Lipid deprivation increases surfactant phosphatidylcholine synthesis via a sterol-sensitive regulatory element within the CTP:phosphocholine cytidylyltransferase promoter

2002 ◽  
Vol 362 (1) ◽  
pp. 81-88 ◽  
Author(s):  
Rama K. MALLAMPALLI ◽  
Alan J. RYAN ◽  
James L. CARROLL ◽  
Timothy F. OSBORNE ◽  
Christie P. THOMAS
Keyword(s):  
Specific Binding ◽  
Core Promoter ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Luciferase Reporter ◽  
Standard Diet ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Ctp:Phosphocholine Cytidylyltransferase

Lipid-deprived mice increase alveolar surfactant disaturated phosphatidylcholine (DSPtdCho) synthesis compared with mice fed a standard diet by increasing expression of CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme for DSPtdCho synthesis. We previously observed that lipid deprivation increases mRNA synthesis for CCT [Ryan, McCoy, Mathur, Field and Mallampalli (2000) J. Lipid Res. 41, 1268–1277]. To evaluate regulatory mechanisms for this gene, we cloned the proximal ∼ 1900bp of the 5′ flanking sequence of the murine CCT gene, coupled this to a luciferase reporter, and examined transcriptional regulation in a murine alveolar epithelial type II cell line (MLE-12). The core promoter was localized to a region between −169 and +71bp, which exhibited strong basal activity comparable with the simian virus 40 promoter. The full-length construct, from −1867 to +71, was induced 2–3-fold when cells were cultured in lipoprotein-deficient serum (LPDS), similar to the level of induction of the endogenous CCT gene. By deletional analysis the sterol regulatory element (SRE) was localized within a 240bp region. LPDS activation of the CCT promoter was abolished by mutation of this SRE, and gel mobility-shift assays demonstrated specific binding of recombinant SRE-binding protein to this element within the CCT promoter. These observations indicate that sterol-regulated expression of CCT is mediated by an SRE within its 5′ flanking region.

scholarly journals Interactions Required for Binding of Simian Virus 40 T Antigen to the Viral Origin and Molecular Modeling of Initial Assembly Events

2004 ◽  
Vol 78 (6) ◽  
pp. 2921-2934 ◽  
Author(s):  
Danielle K. Reese ◽  
Kodangattil R. Sreekumar ◽  
Peter A. Bullock
Keyword(s):  
Structural Information ◽  
Specific Binding ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Binding Domain ◽  
Helicase Domain ◽  
Flanking Sequence ◽  
Viral Origin ◽  
Flanking Sequences

ABSTRACT The purified T-antigen origin binding domain binds site specifically to site II, the central region of the simian virus 40 core origin. However, in the context of full-length T antigen, the origin binding domain interacts poorly with DNA molecules containing just site II. Here we investigate the contributions of additional core origin regions, termed the flanking sequences, to origin recognition and the assembly of T-antigen hexamers and double hexamers. Results from these studies indicate that in addition to site-specific binding of the T-antigen origin binding domain to site II, T-antigen assembly requires non-sequence-specific interactions between a basic finger in the helicase domain and particular flanking sequences. Related studies demonstrate that the assembly of individual hexamers is coupled to the distortions in the proximal flanking sequence. In addition, the point in the double-hexamer assembly process that is regulated by phosphorylation of threonine 124, the sole posttranslational modification required for initiation of DNA replication, was further analyzed. Finally, T-antigen structural information is used to model various stages of T-antigen assembly on the core origin and the regulation of this process.

scholarly journals Early Polyadenylation Signals of Human Papillomavirus Type 31 Negatively Regulate Capsid Gene Expression

2001 ◽  
Vol 75 (17) ◽  
pp. 8147-8157 ◽  
Author(s):  
Scott S. Terhune ◽  
Walter G. Hubert ◽  
Jennifer T. Thomas ◽  
Laimonis A. Laimins
Keyword(s):  
Gene Expression ◽  
Human Papillomavirus ◽  
Binding Sites ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Capsid Gene ◽  
Keratinocyte Differentiation ◽  
Luciferase Reporter ◽  
Genome Replication

ABSTRACT The L1 and L2 capsid genes of human papillomavirus type 31 (HPV-31) are expressed upon keratinocyte differentiation from a promoter located in the E7 open reading frame (ORF) of the early region. Late transcripts must therefore pass through and ignore the early polyadenylation sequences to use the downstream late AAUAAA element located at the end of the L1 ORF. To identify sequences which modulate downstream capsid gene expression, a variety of substitution mutations were introduced into the early polyadenylation signal and studied first in the context of polycistronic luciferase reporter constructs. Removal of the G/U-rich cleavage stimulation factor (CstF) binding sites and the degenerate cleavage and polyadenylation specificity factor binding sites, UAUAUA, had minimal effect on downstream expression as defined by luciferase activities. This is in contrast to the deletion of the HPV-31 early AAUAAA element, which resulted in a dramatic increase in downstream expression. Additional sequences within the first 800 bp of the L2 ORF were also found to negatively regulate capsid expression in luciferase assays. To determine how these mutations influence gene expression in the context of the complete HPV-31 genome, recombinant genomes were constructed that contained a substitution in the AAUAAA sequence, an inserted strong CstF binding site, an inserted simian virus 40 (SV40) late poly(A) signal, or a substitution of the 5′-most 800 nucleotides of the L2 ORF. Reductions in both transient and stable replication were observed with the recombinant genomes containing the strong CstF site or the late SV40 signal, suggesting that alterations in the strength of the upstream poly(A) signal influence expression of viral replication factors. Similarly, disruption of the L2 ORF resulted in a significant reduction in genome replication and an inability to be maintained stably. In contrast, genomes containing a substitution of the AAUAAA sequence had increased levels of transient and stable replication. Quantitation of late transcripts following keratinocyte differentiation in methylcellulose also showed a reduction in downstream capsid gene expression in lines containing genomes with the strong CstF site or the late SV40 signal mutations, while a significant increase in expression was detected in the lines with genomes lacking the AAUAAA sequence. These studies demonstrate that capsid gene expression in HPV-31 requires an inefficient early poly(A) signal which is defined primarily by the AAUAAA element as well as a major negative regulatory element located within the L2 ORF.

scholarly journals Tumour metastasis suppressor, nm23-β, inhibits gelatinase A transcription by interference with transactivator Y-box protein-1 (YB-1)

2002 ◽  
Vol 366 (3) ◽  
pp. 807-816 ◽  
Author(s):  
Sunfa CHENG ◽  
Maria Alexandra ALFONSO-JAUME ◽  
Peter R. MERTENS ◽  
David H. LOVETT
Keyword(s):  
Mesangial Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Repeat Sequence ◽  
Luciferase Reporter ◽  
Metastasis Suppressor ◽  
Gelatinase A ◽  
Glomerular Mesangial Cells ◽  
Tumour Metastasis ◽  
Β Protein

Gelatinase A transcriptional regulation is the consequence of combinatorial interactions with key promoter and enhancer elements identified within this gene. A potent 40bp enhancer response element, RE-1, located in the near 5′ flanking regions of the rat and human gelatinase A genes drives high-level expression in glomerular mesangial cells (MCs). Southwestern-blot analysis of MC nuclear extracts revealed specific interactions of RE-1 with at least four proteins, of which three have been identified as p53, activator protein 2 and the single-stranded DNA-binding factor Y-box protein-1 (YB-1). In the present study, we report the identification of a fourth 17kDa RE-1-binding protein as the rat homologue (nm23-β) of the human nm23-H1 metastasis suppressor gene. Recombinant nm23-β protein bound only the single-stranded forms of the RE-1 sequence. Mutagenesis revealed direct interaction of nm23-β with a repeat sequence, 5′-GGGTTT-3′, shown previously to specifically interact with YB-1 [Mertens, Harendza, Pollock and Lovett (1997) J. Biol. Chem. 272, 22905—22912], and recombinant nm23-β protein competed for single-stranded YB-1 binding. Transient transfection of MC with an nm23-β expression plasmid within the context of a RE-1/simian virus 40 promoter/luciferase reporter yielded a concentration-dependent repression (80—90%) of luciferase activity in MC and Rat1 fibroblasts. A similar pattern of nm23-β repression was demonstrated within the context of the RE-1/homologous gelatinase A promoter. Co-transfection of nm23-β blocked YB-1-mediated activation of transcription and expression of gelatinase A. Nm23-β may be an important physiological regulator of gelatinase A transcription that acts by competitive interference with the single-stranded transactivator YB-1. Gelatinase A is a key mediator of tumour metastasis, suggesting that competitive suppression of transcription by nm23-β (or the human nm23-H1) may be a component of the reduced metastatic capabilities of cells expressing high levels of this protein.

A specific DNA sequence controls termination of transcription in the gastrin gene

1986 ◽  
Vol 6 (4) ◽  
pp. 1032-1043
Author(s):  
K Sato ◽  
R Ito ◽  
K H Baek ◽  
K Agarwal
Keyword(s):  
Signal Sequence ◽  
Regulatory Element ◽  
Shuttle Vector ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Regulation Of Transcription ◽  
Dependent Manner ◽  
Transcriptional Terminator ◽  
S1 Nuclease ◽  
Nuclease Mapping

We located and characterized a downstream transcriptional regulatory element in the human gastrin gene by transferring the gastrin gene 3' fragment, from which the polyadenylation signal sequence was deleted, into the shuttle vector pSCAT10 at a site located immediately downstream from the chloramphenicol acetyltransferase (CAT) gene and upstream from the simian virus 40 polyadenylation region. Study of CAT RNA derived from the hybrid plasmids, indicated regulation of transcription on the gastrin gene fragment. Analysis of deletion mutants generated from the 5' region of the fragment by CAT assay and by S1 nuclease mapping of mRNAs indicated the possible involvement of an oligothymidylate-rich sequence in transcription regulation. Mapping of gastrin gene RNA 3' ends to the 5' side proximal to the oligothymidylate-rich sequence clearly demonstrated that this sequence is a transcriptional terminator element. This unique sequence, interspersed with one or two adenines, which also functions in an orientation-dependent manner, is located 192 nucleotides downstream from the gastrin gene polyadenylation site, and serves as a transcriptional termination signal.

Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene

1988 ◽  
Vol 8 (7) ◽  
pp. 2896-2909 ◽  
Author(s):  
E A Sternberg ◽  
G Spizz ◽  
W M Perry ◽  
D Vizard ◽  
T Weil ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Simian Virus ◽  
Cell Type ◽  
Specific Expression ◽  
Developing Muscle

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

Genetic Test for Involvement of Intervening Sequences in Transport of Nuclear RNA

1982 ◽  
Vol 2 (12) ◽  
pp. 1550-1557
Author(s):  
Luis P. Villarreal ◽  
Susan Carr
Keyword(s):  
Simian Virus 40 ◽  
Helper Virus ◽  
Simian Virus ◽  
Transcriptional Analysis ◽  
Dna Transfection ◽  
Flanking Sequence ◽  
Intervening Sequences ◽  
16S Rna ◽  
Nuclear Rna ◽  
Late Region

The construction of a recombinant virus in the late region of simian virus 40 is presented. The small intervening sequence of late 19S RNA (0.760 to 0.765 map unit) was cloned and inserted into the Eco RI site (1.0 map unit) in the late region of simian virus 40. This is a mutant virus that now has two intervening sequences, one at the normal position (0.760 map unit) and another out of the context of its flanking sequence and now at 1.0 map unit. The recombinant appears poisonous, as repeated attempts to plaque it as a virus with a standard helper virus were unsuccessful. The transcription of this recombinant was, therefore, studied after direct DNA transfection onto CV-1 cells. Nuclease S1 analysis of mutant RNA indicates that the major nuclear transcript was a spliced but nuclear 16S RNA species. Normally, 16S RNA is not found in the nucleus. This result was shown to be an artifact of the DNA transfection protocol. When the glycerol shock was done after infection with virus, a similar alteration in the makeup of nuclear RNA was seen. A transient stock of this double-intron mutant was finally obtained, using a nonrevertable helper virus. The transcriptional analysis of this mutant showed that unspliced 19S RNA was not transported and remained within the nucleus, whereas spliced 19S and 16S RNAs were transported. We conclude that the retention of nuclear transcripts within the nucleus is not simply due to the presence of intronic sequences, as spliced 19S and 16S RNAs which contain the second intron were efficiently transported.

scholarly journals Quantitative Analysis of the Binding of Simian Virus 40 Large T Antigen to DNA

2007 ◽  
Vol 81 (17) ◽  
pp. 9162-9174 ◽  
Author(s):  
Amélie Fradet-Turcotte ◽  
Caroline Vincent ◽  
Simon Joubert ◽  
Peter A. Bullock ◽  
Jacques Archambault
Keyword(s):  
Specific Binding ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Sequence Specificity ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Viral Origin ◽  
Single Stranded Dna ◽  
Terminal Domain

ABSTRACT SV40 large T antigen (T-ag) is a multifunctional protein that successively binds to 5′-GAGGC-3′ sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequence-specific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double- and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5′-GAGGC-3′ sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an ∼10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.

scholarly journals Sequence-specific binding of simian virus 40 A protein to nonorigin and cellular DNA.

1984 ◽  
Vol 4 (12) ◽  
pp. 2631-2638 ◽  
Author(s):  
P J Wright ◽  
A L DeLucia ◽  
P Tegtmeyer
Keyword(s):  
Specific Binding ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Binding Affinities ◽  
Sequence Variations ◽  
Origin Region ◽  
Cellular Dna ◽  
Many Sources

The simian virus 40 A protein (T antigen) recognized and bound to the consensus sequence 5'-GAGGC-3' in DNA from many sources. Sequence-specific binding to single pentanucleotides in randomly chosen DNA predominated over binding to nonspecific sequences. The asymmetric orientation of protein bound to nonorigin recognition sequences also resembled that of protein bound to the origin region of simian virus 40 DNA. Sequence variations in the DNA adjacent to single pentanucleotides influenced binding affinities even though methylation interference and protection studies did not reveal specific interactions outside of pentanucleotides. Thus, potential locations of A protein bound to any DNA can be predicted although the determinants of binding affinity are not yet understood. Sequence-specific binding of A protein to cellular DNA would provide a mechanism for specific alterations of host gene expression that facilitate viral function.

scholarly journals Promoter analysis of the DHCR24 (3β-hydroxysterol Δ24-reductase) gene: characterization of SREBP (sterol-regulatoryelement-binding protein)-mediated activation

2012 ◽  
Vol 33 (1) ◽  
Author(s):  
Lidia A. Daimiel ◽  
María E. Fernández-Suárez ◽  
Sara Rodríguez-Acebes ◽  
Lorena Crespo ◽  
Miguel A. Lasunción ◽  
...  
Keyword(s):  
Cellular Response ◽  
Binding Protein ◽  
Regulatory Element ◽  
Cholesterol Biosynthesis ◽  
Regulatory Elements ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Gene Characterization ◽  
Cis Acting

DHCR24 (3β-hydroxysterol Δ24-reductase) catalyses the reduction of the C-24 double bond of sterol intermediates during cholesterol biosynthesis. DHCR24 has also been involved in cell growth, senescence and cellular response to oncogenic and oxidative stress. Despite its important roles, little is known about the transcriptional mechanisms controlling DHCR24 gene expression. We analysed the proximal promoter region and the cholesterol-mediated regulation of DHCR24. A putative SRE (sterol-regulatory element) at −98/−90 bp of the transcription start site was identified. Other putative regulatory elements commonly found in SREBP (SRE-binding protein)-targeted genes were also identified. Sterol responsiveness was analysed by luciferase reporter assays of approximately 1 kb 5′-flanking region of the human DHCR24 gene in HepG2 and SK-N-MC cells. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated cholesterol-dependent recruitment and binding of SREBPs to the putative SRE. Given the presence of several CACCC-boxes in the DHCR24 proximal promoter, we assessed the role of KLF5 (Krüppel-like factor 5) in androgen-regulated DHCR24 expression. DHT (dihydrotestosterone) increased DHCR24 expression synergistically with lovastatin. However, DHT was unable to activate the DHCR24 proximal promoter, whereas KLF5 did, indicating that this mechanism is not involved in the androgen-induced stimulation of DHCR24 expression. The results of the present study allow the elucidation of the mechanism of regulation of the DHCR24 gene by cholesterol availability and identification of other putative cis-acting elements which may be relevant for the regulation of DHCR24 expression.

Capturing nuclear sequence-specific DNA-binding proteins by using simian virus 40-derived minichromosomes

1988 ◽  
Vol 8 (2) ◽  
pp. 982-987
Author(s):  
U Nir ◽  
E Fodor ◽  
W J Rutter
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Binding Proteins ◽  
Negative Regulation ◽  
P Element ◽  
Enhancer Function ◽  
Sv40 Dna

We have used recombinant simian virus 40 (SV40) minichromosomes to retrieve sequence-specific DNA-binding proteins derived from the cell nucleus of COS-7 cells. We showed that the transcription factors AP-1 and Sp1 are stably bound to the SV40 DNA late in viral infection. Under similar conditions, minichromosomes carrying the rat insulin (rINS1) enhancer, which is under negative regulation in COS-7 cells, bound two proteins which mapped to distinct regions of the rINS1 enhancer. The SV40 P element competed for one of these proteins which bound to the region from -198 to -230. This factor may be related to AP-1. The other factor selectively bound a regulatory element in the region from -92 to -124 of the insulin enhancer. These proteins may play a role in regulating the rINS1 enhancer function.

Sign in / Sign up

Export Citation Format

Share Document