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Unravelling the effect of origin number on the success of genome replication

Periodic Reporting for period 1 - ORI NUMBER (Unravelling the effect of origin number on the success of genome replication)

Reporting period: 2017-12-01 to 2019-11-30

The control of cell division underpins the multicellular basis of mammalian development. To avoid deleterious re-replication events, the process of DNA replication is divided into two non-overlapping steps, first the “licensing” of potential replication origins during G1 and then their sequential “firing” during only S phase. The “pre-replicative complex” (CDC6, CDT1and ORC proteins), license origins in late G1 phase, loading MCM2-7 (mini chromosome maintenance) double hexamers onto the DNA.

However, due to the size of their genome, mammalian cells have a high risk that a replication failure occurs somewhere in the genome. MCM-driven replication forks can irreversibly stall when they encounter DNA damage or tightly bound protein-DNA complexes, resulting in gross chromosomal defects and ultimately, cell death. To minimise the probability of these catastrophic events, two mechanisms are in place: the licensing of “dormant origins” and the existence of a “licensing checkpoint” in late G1. At the moment, the exact regulation of the licensing system and these two safety mechanisms in primary cells is largely unknown. Therefore, a greater understanding of the threshold value of licensing that is needed to activate the licensing checkpoint is important to identify new highly selective anticancer targets.

Meier-Gorlin syndrome (MGS) is a human syndrome characterised by developmental defects, microcephaly and dwarfism but without evidence of chromosomal instability. It is caused by mutations in pre-RC proteins, especially in the ORC complex. However, a number of different cellular and phenotypic abnormalities have been observed in mouse, in vitro human models and patients, with reduced licensing (MCM mutants). These show proliferation defects, genome instability and cancer susceptibility. Therefore, a deeper understanding of pre-RC mutations in primary cell models is required to clarify how these proteins interact with other regulatory systems. This knowledge has significant potential to uncover cell-specific differences in replication licensing and the maintenance of mammalian genome stability. Such findings are vital to the development and refinement of anti-cancer therapeutics.

In this project I investigated how to generate recombinant human iPS cells (hiPSCs) using Crispr/Cas9 technology for MGS-relevant ORC1 mutations and more broadly the licensing system in MGS primary cells versus cancer cell lines.

In this context, the objectives of this project were:
1) To determine if MGS mutations impair origin licensing and licensing checkpoint activation in hiPSCs
2) To clarify different effects of mutant ORC and MCM on the molecular pathways of the licensing checkpoint in genetically modified hiPSCs
3) To perform comparative analyses of these effects on origin licensing and licensing checkpoint activation in hiPS-derived clinically-relevant lineages (neural, cardiac, pulmonary) and mammalian cancer cell lines.
Objective 1: To determine if MGS mutations impair origin licensing and licensing checkpoint activation in hiPSCs.
Different commercially available hiPSCs, including Chips4 and Wibj2 were transfected in order to generate knockin point mutants for the clinically relevant mutations R105Q in exon 2 of ORC1 protein. Attempts were made to introduce the ORC1(R105Q) mutation into hiPSCs using a variety of strategies. None yielded positive knockin clones despite multiple rounds of screening. Finally, a new approach was tested in collaboration with the MRC PPU at Dundee University, called L-CIGA-R. Unfortunately, this method yielded only heterozygous clones for the ORC1 (R105Q), with one recombinant allele and the other subjected to random truncations that made them unusable for further investigations.

Collectively, these results indicated that the ORC locus is resistant to homologous recombination in hiPS cells via Crispr/Cas9, underlining the importance and relevance of pre-RC complex proteins in maintaining human genome integrity.

Objective 2: To clarify different effects of mutant ORC and MCM on the molecular pathways of the licensing checkpoint in genetically modified hiPSCs.
In order to compensate for the negative results from objective 1, we used primary cells for ORC1 mutations, including mouse embryonic fibroblasts (MEFs) and mouse embryonic stem cells (mESCs), generated in Prof. Andrew Jackson's laboratory at University of Edinburgh. We used a combination of techniques to investigate the licensing checkpoint of these mutant lines. Flow cytometry, high-throughput microscopy and DNA combing, highlighted substantial differences in origin licensing (MCM2-7 loading) in MGS R105Q/R105Q and R105Q/del mutant MEFs. Moreover, origin firing and replication initiation at the beginning of S phase was also affected in R105Q/R105Q mutants when compared to controls. This suggests that MGS mutations can have observable effects on genome stability in fibroblasts.
Surprisingly, these differences were not found in mESCs. This suggested that ORC1 mutations on MGS have a bigger impact in somatic cells compared to pluripotent ones. This finding could explain some of the unexpected phenotypes seen in MGS.

Objective 3: To perform comparative analyses of these effects on origin licensing and licensing checkpoint activation in hiPS-derived clinically-relevant lineages (neural, cardiac, pulmonary) and mammalian cancer cell lines.
Differentiation of hiPSCs proved impossible due to failure of suitable mutant clones for ORC1. The absence of a phenotype also in mESCs and variability of differentiation protocols for this cellular model, prompted us to re-orientate our research toward the licensing difference between somatic primary cells as MEFs and IMR90 and mammalian cancer cell lines. Normal and cancer cells were treated with different inhibitors for CDK4/6, checkpoint kinases, and analysed by flow cytometry and western blotting. The results showed how in normal somatic cells, the licensing checkpoint is activated via CDT1 and CDC6 expression in a cell-type specific mechanism.

This supported the idea that CDT1 and CDC6 control licensing checkpoint in primary cells, but not in cancer lines where CDT1 and CDC6 expression appears to be constitutive.

Exploitation and Dissemination:
At present the results from this study have been presented at scientific conferences both locally and internationally. We are in the process of assembling these results into a manuscript that we will submit for publication in a scientific journal.
Collectively, this project has demonstrated that genetic manipulation via homologous recombination of ORC1 locus in hiPSCs is extremely difficult. The only viable mutants were truncated heterozygous for ORC1 R105Q, with no discernible phenotype from the wildtype. However, investigation of other primary mutant cells for MGS syndrome as mutant MEFs show significant origin licensing alterations and differential activation of the licensing checkpoint. This is an important step forward in our understanding of the licensing system in primary cellular models and for cell biology of mammalian development. The pharmacological impact is extensive as it suggests that the licensing system can be a valid target for more precise anticancer therapies as well as developmental syndromes. This has the potential to save considerable time, money and effort in the development of effective and accurate cell therapies.