Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing
Summary
Xenopus eggs can induce the reversal of differentiation processes of somatic cells. Yet, the egg is not fully efficient in reprogramming a differentiated nucleus, as certain genes retain a memory of gene expression of their somatic cell of origin. This is thought to be a reason for the low success rate of current cloning and reprogramming strategies. While previous studies addressed extensively the mechanisms that maintain an inactive state of genes (OFF-memory), we investigated the importance of memory of an active transcriptional state (ON-memory) in maintaining cell fate identity and on resistance to reprogramming. We find that donor cell-type specific ON-memory gene-expression in the wrong cell-type of nuclear transfer (NT)-embryos is as common as OFF-memory gene-expression. When compared to properly reprogrammed genes, we find that ON-memory genes show an elevated level of the active histone mark H3K4me3 in endoderm donor cells. Importantly, we show that a reduction of H3K4 methylation level in donor cells decreases the extent of ON-memory gene expression, globally improves transcriptional reprogramming, and enhances the development of NT-embryos. Therefore, our study reveals that H3K4 methylation safeguards endoderm cell identity and acts as a major barrier for efficient reprogramming in NT-embryos. Furthermore, our results suggest that efficient cell fate reprogramming not only relies on the erasure of epigenetic modifications conferring OFF-memory but also crucially depends on the removal of H3 lysine 4 methylation-mediated memory of an active state of gene expression.
Overall design
73 samples, single-ended RNA-seq libraries from neurula stage 18 or 21 endoderm and gastrula stage 11 ectoderm samples; 2 single-ended ChIP-seq libraries from endoderm cells of neurula (stage 21) embryos with antibody for H3K4me3, 2 replicates for each histone modification pull-down.
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