DNA replication is an essential process that ensures a duplication of the genome before each cell division. All DNA within a cell is not replicated simultaneously, instead some regions are replicated earlier than others. There is a known correlation between positioning within the nucleus and timing of replication, where late replicating regions tend to be close to the periphery of the nucleus and the early replicating regions are more towards the centre of the nucleus. This is suggesting that the organisation of the DNA within the cell could play a role in the regulation of the replication timing. We have previously identified Rif1 as a regulator of the temporal order of replication as well as a regulator of higher-order DNA structures. Here, I have here discovered that regions in which the timing of replication depends on Rif1 will change position as well as change replication timing when Rif1 is removed. Further, we find that regions that replicate late independently of Rif1 maintain their peripheral localisation also after Rif1 is removed. Together this suggests that, at least for the regions analysed, replication timing and nuclear positioning cannot be uncoupled. My aim has also been to understand the role of the nuclear periphery in replication timing control, independently on Rif1. To do so, I have generated cell lines specific for this project. These cells contain specific modifications designed so I can remove key structural components of the nucleus and study the effect this has on replication timing control.
Females have two X chromosomes compared to males who have only one. To deal with this chromosomal imbalance between the sexes, nature has developed as system in which one of the two X chromosomes in mammalian females is silenced. The silencing of an X chromosome is carried out in a complex process, commonly known as the X-inactivation process. This process takes place very early during mouse embryonic development and we find that mouse embryos that lack Rif1 die as a consequence of an inability to perform X-chromosome inactivation. I have been studying X-chromosome inactivation in cells and the effect of removing Rif1, aiming at understanding the mechanism behind Rif1’s role in this fundamental process.
Activators are factors that boost gene transcription while repressors are factors that decrease gene transcription. A general way in which genes are regulated is by fine-tuning the balance between activators and repressors, keeping a gene expressed or repressed respectively. I have discovered that Rif1 plays an important role in the regulation of X-chromosome inactivation by working as an activator in this process’ very first initiating step. Specifically, I find that Rif1 is required to activate the expression of Xist, a key component of this process. Upon Rif1 removal, the regulation of Xist is taken over by a repressor, now inhibiting its expression. A failure in activating the expression of Xist results in a complete block and inability to proceed through the X-chromosome inactivation process.
I have presented both projects at conferences and workshops all over Europe. In addition, we have built invaluable collaborations with excellent European researchers, with whom we have been discussing results as well as future directions of these projects.
