Using luciferase-expressing constructs containing the KIAA0319 upstream region, we characterized the minimal promoter and additional putative transcriptional regulator regions. This revealed that the minor allele of rs9461045, which shows the strongest association with dyslexia in our sample (max p-value = 0.0001), confers reduced luciferase expression in both neuronal and non-neuronal cell lines. Additionally, we found that the presence of this rs9461045 dyslexia-associated allele creates a nuclear protein-binding site, likely for the transcriptional silencer OCT-1. Knocking down OCT-1 expression in the neuronal cell line SHSY5Y using an siRNA restores KIAA0319 expression from the risk haplotype to nearly that seen from the non-risk haplotype. Our study thus pinpoints a common variant as altering the function of a dyslexia candidate gene and provides an illustrative example of the strategic approach needed to dissect the molecular basis of complex genetic traits.

Transcriptional Regulation of Pituitary POMC Is Conserved at the Vertebrate Extremes Despite Great Promoter Sequence Divergence

The stress response involves complex physiological mechanisms that maximize behavioral efficacy during attack or defense and is highly conserved in all vertebrates. Key mediators of the stress response are pituitary hormones encoded by the proopiomelanocortin gene (POMC). Despite conservation of physiological function and expression pattern of POMC in all vertebrates, phylogenetic footprinting analyses at the POMC locus across vertebrates failed to detect conserved noncoding sequences with potential regulatory function. To investigate whether ortholog POMC promoters from extremely distant vertebrates are functionally conserved, we used 5'-flanking sequences of the teleost fish Tetraodon nigroviridis POMCalpha gene to produce transgenic mice. Tetraodon POMCalpha promoter targeted reporter gene expression exclusively to mouse pituitary cells that normally express Pomc. Importantly, transgenic expression in mouse corticotrophs was increased after adrenalectomy. To understand how conservation of precise gene expression mechanisms coexists with great sequence divergence, we investigated whether very short elements are still conserved in all vertebrate POMC promoters. Multiple local sequence alignments that consider phylogenetic relationships of ortholog regions identified a unique 10-bp motif GTGCTAA(T/G)CC that is usually present in two copies in POMC 5'-flanking sequences of all vertebrates. Underlined nucleotides represent totally conserved sequences. Deletion of these paired motifs from Tetraodon POMCalpha promoter markedly reduced its transcriptional activity in a mouse corticotropic cell line and in pituitary POMC cells of transgenic mice. In mammals, the conserved motifs correspond to reported binding sites for pituitary-specific nuclear proteins that participate in POMC transcriptional regulation. Together, these results demonstrate that mechanisms that participate in pituitary-specific and hormonally regulated expression of POMC have been preserved since mammals and teleosts diverged from a common ancestor 450 million years ago despite great promoter sequence divergence.

The Laboratory of Human Genes Structure and Functions

BioTechniquesVol. 41, No. 1 About the AuthorsOpen AccessThe Laboratory of Human Genes Structure and FunctionsShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, RussiaPublished Online:21 May 2018https://doi.org/10.2144/000112215AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail The ResearchThe publication of the human and other metazoan genome sequences has greatly facilitated positioning and analysis of various genomic functional elements, with initial emphasis on coding sequences. However, a complete functional map of sequenced eukaryotic genomes should include positions of all noncoding regulatory elements. Unfortunately, experimental data on genomic positions of a multitude of regulatory sequences, such as enhancers, silencers, insulators, transcription terminators, and replication origins are very limited—especially at the whole genome level. As most genomic regulatory elements (e.g., enhancers) are generally gene-, tissue-, or cell-specific, the prediction of these elements by computational methods is difficult and often ambiguous. Therefore, the development of high-throughput experimental approaches for identification and mapping of genomic functional elements is highly desirable. Our research is focused on the development of experimental approaches for selection of relatively short functionally active DNA fragments from long genomic sequences. The resulting short fragment libraries can be used further for construction of functional maps, either by direct sequencing-mapping, or with the help of microarrays. Recently, we developed methods for identification of nuclear matrix/scaffold attachment regions, potential insulators, open chromatin regions, and binding sites of nuclear proteins.humgen.siobc.ras.ru/research/sel.htmlThe People:From left to right: Lev Nikolaev (Research Scientist), Dmitry Didych (M.Sc. Student), Eugene Snezhkov (Research Scientist), Svetlana Bulanenkova (Ph.D. Student), Sergey Akopov (Research Scientist), Igor Chernov (Research Scientist), Anna Vetchinova (Ph.D. Student), and Eugene Sverdlov (Laboratory Head).The TechniqueThe technique we describe allows for the identification and mapping of in vitro nuclear protein binding sites within long genomic sequences. Unlike chromatin immunoprecipitation, this technique doesn't require any preliminary knowledge of proteins whose binding sites are to be mapped. The data obtained by the use of this and other techniques (published and unpublished) developed by our group will be integrated into functional maps of long (several hundred kilobases) human genome regions or, in a long run perspective, of whole genomes. The resulting maps of genome regulatory elements will help to understand the mechanisms of large-scale regulation of genome activity.Identification and mapping of DNA binding proteins target sequences in long genomic regions by two-dimensional EMSA, p. 90.FiguresReferencesRelatedDetails Vol. 41, No. 1 Follow us on social media for the latest updates Metrics Downloaded 153 times History Published online 21 May 2018 Published in print July 2006 Information© 2006 Author(s)PDF download

Identification of zinc finger binding protein 89 (ZBP-89) as a transcriptional activator for a major bovine growth hormone receptor promoter

The objective of this study was to identify the transcription factors that regulate the expression of growth hormone receptor (GHR) 1A mRNA, a major GHR mRNA variant in the bovine liver. A deoxyribonuclease I footprint analysis revealed that the GHR1A promoter region −69 to −30 (relative to the transcription start site for GHR1A mRNA) contained binding sites for bovine liver nuclear proteins. Using a yeast one-hybrid analysis, zinc finger binding protein 89 (ZBP-89) was identified as a binding protein to this promoter region. Binding of ZBP-89 to the GHR1A promoter region −69 to −30 was further confirmed by an electrophoretic mobility shift assay. In cotransfection analyses, overexpression of ZBP-89 enhanced ( P < 0.01) the activity of the GHR1A promoter and this enhancement was dependent on the putative ZBP-89 binding site in the promoter. These results together indicate that ZBP-89 is a transcription factor that regulates the expression of GHR1A mRNA.

Transcriptional Regulation of the Osterix (Osx, Sp7) Promoter by Tumor Necrosis Factor Identifies Disparate Effects of Mitogen-activated Protein Kinase and NFκB Pathways

Osteoblast (OB) differentiation is suppressed by tumor necrosis factor-α (TNF-α), an inflammatory stimulus that is elevated in arthritis and menopause. Because OB differentiation requires the expression of the transcription factor osterix (Osx), we investigated TNF effects on Osx. TNF inhibited Osx mRNA in pre-osteoblastic cells without affecting Osx mRNA half-life. Inhibition was independent of new protein synthesis. Analysis of the Osx promoter revealed two transcription start sites that direct the expression of an abundant mRNA (Osx1) and an alternatively spliced mRNA (Osx2). Promoter fragments driving the expression of luciferase were constructed to identify TNF regulatory sequences. Two independent promoters were identified upstream of each transcription start site. TNF potently inhibited transcription of both promoters. Deletion and mutational analysis identified a TNF-responsive region proximal to the Osx2 start site that retained responsiveness when inserted upstream of a heterologous promoter. The TNF response region was a major binding site for nuclear proteins, although TNF did not change binding at the site. The roles of MAPK and NFκB were investigated as signal mediators of TNF. Inhibitors of MEK1 and ERK1, but not of JNK or p38 kinase, abrogated TNF inhibition of Osx mRNA and promoter activity. TNF action was not prevented by blockade of NFκB nuclear entry. The forced expression of high levels of NFκB uncovered a proximal promoter enhancer; however, this site was not activated by TNF. The inhibitory effect of TNF on Osx expression may decrease OB differentiation in arthritis and osteoporosis.

Transcriptional regulation of the mouse deoxycytidine kinase: identification and functional analysis of nuclear protein binding sites at the proximal promoter

Deoxycytidine kinase (EC 2.7.1.74, dCK) catalyzes the phosphorylation of deoxynucleosides and several nucleoside analogues that are important in antiviral and cancer chemotherapy. The enzyme is predominantly expressed in lymphoid tissue by as yet poorly defined mechanisms. In this work, we have studied the mouse dCK regulatory region to understand the molecular details of the tissue specific expression of the enzyme. DNase I footprinting and electrophoretic mobility shift assays using nuclear extracts from mouse lymphocytes (EL-4, T cells; J558, B cells) and non-lymphoid cells (L929, fibroblasts) demonstrated the existence of at least six cis-acting elements (FP-1–FP-6) within the proximal promoter region. Functional analysis revealed that all the elements necessary to promote high level transcription of the mdCK gene are located downstream the transcription start site. 5′-Deletion and site-directed mutagenesis assays demonstrated the importance of four GC-rich regions, which bind Sp-1 and Sp-3 transcription factors. In addition, we identified a site (FP-3) located at the −282 to −310 nucleotide region of the promoter, which binds NF-1, only in B cells. Analysis of point mutations introduced at the different regions revealed functional differences in their role in mdCK transcription in the cell lines used.

Characterization of the upstream region that regulates the transcription of the gene for the precursor to EGF-related peptides, exogastrula-inducing peptides, of the sea urchin Anthocidaris crassispina

The EGIP gene for exogastrula-inducing peptides (EGIPs) of the sea urchin Anthocidaris crassispina, which are structurally related to the epidermal growth factor, is activated at the onset of gastrulation in subdomains of the embryonic ectoderm. We showed in our previous study that the spatial and temporal regulation of EGIP is conducted by the upstream region from −372 to +194, and that there is an enhancer element between −372 and −210. In this study, we introduced into sea urchin embryos PCR-amplified DNA containing differently truncated EGIP flanking region that was ligated to the GFP reporter gene, and examined the transient expression of the reporter gene, showing that both the −270/−238 and −249/−210 regions were essential for the enhancer activity. We further showed that there is another activating element between −65 and −21, and that even the region between −65 and +194 is sufficient for ectoderm-specific expression of the EGIP gene. The electrophoretic mobility shift assay showed that the −270/−210 enhancer region and the proximal −61/+30 region include specific binding sites for nuclear proteins of sea urchin embryos. Besides these unique sites, the presence of multiple binding sites for GCF1-like nuclear proteins have been revealed in the upstream DNA.

Promoter identification of CIKS, a novel NF-κB activating gene, and regulation of its expression

We have recently identified a novel gene, named CIKS (Connection to IKK-complex and SAPK), able to activate the transcription factor NF-κB, after interaction with the regulatory subunit NEMO/IKKγ of IKK complex, and the stress-activated protein kinase (SAPK)/JNK. CIKS mRNA is ubiquitously expressed, although its levels differ greatly among different tissues. The aim of this study is to identify and characterize the promoter region of CIKS gene and to analyse the regulation of its expression by different cytokines. The transcription start site of CIKS mRNA was mapped both by primer extension and by a polymerase chain reaction (PCR)-based strategy. The proximal 5′-flanking region of CIKS gene was ‘TATA-less’, but contained other consensus promoter elements including an initiator (Inr), ‘GC’ and ‘CAAT’ boxes. Transfection of luciferase reporter plasmids containing 1.8 kb of the 5′-flanking region increased luciferase activity in epithelial MDCK cells, but not in endothelial HUVEC cells. Deletion analysis identified a sequence from −464 to −220 bp of the 5′-flanking region of CIKS gene essential for basal promoter activity in MDCK cells. Competitive reverse transcriptase–PCR, Northern and Western blot assays showed that different cytokines, such as tumor necrosis factor (TNF)-α, Interleukin (IL)-1β and transforming growth factor (TGF)-β, dramatically increased CIKS mRNA expression in HeLa cells. We conclude that the proximal 5′-flanking region of CIKS gene contains a functional promoter and binding sites for nuclear proteins leading to its basal transcription. Moreover, we demonstrate that the expression of CIKS is up-regulated by different cytokines.

Haplotypes of alpha-globin gene regulatory element in two Brazilian native populations.

The alpha-major regulatory element (alpha-MRE), located 40 Kb far upstream of the alpha-globin gene cluster on chromosome 16, is involved in the regulation of human alpha-globin genes expression. The activity of this element is restricted to a 350-bp fragment in which several nuclear protein binding sites have been identified. This element is genetically polymorphic and different haplotypes, named A-F, have been determined in seven populations of Europe, Africa, Asia, and Oceania. We describe here the alpha-MRE haplotypes found in native Indians from two nonmiscegenated tribes of the north region of Brazil, in Amazonia, the Parakanã and the Xikrin. The A haplotype was predominant in both (70% and 87%, respectively), followed by the B haplotype (30% and 13%, respectively). The haplotype frequency distribution among the Parakanã was similar to that reported for Indonesians and Southeast Asian populations, while the distribution among the Xikrin showed higher similarity to that observed in Indonesians. These results corroborate the existence of genetic affinities between Brazilian Indians and Southeast Asian and Oceanic populations. This was the first investigative work on the alpha-MRE polymorphism in South American native populations in general or Brazilian native populations in particular.

A splicing alteration of 4.1R pre-mRNA generates 2 protein isoforms with distinct assembly to spindle poles in mitotic cells

The C-terminal region of erythroid cytoskeletal protein 4.1R, encoded by exons 20 and 21, contains a binding site for nuclear mitotic apparatus protein (NuMA), a protein needed for the formation and stabilization of the mitotic spindle. We have previously described a splicing mutation of 4.1R that yields 2 isoforms: One, CO.1, lacks most of exon 20-encoded peptide and carries a missense C-terminal sequence. The other, CO.2, lacks exon 20-encoded C-terminal sequence, but retains the normal exon 21-encoded C-terminal sequence. Knowing that both shortened proteins are expressed in red cells and assemble to the membrane skeleton, we asked whether they would ensure 4.1R mitotic function in dividing cells. We show here that CO.2, but not CO.1, assembles to spindle poles, and colocalizes with NuMA in erythroid and lymphoid mutated cells, but none of these isoforms interact with NuMA in vitro. In microtubule-destabilizing conditions, again only CO.2 localizes to the centrosomes. These data suggest that the stability of 4.1R association with centrosomes requires an intact C-terminal end, either for a proper conformation of the protein, for a direct binding to an unknown centrosome-cytoskeletal network, or for both. We also found that 4.1G, a ubiquitous homolog of 4.1R, is present in mutated as well as control cells and that its C-terminal region binds efficiently to NuMA, suggesting that in fact mitotic spindles host a mixture of the two 4.1 family members. These findings led to the postulate that the coexpression at the spindle poles of 2 related proteins, 4.1R and 4.1G, might reflect a functional redundancy in mitotic cells.

NTF2 monomer-dimer equilibrium

Nuclear transport factor 2 (NTF2) mediates nuclear import of RanGDP, a central component of many nuclear trafficking pathways. NTF2 is a homodimer and each chain has independent binding sites for RanGDP and nuclear pore proteins (nucleoporins) that contain FxFG sequence repeats. We show here that the monomer-dimer dissociation constant for NTF2 obtained by sedimentation equilibrium ultracentrifugation is in the micromolar range, indicating that a substantial proportion of cellular NTF2 may be monomeric. To investigate the functional significance of NTF2 dimerization, we engineered a series of point mutations at the dimerization interface and one of these (M118E) remained monomeric below concentrations of 150 μM. CD spectra and X-ray crystallography showed that M118E-NTF2 preserved the wild-type NTF2 fold, although its thermal stability was 20 deg. C lower than that of the wild-type. M118E-NTF2 bound both RanGDP and FxFG nucleoporins less strongly, suggesting that dissociation of the NTF2 dimer could facilitate RanGDP release and thus nucleotide exchange after it had been transported into the nucleus. Moreover, colloidal gold coated with M118E-NTF2 showed reduced binding to Xenopus oocyte nuclear pores. Overall, our results indicate that dimer formation is important for NTF2 function and give insight into the formation of heterodimers by mRNA export factors such as TAP1 and NXT1 that contain NTF2-homology domains.

Molecular regulation of class switch recombination to IgE through epsilon germline transcription.

Molecular regulation of class switch recombination to IgE through epsilon germline transcription.

NF-kappa B p50-dependent in vivo footprints at Ig S gamma 3 DNA are correlated with mu-->gamma 3 switch recombination.

NF-kappa B has been demonstrated to play critical roles in multiple aspects of immune responses including Ig H chain isotype switching. To better define the specific roles the p50 subunit of NF-kappa B plays in mu-->gamma 3 switch recombination (SR), we systematically evaluated p50-deficient B cells for activities that are strongly correlated with SR. B cell activation with LPS plus anti-IgD-dextran plus IL-5 plus IL-4 plus TGF-beta produced normal levels of proliferation and gamma3 germline transcripts in p50-deficient B cells, but mu-->gamma 3 SR was impaired. In vitro binding studies previously showed that NF-kappa B p50 homodimer binds the switch nuclear B-site protein (SNIP) of the S gamma 3 tandem repeat. Ligation-mediated PCR in vivo footprint analysis demonstrates that the region spanning the SNIP and switch nuclear A-site protein (SNAP) binding sites of the S gamma 3 region are contacted by protein in normal resting splenic B cells. B cells that are homozygous for the targeted disruption of the gene encoding p50 (-/-) show strong aberrant footprints, whereas heterozygous cells (+/-) reveal a partial effect in S gamma 3 DNA. These studies provide evidence of nucleoprotein interactions at switch DNA in vivo and suggest a direct interaction of p50 with S gamma 3 DNA that is strongly correlated with SR competence.

Sequence and functional properties of African green monkey CD4 silencer

We reported previously that in African green monkey (AGM) CD4 lymphocytes, CD4 mRNA expression undergoes a decrease following in vitro activation, and CD4 cells are therefore subject to loss of CD4 expression on the cell surface. To examine the transcriptional regulation of the CD4 gene in this species, we analyzed the CD4 silencer, which has been identified as a regulatory element responsible for the down regulation of CD4 transcription in CD8 cell lineage cells. Sequence analysis indicated that the CD4 silencer of the AGM was highly homologous to that of humans. However, two nucleotide substitutions were present in one of the nuclear protein binding sites, which was characterized as the FP II site having a strong enhancing effect on transgene expression in CD4 cells. By performing transient transfection assays, we found that the enhancing activities of the CD4 silencer or FP II-containing fragment of the AGM were greatly reduced in a human CD4 cell line as compared to those of human materials. The CD4 mRNA level was significantly decreased in the human CD4 cell line when synthetic oligonucleotide corresponding to the human FP II sequence was added to the culture. These observations imply that FP II-protein interaction might be required for the maintenance of sufficient expression of the CD4 gene, and the enhancing activity mediated by the above interaction might be decreased in the AGM CD4 silencer, due probably to the nucleotide changes occurring at the FP II site.

An Open Chromatin Structure in a Liver-Specific Enhancer That Confers High Level Expression to Human Apolipoprotein B Transgenes in Mice

A number of DNaseI-hypersensitive (DH) sites have been mapped within a regulatory region situated upstream of the human apolipoprotein B (apoB) promoter (−5262 to −899) that is required for high level expression of human apoB transgenes in the livers of mice. These DH sites were observed in nuclei from transcriptionally active liver-derived HepG2 cells, but were absent from transcriptionally inactive HeLa cell nuclei. Several nuclear protein binding sites were detected in the DNaseI-hypersensitive region by DNaseI footprinting with HepG2 nuclear extracts, representing putative binding sites for the liver-specific activators. The locations of binding sites for these transcription factors were revealed via computer analysis of the DNA sequence of this region against a transcription factor database. Many micrococcal nuclease hypersensitive (MH) sites were also observed in nuclei from HepG2 cells but not in HeLa cell nuclei, implying that in hepatic cells, nucleosomes are either absent or have been displaced from this region by the liver-specific transcriptional activators, as inferred by the correspondence between the DH sites, the MH sites and the footprints.

Functional role of a novel cis-acting element (GAGA box) in human type-1 angiotensin II receptor gene transcription.

GH/growth factors have been shown to increase angiotensin type 1 receptor expression. In the present study we determined the cis-acting regulatory region controlling GH-induced transcription of the human type-1 angiotensin receptor (hAT(1)). In human proximal tubule cells transfected with a chloramphenicol acetyl transferase (CAT) reporter plasmid under the control of the hAT(1) promoter, GH induced CAT activity. Serial deletions of the hAT(1) promoter region indicated that an area between -314 bp and -70 bp upstream of the 5'-end of the cDNA sequence was essential for this activation to occur. Although sequence analysis identified putative multiple nuclear protein binding sites in this region, we determined that a 12 bp sequence (5'-GAGAGGGAGGAG-3', GAGA box) located between -161 bp and -149 bp was important for GH-mediated activation. Using mobility shift assays we demonstrated increased DNA binding activity to the labeled GAGA box in nuclear extracts treated with GH, suggesting this sequence is a GH response element. Southwestern analysis identified an 18 kDa GAGA box-binding protein (GAGA-BP). GH-induced activity of the GAGA-BP occurred within 2.5 min and reached a maximum at 5 min. Activation did not require de novo protein synthesis. Removal of the GAGA box abolished GH-induced transcription as well as basal transcription of the hAT(1) gene. Additional studies demonstrated that epidermal growth factor, platelet-derived growth factor and insulin activate the GAGA-BP, suggesting these growth factors can also regulate the transcription of the hAT(1) gene through the GAGA box. Our data show that the GAGA-BP acts as a trans-acting factor binding to the cis-acting regulatory element in the hAT(1) promoter, which is necessary for the basal and growth factor(s)-mediated transcriptional activation of the hAT(1) gene.

Identification of a Nuclear Respiratory Factor-1 Binding Site within the Core Promoter of the human polio virus receptor/CD155 Gene

In this report we describe a cis-acting element within the core promoter of the CD155 gene specifying the polio virus receptor that is bound by the nuclear respiratory factor-1 (NRF-1) transcription factor. DNase I footprint analysis identified a nuclear protein binding site from -282 to -264 nucleotides upstream of the translation initiation codon of the CD155 gene, which we have called foot print IV (FPIV). Linker scanning mutagenesis revealed that a tandem repeat motif, GCGCAGGCGCAG, located within FPIV was essential for the basal activity of the CD155 core promoter. The results of the electrophoretic mobility shift assay experiments suggested that identical FPIV binding activities were present in a variety of nuclear extracts and that the tandem repeat was essential for binding. A one-hybrid screen was then carried out using FPIV as bait to clone the cDNA of the FPIV binding factor. The sequences of the cDNAs that were cloned from the screen were identical to NRF-1, a result that was confirmed by further electrophoretic mobility shift assay experiments. Overexpression of full-length NRF-1 and a dominant-negative form of NRF-1 modulated reporter gene expression driven by the core promoter. Remarkably, CD155 is the first gene shown to be regulated by NRF-1 that possesses an expression profile during embryogenesis correlating with this factor's proposed role in the development of the vertebrate optic system. We propose that NRF-1, which has been shown by others to be expressed during embryogenesis in animal systems, may be involved in regulating the expression of CD155 at specific stages of central nervous system development.