title: Functional analysis of the upstream regulatory region of chicken miR-17-92 cluster.
abstract: miR-17-92 cluster plays important roles in cell proliferation, differentiation, apoptosis, animal development and tumorigenesis. The transcriptional regulation of miR-17-92 cluster has been extensively studied in mammals, but not in birds. To date, avian miR-17-92 cluster genomic structure has not been fully determined. The promoter location and sequence of miR-17-92 cluster have not been determined, due to the existence of a genomic gap sequence upstream of miR-17-92 cluster in all the birds whose genomes have been sequenced. In this study, genome walking was used to close the genomic gap upstream of chicken miR-17-92 cluster. In addition, bioinformatics analysis, reporter gene assay and truncation mutagenesis were used to investigate functional role of the genomic gap sequence. Genome walking analysis showed that the gap region was 1704 bp long, and its GC content was 80.11%. Bioinformatics analysis showed that in the gap region, there was a 200 bp conserved sequence among the tested 10 species (Gallus gallus, Homo sapiens, Pan troglodytes, Bos taurus, Sus scrofa, Rattus norvegicus, Mus musculus, Possum, Danio rerio, Rana nigromaculata), which is core promoter region of mammalian miR-17-92 host gene (MIR17HG). Promoter luciferase reporter gene vector of the gap region was constructed and reporter assay was performed. The result showed that the promoter activity of pGL3-cMIR17HG (-4228/-2506) was 417 times than that of negative control (empty pGL3 basic vector), suggesting that chicken miR-17-92 cluster promoter exists in the gap region. To further gain insight into the promoter structure, two different truncations for the cloned gap sequence were generated by PCR. One had a truncation of 448 bp at the 5'-end and the other had a truncation of 894 bp at the 3'-end. Further reporter analysis showed that compared with the promoter activity of pGL3-cMIR17HG (-4228/-2506), the reporter activities of the 5'-end truncation and the 3'-end truncation were reduced by 19.82% and 60.14%, respectively. These data demonstrated that the important promoter region of chicken miR-17-92 cluster is located in the -3400/-2506 bp region. Our results lay the foundation for revealing the transcriptional regulatory mechanisms of chicken miR-17-92 cluster.
authors.affiliation: 1. Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture, Harbin 150030, China; 2. Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030, China; 3. College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China;
authors.forename: Min
authors.initials: M
authors.lastname: Cheng
authors.affiliation: 1. Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture, Harbin 150030, China; 2. Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030, China; 3. College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China;
authors.forename: Wen-jian
authors.initials: WJ
authors.lastname: Zhang
authors.affiliation: 1. Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture, Harbin 150030, China; 2. Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030, China; 3. College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China;
authors.forename: Tian-yu
authors.initials: TY
authors.lastname: Xing
authors.affiliation: 1. Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture, Harbin 150030, China; 2. Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030, China; 3. College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China;
authors.forename: Xiao-hong
authors.initials: X
authors.lastname: Yan
authors.affiliation: 1. Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture, Harbin 150030, China; 2. Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030, China; 3. College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China;
authors.forename: Yu-mao
authors.initials: YM
authors.lastname: Li
authors.affiliation: 1. Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture, Harbin 150030, China; 2. Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030, China; 3. College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China;
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.affiliation: 1. Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture, Harbin 150030, China; 2. Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030, China; 3. College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China;
authors.forename: Ning
authors.initials: N
authors.lastname: Wang
mesh_terms: D000818:Animals; D001483:Base Sequence; D002645:Chickens / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D005786:Gene Expression Regulation / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D035683:MicroRNAs