Genomic Imprinting Of Ascl2, Tssc4, Osbpl5 And DNA Methylation Status Of Ascl2 Gene In Promoter

Genomic imprinting is an epigenetic mechanism that can lead to monoallelic expression of genes depending on parent of origin of the allele. Imprinted genes play significant roles in embryonic and placental growth, development and have close relationship in the process of some diseases. Many imprinted genes show distinct tissue-specific and developmental-stage-specific expression patterns. In Cdkn1c/Kcnq1ot1 cluster, at least eight protein-coding genes(Ascl2, Tssc4, Cd81, Kcnq1, Cdkn1 c, Slc22a18, Phlda2, and Osbpl5) are known to be paternally silenced by the production and elongation of the large nc RNA Kcnq1ot1, transcribed from the Kcnq1ot1 gene.In this study, we selected Ascl2, Tssc4 and Osbpl5 in Cdkn1c/Kcnq1ot1 cluster and analyzed the expression and imprinting status of Ascl2, Tssc4 and Osbpl5 in seven organs(heart, liver, spleen, lung, kidney, muscle and subcutaneous fat) of calves. Ascl2 and Tssc4 genes were found to be expressed in all the examined tissues. While Osbpl5 gene was detected in heart, liver, lung, kidney and fat tissues. Then the RT-PCR products were directly sequenced, Ascl2 gene showed monoallelic expressions in lung and liver tissues while biallelic expressions in heart, spleen, kidney, muscle and subcutaneous fat tissues. Tssc4 gene and Osbpl5 gene showed biallelic expression in all the tissues that detected expression. The results indicate that Ascl2 gene presents distinct tissue-specific imprinted pattern. Tssc4 and Osbpl5 gene are not imprinted in adult cattle tissues.Since DNA methylation is a major epigenetic modification of genomic imprinting, then the DNA methylation statuses in promoter region of Ascl2 gene were analyzed in lung, liver and kidney tissues of cattle using bisulfite sequencing analysis. The methylation level of two parental strands exhibited extremely significant difference(P < 0.01) in lung and liver tissues which is monoallelic expressions. The mean methylation levels of A-strands(61.21%、87.28%) showed seriously hypermethylated compared with G-strands( 24.70%、25.61%). However, in kidney tissues which is biallelic expressions, A-strand(54.70%) and G-strand(49.55%) had no significant difference(P >0.05). The results suggest that the differentially methylated Cp G island in promoter of Ascl2, which is required for the imprinting of lung and liver tissues.