Histone Deacetylase Inhibition Improves Activation of Ribosomal RNA Genes and Embryonic Nucleolar Reprogramming in Cloned Mouse Embryos1

Abstract

Our group found that the treatment of embryos with histone deacetylase inhibitors (HDACi), including trichostatin A, Scriptaid, suberoylanilide hydroxamic acid, and oxamflatin, after cloning by somatic cell nuclear transfer (SCNT) resulted in significantly improved efficiency. Although many researchers have investigated the use of HDACi treatment to improve the quality of cloned mouse embryos, the mechanism underlying this treatment has not been completely understood. We believe that the effect of HDACi on embryonic gene activation (EGA) is important for normal development of cloned embryos. In the present study, using highly sensitive fluorescence in situ hybridization (FISH) with probes complementary to mouse rDNA, the effect of Scriptaid on the onset of rRNA synthesis was examined in cloned embryos. In addition, to determine how Scriptaid affects pre-rRNA processing machinery in SCNT embryos with activated rDNA transcription, functional nucleolar formation was analyzed in detail by combined assessment of rRNA synthesis and nucleolar protein allocation in preimplantation embryos. In this experiment, at least part of the rRNA localization by FISH was substituted by 5-bromouridine 5'-triphosphate staining after alpha-amanitin treatment. The results show that in the late 2-cell stage, a number of SCNT embryos initiated transcriptional activation while having one blastomere showing inactivated rRNA transcription and another blastomere showing activated rRNA transcription and despite both nuclei being in interphase. In addition, in some SCNT embryos, the same nuclei contained a mixture of inactively and actively transcribed rRNA, which was rarely observed in intracytoplasmic sperm injection embryos. This asynchronous transcription induced a delay of one cell cycle in SCNT embryo activation of functional nucleoli. Scriptaid can overcome this failure in the timely onset of embryonic gene transcription by activation of rRNA genes and promotion of nucleolar protein allocation during the early phase of EGA.

rRNA Genes Are Not Fully Activated in Mouse Somatic Cell Nuclear Transfer Embryos

“…The histone deacetylase inhibitor can increase the overall histone acetylation level of donor cell chromatins, then make the genes more reachable to maternal factors, therefore, promoting reprogramming. In a recent study, scriptaid (a kind of histone deacetylase inhibitor) helped NT embryos overcome the asynchronous rRNA transcription at the 2-cell stage by activation of rDNA and promotion of nucleolar protein allocation (56). This study revealed how histone deacetylase inhibitor could improve NT embryo development in rDNA activation levels.…”

Scriptaid affects histone acetylation and the expression of development-related genes at different stages of porcine somatic cell nuclear transfer embryo during early development

“…Studies have shown that treating embryos with Scriptaid inhibits histone deacetylase and results in hyperacetylation, which can expose DNA active binding sites and decrease DNA methylation that are important for gene activation [29]. Therefore, gene activation in embryos could transform the transcriptional quiescence genome into a transcriptional activated state [30].…”

Reprogramming mammalian somatic cells

“…However, acetylation of other lysine residues on core histones (H4K8, H4K12) persisted in the genome of cloned embryos [66]. In somatic cells, transcriptionally active 5S rRNA genes are packaged with hyperacetylated histone H4; in the transcriptionally silent oocyte, these residues are hypoacetylated [67,68]. It could be argued that after SCNT, the cloned embryo establishes a histone acetylation pattern that partially resembles that of embryos produced by fertilization.…”