Breed Dependent Ranscriptional Regulation Of GR Expression In The Liver Of Piglets

Glucocorticoids regulate an array of physiological functions by binding to the ubiquitously expressed glucocorticoid receptor (GR). Chinese indigenous pig, Erhualian(EHL), showed more cortisol in blood and expressed higher GR mRNA in liver compared with Large White pig(LW). Previous study in our laboratory have studied cortisol synthesis pathway in LW and EHL piglets, however different GR mRNA expression in liver and investigation into the breed-dependent GR transcriptional regulation is hampered by lacking porcine GR promoter information. In this study, we cloned and sequenced proximal promoter of pig GR gene and detected seven alternative first exons of GR gene.5’and3’ boundary of alternative first eoxns were determined on genomic position. We studied GR mRNA and GR alternative first exons in liver of newborn piglets, and found exon1-4and1-5were significantly higher in LW than that in EHL piglets, methylation of GR promoter had no difference between two breeds of piglets. Trans-acting factors, p-CREB and GR took part in transcriptional regulation of exon1-4and exon1-5and deduced different expression of total GR mRNA. However, hepatic GR mRNA and exon1-9,10in LW were significantly lower than that in EHL in preweaning piglets, Spl and p53were involved in exon1-9,10transcriptional regulation. In vitro experiment we used doxorubicin (DOX) to activate p53and found that p53protein in HepG2cells increased concominant with enhanced GR protein. In vivo and in vitro studies proved that Sp1directedly regulated GR exon1-9,10transcription, while p53regulated GR mRNA through protein-protein mechanism with Spl.1Sequence of proximal promoter of glucocorticoid receptor and first alternative exonsWe first compared the published GR promoter sequences of human (AY436590), rat (AJ271870) and mouse (X66367), and identified the highly conserved regions. Using these highly conserved sequences and the porcine GR cDNA sequence (AY779185.1) as probes, we screened the porcine bacterial artificial chromosome(BAC) library and hit a positive clone (CH242-105G5). The BAC clone was then priority sequenced by the Sanger Institute upon our request.Within the complete sequence (CU928713) of the clone, we verified, by sequencing the overlapping PCR products amplified from porcine genomic DNA extracted from liver, a5300bp59flanking sequence of porcine GR exon2. We designed the upstream primers according to the sequence homology of pig with human and rat,which were located in various first exons, while the downstream primers were located in coding region of exon2. Seven pairs of primers were used to amplify the alternative first exons with cDNAs, then the PCR products were detected in gel and the expected bands were cut out and cloned into the pMDH18-T Vector, after that the clones were sequenced. All the alternative first exons were spliced to the same nocleotide sequence located in exon2, thus the3’end of the first exons were determined. The5’boundaries were identified by bioinformatic prediction referring to the5’boundary sequences of the first exons in human and rat GR. Homologous analysis of GR gene of pig, human, rat and mouse, revealed that pig GR sequence shared more homologous with human than rat and mouse. Sequence of pig GR gene provided basis for further study of pig GR gene transcription and expression in different tissue and different breeds of pigs.2Analysis of methylaiton of promoter of GR in liver of LW and EHL pigsIn this study, we used six newborn male LW and EHL pig and studied total GR mRNA and seven alternative5’-untranslated first exons1-4,1-5,1-6,1-7,1-8,1-9,10and1-11of GR gene. Among all these GR mRNA variants, exons1-4and1-5, as well as the total GR were expressed in a breed-dependent manner in the liver, newborn piglets of LW showing significantly (P<0.05) higher expression compared with EHL. Since promoter of GR gene are GC rich and are located in a CpG island, it was speculated that methylation of promoter will account for GR1-4and1-5mRNA different expression. Genomic DNA of liver was treated with bisulfite and methylation of promoter was analysed by the method of Mass-array ARRAY. Result showed that overall average methylaiton of GR promoter and promoter of exon1-4and1-5had no difference between two breeds of piglets, methylation of several sites of CpG island had difference, bioinformatics analysis revealed that these CpG positions were located in binding sites for transcription factors, however methylation of these positions had on effect on exon transcription, implying that mechanism of transcription regulation involved in trans-acting factors will account for different GR mRNA expression in liver.3Breed-dependent transcriptional regulation of5’-untranslated GR Exon I mRNA variants in the liver of newborn pigletsMethylation of promoter of GR gene were not the reason for different GR mRNA transcription in two breeds of newborn piglets, therefore we studied GR transcription regulation in aspect of trans-acting factors. Bioimformatics anslysis revealed that CREB, Spl and GR harbored binding sites on promoter1-5of GR gene, what is more, promoter1-4harbored two YY1(Ying yang1) binding sites. Nuclear content of Spl, p-CREB and GR in the liver was significantly higher in LW piglets(P<0.05), associated with enhanced binding of p-CREB, as well as higher level of histone H3acetylation in1-4and1-5promoters^<0.05, or P<0.01). In contrast, GR binding to promoters of exons1-4and1-5was significantly diminished in LW piglets, implicating the presence of negative GREs. YY1had no difference in hepatic nuclear content and showed no binding difference to promoter1-4. These results indicated p-CREB and GR were involved in positive and negative transcriptional regulation of GR mRNA, respectively. The difference in the hepatic expression of GR transcript variants between two breeds of pigs was determined, at least partly, by the disparity in the binding of transcription factors and the enrichment of histone H3acetylation to the promoters.4Breed-dependent transcriptional regulation of5’-untranslated GR Exon I mRNA variants in the liver of preweaning pigletsIn this study we studied hepatic GR mRNA expression in preweaning piglets, and found that LW pig express lower GR mRNA than EHL piglets. We studied seven GR alternative first exons and found1-9,10showed breed depended difference, while other exons had no difference. Exon1-9,10was the most abundant expression in GR first exons and deduced total GR mRNA difference in two breeds of piglets. Nuclear content of Sp1in the liver was significantly (P<0.05) higher in LW piglets, however, LW piglets showed decreased binding of Spl to promoter1-9,10, as well as lower level of histone H3acetylation in1-9,10promoter. Co-Immunoprecipitation analysis revealed that Sp1interacted with p53, and vice versa. Hepatic p53mRNA was significantly correlation with GR mRNA(P=0.001), in order to study the mechanism of p53regulating transcription of GR mRNA, we cultured HepG2cell. Dox was added to the culture and activated p53pathway, it significantly increased p53and GR protein (concominant with enhanced GR1-9,10mRNA expression(P<0.05). Promoter-luciferase reporter gene construct containing the sequence between-3149and-2724of pig GR gene (located in promoter1-9,10) was generated, construct was transiently transfected into HepG2cells,100nmol/L DOX significantly increased promoter luciferase activity(P<0.01). The present study showed that Sp1directedly increased transcription of GR, while p53increased GR transcription by interaction with Spl protein.