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Nuclear foundations of cellular potency

Final Report Summary - NUCLEARPOTENCY (Nuclear foundations of cellular potency)

Histone modifications and DNA methylation patterns are dramatically remodelled during early development. Nuclear architecture has recently emerged as a key epigenetic factor, but a role for nuclear architecture in regulating reprogramming and early development has not been established. To investigate the dynamics of nuclear and chromosomal organization throughout preimplantation development, we have undertaken a number of research lines.
We addressed the global dynamics of heterochromatin during pre-implantation development, and in particular its changes in nuclear localization, with the aim to identifying the molecular players involved in regulating its nuclear localization. We used transcription activator-like effector (TALE) technology to visualize endogenous repetitive genomic sequences assembled in heterochromatin and follow nuclear positioning and chromatin dynamics in cultured mouse cells and in the living organism. We manipulated the nuclear position of such regions, demonstrating that the nucleus of the early embryo is functionally regionalised. We also manipulated the expression of specific retrotransposon families, and showed that activation of LINE-1 regulates global chromatin accessibility at the beginning of development and indicate that retrotransposon activation is integral to the developmental program. Our findings provide a framework to address retrotransposon function and the function of genome architecture through visualization and manipulation of nuclear dynamics in vivo.

To address the dynamics and mechanisms of heterochromatin formation, we chose to use repetitive elements, including retrotransposons, as model loci to address how and when heterochromatin forms during development. Using RNA sequencing we showed that repetitive-element expression is dynamic and stage specific. Chromatin immunoprecipitation for H3K4me3 and H3K9me3 in 2- and 8-cell embryos indicated that their developmental silencing follows loss of activating marks rather than acquisition of conventional heterochromatic marks. We have manipulated the expression of key chromatin modifiers in the embryo, and show that heterochromatin remodelling is essential for natural reprogramming at fertilisation. This work also allowed us to uncover two novel mechanisms of RNA regulation, which we studied in vitro and in vivo.

We also implemented novel experimental approaches using live imaging to address global chromatin states in the embryo and cell potency. Our results suggest that changes in cellular plasticity correlate with global changes in chromatin architecture, in particular, with histone mobility. We also showed that the transition from totipotencytowards pluripotency is accompanied by a decrease in histone mobility, and, upon lineage allocation, pluripotent cells retain higher mobility than the differentiated trophectoderm. Importantly, this work led to the suggestion that totipotent cells, both in vitro and in vivo, display particularly high histone core mobility. In line with the above, we identified chromatin assembly, as a major pathway inducing totipotent-like features in vitro, and identified additional epigenetic pathways that induce cells with resemblance to the totipotent 2-cell stage embryo in vitro.

Overall, our work during this ERC has significantly contributed to shed light into the mechanisms governing heterochromatin formation and how global chromatin states can impact cellular plasticity, totipotency, epigenetic reprogramming and cell fate.