Epigenetic Status of H19-Igf2 Imprinted Genes and Loss of 5-Hydroxymethylcytosine in the Brain of Cloned Goats.

In mammals, the imprinted genes play vital roles in development and are generally controlled by DNA methylation at imprinting control regions (ICRs). Recently, it was discovered that 5-hydroxymethylcytosine (5-hmC) is a stable epigenetic modification; however, its functions in cloned animal genomes have not yet been fully elucidated. In this study, we interrogated and quantified the 5-hmC levels in the brain of cloned goats and discovered upregulation of Uhrf1 (p < 0.001), Dnmt1 (p < 0.05), Dnmt3a (p < 0.05), Igf2 (p < 0.01), and H19 (p < 0.05) and downregulation of Dnmt3b (p < 0.001), Tet1 (p < 0.001), Tet2 (p < 0.05), Tet3 (p < 0.001), Mecp2 (p < 0.05), and Igf2r (p < 0.05) in deceased cloned goat tissues compared with the normal controls. We demonstrated that DNA methylation was increased at H19 ICR (51.33% ± 2.03% vs. 93.07% ± 3.06%; p < 0.01) and that DNA was hypomethylated at Igf2 ICR (4.57% ± 1.48% vs. 7.63% ± 1.83%; p > 0.05) in the brain of deceased cloned goats. Finally, we showed that within the cloned goat brain genome, the amount of genome-wide 5-hmC was significantly decreased (0.083% ± 0.026% vs. 0.024% ± 0.007%; p < 0.05), whereas the 5-hmC levels within H19 and Igf2 CCGG sites were not significantly altered (0.17% ± 0.09% vs. 0.03% ± 0.01%; p > 0.05) in the brain of deceased cloned goats. Our data bring further experimental evidence regarding the abnormalities in 5-hmC and advance our current understanding of the role of 5-hmC in cloned animals.