Aneuploidy as a mechanism driving spontaneous loss of heterozygosity in Drosophila intestinal stem cells

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Loss of heterozygosity (LOH): a common cause of genome alteration in somatic cells

As the cells in our tissues are being renewed throughout life, our somatic cells should faithfully preserve the integrity of DNA. In the previous chapter, I highlighted that the integrity of DNA is under constant threat, leading to many possible genome alterations in somatic cells. I also mentioned that despite the threats, there are a number of mechanisms at the cellular- and tissue-level maintaining a degree of protection, especially for the stem cell. I would like to mention here that at the broader organismal level, a degree of protection is also provided through diploidy, where having two copies of each gene, protects against the effects of somatic mutation (Crow and Kimura 1965; Perrot et al. 1991; Otto and Goldstein 1992; Mable and Otto 2001). If a mutation arises in one copy, the second wild-type copy provides a backup, maintaining function. Thus, the heterozygous state masks the effects of recessive deleterious mutations, (with the notable exception of haploinsufficient genes). In this chapter, I explain the loss of the protective heterozygous state, which can lead to cancers, pathological disorders but also occurs in normal human tissues. In particular, I detail what is known about the mechanisms that can lead to the loss of heterozygosity (LOH). Importantly my PhD work aimed to elucidate some of how LOH occurs in adult stem cells.

Mitotic recombination-driven LOH

Mitotic recombination (MR), is homologous recombination (HR) that takes place during interphase of the mitotic cell cycle and not during mitosis, as the name suggests. It is defined as the homology-directed DNA exchange between sister or homologous chromosomes and comes into play to repair DNA double strand breaks (DSBs) using the intact chromosome as a template. The use of a template makes MR a high-fidelity DNA repair mechanism compared with alternative pathways of DSB repair such as NHEJ and alt-EJ, which introduce deletions along with the repair (explained in chapter 1.1). MR however, in spite of the more accurate repair, is clearly a double-edged sword. In cells heterozygous for a tumour suppressor gene (as mentioned above), MR corrects the DSB, but at the cost of recombining out the functional wild-type, thus leading to tumour suppressor gene inactivation establishing the very first steps of cancer.

Cancer initiation and mitotic recombination-driven LOH

In the particular case of adenomatous polyposis coli (APC) mutation carriers, patients develop “familial adenomatous polyposis” as the germline mutation in APC usually manifests in the appearance of polyps, which are small abnormal tissue growths on the surface of the colon. APC regulates β-catenin, a multifunctional protein that plays a vital role in cell-cell communication, growth and signaling. Although these polyps are benign, it is the second hit LOH of the remaining APC copy that facilitates the generation of polyps and initiates the cascade of events attributed to the multistep carcinogenesis of the colon, where the oncogene KRAS gets activated leading to further subsequent inactivations of other tumour suppressor genes such as P53. Studies from human cell lines derived from familial polyposis patient tumours identified that the LOH of APC occurs via MR (Cottrell et al. 1992; Haigis et al. 2002; Thiagalingam et al. 2001; Howarth et al. 2009). Mouse models been developed modeling intestinal cancers using (ApcMin/+) mice also show MR as a mechanism driving APC LOH (Haigis et al. 2002). Additional studies on human cell lines have also provided evidence of MR-driven LOH of other tumour suppressor genes such as retinoblastoma (Rb) (Cavenee et al. 1983), and neurofibromatosis NF1 (Serra et al. 2001), also reviewed in (Tuna et al. 2009; Lapunzina and Monk 2011; Siudeja and Bardin 2017). MR is therefore a frequent means of tumour suppressor gene inactivation, particularly in familial cancers where a germline mutation is pre-existing.

Restoration of the wild-type genotype through MR-driven LOH

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Interestingly, in the same way that MR can lead to the loss of the wild-type allele, it can also have a beneficial role in cases where a dominant mutant allele is lost instead, rescuing mutant phenotypes in heterozygotes. This phenomenon has been observed in Icthyosis (Choate et al. 2010), an autosomal dominant disease causing dry and scaly skin patches where the KRT10 dominant mutation is spontaneously inactivated via MR and the wild-type is restored. The MR-driven spontaneous elimination of the mutant KRT10, and the restoration of wild-type, gives rise to “revertant patches” leading to a natural form gene therapy. This phenomenon has been observed in other skin diseases too (Kiritsi et al. 2012; Jonkman et al. 1997). MR-mediated somatic reversion has also been well described in a metabolic disorder causing immunodeficiency (Hirschhorn et al. 1996) as well as blood disorders (Revy et al. 2019; Jongmans et al. 2012) including Diamond Blackfan Anemia (DBA) where case studies reveal disappearing features of anemia in patients as a result of MR-based somatic reversion to wild-type phenotypes (Jongmans et al. 2018; Venugopal et al. 2017) and a subsequent clonal expansion of the revertant cell, which lost its dominant mutation through MR.

Table of contents :

Table of Figures
Abbreviations
Chapter 1 : Introduction
1.1 DNA damage and mutation in stem and progenitor cells in the context of aging and cancer
Stem cells and tissue dynamics
DNA damage and how it leads to mutation
Mechanisms protecting the stem cell and tissue from the effects of DNA damage
When protection mechanisms fail: acquisition of mutation
DNA damage and somatic mutation in adult tissues: roles in cancer initiation and aging
Contributions of persistent DNA damage to stem cell decline
Towards an understanding of DNA damage and mutation in adult tissues
Concluding remarks
1.2 Loss of heterozygosity (LOH): a common cause of genome alteration in somatic cells
Mitotic recombination-driven LOH
DSBs: Drivers of MR
Yeast: a paradigm for studying mechanisms of MR
Another cause of LOH: Aneuploidy
Concluding remarks
1.3 The Drosophila intestine: A model to study genome alterations such as LOH in stem cells
A dynamic tissue in a powerful in vivo model
Structure of the Drosophila intestine
The role of Notch in regulating cell fate
The aging gut
Impact of the environment on the Drosophila midgut
Advantages of using the Drosophila midgut as a model system to study genome instability in adult stem cells
Chapter 2 : Results
Results Overview
2.1 Mitotic Recombination as a Mechanism Driving Spontaneous Loss of Heterozygosity in Drosophila Intestinal Stem Cells (article in preparation).
Abstract
Introduction
Results
Spontaneous loss of heterozygosity increases with age
Whole genome sequencing to determine the mechanism of LOH
LOH arises through mitotic recombination in both males and females.
LOH through mitotic recombination also happens on other chromosome arms
Rad51 promotes loss of heterozygosity
Whole-genome sequencing data supports cross over via a double-Holliday structure
Mapping of LOH initiation regions provides insight into potential sequence drivers of MR…. 105
Infection with the pathogenic enteric bacteria Ecc15 increases loss of heterozygosity
Discussion
2.2 Aneuploidy as a mechanism driving spontaneous loss of heterozygosity in Drosophila intestinal stem cells
Context
Results
Sequencing evidence for aneuploidy-driven LOH
The H4K16ac histone mark is a good readout for activation of dosage compensation in the Drosophila intestine
Loss of X (aneuploidy) is detected in aging N55E11/+ females
Discussion
Chapter 3: Discussion
Discussion Overview
3.1 Technical evaluation of the work and experimental caveats
Sample size of sequenced tumours
Tumour purity
Controversy surrounding R-loops
Additional biological repeats and RNAi lines
3.2 Discussion of results
Exploiting the clonal nature of LOH neoplasia in the Drosophila intestine: the novelty
Mechanisms of LOH
Genomic Drivers of MR
Impact of the stem cell niche and environment on LOH
LOH with age
3.3 Implications of the research and conclusions
Implications of the research and perspectives
Conclusions
Materials and Methods
Bibliography

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