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

Jerome Jullien,Munender Vodnala,Vincent Pasque,Mami Oikawa,Kei Miyamoto,George Allen,Sarah Anne David,Vincent Brochard,Stan Wang,Charles Bradshaw,Haruhiko Koseki,Vittorio Sartorelli,Nathalie Beaujean,John Gurdon

doi:10.1016/j.molcel.2017.01.030

Abstract

SummaryUnderstanding 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.

Highlights

Complete nuclear reprogramming is an inefficient process, irrespective of the procedure used (Gurdon, 1960; Pereira et al, 2008; Takahashi and Yamanaka, 2006)
We have recently described a nuclear transfer procedure in which mouse nuclei are transcriptionally reprogrammed toward an oocyte type of transcription (Jullien et al, 2014)
To investigate the sensitivity of resistance to multiple treatments, we focused first on the effect of single chromatin modifier treatment, and we evaluated how often overexpression of Kdm4d, Kdm6b, and USP21 as well as inactivation of Dnmt1 could affect the expression of the same resistant genes (Figure 4A)

Summary

Introduction

Complete nuclear reprogramming is an inefficient process, irrespective of the procedure used (Gurdon, 1960; Pereira et al, 2008; Takahashi and Yamanaka, 2006). We have recently described a nuclear transfer procedure in which mouse nuclei are transcriptionally reprogrammed toward an oocyte type of transcription (Jullien et al, 2014). This transcriptional reprogramming is directly mediated by Xenopus oocyte factors and in the absence of cell division. Using this direct assay, we first identify genes that are resistant to transcriptional reprogramming. Increased expression of these genes upon interference with the chromatin status of transplanted nuclei reveals how resistance is achieved on a single-gene basis. We provide evidence that resistance to transcriptional reprogramming following nuclear transfer to egg can be alleviated by USP21 expression

Methods

Findings

Discussion

Conclusion