Functional analysis of human alpha 1(I) procollagen gene promoter. Differential activity in collagen-producing and -nonproducing cells and response to transforming growth factor beta 1.

To gain a further understanding of the regulation of human type I collagen gene expression under physiologic and pathologic conditions, we characterized 5.3 kilobase pairs (kb) of the human alpha 1(I) procollagen gene promoter. A series of deletion constructs containing portions of the alpha 1(I) procollagen 5\u27-flanking region (with end points from -5.3 kb to -84 base pairs (bp)) ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into NIH/3T3 cells. Maximal CAT activity was observed with constructs having 5\u27 end points from -804 to -174 bp. A further 5\u27 deletion to -84 bp caused a marked reduction in CAT activity. Cells transfected with plasmids containing longer 5\u27-flanking fragments of the alpha 1(I) procollagen gene (-2.3 or -5.3 kb) showed reduced CAT activity compared with the -804 bp construct. The activity of the alpha 1(I) procollagen promoter was much lower in cells that do not normally express type I collagen (HeLa cells) compared with collagen-producing NIH/3T3 cells. The CAT activity of deletion constructs containing longer 5\u27 regions than -84 bp was increased by approximately 2-fold in NIH/3T3 cells treated with transforming growth factor beta 1 (TGF beta 1). Electrophoretic mobility shift assays indicated that protein-DNA complex formation with a probe corresponding to the -170 to -80 bp fragment of the alpha 1(I) procollagen promoter was markedly enhanced in nuclear extracts prepared from TGF beta 1-treated fibroblasts as compared with untreated fibroblasts. The DNA binding activity stimulated by TGF beta 1 was specific for an Sp1-like sequence at positions -164 to -142 bp in the promoter. These results demonstrate that 1) there are both positive and negative cis-acting regulatory elements in the human alpha 1(I) procollagen promoter, 2) these regulatory regions function differently in collagen-producing and -nonproducing cells, 3) the alpha 1(I) procollagen promoter contains TGF beta 1-responsive sequences located between -174 and -84 bp from the transcription start site, and 4) TGF beta 1 caused marked stimulation of the DNA binding activity of a nuclear factor interacting with an Sp1-like binding site located within a region encompassing -164 to -142 bp of the alpha 1(I) procollagen promoter

Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter.

Lentiviral vectors almost universally use heterologous internal promoters to express transgenes. One of the most commonly used promoter fragments is a 1.2-kb sequence from the human ubiquitin C (UBC) gene, encompassing the promoter, some enhancers, first exon, first intron and a small part of the second exon of UBC. Because splicing can occur after transcription of the vector genome during vector production, we investigated whether the intron within the UBC promoter fragment is faithfully transmitted to target cells. Genetic analysis revealed that more than 80% of proviral forms lack the intron of the UBC promoter. The human elongation factor 1 alpha (EEF1A1) promoter fragment intron was not lost during lentiviral packaging, and this difference between the UBC and EEF1A1 promoter introns was conferred by promoter exonic sequences. UBC promoter intron loss caused a 4-fold reduction in transgene expression. Movement of the expression cassette to the opposite strand prevented intron loss and restored full expression. This increase in expression was mostly due to non-classical enhancer activity within the intron, and movement of putative intronic enhancer sequences to multiple promoter-proximal sites actually repressed expression. Reversal of the UBC promoter also prevented intron loss and restored full expression in bidirectional lentiviral vectors

An investigation of herpes simplex virus promoter activity compatible with latency establishment reveals VP16-independent activation of immediate-early promoters in sensory neurones

Herpes simplex virus (HSV) type-1 establishes lifelong latency in sensory neurones and it is widely assumed that latency is the consequence of a failure to initiate virus immediate-early (IE) gene expression. However, using a Ore reporter mouse system in conjunction with Ore-expressing HSV-1 recombinants we have previously shown that activation of the IE ICPO promoter can precede latency establishment in at least 30 % of latently infected cells. During productive infection of non-neuronal cells, IE promoter activation is largely dependent on the transactivator VP16 a late structural component of the virion. Of significance, VP16 has recently been shown to exhibit altered regulation in neurones; where its de novo synthesis is necessary for IE gene expression during both lytic infection and reactivation from latency. In the current study, we utilized the Ore reporter mouse model system to characterize the full extent of viral promoter activity compatible with cell survival and latency establishment. In contrast to the high frequency activation of representative IE promoters prior to latency establishment, cell marking using a virus recombinant expressing Ore under VP16 promoter control was very inefficient. Furthermore, infection of neuronal cultures with VP16 mutants reveals a strong VP16 requirement for IE promoter activity in non-neuronal cells, but not sensory neurones. We conclude that only IE promoter activation can efficiently precede latency establishment and that this activation is likely to occur through a VP16-independent mechanism

Genetic analysis of the human tumor necrosis factor alpha/cachectin promoter region in a macrophage cell line.

The 615-bp 5' flanking region of the human TNF-alpha/cachectin gene was isolated and ligated to the luciferase reporter gene. In addition, a series of truncated promoter constructs was generated by exonuclease III digestion. The promoter activity of these constructs was studied in a transient transfection system using the TNF-alpha-producing U937 cell line. Full-length and truncated TNF promoter constructions extending from -615 to -95 bp relative to the transcription start site (TSS) could be induced by phorbol esters. A construct truncated to within 36 bp of the TSS (and within 11 bp of the TATAA box) was inactive. Therefore, the phorbol ester responsive is localized in the TNF/cachectin promoter to a relatively short region proximal to the TATAA box

GC-selective DNA-binding antibiotic, Mithramycin A, reveals multiple points of control in the regulation of Hdm2 protein synthesis

The primary role of the Hdm2/Mdm2 oncoprotein is to regulate the levels and activity of the transcription factor p53. Hdm2 synthesis is itself tightly controlled and, as demonstrated by a recently described SNP (SNP309) in the hdm2-P2 promoter, minor variations in Hdm2 expression have phenotypic consequences on radiation sensitivity and cancer predisposition. To further define mechanisms regulating Hdm2 expression, we have investigated the effects of the GC-selective DNA-binding drug, Mithramycin A (MA) on hdm2 mRNA transcription, trafficking, and translation. Firstly we show that the constitutive hdm2-P1 promoter is inhibited by MA. We define, for the first time, the minimal sequence elements that are required for P1-promoter activity and identify those which confer MA sensitivity. Secondly, MA induces p53-dependent transcription from the hdm2-P2 promoter. Thirdly, and critically, MA also inhibits Hdm2 synthesis at the post-transcriptional level, with negative effects on hdm2 mRNA nuclear export and translation. This study highlights the complex interplay between the pathways that regulate Hdm2 protein synthesis in cancer cells, and furthermore emphasizes the export of hdm2 mRNA from the nucleus to the cytoplasm as a key point of control in this process.<br/><br/

Common promoter variant in cyclooxygenase-2 represses gene expression: evidence of role in acute-phase inflammatory response

Objective: Cyclooxygenase (COX)-2 is a key regulatory enzyme in the synthesis of prostanoids associated with trauma and inflammation. We investigated the COX-2 gene for functional variants that may influence susceptibility to disease. Methods and results: The promoter of COX-2 was screened for variants in healthy subjects by use of polymerase chain reaction-based methods. Promoter activity was investigated by using reporter expression experiments in human lung fibroblasts. Patients undergoing coronary artery bypass graft surgery, with measurements of plasma markers linked to COX-2 activity, were genotyped for association studies. A common COX-2 promoter variant, -765G>C, was found and shown to be carried by >25% of a group of healthy UK subjects. The -765C allele had significantly lower promoter activity compared with -765G, basally (28±3% lower, P<0.005) and in serum-stimulated cells (31±2% lower, P<0.005). In patients subjected to coronary artery bypass graft surgery, the magnitude of rise in levels of C-reactive protein (CRP) was strongly genotype dependent. Compared with -765G homozygotes, patients carrying the -765C allele had significantly lower plasma CRP levels at 1 to 4 days after surgery (14% lower at the peak of CRP levels on day 3, P<0.05 for all time points). Conclusions: For several acute and chronic inflammatory diseases, -765G>C may influence the variability of response observed

Distinct promoter elements mediate the co-operative effect of Brn-3a and p53 on the p21 promoter and their antagonism on the Bax promoter

Although the promoters of both the Bax and p21 genes are activated by p53, they differ in the effect on this activation of the POU family transcription factor Brn-3a. Thus, Brn-3a inhibits activation of the Bax promoter by p53 but enhances the ability of p53 to activate the p21 promoter. We demonstrate that repression of p53-mediated activation of the Bax promoter involves a complex upstream sequence in which two Brn-3a response elements flank the p53 response element. In contrast, a minimal p21 promoter is activated by Brn-3a and such activation cannot be abolished without abolishing basal promoter activity. Moreover, synergistic activation by Brn-3a and p53 continues to be observed when the p53-binding sites in the p21 promoter are substituted by the Bax p53 site or by the region of the Bax promoter essential for Brn-3a-mediated repression, indicating that the p21 core promoter plays a central role in this response. The significance of these effects is discussed in terms of the different responses of the Bax and p21 promoters and the overlapping but distinct roles of Brn-3a and p53 in neuronal growth arrest and apoptosis

An open reading frame upstream from the nifH gene of Klebsiella pneumoniae

An open reading frame upstream from nifHDK operon of Klebsiella pneumoniae had been described. The orientation of this open reading frame is opposite to that of nifHDK and sequence homology was found between the open reading frame promoter and the promoter of nifHDK operon. A recombinant plasmid carrying the promoter region of the open reading frame fused to the beta-galactosidase gene was constructed. Strains of E.coli were transformed with the plasmid containing this open reading frame promoter-lacZ fusion or co-transformed with it and a plasmid carrying the nifA gene. An appreciable activity of beta-galactosidase was found in strains which received both plasmids, indicating that the promoter of the open reading frame can be activated by the product of nifA gene. Thus, the open reading frame found between nifHDK operon and nifJ behaves just like other nif genes of K.pneumoniae in requiring the product of nifA as the positive effector for expression