LIVENUCESC

Live imaging of nuclear dynamics in embryonic stem cell differentiation

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 Obiettivo del progetto (Objective)

'Owing to their unique ability to self-renew indefinitely, as well as their capacity to differentiate into multiple cell types of all three germ layers, embryonic stem (ES) cells hold great promise both as therapeutic agents in the clinic and as research tools in the lab. One of the main challenges in the field is understanding how stem cells achieve their remarkable potential. Recent efforts by us and others have shown that chromatin itself serves as a major contributor to ES cell identity and plasticity. While most of the supporting data emerges from biochemical and molecular studies, I propose here to use live cell imaging techniques and advanced microscopy to probe the transcriptional machinery and chromatin dynamics in living differentiating ES cells. I aim to elucidate the dynamic changes that occur in chromatin structure and function during early ES cell differentiation events. Using photobleaching methods (i.e. FRAP) complemented by biochemical approaches, I will study the dynamic interplay of both transcriptional activators (e.g. Oct-4, Nanog) and transcriptional repressors (e.g. Polycomb Group proteins) with chromatin. Also, using the spinning disk confocal technology I will monitor in real time, changes in chromatin structure, as well as the dynamics of chromatin-protein interactions in living cells. Finally, using ES cell lines carrying a labeled genomic locus at random sites I will be able to directly visualize chromatin motion in living cells. These experiments will provide new insights into the mechanisms that govern chromatin-regulated differentiation events and chromatin dynamics in living cells as well as stem cell identity and pluripotency.'

Introduzione (Teaser)

Embryonic stem (ES) cells have a unique ability to self-renew indefinitely as well as to differentiate into multiple cell types. This makes embryonic stem cells very attractive as laboratory research tools and clinical therapeutic agents.

Descrizione progetto (Article)

The 'Live imaging of nuclear dynamics in embryonic stem cell differentiation' (Livenucesc) project is using live cell imaging techniques and advanced microscopy to investigate the transcriptional processes and chromatin dynamics in living differentiating embryonic stem (ES) cells. The EU-funded work aims to shed light on the changes taking place in chromatin structure and function during early ES cell differentiation events.

Chromatin is the combination of DNA and proteins that constitute the contents of the nucleus and condense to form chromosomes. The interplay of chromatin with both transcriptional activators and repressors is being studied with photobleaching methods and enhanced with biochemical approaches.

Project partners have described the molecular mechanisms underlying chromatin-protein interactions in living cells, and analysed chromatin plasticity in ES cells that were depleted for different nuclear factors. Study results showed that chromatin-remodelling proteins are one of the most strongly expressed groups of genes in ES cells. Study experiments were able to outline the mechanisms regulating chromatin plasticity in ES cells, with results indicating that the epigenetic state of the genome modulates the differentiation potential of ES cells.

The successful creation of specific cell lines achieved in work on another project aim will allow direct visualisation of cellular processes in living cells.

As work continues, Livenucesc researchers are looking to discover proteins common to both fibroblasts and ES cells so as to compare the two with regard to random gene expression. The libraries of clones being generated will be available for use to researchers worldwide.