Cloning and Transcriptional Activity Analysis of the Porcine Abcb1 Gene Promoter: Transcription Factor Sp1 Regulates the Expression of Porcine Abcb1.
ABSTRACT: P-Glycoprotein (P-gp, Abcb1) plays a crucial role in drug disposition and functions by hydrolyzing ATP. However, little is known about the regulatory elements governing the transcription of the porcine Abcb1 gene. In this study, the transcription start site of the pig Abcb1 gene was identified by 5'-RACE. A 1.9-kb fragment of the 5'-flanking region of the Abcb1 gene was cloned from pig genomic DNA and sequenced. The region critical for its promoter activity was investigated via progressive deletions. Further, using mutation assays, two proximal Sp1 binding sites within the 5'-flanking region of Abcb1 were proven to be important cis-regulatory elements involved in regulating the constitutive expression of porcine Abcb1. RNA interference experiments showed that Sp1 regulated the expression of the porcine P-gp at both mRNA and protein levels. Hence, the current work provides valuable information on the regulatory mechanisms of pig Abcb1.
Project description:P-glycoprotein (P-gp) is one of the best-known ATP-dependent efflux transporters, contributing to differences in pharmacokinetics and drug-drug interactions. Until now, studies on pig P-gp have been scarce. In our studies, the full-length porcine P-gp cDNA was cloned and expressed in a Madin-Darby Canine Kidney (MDCK) cell line. P-gp expression was then determined in tissues and its role in the pharmacokinetics of oral enrofloxacin in pigs was studied. The coding region of pig Abcb1 gene was 3,861?bp, encoding 1,286 amino acid residues (Mw?=?141,966). Phylogenetic analysis indicated a close evolutionary relationship between porcine P-gp and those of cow and sheep. Pig P-gp was successfully stably overexpressed in MDCK cells and had efflux activity for rhodamine 123, a substrate of P-gp. Tissue distribution analysis indicated that P-gp was highly expressed in brain capillaries, small intestine, and liver. In MDCK-pAbcb1 cells, enrofloxacin was transported by P-gp with net efflux ratio of 2.48 and the efflux function was blocked by P-gp inhibitor verapamil. High expression of P-gp in the small intestine could modify the pharmacokinetics of orally administrated enrofloxacin by increasing the Cmax, AUC and Ka, which was demonstrated using verapamil, an inhibitor of P-gp.
Project description:The Multidrug Resistance 1 (MDR1; alternatively ABCB1) gene product P-glycoprotein (P-gp), an ATP binding cassette transporter, extrudes multiple endogenous and exogenous substrates from the cell, playing an important role in normal physiology and xenobiotic distribution and bioavailability. To date, the predominant animal models used to investigate the role of P-gp have been the mouse and rat, which have two distinct genes, Abcb1a and Abcb1b. In contrast, the human has a single gene, ABCB1, for which only a single isoform has been validated. We and others have previously shown important differences between Abcb1a and Abcb1b, limiting the extrapolation from rodent findings to the human. Since the guinea pig has a relatively long gestation, hemomonochorial placentation and neuroanatomically mature offspring, it is more similar to the human, and may provide a more comparable model for investigating the regulation of P-gp in the brain and placenta, however, to date, the Abcb1 gene in the guinea pig remains to be characterized. The placenta and fetal brain are barrier sites that express P-gp and that play a critical role of protection of the fetus and the fetal brain from maternally administered drugs and other xenobiotics. Using RNA sequencing (RNA-seq), reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (QPCR) to sequence the expressed isoforms of guinea pig Abcb1, we demonstrate that like the human, the guinea pig genome contains one gene for Abcb1 but that it is expressed as at least three different isoforms via alternative splicing and alternate exon usage. Further, we demonstrate that these isoforms are more closely related to human than to rat or mouse isoforms. This striking, overall similarity and evolutionary relatedness between guinea pig Abcb1 and human ABCB1 indicate that the guinea pig represents a relevant animal model for investigating the function and regulation of P-gp in the placenta and brain.
Project description:The beta-2 adrenergic receptor (beta-2 AR) modulates metabolic processes in skeletal muscle, liver, and adipose tissue in response to catecholamine stimulation. We showed previously that expression of the porcine beta-2 AR gene (ADRB2) is affected by cis-regulatory polymorphisms. These are most likely responsible for the association of ADRB2 with economically relevant muscle-related traits in pigs. The present study focused on characterization of promoter elements involved in basal transcriptional regulation of the porcine ADRB2 in different cell types to aid identification of its cis-regulatory polymorphisms. Based on in silico analysis, luciferase reporter gene assays and gel shift assays were performed using COS-7, HepG2, C2C12, and 3T3-L1 cells. Deletion mapping of the 5´ flanking region (-1324 to +33) of ADRB2 revealed the region between -307 and -269 to be the minimal promoter, including regulatory elements essential for the basal transcriptional activity in all four tested cell types. Directly upstream (-400 to -323) we identified an important enhancer element required for maximal promoter activity. In silico analysis and gel shift assays revealed that this GC-rich element harbors two evolutionarily conserved binding sites of Sp1, a constitutive transcriptional activator. Significant transcriptional activation of the porcine ADRB2 promoter was demonstrated by overexpression of Sp1. Our results demonstrate, for the first time, an important role of Sp1 and of the responsive enhancer element in the regulation of ADRB2 expression. Polymorphisms located in this domain of the porcine ADRB2 promoter represent candidate causal cis-regulatory variants.
Project description:Pigs are commonly used as an animal model to evaluate the toxic effects of exogenous compounds. Cytochrome P450 1A1 (CYP1A1) metabolizes numerous exogenous compounds and is abundantly expressed in the liver, kidneys, and intestines. The high amino acid similarity between human and porcine CYP1A1 indicates that they probably have the same metabolic characteristics. Therefore, understanding the regulatory mechanism of CYP1A1 expression in pigs is particularly important for predicting the toxicology and metabolic kinetics of exogenous chemicals. Currently, the transcriptional regulation of porcine CYP1A1 has rarely been studied, especially regarding basal transcription. In this study, we first confirmed that the key regulatory elements of porcine CYP1A1 basal transactivation are in the proximal promoter region using promoter truncation analysis via a dual luciferase assay in a porcine kidney cell line LLC-PK1. Two overlapping cis-elements, the xenobiotic response element (XRE) and GC box, in this proximal region potentially play key roles in the basal transactivation of porcine CYP1A1. Furthermore, using electrophoretic mobility shift assay and chromatin immunoprecipitation, the GC box binding protein Sp1 was confirmed to bind to the proximal promoter of porcine CYP1A1, instead of AhR, the XRE binding protein. In LLC-PK1 cells, by knocking down either Sp1 or AhR, the expression of porcine CYP1A1 at the mRNA level and protein level was significantly downregulated, suggesting both proteins are important for porcine CYP1A1 expression. However, promoter activity analysis in LLC-PK1 cells treated with an AhR agonist and antagonist confirmed that AhR does not participate in the basal regulation of porcine CYP1A1 at the proximal promoter. In conclusion, our study revealed that the proximal promoter is the key regulatory region for porcine CYP1A1 basal expression. Although AhR plays an important role in the transactivation of porcine CYP1A1 expression, the key determinant transcription factor for its basal transactivation is Sp1 at the proximal promoter of porcine CYP1A1.
Project description:Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) gene plays a crucial role in maintaining genomic stability, tumorigenesis and myogenesis. However, little is known about the regulatory elements governing the transcription of porcine ROCK1 gene. In the current study, the transcription start site (TSS) was identified by 5'-RACE, and was found to differ from the predicted one. The region in ROCK1 promoter which is critical for promoter activity was investigated via progressive deletions. Site-directed mutagenesis indicated that the region from -604 to -554 bp contains responsive elements for Sp1. Subsequent experiments showed that ROCK1 promoter activity is enhanced by Sp1 in a dose-dependent manner, whereas treatment with specific siRNA repressed ROCK1 promoter activity. Electrophoretic mobility shift assay (EMSA), DNA pull down and chromatin immunoprecipitation (ChIP) assays revealed Sp1 can bind to this region. qRT-PCR and Western blotting research followed by overexpression or inhibition of Sp1 indicate that Sp1 can affect endogenous ROCK1 expression at both mRNA and protein levels. Overexpression of Sp1 can promote the expression of myogenic differentiation 1(MyoD), myogenin (MyoG), myosin heavy chain (MyHC). Taken together, we conclude that Sp1 positively regulates ROCK1 transcription by directly binding to the ROCK1 promoter region (from -604 to -532 bp) and may affect the process of myogenesis.
Project description:This study examined how the 1199G > A polymorphism in the ABCB1 gene encoding P-glycoprotein (P-gp) affects the protein's expression, ATPase activity, and ability to pump female steroid sex hormones out of LLC-PK1 cells. The ABCB1 (1199G) or ABCB1 (1199A) allele was transfected into cells, which were incubated for 48 h with various hormone concentrations, then analyzed by Western blotting to examine expression of P-gp protein and by reverse transcription-polymerase chain reaction (RT-PCR) to examine expression of mRNA. Cells were also compared in terms of their transepithelial permeability to steroid sex hormones in the presence and absence of the specific P-gp inhibitor GF120918. P-gp ATPase activity induced by steroid sex hormones was also assayed. Estriol and ethynyl estradiol up-regulated levels of ABCB1 mRNA in a concentration-dependent manner, with ABCB1 (1199A) mRNA showing greater up-regulation than ABCB1 (1199G) mRNA. Estrone, estriol, and ethynyl estradiol were substrates of both types of P-gp in transepithelial permeability assays, and the ABCB1 (1199A) protein showed a significantly higher net efflux ratio for estrone (13.4 vs. 7.4, p < 0.005), estriol (5.6 vs. 3.3, p < 0.05), and ethynyl estradiol (12.7 vs. 5.3, p < 0.005). Induction of P-gp ATPase activity by ethynyl estradiol and progesterone increased with increasing hormone concentration, and the magnitude of stimulation was greater for ABCB1 (1199A) P-gp than for ABCB1 (1199G) P-gp. These results indicate that the ABCB1 (1199G > A) polymorphism influences steroid sex hormone-induced expression and function of P-gp, which may help to explain inter-patient differences in P-gp-mediated chemotherapy resistance in vivo.
Project description:The production of therapeutic proteins from transgenic animals is one of the most important successes of animal biotechnology. Milk is presently the most mature system for production of therapeutic proteins from a transgenic animal. Specifically, ?-casein is a major component of cow, goat and sheep milk, and its promoter has been used to regulate the expression of transgenic genes in the mammary gland of transgenic animals. Here, we cloned the porcine ?-casein gene and analyzed the transcriptional activity of the promoter and intron 1 region of the porcine ?-casein gene. Sequence inspection of the 5'-flanking region revealed potential DNA elements including SRY, CdxA, AML-a, GATA-3, GATA-1 and C/EBP ?. In addition, the first intron of the porcine ?-casein gene contained the transcriptional enhancers Oct-1, SRY, YY1, C/EBP ?, and AP-1, as well as the retroviral TATA box. We estimated the transcriptional activity for the 5'-proximal region with or without intron 1 of the porcine ?-casein gene in HC11 cells stimulated with lactogenic hormones. High transcriptional activity was obtained for the 5'-proximal region with intron 1 of the porcine ?-casein gene. The ?-casein gene containing the mutant TATA box (CATAAAA) was also cloned from another individual pig. Promoter activity of the luciferase vector containing the mutant TATA box was weaker than the same vector containing the normal TATA box. Taken together, these findings suggest that the transcription of porcine ?-casein gene is regulated by lactogenic hormone via intron 1 and promoter containing a mutant TATA box (CATAAAA) has poor porcine ?-casein gene activity.
Project description:BACKGROUND:Alpha1,3-galactosyltransferase (alpha1,3GT) is an enzyme that produces carbohydrate chains termed alphaGal epitopes found in most mammals, although some species of higher primates, including human, are notable exceptions. The evolutionary origin of the lost alpha1,3GT enzyme activity is not yet known, although it has been suggested that the promoter activity of this gene in the ancestors of higher primates was inactivated. METHODS:We used 5'-or 3'-RACE, GenomeWalking, reverse transcriptase polymerase chain reaction (RT-PCR) and dual Luciferase reporter assay for identification of the full-length cDNA, which includes the transcription initiation site and the promoter region of porcine alpha1,3GT gene. RESULTS:The region around exon 1 is guanine and cytosine (GC)-rich (about 70%), comprising a CpG island spanning more than 1.5 kbp. The 5'-flanking region of exon 1 contains multiple transcription factor consensus motifs, including GC-box, SP1, AP2, and GATA-box sites, in the absence of TATA or CAAT-box sequences. The entire gene consists of three 5' noncoding and six coding region exons spanning more than 52 kbp. Detailed analysis of alpha1,3GT transcripts revealed two major alternative splicing patterns in the 5'-untranslated region (5'-UTR) and evidence for minor splicing activity that occurs in a tissue-specific manner. Interspecies comparison of 5'-UTR shows minimal homology between porcine and murine sequences except for exon 2, which suggests that the regulatory regions differ among species. CONCLUSIONS:These observations have important implications for experiments involving genetic manipulation of the alpha1,3GT gene in transgenic animals in terms of promoter utilization, and particularly in genetically engineering cells for the animal cloning technology by nuclear transfer.
Project description:Peroxiredoxin6 (Prdx6) is one of the peroxiredoxin (Prdxs) family members that play an important role in maintaining cell homeostasis. Our previous studies demonstrated that Prdx6 was significantly associated with pig meat quality, especially meat tenderness. However, the transcriptional regulation of porcine Prdx6 remains unclear. In this study, we determined the transcription start site (TSS) of porcine Prdx6 gene by 5' rapid-amplification of cDNA ends (5' RACE). Several regulatory elements including CCAAT/enhancer-binding protein? (C/EBP?), Myogenic Differentiation (MyoD), cAMP response element binding protein (CREB), stimulating protein1 (Sp1) and heat shock factor (HSF) binding sites were found by computational analyses together with luciferase reporter system. Overexpression and RNA interference experiments showed that C/EBP? or CREB could up-regulate the expression of porcine Prdx6 gene at both mRNA and protein level. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation assays (ChIP) confirmed that C/EBP? and CREB could interact with Prdx6 promoter. Immuoprecipitation results also showed that C/EBP? could interact with Prdx6 in vivo. Taken together, our findings identified C/EBP? and CREB as the important regulators of porcine Prdx6 gene expression, and offered clues for further investigation of Prdx6 gene function.
Project description:Enterovirus G (EV-G) belongs to the family of Picornaviridae. Two types of recombinant porcine EV-Gs carrying papain-like cysteine protease (PLCP) gene of porcine torovirus, a virus in Coronaviridae, are reported. Type 1 recombinant EV-Gs are detected in pig feces in Japan, USA, and Belgium and carry the PLPC gene at the junction site of 2C/3A genes, while PLPC gene replaces the viral structural genes in type 2 recombinant EV-G detected in pig feces in a Chinese farm. We identified a novel type 2 recombinant EV-G carrying the PLCP gene with flanking sequences in place of the viral structural genes in pig feces in Japan. The ~0.3?kb-long upstream flanking sequence had no sequence homology with any proteins deposited in GenBank, while the downstream ~0.9?kb-long flanking sequence included a domain having high amino acid sequence homology with a baculoviral inhibitor of apoptosis repeat superfamily. The pig feces, where the novel type 2 recombinant EV-G was detected, also carried type 1 recombinant EV-G. The amount of type 1 and type 2 recombinant EV-G genomes was almost same in the pig feces. Although the phylogenetic analysis suggested that these two recombinant EV-Gs have independently evolved, type 1 recombinant EV-G might have served as a helper virus by providing viral structural proteins for dissemination of the type 2 recombinant EV-G.