CHROMATIN REMODELING DURING DEVELOPMENT AND AGING

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DEBATABLE PHYSIOLOGICAL LEVELS: TECHNICAL ISSUES FOR ANEUPLOIDY DETECTION

Many studies have investigated the karyotype of cells, organs and tissues and it was initially proposed that a certain level of aneuploidy could be tolerated in healthy tissues. Conflicting results emerged and the frequency of abnormal karyotypes in normal organisms remain under debate. It appeared that the variety of different technics used to map aneuploidy might be the issue.
Major organs of debate are brain and liver. Initial studies using karyotype analysis on metaphase spreads reported from 25 to 70% of aneuploid hepatocytes in the developing mouse liver (Faggioli et al., 2011a) and 30% in mouse neural stem cells (NSC) (Rehen et al., 2001; Yang et al., 2003; Kaushal et al., 2003). Fluorescent in situ hybridization analyses on interphase nuclei confirmed high levels of aneuploidy in hepatocytes (Faggioli et al., 2011a) and similar rates in post-mitotic neurons (Rehen et al., 2001; Kaushal et al., 2003) which were shown to participate to the adult neural circuit (Kingsbury et al., 2005). Same trends were observed in humans for both tissues using the same technics of chromosome labelling (Rehen et al., 2005; Pack et al., 2005; Yurov et al., 2007). However, the emergence of new technologies such as single-cell sequencing challenged these data and demonstrated that abnormal karyotypes are rare in normal mammalian tissues (Knouse et al., 2014; Douville et al., 2020). For example, the revisited rate of aneuploid neurons was reduced to < 5% in human brains (Knouse et al., 2014; Cai et al., 2014; van den Bos et al., 2016). Interestingly, low aneuploidy levels were also recently described in wild type (WT) Drosophila tissues (Gogendeau et al., 2015; Sabino et al., 2015; Resende et al., 2018).

Unprogrammed variation in genome content: a double-edged sword

Aneuploidy is infrequent in physiological context because chromosome imbalance affects cellular physiology and consequent organism homeostasis. When out of control, unprogrammed variations to the genome content are associated with pathological conditions such as cancer (Beroukhim et al., 2010), microcephaly (Marthiens et al., 2013) or miscarriage (Jia et al., 2015) and consequences differ whether ploidy variations occur during development or aging.

Ploidy alterations: growth defects and aging

More than 100 hundred years ago, in its dispermic experiment in sea urchin, Boveri linked abnormal karyotype with lethality. He observed that aneuploid embryos, originating from multipolar divisions of the polyploid zygote, presented strong developmental disorders and ultimately died (Boveri, 1902). Several decades later, Bridges made same conclusions in Drosophila. A Drosophila strain carrying an extra-copy of the small chromosome IV were viable but presented developmental defects, decrease body size and sterility (Bridges, 1921a; b). Constitutional aneuploidy, defined as a condition where all cells of the body are aneuploid, originates in gametes from errors in meiosis. In humans, the majority of trisomies and monosomies are embryonic lethal. One autosomal trisomy viable to adulthood is the trisomy of the chromosome 21, however patients present a Down syndrome, characterised by important developmental growth disorders and mental retardation (Lejeune et al., 1959).
Most trisomies are also lethal in mouse (Dyban and Baranov, 1987). Interestingly, somatic aneuploidy, by opposition, concerns a fraction of cells within the whole organism and differentially impacts tissue development and homeostasis. A rare condition, mosaic variegated aneuploidy (MVA) where nearly 25% of cells are aneuploidy, is associated with developmental delay and notably a microcephaly phenotype (Warburton et al., 1991). Consistently with the fact that the brain is susceptible to aneuploidy, induction of chromosome mis-segregation in mice causes drastic reduction in brain size due to loss of neural progenitor pool (Marthiens et al., 2013).

PLOIDY VARIATIONS IN AGING

Whether aneuploidy increases with age and whether this is associated with age-related pathologies are long-term debates. Several studies reported the accumulation of abnormal karyotypes in aging cells of mammalian tissues (Baker et al., 2013), such as in the brain (Iourov et al., 2009; Yurov et al., 2014, 2018; Faggioli et al., 2011b, 2012), liver (Duncan et al., 2010; Faggioli et al., 2011a; Duncan et al., 2012), blood lymphocytes (Jacobs et al., 1961) and oocytes (Jones, 2008). In the brain, for example, the increase of chromosome-specific aneuploidy was proposed to be at the origin of age-related neurodegeneration (Iourov et al., 2009; Shepherd et al., 2018) as in Alzheimer’s disease (AD) (Yurov et al., 2014) but this assumption remains controversial (van den Bos et al., 2016). Polyploidy is also believed to increase rate with age, as recently reported in the Drosophila (Nandakumar et al., 2020) and mammalian brain (López-Sánchez et al., 2017). Another striking example is the exponential increase of aneuploid oocytes with maternal aging in mouse and humans (reviewed in (Ma et al., 2020)). In addition to the potential increase of aneuploidy frequency with age, one can mention the opposite, namely aneuploidy as a source of aging. One of the best examples is MVA which is also associated with premature aging known as progeroid syndromes. This link was further confirmed in mice carrying a BUBR1 mutation- the most affected gene in MVA patients – which presented high levels of aneuploidy and consequent increase in senescence and aging (Sieben et al., 2020). Importantly, gene expression comparison between young and old mice revealed age-associated down-regulation of SAC and centromere proteins (Zahn et al., 2007; Andriani et al., 2017), such as BUBR1. Conversely, BUBR1 OE expands mice healthy lifespan (Baker et al., 2013).

The aneuploid and polyploid paradoxes

Initial studies on budding yeast were crucial for understanding of detrimental effects of aneuploidy on cell physiology. Torres and colleagues generated a collection of yeast strains carrying extra-copies of single chromosomes and showed that extra-chromosomes were actively transcribed and that overall chromosome gain negatively impacts cell growth and proliferation, mainly by extended G1 phase (Torres et al., 2007). Authors proposed that transcriptome changes perturb the protein imbalance causing proteotoxic stress that in turn, deregulates cellular processes and decreases cellular fitness (Torres et al., 2007, 2008). Several proteomic analyses in aneuploid yeast confirmed the dysregulation of the proteasome (Pavelka et al., 2010; Dephoure et al., 2014), such as formation of protein aggregates (Oromendia et al., 2012) as a source of toxicity, in addition to perturbations in redox homeostasis (Dephoure et al., 2014). Interestingly, aneuploidy in mammalian cells similarly affects cell metabolism at the transcriptome (Upender et al., 2004) and proteome level (Stingele et al., 2012; Tang et al., 2011), and reduces proliferation capacity (Williams et al., 2008). A comparative study in yeast, plant, mouse and human cells confirmed that aneuploidies of different chromosomes and in different organisms involve similar cellular pathways causing an anti-proliferative response (Sheltzer et al., 2012). Aneuploidy also interferes with cell survival. The increase production of reactive oxygen species mediated by metabolic and energetic stresses (Dephoure et al., 2014), activates the ATM kinase (Guo et al., 2010) which in turn triggers P53-dependent cycle arrest or apoptosis of aneuploid cells (Li et al., 2010). In agreement, oxidative stress causes P53-dependent senescence of aneuploid human mesenchymal stem cells (Estrada et al., 2013). Chromosome mis-segregation and resulting DNA damage also signals the ATM-P53 pathway activation and induces aneuploid cell death (Jeganathan et al., 2007; Thompson and Compton, 2010; Janssen et al., 2011). In Drosophila unlike mammalian cells, the elimination of aneuploid cells does not involve a p53- dependant mechanism and differs between tissue. In wing discs, aneuploidy is eliminated by epithelial cell delamination and p53-independent death, while aneuploid NBs prematurely differentiate into neurons (Dekanty et al., 2012; Gogendeau et al., 2015). However, current knowledge on the precise signaling pathways involved in aneuploidy elimination is limited.

Table of contents :

List of Abbreviations
Chapter 1 – Introduction
1. The cell cycle
1.1. Cell cycle phases and control
1.2. Mitosis in more detail
1.2.1. Mitotic phases
1.2.2. Spindle apparatus
CHROMOSOMES
TUBULIN AND MICROTUBULES
CENTROSOMES
1.2.3. Several pathways for MT-nucleation and chromosome capture
FROM CENTROSOMES – THE CENTROSOME PATHWAY
FROM CHROMOSOMES – THE CHROMATIN-MEDIATED PATHWAY (CMP)
FROM PRE-EXISTING MTS – THE AUGMIN PATHWAY
1.2.4. Models for chromosome capture and bi-orientation
1.2.5. Spindle assembly checkpoint and mitotic defects
1.3. Centrosome number alterations and consequences
1.3.1. Centrosome loss
1.3.2. Centrosome amplification
2. Variations in genome content
2.1. Physiological variations to the genome content: How, where and why?
2.1.2. Polyploidy
ROUTES TO POLYPLOIDY
POLYPLOIDIZATION FOR FUNCTION
2.1.1. Aneuploidy
ROUTES TO ANEUPLOIDY
DEBATABLE PHYSIOLOGICAL LEVELS: TECHNICAL ISSUES FOR ANEUPLOIDY DETECTION
2.2. Unprogrammed variation in genome content: a double-edged sword
2.2.1. Ploidy alterations: growth defects and aging
CONSEQUENCES OF CONSTITUTIONAL AND MOSAIC ANEUPLOIDY ON TISSUE DEVELOPMENT
PLOIDY VARIATIONS IN AGING
2.2.1. The aneuploid and polyploid paradoxes
THE ANEUPLOID PARADOX
THE POLYPLOID PARADOX
2.2.3. Whole genome duplication and aneuploidy : sources of CIN and GIN
3. Gene expression plasticity: differentiation and cell identity
– lessons from Drosophila
3.1. Regulation of gene expression and epigenetic marks
BRIEF INTRODUCTION TO DROSOPHILA GENOME
DIRECT REGULATION: PROMOTORS AND TRANSCRIPTION FACTORS
CHROMATIN STATE: EUCHROMATIN VERSUS HETEROCHROMATIN
3D ORGANIZATION: CHROMATIN FOLDING AND CHROMOSOME TERRITORIES
3.2. Epigenetic landscape establishment: from stem to differentiated state
3.2.1 The balance between proliferation and differentiation
3.2.2. Gene expression pattern establishment
SPATIAL AND TEMPORAL PATTERNING OF NEURONAL DIFFERENTIATION.
CHROMATIN REMODELING DURING DEVELOPMENT AND AGING
3.3. Cellular memory: Epigenetic stability versus plasticity
Chapter 2 – Results – Section A
Article – Goupil, Nano et al. 2020 – Journal of Cell Biology.
“Chromosomes function as a barrier to mitotic spindle bipolarity in polyploid
cells”
Abstract
Introduction
Results
Discussion ..
Materials and Methods
Supplemental material
Chapter 2 – Results – Section B
Article – Goupil et al. 2021 – preprint version on BioRxiv
“Drosophila neural stem cells show a unique dynamic pattern of gene
expression that is influenced by environmental factors”
Abstract
Introduction
Results
Discussion
Materials and Methods
FLY HUSBANDRY AND FLY STOCKS
GAL80 DROSOPHILA LINES ESTABLISHMENT
IMMUNOFLUORESCENCE OF DROSOPHILA LARVAL WHOLE MOUNT TISSUES
LIVE IMAGING OF DROSOPHILA LARVAL BRAINS
DNA FLUORESCENT IN SITU HYBRIDIZATION
RNA FLUORESCENT IN SITU HYBRIDIZATION
POLYMERASE CHAIN REACTION (PCR) OF DROSOPHILA LINES
Chapter 3 – Discussion and Perspectives
Multipolar divisions in polyploid cells: a problem of scaling?
Low frequency of chromosome loss in WT tissues: not happening or not tolerated? .
Genetic and epigenetic regulations: why is the brain so different?
Bibliography

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