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Tesi etd-01252018-181316

Thesis type
Tesi di laurea magistrale
Single cell assay for measuring replication timing and determining the localisation of specific domains
Corso di studi
relatore Prof. Campa, Daniele
Parole chiave
  • proximity ligation
  • Rif1
  • replication timing
Data inizio appello
Data di rilascio
Riassunto analitico
DNA replication is highly organized, both spatially and temporally. In the eukaryotic genomes there are many origins of replication that fire in a specific temporal order. Contiguous regions, whose origins fire simultaneously, form replication domains. Although replication is continuous, for simplicity the eukaryotic genome is classified into early and late replicating domains. There is a strong relationship between replication timing and nuclear three-dimensional organization. For example, late replicating domains are generally localised either to the nuclear periphery or in the peri-nucleolar region. In addition, different S phase sub-stages can be identified on the basis of the spatial distribution of nucleotides incorporated at the replication fork, with each sub-stage displaying a characteristic spatial distribution of replication forks.
One of the major regulators of replication timing in mammalian cells is the protein RIF1. Loss of RIF1 affects the temporal order of DNA replication, and many regions switch their replication timing either from late to early or from early to late. RIF1 coats the late replicating genome, forming large Rif1 associated domains (RADs), mainly located to the nuclear periphery. RADs show a large degree of overlap with Lamina associated domains (LADs), the regions of the genome that are located to the nuclear periphery and are bound by Lamin B1. However, beside these RADs- Lamin B1 positive (RADs-LB+), there are also RADs that, although peripheral, are not bound by Lamin B1 (RADs-LB-). These two types of RADs react differently to RIF1 deficiency. While RADs-LB+ are unperturbed by Rif1 deletion, RADs-LB- switch their replication timing from late to early S phase, simultaneously relocating away from the nuclear periphery. These observations suggest that RIF1 could control both peripheral localisation and replication timing. In support of this hypothesis, it has been shown that deletion of Rif1 also determines a rearrangement of chromatin’s architecture, as well as inducing an alteration of the spatial distribution of replication forks within the nucleus.
In order to test if RIF1 controls replication timing at least in part through determining peripheral localisation of Lamin B1 negative late replicating domains, we needed to set up a single cell assay for measuring simultaneously replication timing and nuclear position. We intend to determine if changes of replication timing in Rif1 null cells correspond to changes in nuclear position of specific loci in the same cell.
To this end we have employed a proximity ligation-based assay (PLA) to determine when a specific locus, labelled by a fluorescence in situ hybridisation (FISH) probe, is close to a BrdU molecule, incorporated during DNA replication in the newly synthetized strand. In a wild type cell, comparison of the PLA signal and the spatial distribution of replication forks would suffice to establish the timing of replication of the single locus. However, since Rif1 null cells display an aberrant spatial organisation of replication forks, we have resolved to attempt the classification of S phase sub-stages based on DAPI intensity, that reflects the DNA content. This assay was originally designed to identify cell cycle stages (G1-S-G2). We therefore needed to test if this method was sensitive enough to discriminate between stages with a much more similar DNA content.
After a first stage of optimisation of the DAPI staining procedure and the acquisition settings, we could establish that the method allowed discrimination not only between the cell cycle phases, but also between S phase sub-stages. We have compared the spatial distribution of replication forks and the integrated DAPI intensity in wild type cells and found that DAPI intensity (aka DNA content) shows a good correspondence to the different patterns of replication forks distribution. Thanks to this, we are therefore able to analyse the progression of the S phase in Rif1 null cells irrespective of the altered forks distribution.
In conclusion, combining the identification of the S phase sub-stages through the measurement of the DAPI intensity and the visualization of the PLA signal, we would be able, for the first time in single cells, to understand if RIF1 controls both replication timing and nuclear localisation of Lamin B1 negative late replicating domains.