LIVENUCESC · Live imaging of nuclear dynamics in embryonic stem cell differentiation

Periodic Report Summary - LiveNucESC (Live imaging of nuclear dynamics in embryonic stem cell differentiation)

A summary description of the project objectives The objectives of this proposal were threefold: (I) to study protein-chromatin interactions in living cells (using photobleaching methods). (II) to study dynamic nuclear processes in living cells. (III) to directly monitor chromatin movement in living embryonic stem cells. - a description of the work performed since the beginning of the project We have essentially completed our first aim and have recently submitted a paper describing the molecular mechanisms that underlie chromatin protein dynamics in living cells using fluorescence recovery after photobleaching [FRAP) (Melcer et al., under revisions in Cell Stem Cell). We have analyzed chromatin plasticity in ES cells that are depleted for different nuclear factors including DNA methyltransferases, histone methyltransferases and lamin A. In addition, we began to work on our second aim and have created cell lines which express GFP-fusion proteins under their own endogenous promoters using exon tagging approach. We inserted YFP exons into the genomes of the cells and FACS-sorted the fluorescing ones, thus creating stable clones which express yellow endogenous proteins. This allows direct visualization of cellular processes in living cells and we now began to perform time lapse microscopy studies. - a description of the main results achieved so far To study the nature of the hypermobility of chromatin proteins in ES cells, we performed a genome-wide expression screen using whole genome tiling arrays. Our analysis revealed chromatin-remodeling proteins as one of the most predominantly expressed groups of genes in ES cells (Efroni et al., Cell Stem Cell, 2008). We showed that at least one of these proteins, Chd1, is essential for ES cell pluripotency and for maintaining an open chromatin conformation (Gaspar-Maia et al., Nature, 2009). We further investigated the mechanisms that regulate chromatin plasticity in ES cells by fluorescence photobleaching methods in living cells. Using epigenetic drugs and mutant ES cells lacking various chromatin binding proteins, we find that histone acetylation enhances chromatin dynamics, while histone H3 lysine 9 (H3K9) methylation and lamin A expression restrict chromatin dynamics exclusively in heterochromatin. Altered chromatin dynamics is associated with perturbed differentiation. These data delineate the mechanisms for chromatin plasticity in ES cells, and indicate that the epigenetic state of the genome modulates the differentiation potential of ES cells (Melcer et al., under revisions). - the expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far) In a year's time, we expect to produce a library of clones that express YFP-fusion proteins under their own endogenous promoters. We hope to be able to find common proteins to both fibroblasts and ES cells and compare the two systems in terms of stochastic gene expression. We also expect to differentiate some of the ES cell clones and track the transcription levels in real-time. Importantly, the libraries that we are generating can be used by researchers world-wide and we anticipate a wide interest in these clones, which can yield new and interesting collaborations or strengthen the dialog between us and additional labs throughout Europe and the rest of the world. In addition, by the end of the project, we also expect to cover our third aim, namely, to track dynamic behaviour of single chromosomal loci in living embryonic stem cells. This has not been done yet in ES cells and we eagerly await to explore this avenue.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз