Final Report Summary - IMMRSRGE (Identifying molecular mechanism responsible for spatial reorganisation of the genome during embryogenesis)
The IMMRSRGE project set out to identify factors that affect to spatial position of specific gene loci in the mammalian nucleus. The focus was on genes that move either away from, or towards, the edge of the nucleus (nuclear periphery) as mouse embryonic stem cells (ESCs) differentiate into epiblast stem cells (EpiSCs). The interest in this period of development is that this corresponds to the period of blastocyst implantation development when substantial nuclear reorganization takes place. The position of genes relative to the nuclear periphery has been associated with the repression of gene expression.
The approach taken was a visual screen using the technique of RNAi to knockdown the levels of specific proteins. Gene localization was then assayed by fluorescence in situ hybridization (FISH). Given the nature of the assay, it was determined that the most feasible strategy would be a targeted RNAi screen, focusing on targets known to be involved in the structure of the nuclear periphery, and targets known to modify the structure and organization of chromatin. To prioritise these targets, we analysed published data on DNA sequences that associate with the lamin B1 – a protein located just under the nuclear envelope. These sequences occur in large domains – lamin-associated domains (LADs) that can be subdivided into “constitutive LADs” which are preferentially associated with lamin B1 in different cell types, and “facultative LADs” whose lamin association is regulated during development. We then correlated this data to ‘epigenomic’ datasets of histone modifications in ESCs and then ESCs that had been differentiated.
This was very successful. We identified a modified histone that, when knocked down, caused some genes to aberrantly relocate to the nuclear periphery in ESCs. In normal development, these same genes would not normally move to the nuclear periphery until the epiblast stage of development. Conversely, we identified other gene loci that inappropriately move to the nuclear periphery when this modified histone is depleted in EpiScs. We have followed this exciting finding up by analysing other chromatin features associated with the candidate histone modification and are investigating whether these other factors also result in the spatial reorganisation of the nucleus.
Though this project has now finished, due to a career change of the Marie-Curie fellow, the findings form this project are being followed up in the host laboratory and are being prepared for publication in a high impact peer-reviewed biomedical journal. The work was also presented at two international meetings in 2013, which gave the fellow important presentation and English language skills. The work from this project represents a significant advance in the understanding of genome function during early embryonic development.
The approach taken was a visual screen using the technique of RNAi to knockdown the levels of specific proteins. Gene localization was then assayed by fluorescence in situ hybridization (FISH). Given the nature of the assay, it was determined that the most feasible strategy would be a targeted RNAi screen, focusing on targets known to be involved in the structure of the nuclear periphery, and targets known to modify the structure and organization of chromatin. To prioritise these targets, we analysed published data on DNA sequences that associate with the lamin B1 – a protein located just under the nuclear envelope. These sequences occur in large domains – lamin-associated domains (LADs) that can be subdivided into “constitutive LADs” which are preferentially associated with lamin B1 in different cell types, and “facultative LADs” whose lamin association is regulated during development. We then correlated this data to ‘epigenomic’ datasets of histone modifications in ESCs and then ESCs that had been differentiated.
This was very successful. We identified a modified histone that, when knocked down, caused some genes to aberrantly relocate to the nuclear periphery in ESCs. In normal development, these same genes would not normally move to the nuclear periphery until the epiblast stage of development. Conversely, we identified other gene loci that inappropriately move to the nuclear periphery when this modified histone is depleted in EpiScs. We have followed this exciting finding up by analysing other chromatin features associated with the candidate histone modification and are investigating whether these other factors also result in the spatial reorganisation of the nucleus.
Though this project has now finished, due to a career change of the Marie-Curie fellow, the findings form this project are being followed up in the host laboratory and are being prepared for publication in a high impact peer-reviewed biomedical journal. The work was also presented at two international meetings in 2013, which gave the fellow important presentation and English language skills. The work from this project represents a significant advance in the understanding of genome function during early embryonic development.