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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - REPTIMORG (DNA replication timing and spatial organization of chromatin)

Teaser

Summary

DNA replication is the essential process that ensures a correct duplication of the DNA before each cell division. This is a highly organised process in both space in time. It is known that all DNA within the cell is not replicated simultaneously, instead some regions are replicated earlier than others. The reason behind this temporal control of replication is not entirely clear but unscheduled replication is a common feature of cancer and malignant cells, highlighting the importance of an understanding of how the timing is regulated. Further, it is commonly known that the DNA is not randomly distributed within the nucleus. Instead, some regions are always positioned close to the periphery of the nucleus while other regions are more in the centre. Regions that are close to the nuclear periphery tend to replicate late while regions towards the centre replicate early. 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 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 Rif1â€™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.

Work performed

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 Rif1â€™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 processâ€™ 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.

Final results

I have discovered novel essential players in the regulation the expression of Xist and the vital process X- chromosome inactivation. I find that the loss of on single factor, an activator, leads to a gain of a repressor and a complete block of a whole process. We expect to be able to generalise what I have discovered about the regulation of Xist to other genes that are important for embryonic development. Further, deregulation of gene expression is also a general feature of disease state. What I discovered has the potential to reveal a novel mechanism leading to pathology.