44 Misregulation of ten-eleven translocation 3 CXXC domain leads to abnormal formation of 5-hydroxymethylcytosine and expression of pluripotency genes in pig embryos
K. Uh A , J. Ryu A , H. Miko A , K. Carey A and K. Lee AVirginia Tech, Blacksburg, VA, USA
Reproduction, Fertility and Development 31(1) 148-148 https://doi.org/10.1071/RDv31n1Ab44
Published online: 3 December 2018
Abstract
Ten-eleven translocation (TET) methylcytosine dioxygenases are considered to play an important role in regulation of DNA methylation patterns by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). TET3 protein, a member of TET family, is enriched in mature oocytes and early stage embryos and contributes to DNA demethylation of the paternal genome in zygotes. N-terminal CXXC domain of TET3 is thought to be important in catalysing 5mC oxidation through its DNA binding potential. However, it is not clear whether specific DNA binding of CXXC domain is required for 5hmC conversion in mammalian embryos. Here, we investigated the role of TET3 CXXC domain in controlling 5hmC formation in fertilized pig embryos by injecting TET3 CXXC domain into mature pig oocytes as a dominant negative to inhibit the direct binding of TET3 to the genome through the CXXC domain. The CXXC domain of pig TET3 was identified through bioinformatics comparison of TET3 sequences among different species and cloned from mature pig oocyte-derived cDNA. To construct the green fluorescent protein (GFP)-CXXC fusion protein, CXXC sequence was subcloned into N-terminal GFP fusion vector, and then mRNA was synthesised by in vitro transcription. The GFP-CXXC mRNA (100 ng/µL) was injected into oocytes matured in vitro for 36 to 37 h. Then, the oocytes were fertilized at 42 h post-maturation. Water-injected oocytes served as a control. At 17 h post-fertilization, zygotes were collected to analyse 5hmC level by immunocytochemistry. The level of 5hmC was analysed using ImageJ (