1141612823Effect of recombinant ovine intereferon tau (IFN) on the ovine Mx1 promoter/enhancer region

Ovine Mx1 (oMx1) is expressed in the uterus during the estrous cycle and is strongly up-regulated during early pregnancy in the uterus and peripheral blood leukocytes. Mx proteins are GTPases which are important elements in innate immunity. Our results showed that steroids are required for oMx1 protein induction by IFN in the uterus. To addresses the role of IFN in regulating oMx1 expression, a 2.1 kb fragment containing 1.7 kb of the promoter/enhancer, exon1 and partial intron A was cloned. Serial deletions were prepared along with a clone that contained the 2 promoter-ISREs (-101 & −145) but not the intronic-ISRE (+224). An ovine uterine cell line was transfected with reporter plasmids driven by the oMx1 promoter deletion constructs. The full-length promoter was induced by IFN in a dose- and time-dependent manner. Treatment with 10,000 AVU/mL IFN increased luciferase activity 5- and 10-fold at 3 and 12 hr, respectively. Promoter deletions showed the 2 proximal ISRE (−101 and −145), but not an intronic ISRE (+244), were required for maximal response. Deletion of a distal region (−920 to −715) resulted in decreased luciferase activity (~4-fold). However, subsequent deletion of the −715 to −437 region restored maximal promoter response (~10-fold). Results suggest that regions −920 to −715 and −715 to −437 have positive and negative regulatory element binding sites, respectively. The importance of the 2 proximal ISRE sites for oMx1 promoter activation is consistent with results for the human MxA promoter. An intronic ISRE is present in the human MxA gene, however, this site may not be required for oMx1 promoter activation by IFN. Identifying positive and negative regulatory regions in oMx1 promoter may help elucidate the unique regulation of Mx1 during early pregnancy.     

The promoter-proximal KCS element of the PKR kinase gene enhances transcription irrespective of orientation and position relative to the ISRE element and is functionally distinct from the KCS-like element of the ADAR deaminase Promoter.

The RNA-dependent protein kinase PKR promoter is interferon (IFN) inducible and possesses a novel 15-base pair (bp) constitutive activator element, designated kinase conserved sequence (KCS), in addition to an IFN-stimulated response element (ISRE). Deletion of the KCS element or point mutations within the KCS element greatly reduce both basal and IFN-inducible PKR promoter activity. The IFN-inducible RNA-specific adenosine deaminase ADAR1 promoter possesses a KCS-like (KCS-l) element. The sequences of the KCS and KCS-l elements and their positions relative to the cognate ISRE element are similar between the PKR and ADAR1 promoters. However, substitution of the ADAR1 KCS-l element for the KCS element of the PKR promoter resulted in significantly reduced basal and IFN-inducible promoter activities comparable to either point mutation or entire deletion of the PKR KCS element. The PKR KCS element selectively bound nuclear proteins more efficiently than did the ADAR1 KCS-l element. Reversing the positions of the KCS and ISRE elements of the PKR promoter relative to one another or reversing the orientation of either element while conserving the naturally occurring 4-bp spacing between the two elements did not significantly reduce basal or IFN-inducible promoter activity. Taken together, these results are consistent with the notion that the KCS and ISRE elements of the PKR promoter function as a unit.     

Characterisation of gamma-interferon responsive promoters in fish

Reporter constructs of three interferon (IFN)-gamma-induced rainbow trout genes were generated to examine specificity to type I or type II IFN. Constructs included gammaIP-10, LMP2 and TAP2 and were used to transfect trout fibroblast cells (RTG-2) which were then exposed to rainbow trout rIFNs. The gammaIP-10 construct showed high reporter activity even in the absence of rIFNs. The LMP2 promoter contained one GAS element and two double ISRE elements, of four constructs made, only those with ISRE elements showed significant reporter activity following rIFN-gamma stimulation. The TAP2 regulatory region contained two GAS, two ISRE and one C/EBP element from which four constructs were made. Reporter expression for the construct containing all five elements showed an 11- and 2-fold increase in response to rIFN-gamma and type I rIFN, respectively. Constructs containing only the GAS elements did not respond to rIFNs. The TAP2 construct with two ISRE and the C/EBP gave the greatest dose-dependent reporter response to rIFN-gamma, with no significant response to type I rIFN. These data suggest that the ISRE elements, or elements nearby, are essential for the induction of type II IFN responsive genes in trout. The TAP2 construct is a candidate to develop a IFN-gamma reporter stable cell line.     

Promoter analysis of genes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements.

We have investigated the functional organization and properties of cis regulatory elements in the promoter regions of two genes from tomato (LAT52 and LAT59) that are preferentially and coordinately expressed during pollen maturation. Promoter deletion analysis in transgenic plants demonstrated that only minimal (less than 200 bp) promoter proximal regions are required for developmentally regulated expression in pollen and in specific cell types of the sporophyte. Cis-acting regulatory regions of these two promoters and of a third pollen-expressed promoter (LAT56) were characterized in detail using a transient expression assay. We identified two upstream activator regions in the LAT52 promoter and further showed that a 19-bp segment from one of those regions enhanced expression of the heterologous CaMV35S promoter in pollen. Similarities in sequence between crucial cis elements provide evidence that shared regulatory elements are involved in the coordinate expression of the LAT genes during microsporogenesis.     

NF-κB结合元件缺失对NOX1启动子转录活性的影响

目的:构建含NADPH氧化酶1(NOX1)近端启动子区的pGL3萤光素酶报告基因载体及缺失NF-κB结合元件的相应载体,分别测定其相应活性,探讨NF-κB结合元件缺失对NOX1启动子区转录活性的影响。方法:采用PCR法克隆NOX1启动子区序列(1 415 bp),将目的片段与pGL3萤光素酶载体分别双酶切、纯化后进行连接(pGL3-NOX1-1415),测序鉴定;应用Alibaba 2.1软件分析NOX1近端启动子区,获取NF-κB结合元件;重叠PCR法将含该元件的启动子区域(88 bp)缺失,并构建相应载体(pGL3-NOX1-1327)。将两载体分别与pRL-TK内参质粒瞬时转染进入A549细胞,采用TNF-α(10μg/L)刺激细胞24 h,萤光酶标仪检测A549细胞的萤光素酶活性。结果:测序鉴定结果提示pGL3-NOX1-1415及NF-κB结合元件缺失的pGL3-NOX1-1327载体构建成功;细胞实验显示,TNF-α刺激后,转染pGL3-NOX1-1415的A549细胞萤光素酶活性明显强于对照组(P0.05),而转染NF-κB结合元件缺失的pGL3-NOX1-1327的细胞萤光素酶活性明显低于转染pGL3-NOX1-1415组(P0.05)。结论:TNF-α诱导A549细胞NOX1基因活化与NF-κB密切相关,NF-κB参与了TNF-α诱导的NOX1基因启动子的转录调控。     

Characterization of IFN-gamma regulation of the complement factor B gene in macrophages.

Complement factor B (Bf) is involved in the activation of the alternative complement cascade. Bf is induced by IFN-gamma; however, the mechanisms of Bf gene regulation have not been well characterized in general, and not in macrophages specifically. Northern analysis reveals that IFN-gamma induces a dose- and time-dependent increase in Bf mRNA expression in primary macrophages and macrophage cell lines. MH-S cells transfected with reporter constructs containing truncated regions of the Bf promoter reveal that IFN-gamma responsiveness lies between -154 and -53 bp on the Bf promoter. This region of the Bf promoter contains both an IFN-gamma-activation site (GAS) and an interferon-stimulated response element (ISRE). Site-directed mutagenesis of the GAS binding site or the ISRE binding site in this region of the Bf promoter partially inhibits IFN-gamma responsiveness. Mutagenesis of both the GAS and ISRE cis elements totally abrogates IFN-gamma responsiveness of the Bf promoter. Electrophoretic mobility shift assays reveal nuclear binding complexes involving both Bf-GAS and Bf-ISRE oligonucleotide sequences upon IFN-gamma stimulation. In competition assays, both Bf-GAS and Bf-ISRE oligonucleotides, but not mutant Bf-GAS nor mutant Bf-ISRE oligonucleotides, compete for the DNA binding. Supershift analysis reveals that Stat1-GAS and IRF-1-ISRE nuclear binding complexes contribute to induction of Bf by IFN-gamma. Western analysis confirms an IFN-gamma-stimulated increase in tyrosine phosphorylation of Stat1. These findings suggest that both GAS and ISRE cis binding sites have an additive effect on IFN-gamma-stimulated Bf gene expression and that both are required for full expression of Bf by IFN-gamma. Stat1 and IRF-1 take part in IFN-gamma-stimulated Bf gene induction in macrophages through their respective cis binding elements.