Perfil epigenético e transcricional de fibroblastos isolados de pele bovina cultivados in vitro com substâncias desmetilantes de DNA

Abstract

Somatic cell nuclear transfer (SCNT) is a commercially available technique of assisted reproductive technology with broad applications. It is still a low-efficiency technique, possibly as a consequence of the epigenetic profile of the somatic genome of the donor cell, which carries characteristic marks of differentiated cells, which hampers correct reprogramming after nuclear transfer. This differentiated cell memory leads to changes in DNA methylation patterns as well as changes in the gene expression profile of important genes during embryonic development. In this study, we analyzed the DNA methylation and gene expression profile of bovine fibroblasts cultured in vitro with DNA toxicity drugs of low toxicity, SAH and procaine. Regarding the epigenetic profile, the global levels of DNA methylation were quantified as well as the levels of methylation in a satellite repetitive region of the treated cells. In addition, embryos were produced and their methylation levels were evaluated for two repetitive satellite regions. Regarding the gene expression profile, we determined the mRNA levels for the DNMT1, DNMT3A, DNMT3B, TET1, TET2, TET3 and OCT4 genes, which are related to the reprogramming of DNA methylation and pluripotency state. Cells treated with DNA demethylating substances showed lower levels of DNA methylation, both globally and in the satellite region. The embryonic clones produced from treated cells had lower levels of methylation compared to control embryos for the Satelite I region. Regarding the transcriptional profile, we observed that in cells treated with SAH + procaine, they presented a general reduction in the levels of DNMTs. On the other hand, for the TET3 enzyme, a higher level of transcripts was observed in comparison to the control. Our results showed that the use of demethylating agents in nucleating donor cells is capable of altering their epigenetic profile, in order to modify at the transcriptional level the production of enzymes related to methylation, which can lead the cell to a state of less differentiation. We have discussed, in this research study, the use of chromatin modulating agents as a perspective for use in cloning, medical research in cancer and other diseases.