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Molecular control of DNA replication timing in mammalian cells

Periodic Reporting for period 3 - RepTime (Molecular control of DNA replication timing in mammalian cells)

Reporting period: 2020-10-01 to 2022-03-31

DNA replication is the essential process that allows the transmission of the genetic information through the cell generations. DNA replication starts from sites of the genome called origins of replication, that are not activated all at once, but follow a temporal order called the DNA replication-timing program. Why are not all the origins of replication activated at once? What is the biological role of the replication-timing program? This program is cell-type specific, dynamically regulated during embryonic development and cell fate determination, and highly conserved among similar species, suggesting a fundamental role. Attempts to increase artificially the number of origins activated at once induces DNA replication stress and the DNA damage response. In agreement with these observations, the only alterations of the DNA replication timing program known to be compatible with life are correlated with cancer. However, very little is known about the molecular machinery that controls the order of activation of the origins of replication and the exact biological role of the DNA replication timing program is still unknown. Moreover, in mammalian cells, even the location of the active origins of replication and the principle guiding their distribution are still unclear. In my proposal I address different aspects of the regulation of DNA replication timing.
1. I propose a novel approach to map the origins of replication (WP1 and 2);
2. I investigate the role of the nuclear periphery in instructing late replication (WP3, 4 and 5);
3. I dissect the molecular mechanism by which RIF1, the only protein known to regulate replication timing genome-wide, works (WP6 and 7).
4. I use the process of X inactivation as a model biological process to understand the biological role of the replication-timing program (WP8).

DNA replication is a defining feature of life. Mistakes during DNA replication are incompatible with organismal development, can induce mutagenesis or activate the DNA damage response, inducing senescence. Ultimately, all these consequences together link problems during DNA replication control to cell transformation or the genesis of neurological developmental disorders.
The understanding of the basic molecular principles controlling DNA replication timing is therefore of value both for the scientific community, to increase our comprehension of the basic rules of life, and to the general public. In the long term, the knowledge of the molecular process guiding DNA replication timing has the potential to lead to novel diagnostic or therapeutic tools.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
WP1: The aim of this WP is to set up a new method to identify in G1 specifically the origins of DNA replication that are going to be active in the following S-phase. From the mouse line we had already created, we have derived mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs) expressing the tagged allele of a protein that is involved in the activation of origins of replication. We have characterised the cells, analysed the expression and the localisation of the tagged protein. Preliminary chromatin immunoprecipitation experiments have been carried out to establish the protocol to isolate and map active origins of replication.
WP2: The aim of this WP is complementary to the goal of WP1, and it is to set up a method to map in G1 origins of replication that will be either active of dormant in the following S-phase. Work to establish the mouse line expressing the tagged allele of the gene that we have chosen as a marker has been undertaken.
WP3: The seclusion to the nuclear periphery of a subset of RIF1-associated, late-replicating genomic regions is RIF1-dependent. The aim of this WP is to assess the role of the peripheral localisation during the determination of replication timing. We have established all the cell lines to artificially re-localise to the nuclear periphery four genomic regions that shift replication timing and subnuclear positioning as a consequence of Rif1 deletion.
WP4 and 5: The goal of these WPs is complementary to the aim of WP3 and it is to understand the role of different lamina-associated proteins in the determination of the replication timing of some late-replicating regions. By multiple mouse crosses, we have derived ESCs of the appropriate genotypes to test the role of the lamina in determining the replication timing of the lamin-associated domains (LADs).
WP6: This WP has been designed to uncouple the role of RIF1 in the control of nuclear architecture and in the determination of the replication timing. We have created cell lines that will enable us to inducibly restore RIF1, in absence of the endogenous protein, in a time-controlled manner. We have also set up a protocol to induce RIF1 expression specifically in G1, to test until what stage of the cell cycle cells are competent to respond to RIF1 restoration.
WP7: The aim of this WP is to understand the role of the interaction between RIF1 and protein phosphatase 1 (PP1) in the RIF1-dependent organisation of the architecture of chromatin contacts. We have analysed the replication timing (Repli-seq) and nuclear organisation (HiC) in cells expressing Rif1 mutants unbale to interact with protein phosphatase 1 (PP1-Rif1PP1), Rif1 null cells, Rif1 hemizygous controls and wild type ESCs. These analyses have yielded unexpected and exciting results. We have found that a. The RIF1-dependent control of the replication-timing program is not entirely executed through PP1. b. Nuclear organisation is entirely controlled through the interaction between RIF1 and PP1 and it is exquisitely sensitive to RIF1 dosage. c. RIF1 entire complement and its interaction with PP1 are necessary to define the compartmentalisation of chromatin contacts (A/B compartmentalisation). d. We have been able to uncouple nuclear organisation and replication timing, taking advantage of Rif1 hemizygotes. We have therefore concluded that nuclear organisation and replication timing are coordinated through RIF1 and that replication timing can still be functional when nuclear compartmentalisation is affected. These results have been summarised in a paper that has been submitted.
WP8: In this WP we proposed to study the role of the switch of replication timing of the future inactive X chromosome from early to late S-phase during the process of X inactivation. We proposed to delete Rif1 in female mouse embryonic stem cells to block the switch during differentiation. However, we have discovered that RIF1 has an earlier function in the context of the process of X chromosome inactivation (XCI), that precedes the switch of replication timing. Rif1 null female ESCs fail to upregulate the long-non-coding RNA (lncRNA) Xist that is the master regulator of XCI. Interestingly, we found that this is linked to the role of RIF1 in protecting Xist promoter from the association of a transcriptional repressor, that we have also identified in the SILAC screen in WP7. Through a double-bookmarking system, where RIF1 identifies the future inactive X during differentiation, we have shown that RIF1 is involved in the choice of the identity of the future active and inactive X chromosome (Xa and Xi). This solves a long-standing question of how, during the first steps of random XCI, the symmetry of the two X chromosomes is broken. These results have been summarised in a paper that has been submitted.
WP 1 and 2: by the end of the project we aim at mapping active origins of replication in multiple tissues in the mouse, and correlate it with the three-dimensional organisation of chromatin contacts of the origins and the state of gene expression of the associated genes. The work proposed in these work packages is based on a novel way of mapping origins of replication.

For WP 3, 4 and 5, we expect to soon be able to perform the experiments to test the role of the nuclear periphery in the regulation of the replication timing. In WP 3, we have devised alternative, new methods to induce the re-localisation of specific genomic regions between different subnuclear compartments. We have created a new protocol to target large repetitive regions.

WP 6. By the end of the project we expect to be able to conclude if chromatin organisation plays an instructive role in the control of replication timing.

WP 7. We have discovered that the role of RIF1 in the control of chromatin organisation is
1. Dosage-sensitive.
2. Mediated by the interaction with Protein Phosphatase 1 (PP1).

WP 8. We have discovered that the earliest function of RIF1 during the X-inactivation program is in the control of the expression of the long-non-coding RNA Xist.