Analysis Of The Imprinting Status Of NTM,ZNF597,NAA60 And GLIS3 In Bovine

Genomic imprinting is an epigenetic phenomenon that leads to genes to be preferentially expressed from the paternal or maternal allele.Imprinted genes play an important role in mammalian growth and development,and the placenta function.Neurotrimin(NTM)plays an important role in the development of nerve.The NTM is an imprinted gene in human(Homo sapiens),but the imprinting state in cattle(Bos taurus)has not been revealed.The purpose of present study is to analyze the sequences of cattle NTM gene,the structure of its coding protein,and to identify the imprinting status of NTM gene in adult tissues and placenta in cattle.A new splice NTMxd(GenBank No.: MH037574)of cattle NTM gene was obtained by the technology of rapid amplification of cDNA ends(RACE),and a more complete transmembrane signal was found in the N-terminal of amino acid sequence encoded by the splice.In order to analyze the imprinting status of NTM gene in cattle,a locus of single nucleotide polymorphism(SNP)was found in cattle NTM gene by direct sequencing of PCR products,the accession number of the SNP locus in NCBI website(www.ncbi.nlm.nih.gov)is rs42185569.The total RNA was extracted from the tissue samples and placentas of heterozygous individuals,and performed expression analysis by reverse transcription-PCR(RT-PCR).Direct sequencing of the RT-PCR amplified products revealed that NTM gene exhbited monoallelic expression in cattle placenta and adult tissues,indicating that NTM gene is an imprinted gene in cattle.ZNF597 and NAA60 are two paternal imprinted genes in human ZNF597-NAA60 imprinted locus,but which orthologues showed biallelical expression in the mouse.In this study,we investigated the allelic expression of ZNF597 and NAA60 in bovine placental and adult tissues.Comparison of the genotypes of mRNA and genomic DNA,ZNF597 gene was identified to be monoallelic expression in placental and seven detected tissues,including brain,heart,liver,spleen,lung,kidney and muscle,nevertheless NAA60 gene exhibited biallelical expression in these tissues.Moreover,imprinting status of ZNF597 was analyzed in bovine placenta and confirmed that the maternal allele was expressed.To determine whether DNA methylation involves in regulating the monoallelic/imprinted expression of bovine ZNF597,the methylation status of two CpG-enriched regions in ZNF597-NAA60 locus on bovine chromosome 25 was analyzed using pyrosequencing method.Ten CpG loci in bovine ZNF597 promoter region showed differential methylation region(DMR)-like methylation patterns.In summary,bovine ZNF597 gene is a maternal expressed imprinting gene regulated by DNA methylation,while NAA60 gene is not imprinted in cattle.GLIS3 gene,as a transcriptional repressor and activator,is involved in the development of pancreatic cells,thyroid,liver and kidney.Studies have confirmed that the GLIS3 gene is imprinted in humans,and that mutations in the gene are associated with neonatal and congenital hypothyroidism,while the GLIS3 gene is rarely studied in cattle.We analyzed the SNP site: rs43549703,sequencing results showed that the GLIS3 gene was biallele expression in all test tissues,and confirmed that the bovine GLIS3 gene was a non-imprinted gene.The results show that NTM gene is imprinted gene in cattle,bovine ZNF597 gene is an imprinted gene expressed regulate and control by DNA methylation,while NAA60 gene and GLIS3 gene have no imprinting in cattle.It provides a reference for the further study of the function of NTM gene and related neurodevelopmental diseases and ZNF597 gene related diseases.