241 GENE EXPRESSION OF DNMT1 ISOFORMS IN PORCINE OOCYTES, EMBRYOS, AND SOMATIC CELLS

Abstract

In human, mouse, and some marsupials, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (DNMT1o) enzyme. The identification and characterization of a similar oocyte transcript variant in farm animals would greatly contribute to the understanding of the methylation processes that occur during nuclear remodeling of in vivo embryos as well as in cloned embryos. The objectives of this study were to identify the alternative splicing variants of DNMT1 in porcine oocytes and determine the gene expression pattern of the different DNMT1 isoforms during embryo development. Total RNA was isolated from a pool of 50 zona free mature porcine oocytes using the Trizol method. A RACE System (Invitrogen, Carlsbad, CA) was used to amplify the 5′ cDNA end of DNMT1. Polymerase chain reaction products were separated by electrophoresis, recovered from the agarose gel, subcloned into a cloning vector, and sequenced. The location of exons and introns of every transcript variant was determined by aligning the 5′RACE-derived sequences to the Sus scrofa DNMT1 genomic sequence located on chromosome 2 (CU462940). RNA levels of the DNMT1 isoforms were analyzed in porcine oocytes and in vivo embryos, as well and in cells from ovary, lung, spleen, liver, heart, and skin by quantitative PCR using the AACT method. DNMT1 protein levels of porcine oocytes and different somatic cells types were analyzed by Western blot. Two new DNMT1o RNA isoforms were identified (EU908730 and EU908731). The previously reported DNMT1s isoform (DQ060156) was expressed at low but constant levels in oocytes, as well as in embryos from the 2-cell to the blastocyst stage. Abundant RNA levels of EU908730 and EU908731 were detected in oocytes and embryos from the 2-cell to the 8- to 16-cell stage. Levels of these DNMT1o transcripts were low at the morula and blastocyst stage. Although DQ060156 was present in all the somatic cell types analyzed, EU908730 and EU908731 were not detected in any somatic tissues. As predicted by the RNA sequence and verified by Western blot analysis, EU908730 and EU908731 RNAs translate one DNMT1o enzyme (ˆ170 KDa). Western blot analysis confirmed that both the oocyte and the somatic forms of DNMT1 protein are present in porcine oocytes and early embryos, while somatic cells produce only DNMT1s protein. In conclusion, this study demonstrates that porcine oocytes express a DNMT1o RNA in addition to the DNMT1s somatic transcript. The 2 newly identified RNA isoforms are produced by alternative splicing of the DNMT1 gene and translate a DNMT1o protein that is unique to oocytes and early embryos. Analysis of the oocyte and somatic DNMT1 isoforms in pre-implantation embryos will determine the expression pattern of this transcript during genomic methylation and its involvement during nuclear reprogramming and cellular differentiation.