Final Report Summary - GRPN (Gene regulation at the nuclear periphery)
The spatial organisation of the genome in the nucleus has a role in the regulation of gene expression. In vertebrates, chromosomal regions with low gene-density, and that are less transcribed, are located close to the nuclear periphery. Correlations have also been made between the transcriptional state of some genes and their location near the nuclear periphery. For example, the proneural Ptn gene relocates away from the nuclear periphery when it is activated during neural differentiation of mouse embryonic stem cells (mESCs).
The mechanisms involved in nuclear reorganisation are poorly understood. In order to find what could be the factors involved, it is important to explore when this reorganisation takes place during mouse embryogenesis. To do this, we used two different in vitro differentiation protocols of mESC. We differentiated the cells into Neural progenitor cell (NPC)s, which recapitulate neurectoderm formation. We also differentiated the cell into epiblast ESC (EpiSC)s, which corresponds to an earlier stage, post-implantation and before the lineage commitment. The observation by Fluorescence hybridisation in situ (FISH) in EpiSC of five loci showed that three are already relocated in nuclear centre while two require neurectoderm differentiation. Investigation of the transcription state showed that the movement was correlated to the gene activation.
This result raises a recurrent question in the field, whether subnuclear movement precedes gene activation or if it is a consequence of transcription. We tried to address this question by two different approaches. To test if nuclear reorganisation is important for correct expression of relocated genes, we tried to impair the relocation of Ptn in mESC by tethering its intergenic region to the nuclear envelope. We used a bacterial operator / repressor system previously set up in the host laboratory. Unfortunately, despite trying two different systems, and targeting 5 different regions we did not obtained correct targeted insertions. On the opposite, we addressed directly the question of the role of transcription activation in gene relocation by activating Ptn in mESC. We engineered a Transcription activator-like (TAL) effector Deoxyribonucleic acid (DNA) binding domain with specificity to 14bp upstream Ptn transcription start site. This domain was fused to a histone acetyltransferase domain (VP64) to yield synthetic gene-specific transcription factors (tAW5). Transient transfection of this TALE-TF induces a 25-fold upregulation of Ptn expression. Interestingly, FISH analysis showed that Ptn specifically relocates to the nuclear centre when activated. These results demonstrate for the first time on an endogenous gene that transcription activation is sufficient to induce subnuclear repositioning.
The mechanisms involved in nuclear reorganisation are poorly understood. In order to find what could be the factors involved, it is important to explore when this reorganisation takes place during mouse embryogenesis. To do this, we used two different in vitro differentiation protocols of mESC. We differentiated the cells into Neural progenitor cell (NPC)s, which recapitulate neurectoderm formation. We also differentiated the cell into epiblast ESC (EpiSC)s, which corresponds to an earlier stage, post-implantation and before the lineage commitment. The observation by Fluorescence hybridisation in situ (FISH) in EpiSC of five loci showed that three are already relocated in nuclear centre while two require neurectoderm differentiation. Investigation of the transcription state showed that the movement was correlated to the gene activation.
This result raises a recurrent question in the field, whether subnuclear movement precedes gene activation or if it is a consequence of transcription. We tried to address this question by two different approaches. To test if nuclear reorganisation is important for correct expression of relocated genes, we tried to impair the relocation of Ptn in mESC by tethering its intergenic region to the nuclear envelope. We used a bacterial operator / repressor system previously set up in the host laboratory. Unfortunately, despite trying two different systems, and targeting 5 different regions we did not obtained correct targeted insertions. On the opposite, we addressed directly the question of the role of transcription activation in gene relocation by activating Ptn in mESC. We engineered a Transcription activator-like (TAL) effector Deoxyribonucleic acid (DNA) binding domain with specificity to 14bp upstream Ptn transcription start site. This domain was fused to a histone acetyltransferase domain (VP64) to yield synthetic gene-specific transcription factors (tAW5). Transient transfection of this TALE-TF induces a 25-fold upregulation of Ptn expression. Interestingly, FISH analysis showed that Ptn specifically relocates to the nuclear centre when activated. These results demonstrate for the first time on an endogenous gene that transcription activation is sufficient to induce subnuclear repositioning.