Special Report

The epigenetic landscape of aneuploidy: constitutional mosaicism leading the way?

The role of structural genetic changes in human disease has received substantial attention in recent decades, but surprisingly little is known about numerical chromosomal abnormalities, even though they have been recognized since the days of Boveri as partaking in different cellular pathophysiological processes such as cancer and genomic disorders. The current knowledge of the genetic and epigenetic consequences of aneuploidy is reviewed herein, with a special focus on using mosaic genetic syndromes to study the DNA methylation footprints and expressional effects associated with whole-chromosomal gains. Recent progress in understanding the debated role of aneuploidy as a driver or passenger in malignant transformation, as well as how the cell responds to and regulates excess genetic material in experimental settings, is also discussed in detail.

Josef Davidsson Division of Molecular Medicine & Gene Therapy, Lund Stem Cell Center, Lund University, SE-221 84 Lund, Sweden [email protected] med.lu.se

Keywords:  aneuploidy • cancer • epigenetics • hydroxymethylation • imprinting • methylation • trisomy

Mosaic aneuploidy influence genetic & epigenetic variation Imbalances in the relative quantity of geneproducts as a result of a rearrangement of the human genome, rather than from DNA base alterations, have long been known to be the cause of such emblematic genomic disorders as the Down syndrome (DS) and the Turner and Klinefelter sex chromosome syndromes [1] . If an aberrant genomic rearrangement occurs in the germline, but postzygotically, only a subset of the affected individuals adult cells will be propagated with the change – creating a bona fide constitutional genetic mosaic. The aberrations involved in creating coexistence of cells with different genetic composition do include both point mutations and small rearrangements, however, the most commonly observed form of somatic mosaicism is an abnormal number of chromosomes – aneuploidy. In fact, it has been widely reported that aging women tend to lose their active or, more commonly, inactive X chromosome (Xi) in a lineage-restricted fashion [2,3] , possibly due to a propensity of X chromosomal anaphase

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lagging [4] . Pre- and peri-natal whole-chromosome mosaicism can be found in approximately 50% of preimplantation embryos, 1% of chorionic villous samples, 0.2–0.3% of amniotic fluids and 0.1% of neonates [5,6] . In adult individuals the frequency of autosomal large chromosomal abnormalities (duplication, deletions and uniparental isodisomies) have been measured around 0.5% until 50 years of age, then rapidly rising to approximately 2% in the elderly [7,8] . Until recently, with the exception of a handful of well-defined syndromes, conditions with congenital gains or losses of entire chromosomes have neither as complete constitutional aneuploids nor as genetic mosaics been regarded as compatible with a viable phenotype in humans. However, with the recent detection of acquired mosaic cellular aneuploidy present in normal adults at a high frequency calls for a revised view in which somatic mosaicism could be considered more as a contributor to inter- and intra-individual genetic variation than solely a pathogenetic event. Notwithstanding this view, clonal

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Special Report  Davidsson mosaicism for large chromosomal anomalies – both acquired and constitutional – most likely in many instances contribute to time-dependent somatic events such as cancer and other late-onset disorders [7–9] . Aneuploidy & cancer Genetic instability has long been regarded as an enabling characteristic in reaching the classic hallmarks of cancer [10] , and it is most commonly manifested by the acquisition of structural and/or numerical chromosomal aberrations in the neoplastic cell. Structural chromosomal changes deregulating oncogenes or eliminating tumor suppressor genes have been studied in abundance but, to date, surprisingly little is known about the oncogenic role of numerical abnormalities [11] – the overall most common acquired genetic aberration observed in neoplasia [12] . In the literature, cytogenetically defined aneuploidy caused by chromosomal instability (CIN) in cancer, resulting in a high rate of gain and/or loss of chromosomes, is not always distinguished from aneuploidy in cells displaying uniform karyotypes over time. This might be unfortunate, since far from all aneuploid cells are actually a result of CIN – not even in neoplasia [13] . For example, acquired trisomy 8 is one of the most common, and occasionally the sole, recurrent karyotypic findings in myeloid malignant conditions, Ewing’s sarcomas and desmoid tumors [14] . The causative role of aneuploidy as not merely a passenger mutation caused by genetic instability, but as a true contributor to the process of neoplastic transformation has been a long-standing debate [15] . One possibility is that aneuploidy simply is indirect damage caused by the process of malignant transformation, and that the different recurrent aneuploidies are the ones tolerated in the context of continued cell growth and division. This hypothesis has been strengthened by murine experimental systems, wherein aneuploidy generally inhibited tumorigenesis by being associated with decreased proliferation and where oncogenesis only occurred in conjunction with gains of specific chromosomes [16,17] . The alternate view, although not mutually excluding or contrasting the passenger theory, is that aneuploidy is not always an artifact, but can act as a true driver of neoplasia if it occurs under specific cellular circumstances. There, it conveys a fitness advantage and may be positively selected for in the process of tumor evolution [13] . Supporting such a view is the fact that there is a well-known association between the predisposition to malignant disease and constitutional aneuploidy [18] . Regardless of the outcome of this issue, currently very little is known about the genetic effects and the epigenomic consequences of gains or losses of whole chromosomes. Thus, although rare disorders, the

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constitutional aneuploidy syndromes (Table 1) offer a unique opportunity not only to study neoplastic evolution in situ, but also to investigate the direct functional outcome of chromosomal imbalances on epigenetic regulation and gene expression. Lessons learned from DS DS, caused by either whole or partial trisomy 21, is associated with a plethora of phenotypes including mental retardation, heart defects and early-onset Alzheimer’s disease [20] . Occurring in approximately one of 700 live births, DS and the sex chromosome syndromes constitutes the most common aneuploidies found in humans today (Table 1) [21] . Being such a common genomic disorder, DS and gain of chromosome 21 (chr21) has served as the prototype chromosomal abnormality when studying aneuploidy and copy-number variation in humans for several decades. But still, the majority of genes responsible for most DS phenotypes remain unknown. However, with the advent of transgenic and transchromosomal mouse models, as well as high throughput techniques of mapping gene expression, some progress have been made. Furthermore, in DS it has been demonstrated that not all genes on chr21 are dosage sensitive. Only a subset of alleles are overexpressed in DS with complete trisomy 21 and this aneuploidy affect also other chromosomes, showing consistently altered expression of non-chr21 genes [22–25] . In addition, altered gene-specific DNAmethylation on a wide variety of other autosomes than chr21, is a recurring event associated with trisomy 21 in DS [26] and these epigenetic changes occur early in development [27] . Recently, it was demonstrated in a landmark study that the gene dose imbalance in DS embryonic stem cells could be corrected by insertion of an inducible XIST transgene into one of the three chr21 copies [28] , inducing stable heterochromatin modifications and chromosome-wide transcriptional silencing of the recipient chromosome. Interestingly, the study also investigated clones carrying XIST on two or all three copies of chr21 and found that after 20 days of induction culturing, the cells lost localization or expression of the transgene and the transcriptional repression of the targeted chromosome was no longer present. This suggests epigenetic adaptation to avoid functional mono- or nulli-somy followed by in vitro clonal selection. These findings are consistent with the fact that autosomal monosomic clones, compared to autosomal trisomic ones, rarely create genetic mosaicism in humans [7,8] . Using constitutional genetic mosaics to model partial & complete aneuploidy Gene expression patterns in patients with constitutional genomic disorders have, until recently, exclusively been

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The epigenetic landscape of aneuploidy: constitutional mosaicism leading the way? 

Special Report

Table 1. Human genomic disorders displaying constitutional chromosomal mosaicism. Chromosome

Type

Name

Prevalence

OMIM

Orpha number

8

Trisomy

CT8M





96061

8p

Isochromosome









9

Trisomy







99776

9p

Isochromosome







3310

12p

Isochromosome

Pallister– Killian syndrome

95% cells in culture)

Fluorescent light Detector

Hematopoietic cells in suspension culture

Aneuploidy (>95% cells in culture)

Diploid culture Expansion stage DNA + RNA Adherent cells

Cell counting and dilution

24 wells = 1 cell/well

6 wells Aneuploid culture

96 wells

Figure 1. Schematic overview of single-cell cloning of constitutionally aneuploid mosaic cells. Hematopoietic cells can be cultured in suspension and seeded as one cell per well using simple FACS methods. Adherent cells can simply be counted and diluted so that approximately one cell can be plated per well in 96-well microtiter plates. Viable cell cultures are used for further passages at a ratio of 1:1 to 24-well and subsequently 6-well microtiter plates. Cultures are then resuspended, after which a fraction from each are used for interphase FISH analysis with probes hybridizing to the chromosome of interest. At least a minimum of 95% of normal or abnormal nuclei should be used as a cut-off to designate a culture as being either diploid or aneuploid. The selected cultures can then be transferred to cell culture flasks for RNA and DNA extraction at selected time points.

through a mitotic error (Figure 2C) . Trisomic chromosomes originating from a mitotic event theoretically are expected to have the same percentage of heterozygosity as non-gained autosomes. Notably though, a higher calling rate when analyzing SNPs and/or microsatellite markers causes the level of heterozygosity to appear significantly lower for a chromosome gained by such an error. CT8M displays reduction to homozygosity and, in addition, demonstrates no preferential parental origin of the gained chromosome 8 (chr8) [36–40] . However, a meiotic II error (Figure 2B) cannot be totally excluded using heterozygosity data alone, not until the recombination frequency have been measured in a larger series of CT8M patient-parent pairs. This since a reduced recombination rate has been reported to be associated with meiosis II nondisjunction events [41] . The consequences logically attributed to CT8M and to other partial and total aneuploids, are direct gene dosage effects. This likely causes deregulation of proteins that in turn may influence the transcription of key genes situated on the gained, as well as on other, chromosomes [42] . Before our study, no genome-wide gene expression analyses of CT8M cases had been performed; however, it had been demonstrated that acute myeloid leukemia (AML) cells with acquired trisomy 8 overexpress most genes on this chromosome

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[43–45] .

As mentioned previously, acquired trisomy 8 is one of the most common cytogenetic finding in myeloid malignant conditions: such as AML, myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) [14] . CT8M is clearly associated with an increased risk of developing one of these hematologic disorders, as demonstrated by a plethora of studies describing AML, MDS or MPN in patients with this syndrome [39,46–60] . Maserati et al. even proposed that the apparent ‘acquired’ trisomy 8 so frequently found in myeloid malignancies instead could represent an unrecognized CT8M in a not insignificant proportion of cases. However, this study was performed on a small series of patients, and therefore needs to be verified or rebutted by larger cohorts [61] . Expressional effects of trisomes are not confined to gained chromosomes Utilizing the abovementioned single cell cloning approach on cells derived from a CT8M patient, we compared the global gene expression signature of trisomic fibroblasts to the their disomic counterparts. Not surprisingly, the former displayed a characteristic global gene expression pattern readily separable from the latter when performing different hierarchical clustering analyses [32] . Notably, the majority of chr8

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The epigenetic landscape of aneuploidy: constitutional mosaicism leading the way? 

genes in the constitutional trisomy 8 cells were overexpressed, like previous findings in AML cells harboring acquired trisomy 8. However, it should be stressed that not all differentially expressed genes were located on this chromosome. This is similar to what has been

Special Report

reported in DS, where the deregulation of genes wasn’t confined to the gained chromosome as such. So a trisomy, constitutional or acquired, thus affects genes situated on chromosomes other than the gained one. A cooperation between a direct gene dosage effect, as Maternal chromatid

Meiosis I

Paternal chromatid Partner chromatid

Meiosis II

Meiotic II nondisjunction

Gametes n+1

n+1

n-1

n-1

n+1

n-1

n

n

Zygotes

×2

×2

2n + 1

×2

2n + 1

2n - 1

2n - 1

2n

DNA replication

Mitosis

Mitotic nondisjunction Cell proliferation

2n + 1

2n - 1

Figure 2. The nondisjunctional origin of trisomic chromosomes in human cells. (A) The origin of a trisomy from a meiosis I error. This will create an n + 1 gamete with one chromatid from each parent, increasing the heterozygosity in the postzygotic trisomic cells as compared with other autosomes. (B) A meiosis II error will create an n + 1 gamete with two chromatids from either of the parents. (C) A postzygotic mitotic error creating a trisomic n + 1 gamete with two chromatids from either of the parents. The trisomic origin from either a meiosis II error or a post-zygotic mitotic event can thus not be determined by heterozygosity data alone.

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Special Report  Davidsson well as a more global perturbation of gene expression, are thus likely factors in creating the pathophenotypes associated with CT8M and other genomic disorders that carry large chromosomal imbalances. Regarding the elevated risk for AML, MDS and MPN seen in CT8M carriers, pathway analyses of the differentially expressed genes cells revealed that ‘cancer’ as well as ‘hematological system development and function’ and ‘hematopoiesis’ were top ranked features in our study [32] . This aids the understanding of the increased incidence of myeloid neoplasms seen in patients with this syndrome. However, based on current evidence, acquired trisomy 8 seems to be an early and important event, but not by itself sufficient to generate leukemogenesis in myeloid neoplasms [62] . A constitutional trisomy 8 could therefore represent an inherent necessary ‘hit’ in a stochastic process that may lead to malignancy, instead of being a directly initiating event. This argument is consistent with the fact that not all CT8M individuals develop cancer, although no formal estimate of the actual incidence yet exists. Epigenetic hallmarks of aneuploidy DNA methylation

Already in the mid-1970s, researchers suggested that methylation of cytosine in CpG dinucleotides, creating a 5-methylcytosine (5mC), might be responsible for the stable maintenance of gene expression patterns through several mitotic cell divisions [63,64] . This DNA methylation was later demonstrated to be preferentially situated at genomic regions aptly named CpG islands, since they were motifs scattered around the human genome as isolated atolls, unusually rich in CpG dinucleotides [65] . A CpG island is commonly defined as a region consisting of at least 200 bp, with a C+G nucleotide content >50% and often with an observed/expected CpG ratio >0.6 [66] . These motifs are frequently located within, or in close proximity to, gene promoters [67] . When a promoter is methylated, the gene becomes silenced, whereas the opposite is true when it is demethylated. DNA methylation is believed to impact the transcription of genes in two ways: first, the chemical modification may itself physically hinder the binding of transcription factors to DNA; and second, methylated DNA may be preferentially bound to by proteins that recruit repressive complexes to the locus (e.g., histone deacetylases, inducing a closed chromatin structure) [68–70] . However, the straightforward paradigm of gene silencing and promoter hypermethylation far from always hold true, since there are many examples where promoter methylation have been associated with gene expression [71] . In fact, it has been reported that most methylation associated with tran-

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scriptional downregulation occur not in promoters, but in up to 2 kb distant non-CpG island sequences, named ‘CpG island shores’ [72] . Previously, when studying global methylation signatures in high hyperdiploid acute lymphoblastic leukemia (ALL), we demonstrated that the majority of tri- and tetra-somic chromosomes in this ALL subtype were significantly less methylated in gene-poor regions compared to their disomic autosomal counterparts [73] . Presaging these findings, trisomy 7 and 14 in colon cancer had also been reported to exhibit this pattern of hypomethylation [74] . Logically, an intriguing question following these observations was if hypomethylation could be a general epigenetic phenomenon associated with gains of chromosomes, irrespective of whether constitutional or acquired. This was an incentive to investigate global large-scale methylation patterns using our single cell-derived trisomy 8-positive and -negative cells. When investigating the 5mC content of individual CpG islands and promoters we couldn’t detect any elevated occurrence of chr8 genespecific hypermethylation in the trisomy 8 cells. However, comparing the methylation content of gene-poor regions in the trisomy 8 cells to that of the disomic ones, the former did in fact display a general depletion of 5mC on chr8, but not on other autosomes [32] . This suggests that a more general association between aneuploidy and globally lowered levels of 5mC of gene-poor regions on both constitutional and acquired gained chromosomes could be a plausible epigenetic hallmark for all types of numerical chromosomal changes. DNA hydroxymethylation

5mC has illustriously been called the ‘fifth base’, due to its role as a dynamic regulator of gene expression. Quite recently a ‘sixth base’ was implicated, when it was demonstrated that a substantial amount of 5-hydroxymethylcytosine (5hmC), previously only detected in bacteriophages [75] and spuriously in mammalian tissue [76] , was unambiguously present in murine and human DNA. Approximately 5% of all CpG cytosines present at MspI and TaqαI restriction sites in embryonic stem cell DNA and approximately 20% of all CpG cytosines in Purkinje cell DNA consists of 5hmC [77,78] . Simultaneously it was demonstrated that the TET gene family was responsible for the oxidation of 5mC to 5hmC [78 ,79] using α-ketoglutarate, a cofactor produced by the IDH gene family [80] . Although the exact function for 5hmC is not yet known, several different and possibly cooperating scenarios are possible. The first one is that hydroxymethylation facilitates passive DNA demethylation by interfering with the maintenance methylation performed by DNMT1, since this protein doesn’t recognize the 5hmC base [81] . The second sce-

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The epigenetic landscape of aneuploidy: constitutional mosaicism leading the way? 

nario also involves passive demethylation, since 5mCbinding proteins such as MBD1, MBD2 and MBD4 are unable to ligate to 5hmC, hydroxymethylation thus abrogating the function of these proteins on methylated DNA [82] . However, it has been demonstrated that 5hmC is able to bind to MECP2, a methyl-CpG-binding protein abundantly expressed in the brain. Interestingly, MECP2 is mutated in the neurological disorder Retts syndrome, where the R133C mutation preferentially inhibits 5hmC binding [83] , implicating that cell type-specific expression of different 5hmC-binding proteins could be responsible for the marked difference of hydroxymethylation levels between disparate tissues [84] . The last putative function of hydroxymethylation implies that 5hmC actively recruit specific chromatinmodifying proteins or macromolecular complexes that alter DNA configuration status as well as regional gross methylation levels through deamination and/or stepwise oxidation. To date, however, the functions included in an active deamination pathway remain to be fleshed out and no protein specifically binding to 5hmC alone has yet been found in vivo [85] . This argues against a current status of 5hmC as an actual epigenetic mark in its own right. Whether the only cellular role of 5hmC is as a passive intermediate or not, the modification is most commonly detected in the bodies of actively transcribed genes [86] , affecting both cellular lineage commitment as well as tissue-specific gene expression [87,88] . In fact, the most important predictor of 5hmC content in a cell is the tissue type from which it is derived [84] . Moreover, there is a negative correlation between proliferating cells and hydroxymethylation [89] and, logically, the levels of 5hmC decrease rapidly over time in in vitro cultured cells, creating huge interpretation difficulties when using nonmatched samples. However, this caveat is easily circumvented by using our single cell cloning approach and thus comparing identical cell types with identical rates of proliferation. Global loss of 5hmC compared to surrounding tissue is seen in an array of human malignancies [85,89] . In addition, reintroduction of TET2 and IDH2 in murine experimental systems have demonstrated restored 5hmC levels and decreased metastatic potential of melanoma cells, making gross loss of hydroxymethylation a functionally validated epigenetic hallmark of malignant transformation [90] . Mutually exclusive mutations in TET and IDH genes are frequent in myeloid neoplasms, in fact TET2 is the most commonly mutated gene detected in MDS overall [91] . Linking this to the CT8M syndrome where the patients have an increased risk for developing AML, MDS or MPN is purely speculative, but the finding of decreased gene promoter/CpG island 5hmC content on chr8 in the tri-

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Special Report

somy 8 cells is intriguing. Is this finding related to the hypomethylation of the gained chromosome by means of passive demethylation, thus depleting it of 5hmC? Or is it an indicative of local hydroxymethylation dysregulation, similar to the effects caused by TET and IDH gene disruption in myeloid malignancies? The epigenetic context and the cellular role of the decreased levels of 5hmC on the X chromosome remain to be studied further. Interestingly, when comparing the 5hmC levels of chr8 in the trisomic cells to those in the disomic ones in CT8M, we detected a widespread hypohydroxymethylation on this chromosome [32] . This can be compared to the Xi that, unlike its active counterpart, exhibits hypomethylation of gene-poor regions [32 ,73,74] and hypermethylation of a substantial proportion of promoters, where the latter is considered to be a part of the sex chromosome dosage compensation machinery [92–94] . In agreement with a previous study [95] , we observed a general depletion of 5hmC on the X chromosome compared to all autosomes in both males and females [32] . Thus, the gained chromosome in CT8M partly mimics the epigenomic signature of Xi regarding methylation of gene-poor regions and hydroxymethylation of promoter regions, but with the exception that no elevated level of promoter/CpG island 5mC content on chr8 was detected. A possible explanation of the observed overexpression of chr8 genes in the trisomy 8 cells could thus be that dosage compensation is incomplete or absent for the majority of genes on the gained chromosome in CT8M. The occurrence of 5hmC as another component in the intricate web of epigenetic regulators is a major breakthrough in epigenetics, and a more lucid picture of its exact function remains to be described. Aneuploidy & genomic imprinting Genomic imprinting refers to the selective methylation of maternal and paternal genomes during gametogenesis, so that a parent-of-origin-specific expression of certain genes occurs in the organism. Established as key player in mammalian embryogenesis in the 1980s [96 ,97] , imprinting defects were soon linked to the presupposed concept of uniparental isodisomy (UPD), (i.e., having the same parental origin for one or several pairs of chromosomes) [98] . The sister disorders, Prader–Willi syndrome (PWS) and Angelman syndrome (AS), clearly illustrate the connection between imprinting and UPD. These conditions are commonly caused by deletions of paternal (in PWS) and maternal (in AS) gene copies in the 15q11–13 region. This induces transcriptional downregulation of critical loci, since the remaining alleles are imprinted. However, both of these syndromes can also be created by

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Special Report  Davidsson chromosome 15-specific maternal UPD in PWS and chromosome 15-specific paternal UPD in AS [99] , creating homozygosity of imprinted genes. Relating UPD to aneuploidy, a gain or loss of an entire chromosome may not only induce pathogenesis by gross copy number change, but also have the potential to perturb and dysregulate the finely tuned selective monoallelic expression of imprinted genes. Loss or gain of genomic imprinting by aneuploidy has mainly received attention in neoplasia; it has been suggested to influence the development of pediatric leukemia. It was proposed that a high number of gains or losses of preferentially maternally or paternally derived chromosomes could affect the pathogenesis of highhyperdiploid and near-haploid ALL [100] . However, when polymorphic microsatellite markers were analyzed on gained chromosomes in high-hyperdiploid ALL, no significant skewed parental origin was detected, indicating that chromosome-wide loss of imprinting is not common in this leukemic subtype [101,102] . It has been reported that the frequent deletions of chromosome arm 9p seen in childhood ALL, lead to a preferential loss of maternal alleles [103,104] . Any germline event leading to paternal UPD on 9p could thus lead to homozygosity and subsequent downregulation of, for example, a paternally imprinted tumor suppressor gene promoting leukemogenesis. However, these initial results have not been confirmed in any subsequent study, and large-scale genomic studies of childhood ALL have not identified constitutional UPD to be a particularly frequent event on chromosome 9p [105] . Cellular adaptation to aneuploidy An interesting hypothesis fronting epigenetics as a game changer into the discussion of the potential driving role of aneuploidy in tumorigenesis, is that epigenomic dysregulation could promote rapid survival selection of malignant cells. For example, gain of a chromosome may introduce a global epigenetic effect as a cellular response to overcome the detrimental effects of aneuploidy. This may indirectly facilitate the process of neoplastic adaptive evolution by producing a fitness advantage only after a secondary and required genetic event has occurred [106] . But how do aneuploid cells respond to the altered transcriptional and proteomic levels [42] in a broader context? Besides minimizing the effects of the copy number alteration through dosage compensation achieved by epigenetic and post-translational modifications [42] , the possibility of a transcriptional stress response as an adaptation to aneuploidy have been suggested. In fact, experimentally generated aneuploid yeast strains share a common gene expression signature, named environmental stress response (ESR) that includes increased expression

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of genes related to ribosomal biogenesis and nucleic acid metabolism [107] . Since this signature was originally described for euploid yeast grown under stressful conditions and at slow growth rates [108] , this could suggest that aneuploid yeast cells might have the ability to sense the detrimental effects of gain of chromosomes and initiate ESR as a response to this. However, contradicting this, another study on aneuploid yeast only detected ESR using highly stringent analysis criteria and failed to associate it with both growth rate and number of gained chromosomes [109] . In addition, both the aforementioned studies reported gene expression levels that were proportional to the gene dosage increase, but when correlating this to protein abundance their results again differed. Pavelka et al. found minimal dosage compensation of the core complex proteins analyzed [109] , whereas Torres et al. described dosage compensation through degradation in 13 out of 16 multiprotein complexes investigated [107] . It should be noted, however, that the methods used for strain construction and selection, as well as growth and data analysis, were highly divergent in these studies, making a direct comparison difficult and thus warranting further investigations to clarify these issues. Regarding how aneuploid cells handle and regulate the excess chromatin with respect to nuclear architecture, chromosomal crosstalk and higher-order chromatin arrangements is still essentially dark matter in the field of epigenetics [110,111] . Interestingly, the DNA hypomethylation pattern that we and others detected in both constitutionally and acquired gained chromosomes, seems to be restricted to genomic regions harboring no or few genes [32 ,73,74] . The hypomethylation could hence be unrelated to direct gene regulatory mechanisms and instead be connected to chromatin regulation, since it has previously been reported that absence of DNA 5mC influences chromatin compartmentalization by increasing the clustering of pericentric heterochromatin to the nucleus [112] . Furthermore, when Hansen et al. compared high-resolution methylomes of human colorectal cancer to normal matched mucosa cells they discovered discrete blocks of hypomethylation. These regions displayed extreme gene expression variability and covered more than half of the genome [113] . These hypomethylated blocks corresponded to large organized chromatin K9 modifications (LOCKs) and lamina-associated domains (LADs) [113,114] , heterochromatic regions enriched for post-translational modifications (e.g., histone modifications) and associated with proteins of the nuclear lamina, respectively. LOCKs expand during cellular differentiation and are lost in cancer [115] , whereas genes situated in LADs are typically transcriptionally repressed [116] . It is thus possible that the detected hypomethylation of specific regions on the tri-/tetra-

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The epigenetic landscape of aneuploidy: constitutional mosaicism leading the way? 

somic chromosomes directly induces, or is a footprint of, chromatin remodeling and nuclear positioning that enables a cellular adaptation to excess genetic material. However, any direct effect of aneuploidy concerning LOCK and LAD regions remains to be investigated.

Special Report

Conclusion & future perspective In this review the vantages of using a mosaic genetic syndrome to study the mechanisms associated with aneuploidy have been explored in detail. Using a patient’s own diploid cells as a control effectively cir-

Executive summary Mosaic aneuploidy influences genetic & epigenetic variation • If an aberrant genomic rearrangement occurs in the germline, but postzygotically, only a subset of cells will be propagated with the change – creating a constitutional somatic mosaic. • The most common cause of somatic mosaicism is aneuploidy. • Recent detection of acquired mosaic cellular aneuploidy at a high frequency in normal adults indicates that somatic mosaicism could contribute to inter- and intra-individual genetic variation.

Aneuploidy & cancer • The role of aneuploidy either as a passenger mutation or as a driver in neoplasia is currently debated. • In murine experimental systems aneuploidy generally inhibits tumorigenesis and malignant transformation can only occur in conjunction with gains of certain specific chromosomes. • The constitutional aneuploidy syndromes offer a unique opportunity to study neoplastic evolution and the functional outcome of chromosomal imbalances both on a direct gene expressional and on an epigenetic regulatory level.

Lessons learned from Down syndrome • The majority of genes responsible for most Down syndrome phenotypes remain unknown. • Not all genes on chromosome 21 are dosage sensitive, with only a subset overexpressed in Down syndrome. • Trisomy 21 induces changes in expression and DNA methylation of nonchromosome 21 genes. • Silencing of two or all three copies of chromosome 21 in Down syndrome experimental systems leads to epigenetic adaptation to avoid functional mono- or nulli-somy, followed by in vitro selection of clones with disomic expression.

Using constitutional genetic mosaics to model partial & complete aneuploidy • Using single cell cloning of cells from an individual with a constitutional mosaicism enables unbiased analysis of both gene expressional and epigenetic consequences associated with gain of the specific chromosome.

Constitutional trisomy 8 mosaicism syndrome • Constitutional trisomy 8 mosaicism syndrome (CT8M) is a well-defined syndrome with a high phenotypic variability and most likely arises through a postzygotic mitotic error. • Acquired trisomy 8 is one of the most common cytogenetic findings in myeloid malignancies, and CT8M is associated with an increased risk of developing such disorders.

Expressional effects of trisomies are not confined to gained chromosomes • Constitutional as well as acquired trisomies are associated with a direct gene dosage effect, but also affect genes situated on other chromosomes than the gained one. • The gene dosage effects together with a global epigenetic dysregulation are likely factors in creating the pathophenotypes associated with disorders carrying large chromosomal imbalances.

Epigenetic hallmarks of aneuploidy: DNA methylation • The paradigm of gene silencing and promoter hypermethylation does not always hold true, since there are many examples where promoter methylation have been associated with gene expression. • An association between aneuploidy and lowered levels of 5mC in gene-poor regions could be a plausible epigenetic hallmark for all types of numerical chromosomal changes.

Epigenetic hallmarks of aneuploidy: DNA hydroxymethylation • The most important predictor of hydroxymethylation content in a cell is the tissue type from which it is derived from. 5hmC decrease rapidly over time in cultured cells, creating interpretation difficulties when using nonmatched samples. • Global loss of 5hmC is associated with human malignancies. • Decreased levels of 5hmC are found on the X chromosome compared to autosomes. • In CT8M decreased 5hmC content were found on chromosome 8 in the trisomy 8 cells. • The gained chromosome in CT8M mimics the epigenetic signature of the inactivated X chromosome with hypomethylation of gene-poor regions and hypohydroxymethylation of promoter regions.

Aneuploidy & genomic imprinting • Genomic imprinting refers to the selective methylation of maternal and paternal genomes during gametogenesis, so that a parent-of-origin monoallelic expression of certain genes occurs in the organism. • Imprinting imbalances are linked to uniparental isodisomy – having the same parental origin for one or several pairs of chromosomes. • Loss or gain of genomic imprinting through aneuploidy, creating selective uniparental mono- or tri-somies, has received attention mainly in neoplasia.

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Executive summary (cont.). Cellular adaptation to aneuploidy • Aneuploid yeast strains share a common stress-associated gene expression signature, suggesting that the cells might have the ability to sense the detrimental effects of aneuploidy. • The handling of excess chromatin in the context of nuclear architecture, chromosomal crosstalk and higher-order chromatin arrangements is currently unknown.

Conclusion & future perspective • Mosaic genetic syndromes are the best model systems available today to investigate the genetics and epigenetics of constitutional aneuploidy. • Dysregulated epigenetic control mechanism must be taken in account when analyzing chromosomal imbalances in the context of genomic disorders and cancer. • Current knowledge about the cause and consequences of aneuploidy remains to be expanded, especially from an epigenetic standpoint. • The role of aneuploidy as a driver or passenger in neoplasia should also be explored further and the various components of cellular adaptation and regulation of excess chromatin remain to be defined.

cumvents some of the interpretation difficulties introduced when comparing aneuploid cells with unrelated controls. Thus, mosaic genetic syndromes must be considered the best-suited model systems today for investigating the epigenomics of constitutional aneuploidy. The current knowledge about the cause and consequences of aneuploidy remains to be expanded, especially from an epigenetic standpoint. However, it can be concluded that the transcriptional effects of aneuploidy also involve noncopy-number altered genes (i.e., genes situated on chromosomes that are not gained or lost). The take-home message is thus that gene–gene interactions and epigenetic gene regulatory events are most likely stimulated by large-scale copy number gains, but not confined to the gained chromosome as such. The pathogenetic effects of aneuploidy can therefore not solely be considered in the light of the gene dosage imbalance theory; also, the dysregulated epigenetic control mechanism must be taken in account when analyzing chromosomal imbalances in genomic disorders and cancer. Interestingly, both constitutional and acquired gained chromosomes seem to exhibit a specific methylation signature, mimicking the patterns of X chromosome inactivation, with a general depletion of 5hmC and a global hypomethylation of gene-poor regions on Xi. These novel findings must be confirmed in larger

studies, and the functional consequences of such epigenetic hallmarks must also be addressed experimentally. The role of aneuploidy as a driver, passenger and/ or epigenome dysregulator in neoplasia should also be explored further and the various components of cellular adaptation and regulation of excess chromatin remain to be defined. Today it is also unclear whether 5hmC is a passive demethylation intermediate or actually plays an active role in gene expression regulation. The avenues of research are many in the post-genomic era, and without a doubt, the coming years will yield many novel insights in how cells respond to the genetic, and epigenetic effects of aneuploidy.

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Financial & competing interests disclosure J Davidsson is supported by the Swedish Cancer Society and the Swedish Research Council. The author has no other relevant affiliation or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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