Catalytic inhibition of H3K9me2 writers disturbs epigenetic marks during bovine nuclear reprogramming

Alessandra Bridi,Dewison Ricardo Ambrizi,Juliano Coelho Da Silveira,Rafael Vilar Sampaio,Pablo J Ross,Carolina Habermann Macabelli,Felipe Perecin,Maite Del Collado,Ana Clara Faquineli Cavalcante Mend De Ávila,Lilian De Jesus Oliveira,Tiago Henrique Camara De Bem,Fabiana Fernandes Bressan,Flávio Vieira Meirelles,Marcos Roberto Chiaratti,Lawrence Charles Smith,Juliano Rodrigues Sangalli

doi:10.1038/s41598-020-67733-9

Abstract

Orchestrated events, including extensive changes in epigenetic marks, allow a somatic nucleus to become totipotent after transfer into an oocyte, a process termed nuclear reprogramming. Recently, several strategies have been applied in order to improve reprogramming efficiency, mainly focused on removing repressive epigenetic marks such as histone methylation from the somatic nucleus. Herein we used the specific and non-toxic chemical probe UNC0638 to inhibit the catalytic activity of the histone methyltransferases EHMT1 and EHMT2. Either the donor cell (before reconstruction) or the early embryo was exposed to the probe to assess its effect on developmental rates and epigenetic marks. First, we showed that the treatment of bovine fibroblasts with UNC0638 did mitigate the levels of H3K9me2. Moreover, H3K9me2 levels were decreased in cloned embryos regardless of treating either donor cells or early embryos with UNC0638. Additional epigenetic marks such as H3K9me3, 5mC, and 5hmC were also affected by the UNC0638 treatment. Therefore, the use of UNC0638 did diminish the levels of H3K9me2 and H3K9me3 in SCNT-derived blastocysts, but this was unable to improve their preimplantation development. These results indicate that the specific reduction of H3K9me2 by inhibiting EHMT1/2 during nuclear reprogramming impacts the levels of H3K9me3, 5mC, and 5hmC in preimplantation bovine embryos.

Highlights

Orchestrated events, including extensive changes in epigenetic marks, allow a somatic nucleus to become totipotent after transfer into an oocyte, a process termed nuclear reprogramming
These results indicate that the specific reduction of H3K9me[2] by inhibiting EHMT1/2 during nuclear reprogramming impacts the levels of H3K9me3, 5mC, and 5hmC in preimplantation bovine embryos
To test whether the UNC0638 treatment would be suitable for the bovine species, fetal fibroblasts were treated with 250 nM of UNC0638 for 96 h and the levels of H3K9me[2] were analyzed by immunofluorescence (IF) and western blot (WB)

Summary

Introduction

Orchestrated events, including extensive changes in epigenetic marks, allow a somatic nucleus to become totipotent after transfer into an oocyte, a process termed nuclear reprogramming. The use of UNC0638 did diminish the levels of H3K9me[2] and H3K9me[3] in SCNT-derived blastocysts, but this was unable to improve their preimplantation development These results indicate that the specific reduction of H3K9me[2] by inhibiting EHMT1/2 during nuclear reprogramming impacts the levels of H3K9me3, 5mC, and 5hmC in preimplantation bovine embryos. Small molecules have been used to inhibit EHMT1/2 in an attempt to reduce H3K9me[2] and facilitate nuclear reprogramming during induced pluripotent stem cell (iPSC) g eneration[19,20] and SCNT21,22; even with the intense crosstalk between histone modification and DNA methylation, the effects of the inhibition of H3K9me[2] writers on DNA methylation and other epigenetic marks remain unknown. We hypothesized that the inhibition of EHMT1/2 activity either before or after nuclear transfer results in decreased H3K9me[2] levels and improves development of SCNT-derived embryos. We investigated throughout preimplantation development the consequences of EHMT1/2 inhibition on additional epigenetic marks and expression of genes related to epigenetic regulation

Methods

Results

Discussion

Conclusion