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When chromosome segregation fails: consequences of aneuploidy on cellular and organismal physiology.

Aneuploidy results in an unbalanced genome in which chromosomes(s), or pieces of chromosomes are missing or supernumerary. The condition has a profound impact on human health. Aneuploidy is the leading cause of mental retardation and spontaneous abortions and a key characteristic of cancer, with an estimated 70–90% of all solid human tumors harboring aneuploid genomes. Given the dramatic impact of aneuploidy on organismal and cellular fitness it is important to understand how the aneuploid condition impacts cell and organismal physiology. We study the effects of aneuploidy on normal cell physiology and its role in cancer and Down Syndrome.

Cellular models to study aneuploidy:

To study the effects of aneuploidy on cellular and organismal physiology we created two types of aneuploidy models – cells that harbor defined aneuploidies and cells in which the karyotype is continuously changing, a condition known as chromosome instability (CIN).

Defined aneuploidy models:

For our work on aneuploidy in budding yeast we created 20 strains carrying one or two additional chromosomes that are maintained via selection. We have also created budding yeast cells in which we can induce the mis-segregation of specific chromosomes at will, allowing us to analyze the immediate consequences of defined single- and multi-chromosome gains and losses.

To study the effects of defined chromosome gains on mammalian cell physiology in vitro, we generated mouse embryonic fibroblasts (MEFs) carrying four different trisomies (trisomy 1, 13, 16 or 19). Because all autosomal aneuploidies are lethal in the mouse we use hematopoietic reconstitutions to assess the effects of aneuploidy on cellular fitness in vivo. We isolate hematopoietic stem cells (HSCs) from fetal livers and compare the ability of euploid and aneuploid HSCs to reconstitute the hematopoietic system of lethally irradiated recipient mice.

CIN models:

To examine the effects of chromosome instability on yeast and mammalian cell physiology we employ mutations or compounds that interfere with chromosome segregation and hence generate cell populations that harbor multiple random aneuploidies and continuous CIN.

The cellular response to aneuploidy.

To understand the effects of aneuploidy on cellular physiology we first asked whether changes in gene copy number translate into a corresponding change in RNA and protein levels. This appears to be the case. Approximately 80 percent of proteins changes in proportion to gene copy number in yeast (Figure 1).

Studies by others have shown that this is also the case in mammals. Having established that changes in gene copy number lead to a corresponding change in gene products, the question of how aneuploidy affects cellular physiology must be reframed. To understand aneuploidy, we must understand how altering the copy number of hundreds if not thousands of genes simultaneously affects cells and organisms.

What have our studies of aneuploid cells revealed? Our analyses of aneuploid yeast, mouse and human cells suggest that aneuploidy causes chromosome-specific effects that are elicited by the increased (or decreased) number of copies of individual genes and/or combinations of a small number of genes present on the aneuploid chromosome. Our data further revealed a suite of phenotypes that is independent of the identity of the aneuploid chromosome (Figure 2).

These phenotypes are conserved from yeast to mammals and are called the “aneuploidy-associated stresses”. They include cell cycle defects, metabolic alterations, a gene expression signature characteristic of slow growth and stress, genomic instability and proteotoxicity, a cellular stress response elicited by mis- and unfolded proteins.

What is the origin of the myriad of phenotypes observed in aneuploid cells? Our data show that imbalances in gene expression are responsible. Phenotypes of aneuploid cells are attenuated by an increase in base ploidy. In yeast for example, haploid cells carrying an extra copy of a chromosome (disome) have more severe phenotypes than diploid cells harboring an extra copy of that same chromosome (trisomes). This result shows that perturbations in the relative ratios of proteins cause the phenotypes observed in aneuploid cells. In the case of chromosome –specific phenotypes changes in relative expression of specific proteins are responsible for the chromosome-specific phenotype. The general aneuploidy associated stresses are thought to be caused primarily by the sum of many gene imbalances that on their own have little or no phenotype. Proteotoxicity in aneuploid cells is a case in point. Many proteins that function in protein complexes require binding to other components of the complex to achieve a native conformation. Thus it is not surprising that eukaryotic cells have evolved to express subunits of the same complex at equal levels (Figure 3).

In aneuploid cells, this balance is disrupted because dosage compensation does not occur on autosomes. As aneuploidy affects expression of many genes (this is especially true for large chromosomes), many proteins mis-fold, which places a burden on the cells’ protein quality control pathways.

We continue to study the effects of aneuploidy on cell physiology and use genetic approaches to uncover additional phenotypes that are unique to specific aneuploidies and traits that are shared by many different aneuploidies.

The organismal response to aneuploidy.

Given the detrimental effects of aneuploidy on cell physiology and the tumorigenic potential of aneuploid cells we have begun to explore the possibility that aneuploid cells are selected against and/or culled in the organism. Single cell sequencing of various tissues of mice harboring mutations in the chromosome segregation regulator BUB1B revealed that while tissues that primarily proliferate in the embryo (i.e. brain) are highly aneuploid, tissues that continuously regenerate during adulthood harbor little aneuploidy (Figure 4).

This result indicates that aneuploid cells are selected against or perhaps even actively culled in the adult animal and can be explained by the population flush effect principle. This principle states that in rapidly expanding populations – as occurs during embryonic development – purifying selection is relaxed such that less fit individuals can survive and significantly contribute to the population. During tissue regeneration in the adult purifying selection strength forces become relatively stronger, selecting against less fit aneuploid cells.

Hematopoietic reconstitution experiments support the idea that aneuploid cells are selected against in adult tissues. Single cell sequencing of peripheral blood following reconstitution of a lethally irradiated recipient with BUB1B mutant hematopoietic stem cells shows that shortly after transplantation when stem cells and progenitor cells are rapidly expanding to repopulate the hematopoietic compartment of the recipients, aneuploidy levels are high (Figure 5).

As the transplant stabilizes and proliferation rates reach steady state aneuploidy levels drop.

More recently we have obtained evidence that aneuploid cells are in fact actively culled. We observe that cells with highly aneuploid karyotypes senesce and are recognized by the innate immune system in vitro and in vivo. We now want to understand how the innate immune system recognizes aneuploid cells and eliminates them. We are especially intrigued by the possibility that aneuploidy is one reason for immune recognition of cancer cells.

Recent Publications:

Blank HM, Sheltzer JM, Meehl CM, Amon A. Mitotic entry in the presence of DNA damage is a widespread property of aneuploidy in yeast. Mol Biol Cell. 2015 Apr 15; 26(8): 1440-1451. PMCID: PMC4395125

Bonney ME, Moriya H, Amon A. Aneuploid proliferation defects in yeast are not driven by copy number changes of a few dosage-sensitive genes. Genes Dev. 2015 May 1; 29(9): 898-903. PMCID: PMC4421978

Dephoure N, Hwang S, O'Sullivan C, Dodgson SE, Gygi SP, Amon A, Torres EM. Quantitative proteomic analysis reveals posttranslational responses to aneuploidy in yeast. Elife. 2014 Jul 29;3:e03023. PMCID: PMC4129440

Dodgson SE, Kim S, Costanzo M, Baryshnikova A, Morse DL, Kaiser CA, Boone C, Amon A. Chromosome-Specific and Global Effects of Aneuploidy in Saccharomyces cerevisiae. Genetics. 2016 Apr;202(4):1395-1409. doi: 10.1534/genetics.115.185660. Epub 2016 Feb 2.

Dodgson SE, Santaguida S, Kim S, Sheltzer J, Amon A. The pleiotropic deubiquitinase Ubp3 confers aneuploidy tolerance. Genes Dev. 2016 Nov 2. [Epub ahead of print]

Knouse KA, Wu J, Whittaker CA, Amon A. Single cell sequencing reveals low levels of aneuploidy across mammalian tissues. Proc Natl Acad Sci U S A. 2014 Sep 16; 111(37): 13409-11344. PMCID: PMC4169915

Knouse KA, Wu J, Amon A. Assessment of megabase-scale somatic copy number variation using single cell sequencing. Genome Res. 2016 Mar;26(3):376-84. doi: 10.1101/gr.198937.115. Epub 2016 Jan 15. PMCID: PMC4772019

Oromendia AB, Dodgson SE, Amon A. (2012). Aneuploidy causes proteotoxic stress in yeast. Genes Dev. 15, 2696-708.

Pfau SJ, Silberman RE, Knouse KA, Amon A. Aneuploidy impairs hematopoietic stem cell fitness and is selected against in regenerating tissues in vivo. Genes Dev. 2016 Jun 15;30(12):1395-408. doi: 10.1101/gad.278820.116.PMCID:PMC4926863

Santaguida S, Vasile E, White E, Amon A. Aneuploidy-induced cellular stresses limit autophagic degradation. Genes Dev. 2015 Oct 1; 29(19): 2010-2021. doi: 10.1101/gad.269118.115. Epub 2015 Sep 24. PMCID: PMC4604343

Sheltzer JM, Torres EM, Dunham MJ, Amon A. (2012). Transcriptional consequences of aneuploidy. Proc Natl Acad Sci U S A. 109,12644-12649.

Sheltzer JM, Blank HM, Pfau SJ, Tange Y, George BM, Humpton TJ, Brito IL, Hiraoka Y, Niwa O, Amon A. Aneuploidy drives genomic instability in yeast. Science. 2011 Aug 19; 333(6045): 1026-1030. PMCID: PMC Journal In Process

Tang YC, Williams BR, Siegel JJ, Amon A. Identification of aneuploidy-selective antiproliferation compounds. Cell. 2011 Feb 18; 144(4): 499-512.

Thorburn RR, Gonzalez C, Brar GA, Christen S, Carlile TM, Ingolia NT, Sauer U, Weissman JS, Amon A. Aneuploid yeast strains exhibit defects in cell growth and passage through START. Mol Biol Cell. 2013 May; 24(9): 1274-1289. PMCID: PMC3639041

Torres EM, Sokolsky T, Tucker CM, Chan LY, Boselli M, Dunham MJ, Amon A. Effects of aneuploidy on cellular physiology and cell division in haploid yeast. Science. 2007 Aug 17; 317(5840): 916-924.

Torres EM, Dephoure N, Panneerselvam A, Tucker CM, Whittaker CA, Gygi SP, Dunham MJ, Amon A. Identification of Aneuploidy-Tolerating Mutations. Cell. 2010 Oct 1; 143(1): 71-83. Epub 2010 Sep 16. PMCID: PMC2993244

Torres EM, Springer M, Amon A. No current evidence for widespread dosage compensation in S. cerevisiae. Elife. 2016 Mar 7;5. pii: e10996. doi: 10.7554/eLife.10996. PMCID: PMC4798953

Williams BR, Prabhu VR, Hunter KE, Glazier CM, Whittaker CA, Housman DE, Amon A. Aneuploidy affects proliferation and spontaneous immortalization in mammalian cells. Science. 2008 Oct 31; 322(5902): 703-709. PMCID: PMC2701511