Analysis Of The Imprinting Status OfGAB1,SFMBT2,SLC38A4 And AXL In Bovine

Genomic imprinting is an epigenetic phenomenon in which one of the two alleles of the parent is preferentially expressed.Generally,expression of imprinted genes are species specificity,tissue specificity and developmental stage specificity.Most of the imprinted genes are clustered in the genome,but some are isolated in the genome.Imprinted genes commonly regulate the growth and development of the fetus and placenta,and are regulated by imprinting Control Regions(ICR).Epigenetics of local chromosomes(e.g.promoter methylation,long non-coding RNAs expression and histone modification)is regulated by ICR to partake to regulate the transcription of monoallele.At present,imprinting genes are mainly studied in humans and mice,but few imprinting genes have been identified in cattle.GAB1,SFMBT2 SLC38A4 and AXL are four new imprinted genes identified in mice,which are independent in genome and play an important role in the development of fetus and placenta and the growth of animals after birth.In this study,the expression and imprinting status of GAB1,SFMBT2,SLC38A4 and AXL gene in bovine tissues and placenta were analyzed,the full length of cDNA sequence of bovine AXL gene was cloned,and the role of DNA methylation in SLC38A4 and AXL imprinting was analyzed.The expression of GAB1,SFMBT2,SLC38A4 and AXL genes in bovine tissues(heart,liver,spleen,lung,kidney,muscle,fat,brain and placenta)was analyzed by RT-PCR.The results showed that four genes were expressed in all tissues.RT-PCR basing on SNP was used to analyze the imprinting status of bovine GAB1,SFMBT2,SLC38A4 and AXL.It was found that GAB1 and SFMBT2 were both biallelic expression in bovine tissues and placenta.SLC38A4 gene was also expressed biallelically in adult bovine tissues,but polymorphic imprinting in placenta.AXL gene expressed monoallelicly in bovine heart,liver,spleen and fat,but biallelic expression in lung,kidney,muscle and placenta.It suggested that AXL was tissue-specific imprinting in cattle.AXL gene was amplified by RACE(Rapid Amplification of cDNA ends)and RT-PCR.Four 5’ flanking sequences,two 3’ flanking sequences and two RT-PCR variable splicing were obtained.Two amino acid sequences of different translation start sites,named AXL-1 and AXL-2,were obtained after sequence fragment assembly.The protein encoded by human AXL gene was 894 amino acids,was composed of two immunoglobulin-like motifs at the N-terminal,followed by two fibronectin type-III motifs.Analysis of the protein structure AXL-1 and AXL-2 revealed that AXL-1 had the same motifs as human AXL protein,but AXL-2 lacked an immunoglobulin-like motif at the N-terminal.It was speculated that AXL-1 has more bioactivity than AXL-2,because of containing more than one immunoglobulin.The methylation level of the conserved regions of SLC38A4 and AXL genes located in the promoter region was analyzed by bisulfite direct sequencing.The results showed that 69 CGs arrounding the transcriptional start site of SLC38A4 gene showed low methylation level in placental tissues of imprinted placenta and theirs’ male spermatozoa.It is speculated that DNA methylation may not be involved in polymorphic placental imprinting of SLC38A4.We have analyzed DNA methylation status of 13 CGs located in the promoter region of AXL gene and 10 CGs located in the homologous region of the differential methylation region of human in monoallele expression and biallelic expression tissues.The differential methylation region of the promoter region of AXL gene was found in monoallelic expression tissues,which indicated that tissues-specific imprinting expression of AXL gene was regulated by DNA methylation of the promoter region in adult tissues.