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SYSSTEMCELL Report Summary

Project ID: 339431
Funded under: FP7-IDEAS-ERC
Country: Netherlands

Mid-Term Report Summary - SYSSTEMCELL (Regulatory transcriptional networks orchestrating mouse pluripotent embryonic stem cells)

The lack of a quantitative framework around the dynamics of gene expression and its determinants represents a major hurdle for capturing transcription regulation into computational models. Embryonic Stem Cells (ESC) provide an excellent cellular model to quantitatively define the principles of gene regulation at the biochemical and (epi)genetic level. SysStemCell focuses on two distinct but readily interconvertible pluripotent states: ESCs derived from the Inner Cell Mass (ICM) and maintained in serum-free medium complemented with two kinase inhibitors and LIF (‘2i ESC’) and ESCs classically cultured in the presence of serum and LIF (‘serum ESC’). Our initial analysis of the epigenetic and transcriptome make-up of 2i- and serum-grown Embryonic Stem Cells (ESCs) has revealed that the epigenome and transcriptome of these two pluripotent cell states are surprisingly different. Amongst others, the transcriptome revealed a prominent change in the metabolic state, and epigenome profiling showed a strong reduction of H3K27me3, a repressive epigenetic mark that is deposited by the Polycomb complex at so-called bivalent promoters.

We performed whole-genome bisulfite sequencing (WGBS) and showed that 2i ESCs are globally and strongly hypomethylated as compared to conventional ESCs maintained in serum. The important realization from the DNA methylome and our earlier studies is that 2i-grown ESCs are reminiscent of in ICM cells - referred to as naive or ground state - whereas classically serum-grown ESCs reflect pluripotent cells from post-implantation embryo – often referred to as primed. Collaborative follow up studies focusing on the transition from primed hypermethylated ESCs to naive hypomethylated ones revealed a critical role of the replication-coupled maintenance methylation machinery. An extensive and detailed molecular characterization allowed the establishment of a mathematical model that accurately predicts global DNA (de)methylation kinetics. The notion that pluripotency is intimately linked to the lack of G1-check point is based solely on studies performed in serum-grown ESCs. Our analysis shows that G1 control in naive pluripotent 2i ESCs is fundamentally different from that in serum ESCs.

Analysis of the 3D organization of chromatin using Hi-C followed by target enrichment of interactions (CHi-C) revealed extreme long-range intra- and inter-chromosomal interactions (ELRI’s) that occur between a small subset of H3K27me3 marked bivalent promoters solely in serum-grown ESCs. Reverting serum-to-2i ESCs resolved these promoter-promoter interactions in a spatiotemporal manner. The functional implications of the presence or absence of these ELRI’s for pluripotency remains to be elucidated. Deep analysis and integration of the dynamic alterations in transcription regulation, enhancer activation, chromatin accessibility and 3D chromatin organization unveiled critical transcription factor networks and epigenetic mechanisms that drive the interconversion. Surprisingly, the extensive transcriptome and epigenome reprogramming is achieved without extensive rewiring of promoter-enhancer (looping) interactions.

Collectively, SysStemCell is well underway to achieve its ambitious goals and has already provided a deeper insight in the mechanisms and general principles that orchestrate gene expression in pluripotency.

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