Periodic Reporting for period 1 - REPTIMORG (DNA replication timing and spatial organization of chromatin)
Reporting period: 2016-07-01 to 2018-06-30
We have previously identified the protein Rif1 as a regulator of the temporal order of replication. But Rif1 is a complex protein with functions also in controlling higher-order DNA structures. My aim has been to understand if these two functions of Rif1 can be uncoupled or if Rif1 through just one single mechanism is controlling both. My research has also focused on the role of the nuclear periphery and the spatial organisation of DNA, in replication timing control, independently on Rif1. My studies show that Rif1 controls when certain regions are replicated while also positioning these regions to the nuclear periphery. 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.
Females have two X chromosomes compared to males who have only one. To deal with this chromosomal imbalance between the sexes, nature has developed a system in which one of the two X chromosomes in mammalian female cells is being silenced. This means that the vast majority of genes on this chromosome become inactive, in other words repressed. The silencing of an X chromosome is carried out in a complex process, commonly known as the X-chromosome inactivation process. This process takes place very early during embryonic development, in mice as well as in humans, and it is absolutely crucial for the development of the female embryo. We find that mouse embryos that lack Rif1 die as a consequence of an inability to perform X-chromosome inactivation. I have been studying the process of X-chromosome inactivation in cells and the effect of removing Rif1, aiming at understanding the mechanism behind Rif1’s role in this fundamental process. My studies show that Rif1 plays a key role in the very first initiating steps and without Rif1, this whole process is blocked.
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.