Tissue-specific Expression And Methylation Signatures Of The Iodothyronine Deiodinase Type 3 Gene (DIO3) In Goats

Iodothyronine deiodinase type 3 （DIO3） is an important enzyme in the metabolism of thyroid hormones. It plays critical roles in fetal development, neonatal growth, and is especially important for brain development in mammals. For a further understanding of the structure and function of DIO3 gene, DIO3 gene was isolated from Nanjiang yellow goat heart muscle. The difference of expression of DIO3 mRNA in different tissues and different growth stages were detected by real-time fluorescence quantitative PCR method. Meanwhile, location of DIO3 mRNA in different regions in the brain was analyzed by in situ hybridization techniques. Moreover, identification DIO3 gene intragenic CpG island the methylation status in different tissues of Nanjiang yellow goat in neonatal goats using the Mass ARRAY platform. The main results were showed as follows:（1） A 906-bp fragment of the goat DIO3 gene was obtained, which covered the whole coding region of 301 amino acids; The coding sequence of DIO3 was 83.61,84.37,88.52, 90.20,90.95, and 97.46% identical to mouse, rat, human, pig, sheep, and cattle sequences, respectively. Sequence alignment revealed that the encoded amino acid sequence of goat DIO3 was 98.67% identical to cattle DIO3, and 89.47,89.80,92.36,92.76, and 93.11% identical to DIO3 of mouse, rat, sheep, human, and pig, respectively. Strikingly an in-frame TGA encodes a selenocysteine at the active site critically important for catalytic activity.（2） The bioinformatics analysis revealed that the DIO3 protein has a molecular weight of 33.9 kDa with a theoretical isoelectric point （pI） of 6.45. Eleven phosphorylation sites （Ser:7, Thr:1, Tyr:3） were indicated by NetPhos 2.0 neural network predictions. Secondary structure prediction indicated that the caprine DIO3 protein was composed of alpha-helix （41.20%）, extended strand （14.29%）, and random coil （44.52%） structures. Phylogenetic analysis indicated that the goat DIO3 had close evolutionary relationships with sheep and cattle. Capra hircus DIO3 clustered first with that of Ovis arise, while DIO3 from Bos taurus, Sus scrofa, and Canis lupus familiar is were grouped into a cluster. The caprine DIO3 sequence was more similar to the DIO3 sequences of other mammals than to those of Halichoeres trimaculatus and Chrysiptera cyanea.（3） The expression levels of the DIO3 mRNA were detected in different tissues at different stages. Particularly high expression levels of DIO3 were seen in liver, kidney and Longissimus dorsi, low expression levels were seen in heart and brain and very little expression were detected in lung.（4） The ISH probes successfully revealed the spatial expression of DIO3 mRNA in the different brain areas. In the cerebral cortex, weak signals could be observed in the meninges and molecular layer, but signals were particularly striking in the neuropil of the cerebral cortex. Wide distribution of DIO3 transcript is illustrated in neurons and neuropil of the brainstem. In the cerebellum, moderately diffuse expression was observed throughout, in the molecular layer, Purkinje cell layer, and granule cell layer, while the highest concentration of DIO3 mRNA was identified in the white matter. Moreover, DIO3 mRNA was also seen in the medulla oblongata with signal primarily accumulated over the neuropil. Similar to other regions, the hypothalamus and white pulp also showed DIO3 expression in the neuropil.（5） The goat DIO3 gene contains a conserved CpG island spanning almost the entire coding region （g.71-851）. The DIO3 CpG island harbors 51 CpG sites, and their average methylation level had a range of 0.03-0.79. The results showed a conserved pattern of methylation in the different tissues. Methylation profiling of DIO3 showed a difference between the 5’-and 3’-regions of the coding region. DNA methylation of the 5’region （CpGs₁～24） was significantly （P<0.01） lower than that of the 3’region （CpGs₂5～51）. Furthermore, no significant difference in methylation status was observed among six tested tissues with levels in the range of 29.11-33.12%.