combinational-treatment-trichostatin-vitamin-c-improves-efficiency

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of ...

University of Cambridge

Transposable elements in the genome are generally silenced in differentiated somatic cells. However, increasing evidence indicates that some of them are actively transcribed in early embryos and the proper regulation of retrotransposon expression is essential for normal development. Although their developmentally regulated expression has been shown, the mechanisms controlling retrotransposon expression in early embryos are still not well understood. Here, we observe a dynamic expression pattern of retrotransposons with three out of ten examined retrotransposons (1a11, λ-olt 2-1 and xretpos(L)) being transcribed solely during early embryonic development. We also identified a transcript that contains the long terminal repeat (LTR) of λ-olt 2-1 and shows a similar expression pattern to λ-olt 2-1 in early Xenopus embryos. All three retrotransposons are transcribed by RNA polymerase II. Although their expression levels decline during development, the LTRs are marked by histone H3 lysine 4 trimethylation. Furthermore, retrotransposons, especially λ-olt 2-1, are enriched with histone H3 lysine 9 trimethylation (H3K9me3) when their expression is repressed. Overexpression of lysine-specific demethylase 4d removes H3K9me3 marks from Xenopus embryos and inhibits the repression of λ-olt 2-1 after gastrulation. Thus, our study shows that H3K9me3 is important for silencing the developmentally regulated retrotransposon in Xenopus laevis. 

Histone H3 lysine 9 trimethylation is required for suppressing the expression of an embryonically activated retrotransposon in Xenopus laevis.

Understanding the mechanism of resistance of genes to reactivation will help improve the success of nuclear reprogramming. Using mouse embryonic fibroblast nuclei with normal or reduced DNA methylation in combination with chromatin modifiers able to erase H3K9me3, H3K27me3, and H2AK119ub1 from transplanted nuclei, we reveal the basis for resistance of genes to transcriptional reprogramming by oocyte factors. A majority of genes is affected by more than one type of treatment, suggesting that resistance can require repression through multiple epigenetic mechanisms. We classify resistant genes according to their sensitivity to 11 chromatin modifier combinations, revealing the existence of synergistic as well as adverse effects of chromatin modifiers on removal of resistance. We further demonstrate that the chromatin modifier USP21 reduces resistance through its H2AK119 deubiquitylation activity. Finally, we provide evidence that H2A ubiquitylation also contributes to resistance to transcriptional reprogramming in mouse nuclear transfer embryos.

Gene Resistance to Transcriptional Reprogramming following Nuclear Transfer Is Directly Mediated by Multiple Chromatin-Repressive Pathways.

Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency.

Mami Oikawa

Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Zoology

University of Cambridge

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer