Periodic Report Summary 2 - LIVENUCESC (Live imaging of nuclear dynamics in embryonic stem cell differentiation)
The objectives of this proposal were threefold, namely:
(a) to study protein-chromatin interactions in living cells (using photobleaching methods);
(b) to study dynamic nuclear processes in living cells;
(c) to directly monitor chromatin movement in living embryonic stem cells (ESCs).
Description of the work performed since the beginning of the project
The first aim has been completed and a paper describing the molecular mechanisms that underlie chromatin protein dynamics in living cells using fluorescence recovery after photobleaching (FRAP) is currently under revisions (Melcer et al., under revisions in Nature Communications). We have analysed chromatin plasticity in ESCs that are depleted for different nuclear factors including deoxyribonucleic acid (DNA) methyltransferases, histone methyltransferases and lamin A and 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 ESCs, and indicate that the epigenetic state of the genome modulates the differentiation potential of ESCs.
In addition, we also addressed our second aim fully. We created 2 libraries of GFP traps in ESCs, in which green fluorescence protein (GFP) exons were randomly inserted using retroviruses thus endogenously labelling genes. We have used these cells successfully to follow nuclear processes as they occur in living ESCs and during the course of differentiation. Analysing fluorescence levels in differentiating ESCs, we found screened for nuclear proteins which their level decreases during differentiation. We identified one protein (SET) which so far has not been implicated in pluripotency. Its expression level goes down faster than some of the bona fide pluripotency genes. Using knockdown and over-expression studies, we show that SET is important for pluripotency and ESC differentiation.
Finally, to study the nature of the hypermobility of chromatin proteins in ESCs, we performed a genome-wide expression screen using whole genome tiling arrays. Our analysis revealed chromatin-remodelling 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). For the third aim, we have monitored chromatin movement directly using H2B-GFP stable ESC lines and followed the changes in real time using time lapse microscopy. For these experiments, we are using a confocal spinning disk microscope which we have available in our lab allowing long-term imaging and very low photodamage.
Expected final results and their potential impact and use
Our results will be published in the international scientific journals and our library and screens will be made public for general use. Our work has already led to several international collaborations, including Oliver Rando (U-Mass), Michael Bustin (NIH), Prim Singh (Germany), Newman Sze (Singapore), Miguel Ramalho-Santon (UCSF), Christian Seiser (Vienna) and others. In addition, my lab has been invited to be a member of the Nucleosome4D Marie Curie training network, and I am an associate PI in the EUROSYSTEM European Union (EU) network on stem cells and system biology.
(a) to study protein-chromatin interactions in living cells (using photobleaching methods);
(b) to study dynamic nuclear processes in living cells;
(c) to directly monitor chromatin movement in living embryonic stem cells (ESCs).
Description of the work performed since the beginning of the project
The first aim has been completed and a paper describing the molecular mechanisms that underlie chromatin protein dynamics in living cells using fluorescence recovery after photobleaching (FRAP) is currently under revisions (Melcer et al., under revisions in Nature Communications). We have analysed chromatin plasticity in ESCs that are depleted for different nuclear factors including deoxyribonucleic acid (DNA) methyltransferases, histone methyltransferases and lamin A and 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 ESCs, and indicate that the epigenetic state of the genome modulates the differentiation potential of ESCs.
In addition, we also addressed our second aim fully. We created 2 libraries of GFP traps in ESCs, in which green fluorescence protein (GFP) exons were randomly inserted using retroviruses thus endogenously labelling genes. We have used these cells successfully to follow nuclear processes as they occur in living ESCs and during the course of differentiation. Analysing fluorescence levels in differentiating ESCs, we found screened for nuclear proteins which their level decreases during differentiation. We identified one protein (SET) which so far has not been implicated in pluripotency. Its expression level goes down faster than some of the bona fide pluripotency genes. Using knockdown and over-expression studies, we show that SET is important for pluripotency and ESC differentiation.
Finally, to study the nature of the hypermobility of chromatin proteins in ESCs, we performed a genome-wide expression screen using whole genome tiling arrays. Our analysis revealed chromatin-remodelling 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). For the third aim, we have monitored chromatin movement directly using H2B-GFP stable ESC lines and followed the changes in real time using time lapse microscopy. For these experiments, we are using a confocal spinning disk microscope which we have available in our lab allowing long-term imaging and very low photodamage.
Expected final results and their potential impact and use
Our results will be published in the international scientific journals and our library and screens will be made public for general use. Our work has already led to several international collaborations, including Oliver Rando (U-Mass), Michael Bustin (NIH), Prim Singh (Germany), Newman Sze (Singapore), Miguel Ramalho-Santon (UCSF), Christian Seiser (Vienna) and others. In addition, my lab has been invited to be a member of the Nucleosome4D Marie Curie training network, and I am an associate PI in the EUROSYSTEM European Union (EU) network on stem cells and system biology.