nifH promoter activity is regulated by DNA supercoiling in Sinorhizobium meliloti

In prokaryotes, DNA supercoiling regulates the expression of many genes; for example, the expression of Klebsiella pneumoniae nifLA operon depends on DNA negative supercoiling in anaerobically grown ceils, which indicates that DNA supercoiling might play a role in gene regulation of the anaerobic response. Since the expression of the nifH promoter in Sinorhizobium meliloti is not repressed by oxygen, it is proposed that the status of DNA supercoiling may not affect the expression of the nifH promoter. We tested this hypothesis by analyzing nifH promoter activity in wild-type and gyr- Escherichia coli in the presence and absence of DNA gyrase inhibitors. Our results show that gene expression driven by the S.meliloti nifH promoter requires the presence of active DNA gyrase. Because DNA gyrase increases the number of negative superhelical turns in DNA in the presence of ATP, our data indicate that negative supercoiling is also important for nifH promoter activity. Our study also shows that the DNA supercoiling-dependent S. meliloti nifH promoter activity is related to the trans-acting factors NtrC and NifA that activate it. DNA supercoiling appeared to have a stronger effect on NtrC-activated nifH promoter activity than on NifA-activated promoter activity. Collectively, these results from the S. meliloti nifH promoter model system seem to indicate that, in addition to regulating gene expression during anaerobic signaling, DNA supercoiling may also provide a favorable topology for trans-acting factor binding and promoter activation regardless of oxygen status.     

The RFA regulatory sequence-binding protein in the promoter of

The prostate-specific antigen (PSA), which plays an important role during the liquefaction process of semen, is a differentiation marker for human prostate. It has become the most sensitive marker for monitoring and detecting prostate cancer. PSA as a serine protease can activate some growth factors that might be related to the advancement of prostate cancer by hydrolyzing growth factor-binding protein. The PSA gene is expressed specifically in prostate epithelial cells. The ex-pression of…     

Up-regulation of NKX3.1 expression and inhibition of LNCaP cell proliferation induced by an inhibitory element decoy

NKX3.1 is an androgen-regulated prostate-specific homeobox gene that is thought to play an important role in prostate development and cancerogenesis. NKX3.1 acts as a tumor suppressor gene specifically in the prostate. Up-regulation of NKX3.1 gene offers a promising gene therapy for prostate cancer. The decoy strategy has been developed and is considered a useful tool for regulating gene expression and gene therapy. In our previous studies, we identified a 20 bp inhibitory element upstream of the NKX3.1 promoter.In this study, we focused on using the 20 bp inhibitory element decoy to block negative regulation of the NKX3.1 gene and to up-regulate NKX3.1 expression using synthetic double-stranded oligodeoxynucleotides of the 20 bp inhibitory element. We found in an electrophoretic mobility shift assay experiment that the 20 bp inhibitory decoy presented competitive binding to a specific binding protein of the 20 bp inhibitory element in prostate cancer cell line LNCaP. In luciferase reporter gene assays, we found that the 20 bp inhibitory decoy could enhance NKX3.1 promoter activity, and RT-PCR and Western blot analysis revealed that NKX3.1 expression was up-regulated effectively by the transfection with the 20 bp inhibitory decoy. Furthermore,cell proliferation was inhibited by up-regulated NKX3.1 expression induced by the 20 bp inhibitory decoy.     

Three-dimensional genome architectural CCCTC-binding factor makes choice in duplicated enhancers at Pcdhα locus

During development, gene expression is spatiotemporally regulated by long-distance chromatin interactions between distal enhancers and target promoters. However, how specificity of the interactions between enhancers and promoters is achieved remains largely unknown. As there are far more enhancers than promoters in mammalian genomes, the complexities of enhancer choice during gene regulation remain obscure. CTCF, the CCCTC-binding factor that directionally binds to a vast range of genomic sites known as CBSs(CTCF-binding sites), mediates oriented chromatin looping between a substantial set of distal enhancers and target promoters. To investigate mechanisms by which CTCF engages in enhancer choice, we used CRISPR/Cas9-based DNA-fragment editing to duplicate CBS-containing enhancers and promoters in the native genomic locus of the clustered Pcdhα genes. We found that the promoter is regulated by the proximal one among duplicated enhancers and that this choice is dependent on CTCF-mediated directional enhancer-promoter looping. In addition, gene expression is unaltered upon the switch of enhancers. Moreover, after promoter duplication, only the proximal promoter is chosen by CTCF-mediated directional chromatin looping to contact with the distal enhancer. Finally, we demonstrated that both enhancer activation and chromatin looping with the promoter are essential for gene expression. These findings have important implications regarding the role of CTCF in specific interactions between enhancers and promoters as well as developmental regulation of gene expression by enhancer switching.     

Highly efficient transformation of intact yeast-like conidium cells of Tremella fuciformis by electroporation

Tremella fuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. So it is a good recipient cell for exogenous gene expression. In this study, two expression vectors pGlg-gfp containing gpd-Gl promoter and gfp gene and pGlg-hph containing gpd-Gl promoter and hph gene were constructed. The lowest sensitive concentration of hygromycin for the blastospore was determined on three types of media. Our ex- periments showed that the lowest sensitive concentration of hygromycin for the blastospore was 5 μg/mL on MA medium. The intact blastospores were transformed with the expression vector pGlg-hph by electroporation. The putative transformants were obtained by the MA selective medium. Experi- mental results showed that the most effective parameters for the electroporation of intact blastospores were obtained by using STM buffer, 1.0×108 cells/mL of blastospores, 200 μL in transformation volume, 6 μg plasmid, 2.0 kV/cm of electric pulse voltage, stillness culturing on MB liquid medium for 48 h after electroporation. In these transformation conditions, the efficiency reached 277 colonies/μg DNA. Co-transformation of plasmid pGlg-gfp and pGlg-hph with ratio of 1:1 was performed by electroporation with the optimal parameters. The putative co-transformants were obtained by the MA selective medium. Eight randomly selected colonies from the vast putative co-transformants were analyzed by PCR de- tection and Southern blotting. The experiments showed that the gfp was integrated into the genomes of three transformants. The co-transformation efficiency was 37.5%. Green fluorescence was observed under laser scanning confocal microscope in these gfp positive transformants. This indicates that the exogenous gfp can be expressed effectively in the yeast-like conidia of T. fuciformis.     

Identification of a novel nucleus protein involved in the regulation of urokinase in 95D cells

The urokinase-type plasminogen activator (uPA) plays an important role in cellular invasion.By using the downstream part of a 74 bp DNA region called the cooperation mediator (COM) of the uPA promoter as a bait sequence in the yeast one-hybrid screen, a gene called PBK1 was previously cloned from the cDNA library of the 95D lung cancer cell strain. In this study, the intracellular distribution of PBK1 was studied by using the transient transfection of pEGFP-C3-PBK1, and PBK1 was found to be localized in the nucleus. Co-transfection of pEGFP-C3-PBK1 and the deletion mutants of the pGL3-uPA promoter indicated that PBK1 can increase the uPA promoter activity by about 25% and this effect is uPA enhancer-dependent.Western blotting and Enzyme-linked immunoadsordent assay further confirmed that PBK1 can upregulate the expression of uPA. Our results suggest that PBK1 is involved in the regulation of uPA expression, which might provide a new clue to further understanding the regulation mechanism of uPA expression.     

Treatment of hepatitis C virus core-positive hepatocytes with the transfer of recombinant caspase-3 using the 2~,5~-oligoadenylate synthetase gene promoter

Hepatitis C virus （HCV） infection is a leading cause of liver-related morbidity and mortality throughout the world. There is no vaccine available and current therapy is only partially effective. Since HCV infects only a minority of hepatocytes, we hypothesized that induction of apoptosis might be a promising approach for the treatment of hepatitis C. In the present study, recombinant caspase-3 gene （re-caspase-3） was used because it has the ability to induce apoptosis that is independent of the initiator caspases. An HCV-specific promoter is required to regulate the cytotoxic caspase-3 expression in HCV-infected cells. It has been reported that HCV core protein can specifically activate the 21,5~-oligoadenylate synthetase （OAS） gene promoter in human hepatocytes. Therefore, we constructed an expression vector consisting of the re-caspase-3 under the OAS gene promoter （pGL3-OAS-re-caspase-3） and then investigated its effect on HCV core-positive liver cells. It was found that the pGL3-OAS-re-caspase-3 construct induced apoptosis in HCV core-positive liver ceils, but not in normal liver ceils. These results strongly suggested that the transfer of the re-caspase-3 gene under the OAS promoter was a novel targeting approach for the treatment of HCV infection.     

雄激素应答元件假冒DNA对PSA基因启动子的抑制作用

研究雄激素应答元件假冒DNA(AREdecoy)对前列腺特异抗原 (PSA)基因启动子的抑制作用 .联合运用报告基因和假冒DNA策略 ,构建了含PSA基因 5′侧启动子区 6 40bpDNA的萤光素酶表达载体pGL3 PSA ,与人工合成的双链硫代ARE假冒DNA共转染前列腺癌细胞株PC3 M并作用不同的时间 (2 4h、4 8h、72h) .应用双萤光素酶测定系统 ,检测萤光素酶的表达活性 .结果显示 :AREdecoyDNA显著抑制报告基因萤光素酶的表达 ,抑制率可达 95 % ,而对照decoyDNA无此作用 .作用不同的时间对萤光素酶活性的抑制无显著性差异     

Identification of two novel cis-elements in the promoter of the prostate-specific antigen gene that are required to enhance androgen receptor-mediated transactivation.

A monomeric androgen responsive element (ARE) is not sufficient to mediate significant androgen induction of the prostate-specific antigen (PSA) gene. Co-transfection experiments using a series of 5'deletion fragments of the proximal promoter region of the PSA gene linked to bacterial chloramphenicol acetyltransferase (CAT) as a reporter have identified two motif sequences which are indispensable for androgen receptor (AR)-mediated transactivation of the PSA promoter and have been designated as motifs A and B respectively. Of note, motif B alone has very little independent enhancer activity regardless of the presence or absence of androgen, whereas multi-copies of motif A exert androgenic inducibility for a heterologous promoter independent of the presence of ARE. Nucleotide substitutions in either motif significantly decrease the androgen inducibility and the nuclear protein binding ability. Furthermore, gel band shift experiments consistently demonstrate that nuclear proteins can bind these motifs, and they are non-receptor factors. Our data indicate that these two DNA motifs are novel cis -regulatory elements and exhibit different mechanisms in cooperation with ARE for AR-mediated transactivation.