Dnmt3l-knockout donor cells improve somatic cell nuclear transfer reprogramming efficiency

Abstract

Nuclear transfer (NT) is a technique used to investigate the development and reprogramming potential of a single cell. DNA methyltransferase-3-like, which has been characterized as a repressive transcriptional regulator, is expressed in naturally fertilized egg and morula/blastocyst at pre-implantation stages. In this study, we demonstrate that the use of Dnmt3l-knockout (Dnmt3l-KO) donor cells in combination with Trichostatin A treatment improved the developmental efficiency and quality of the cloned embryos. Compared with the WT group, Dnmt3l-KO donor cell-derived cloned embryos exhibited increased cell numbers as well as restricted OCT4 expression in the inner cell mass (ICM) and silencing of transposable elements at the blastocyst stage. In addition, our results indicate that zygotic Dnmt3l is dispensable for cloned embryo development at pre-implantation stages. In Dnmt3l-KO mouse embryonic fibroblasts, we observed reduced nuclear localization of HDAC1, increased levels of the active histone mark H3K27ac and decreased accumulation of the repressive histone marks H3K27me3 and H3K9me3, suggesting that Dnmt3l-KO donor cells may offer a more permissive epigenetic state that is beneficial for NT reprogramming. Reproduction (2015) 150 245-256

Transient DNMT3L Expression Reinforces Chromatin Surveillance to Halt Senescence Progression in Mouse Embryonic Fibroblast

“…The gain-offunction effect of ectopic DNMT3L on TE repression observed in our current study was consistent with our previous DNMT3L loss-of-function study using Dnmt3l-KO embryo-derived MEFs (Liao et al, 2015). MEFs derived from Dnmt3l-KO embryos have an accelerated premature senescence phenotype, which is associated with a global reduction in repressive H3K9me3 and H3K27me3 markers (Liao et al, 2015). We further developed bioinformatic pipelines to quantify the representation of each TE subfamily from our published strand-specific RNA-seq datasets of Dnmt3l−/− and Dnmt3l+/+ littermate-derived MEFs (Liao et al, 2015).…”

“…Therefore, enhancement of the heterochromatic silencing of repeat elements could be key to maintaining the healthspan. The gain-offunction effect of ectopic DNMT3L on TE repression observed in our current study was consistent with our previous DNMT3L loss-of-function study using Dnmt3l-KO embryo-derived MEFs (Liao et al, 2015). MEFs derived from Dnmt3l-KO embryos have an accelerated premature senescence phenotype, which is associated with a global reduction in repressive H3K9me3 and H3K27me3 markers (Liao et al, 2015).…”

“…MEFs derived from Dnmt3l-KO embryos have an accelerated premature senescence phenotype, which is associated with a global reduction in repressive H3K9me3 and H3K27me3 markers (Liao et al, 2015). We further developed bioinformatic pipelines to quantify the representation of each TE subfamily from our published strand-specific RNA-seq datasets of Dnmt3l−/− and Dnmt3l+/+ littermate-derived MEFs (Liao et al, 2015). We found that more than 90% of the differentially expressed TE subfamilies were upregulated in Dnmt3l-KO MEFs ( Supplementary Table S1).…”

Epigenetic Reprogramming During Somatic Cell Nuclear Transfer: Recent Progress and Future Directions

“…Improving DNA methylation reprogramming has been applied in cloned embryos (Enright et al, 2003;Huan et al, 2015a;Liao et al, 2015). One way in which this is accomplished is by recapitulating the DNA methylation pattern of normal fertilized embryos using DNA-demethylating agents or by Dnmts including Dnmt1 and Dnmt3l gene silencing.…”

“…The nucleoside analog 5-aza-2 -deoxycytidine (5-aza-dC) is incorporated into the genome during DNA replication, inhibiting DNMT1 activity and resulting in DNA hypomethylation (Enright et al, 2003). Genomic DNA hypomethylation by 5-aza-dC treatment has been shown to improve the development of cloned embryos, whereas Dnmt1 or Dnmt3l knockdown in somatic cells or cloned embryos increases gene-specific DNA methylation and histone modification reprogramming and, consequently, developmental competence (Diao et al, 2013;Huan et al, 2015a;Liao et al, 2015;Song et al, 2017b). Additionally, the expression level of Tet3 in oocytes has been shown to be positively correlated with the developmental competence, and Tet3 overexpression in donor cells restores normal DNA hypermethylation and increases the full-term development of cloned embryos (Han et al, 2018).…”