Non-random Sister Chromatid Segregation Research Articles

Nonrandom sister chromatid segregation mediates rDNA copy number maintenance in Drosophila.

Although considered to be exact copies of each other, sister chromatids can segregate nonrandomly in some cases. For example, sister chromatids of the X and Y chromosomes segregate nonrandomly during asymmetric division of male germline stem cells (GSCs) in Drosophila melanogaster. Here, we demonstrate that the ribosomal DNA (rDNA) loci, which are located on the X and Y chromosomes, and an rDNA binding protein Indra are required for nonrandom sister chromatid segregation (NRSS). We provide the evidence that NRSS, following unequal sister chromatid exchange, is a mechanism by which GSCs recover rDNA copy number, counteracting the spontaneous copy number loss that occurs during aging. Our study reveals an unexpected role for NRSS in maintaining germline immortality through maintenance of a vulnerable genomic element, rDNA.

Centromere function in asymmetric cell division in Drosophila female and male germline stem cells.

The centromere is the constricted chromosomal region required for the correct separation of the genetic material at cell division. The kinetochore protein complex assembles at the centromere and captures microtubules emanating from the centrosome to orchestrate chromosome segregation in mitosis and meiosis. Asymmetric cell division (ACD) is a special type of mitosis that generates two daughter cells with different fates. Epigenetic mechanisms operating at the centromere have been proposed to contribute to ACD. Recent studies have shown that an asymmetric distribution of CENP-A-the centromere-specific histone H3 variant-between sister chromatids can bias chromosome segregation in ACD. In stem cells, this leads to non-random sister chromatid segregation, which can affect cell fate. These findings support the 'silent sister' hypothesis, according to which the mechanisms of ACD are epigenetically regulated through centromeres. Here, we review the recent data implicating centromeres in ACDs and cell fate in Drosophila melanogaster female and male germline stem cells.

Non-Random Sister Chromatid Segregation in Human Tissue Stem Cells

The loss of genetic fidelity in tissue stem cells is considered a significant cause of human aging and carcinogenesis. Many cellular mechanisms are well accepted for limiting mutations caused by replication errors and DNA damage. However, one mechanism, non-random sister chromatid segregation, remains controversial. This atypical pattern of chromosome segregation is restricted to asymmetrically self-renewing cells. Though first confirmed in murine cells, non-random segregation was originally proposed by Cairns as an important genetic fidelity mechanism in human tissues. We investigated human hepatic stem cells expanded by suppression of asymmetric cell kinetics (SACK) for evidence of non-random sister chromatid segregation. Cell kinetics and time-lapse microscopy analyses established that an ex vivo expanded human hepatic stem cell strain possessed SACK agent-suppressible asymmetric cell kinetics. Complementary DNA strand-labeling experiments revealed that cells in hepatic stem cell cultures segregated sister chromatids non-randomly. The number of cells cosegregating sister chromatids with the oldest “immortal DNA strands” was greater under conditions that increased asymmetric self-renewal kinetics. Detection of this mechanism in a human tissue stem cell strain increases support for Cairns’ proposal that non-random sister chromatid segregation operates in human tissue stem cells to limit carcinogenesis.

Stem cell genetic fidelity.

Stem cell genetic fidelity.

Chromosome-specific nonrandom sister chromatid segregation during stem-cell division

Adult stem cells undergo asymmetric cell division to self-renew and give rise to differentiated cells that comprise mature tissue1. Sister chromatids may be distinguished and segregated non-randomly in asymmetrically dividing stem cells2, although the underlying mechanism and the purpose it may serve remain elusive. We developed the CO-FISH (chromosome orientation fluorescence in situ hybridization) technique3 with single-chromosome resolution and show that sister chromatids of X and Y chromosomes, but not autosomes, are segregated non-randomly during asymmetric divisions of Drosophila male germline stem cells (GSCs). This provides the first direct evidence that two sister chromatids containing identical genetic information can be distinguished and segregated non-randomly during asymmetric stem cell divisions. We further show that the centrosome, SUN-KASH nuclear envelope proteins, and Dnmt2 are required for non-random sister chromatid segregation. Our data suggest that the information on X and Y chromosomes that enables non-random segregation is primed during gametogenesis in the parents. Moreover, we show that sister chromatid segregation is randomized in GSC overproliferation and dedifferentiated GSCs. We propose that non-random sister chromatid segregation may serve to transmit distinct information carried on two sister chromatids to the daughters of asymmetrically dividing stem cells.

Nonrandom sister chromatid segregation of sex chromosomes in Drosophila male germline stem cells

Sister chromatids are the product of DNA replication, which is assumed to be a very precise process. Therefore, sister chromatids should be exact copies of each other. However, reports have indicated that sister chromatids are segregated nonrandomly during cell division, suggesting that sister chromatids are not the same, although their DNA sequences are the same. Researchers have speculated that stem cells may retain template strands to avoid replication-induced mutations. An alternative proposal is that cells may segregate distinct epigenetic information carried on sister chromatids. Recently, we found that Drosophila male germline stem cells segregate sister chromatids of X and Y chromosomes with a strong bias. We discuss this finding in relation to existing models for nonrandom sister chromatid segregation.

DNA asymmetry in stem cells – immortal or mortal?

The immortal strand hypothesis proposes that stem cells retain a template copy of genomic DNA (i.e. an 'immortal strand') to avoid replication-induced mutations. An alternative hypothesis suggests that certain cells segregate sister chromatids non-randomly to transmit distinct epigenetic information. However, this area of research has been highly controversial, with conflicting data even from the same cell types. Moreover, historically, the same term of 'non-random sister chromatid segregation' or 'biased sister chromatid segregation' has been used to indicate distinct biological processes, generating a confusion in the biological significance and potential mechanism of each phenomenon. Here, we discuss the models of non-random sister chromatid segregation, and we explore the strengths and limitations of the various techniques and experimental model systems used to study this question. We also describe our recent study on Drosophila male germline stem cells, where sister chromatids of X and Y chromosomes are segregated non-randomly during cell division. We aim to integrate the existing evidence to speculate on the underlying mechanisms and biological relevance of this long-standing observation on non-random sister chromatid segregation.

Discovering non-random segregation of sister chromatids: the naïve treatment of a premature discovery.

The discovery of non-random chromosome segregation (Figure 1) is discussed from the perspective of what was known in 1965 and 1966. The distinction between daughter, parent, or grandparent strands of DNA was developed in a bacterial system and led to the discovery that multiple copies of DNA elements of bacteria are not distributed randomly with respect to the age of the template strand. Experiments with higher eukaryotic cells demonstrated that during mitosis Mendel’s laws were violated; and the initial serendipitous choice of eukaryotic cell system led to the striking example of non-random segregation of parent and grandparent DNA template strands in primary cultures of cells derived from mouse embryos. Attempts to extrapolate these findings to established tissue culture lines demonstrated that the property could be lost. Experiments using plant root tips demonstrated that the phenomenon exists in plants and that it was, at some level, under genetic control. Despite publication in major journals and symposia (Lark et al., 1966, 1967; Lark, 1967, 1969a,b,c) the potential implications of these findings were ignored for several decades. Here we explore possible reasons for the pre-maturity (Stent, 1972) of this discovery.

SACK-expanded hair follicle stem cells display asymmetric nuclear Lgr5 expression with non-random sister chromatid segregation.

We investigated the properties of clonally-expanded mouse hair follicle stem cells (HF-SCs) in culture. The expansion method, suppression of asymmetric cell kinetics (SACK), is non-toxic and reversible, allowing evaluation of the cells' asymmetric production of differentiating progeny cells. A tight association was discovered between non-random sister chromatid segregation, a unique property of distributed stem cells (DSCs), like HF-SCs, and a recently described biomarker, Lgr5. We found that nuclear Lgr5 expression was limited to the HF-SC sister of asymmetric self-renewal divisions that retained non-randomly co-segregated chromosomes, which contain the oldest cellular DNA strands, called immortal DNA strands. This pattern-specific Lgr5 association poses a potential highly specific new biomarker for delineation of DSCs. The expanded HF-SCs also maintained the ability to make differentiated hair follicle cells spontaneously, as well as under conditions that induced cell differentiation. In future human cell studies, this capability would improve skin grafts and hair replacement therapies.

Molecular Cloaking of H2A.Z on Mortal DNA Chromosomes During Nonrandom Segregation

Although nonrandom sister chromatid segregation is a singular property of distributed stem cells (DSCs) that are responsible for renewing and repairing mature vertebrate tissues, both its cellular function and its molecular mechanism remain unknown. This situation persists in part because of the lack of facile methods for detecting and quantifying nonrandom segregating cells and for identifying chromosomes with immortal DNA strands, the cellular molecules that signify nonrandom segregation. During nonrandom segregation, at each mitosis, asymmetrically self-renewing DSCs continuously cosegregate to themselves the set of chromosomes that contain immortal DNA strands, which are the oldest DNA strands. Here, we report the discovery of a molecular asymmetry between segregating sets of immortal chromosomes and opposed mortal chromosomes (i.e., containing the younger set of DNA template strands) that constitutes a new convenient biomarker for detection of cells undergoing nonrandom segregation and direct delineation of chromosomes that bear immortal DNA strands. In both cells engineered with DSC-specific properties and ex vivo-expanded mouse hair follicle stem cells, the histone H2A variant H2A.Z shows specific immunodetection on immortal DNA chromosomes. Cell fixation analyses indicate that H2A.Z is present on mortal chromosomes as well but is cloaked from immunodetection, and the cloaking entity is acid labile. The H2A.Z chromosomal asymmetry produced by molecular cloaking provides a first direct assay for nonrandom segregation and for chromosomes with immortal DNA strands. It also seems likely to manifest an important aspect of the underlying mechanism(s) responsible for nonrandom sister chromatid segregation in DSCs.

Identification of sister chromatids by DNA template strand sequences

It is generally assumed that sister chromatids are genetically and functionally identical and that segregation to daughter cells is a random process. However, functional differences between sister chromatids regulate daughter cell fate in yeast and sister chromatid segregation is not random in Escherichia coli. Differentiated sister chromatids, coupled with non-random segregation, have been proposed to regulate cell fate during the development of multicellular organisms. This hypothesis has not been tested because molecular features to reliably distinguish between sister chromatids are not obvious. Here we show that parental 'Watson' and 'Crick' DNA template strands can be identified in sister chromatids of murine metaphase chromosomes using CO-FISH (chromosome orientation fluorescence in situ hybridization) with unidirectional probes specific for centromeric and telomeric repeats. All chromosomes were found to have a uniform orientation with the 5' end of the short arm on the same strand as T-rich major satellite repeats. The invariable orientation of repetitive DNA was used to differentially label sister chromatids and directly study mitotic segregation patterns in different cell types. Whereas sister chromatids appeared to be randomly distributed between daughter cells in cultured lung fibroblasts and embryonic stem cells, significant non-random sister chromatid segregation was observed in a subset of colon crypt epithelial cells, including cells outside positions reported for colon stem cells. Our results establish that DNA template sequences can be used to distinguish sister chromatids and follow their mitotic segregation in vivo.

One for You, Two for Me

CELL BIOLOGY Asymmetric cell division, wherein the daughter cell receives an unequal set of contents from the dividing mother cell, drives the formation of the many and diverse cell lineages that populate multicellular organisms. The single-celled yeast Saccharomyces cerevisiae also undergoes asymmetric cell divisions to produce so-called mother and daughter cells, but as this occurs at every cell division, there are no obviously distinct cell lineages formed, as is true for many unicellular organisms. However, a closer look at the way yeast proteins are segregated during meiosis reveals a rather different outcome. Thorpe et al. have fluorescently tagged a number of proteins from the kinetochore (the molecular machine that links the chromosomes to the spindle in dividing cells and thus ensures their proper segregation) and find that they segregate asymmetrically at the first meiotic division, forming the haploid spore, and also in subsequent divisions, so that they are preferentially retained in the mother cell lineage derived from the spore. These same proteins are symmetrically distributed between other dividing “cell types,” and other proteins that are not part of the kinetochore do not show a similar asymmetry. The unequal segregation of the kinetochores may allow for the non-random segregation of sister chromatids, which would thereby maintain an “immortal” DNA strand, or of the centromeric DNA to which the kinetochores bind, which could drive the evolution of the centromeres. — GR Proc. Natl. Acad. Sci. U.S.A. 106 , 10.1073/pnas.0811248106 (2009).

Modeling Stem Cell Asymmetry in Yeast

For adult stem cells to both self-renew and give rise to differentiating progenitors, they must undergo an inherently asymmetric division. This defining model of asymmetric cell division requires either that stem cells preferentially distribute internal factors, thereby maintaining a stem cell phenotype in one lineage, or that extrinsic signals determine the fate of daughter cells, allowing the maintenance of one stem cell lineage. Although microbial systems are often used to model asymmetry, lineage-specific asymmetry has not been characterized in these organisms. Recently, we identified a stem-cell-like lineage-specific pattern of kinetochore asymmetry in postmeiotic yeast spores. Because the function of the kinetochore is to segregate chromosomes, this asymmetry has the potential to segregate sister chromatids nonrandomly. This may be relevant to stem cells because more than 30 years ago, it was proposed that stem cells selectively segregate one strand of their chromosomes into the self-renewing stem cell lineage (Cairns 1975). Although advanced labeling methods have provided evidence to both support and refute this hypothesis, it remains unclear how nonrandom sister-chromatid segregation might be achieved in a stem cell lineage. We have identified a kinetochore-specific mechanism in yeast that could support lineage-specific nonrandom sister-chromatid segregation and we discuss the implications of this observation.

Non-random sister chromatid segregation by cell type

The non-random segregation of the sister chromatids of differentiating cells at mitosis is hypothesized. In a cell with n chromosomes, DNA replication results in n pairs of chromatids that must be segretated to daughter cells via one of 2 n modes. In only one of these 2 n modes do all of the oldest/newest parental DNA strands segregate togehter. It is our hypothesis that the segregation mode employed by the parental cell is fully non-random. The design of a system that can control this segregation is presented. In this control system, cell type specific segregation patterns will be dependent upon the use/non-use of potential replicative origins. As a consequence of controlled segregation, sister chromatid exchanges will, by their mis-segregatioe nature, engender abnormal cell types. Experimental data in the literature is re-interpreted in the light of this hypothesis.

Random segregation of sister chromatids in developing chick retinal cells demonstrated in vivo using the fluorescence plus Giemsa technique.

Experiments were designed to test whether nonrandom segregation of sister chromatids at mitosis has a role in the production of cell diversity during embryogenesis, Segregation was examined in vivo in retinal cells from embryonic chicks. Chromatids were labelled with bromouracil and stained by the fluorescence plus Giemsa technique. No evidence of nonrandom segregation was observed in a frequency distribution of pairs of bifilarly labelled sister chromatids at the third metaphase after the start of labeling. Nor was there evidence that chromatids from homologous chromosomes segregated nonrandomly. Nonrandom segregation is probab;y not a mechanism for cell diversification.

Sequential DNA replication and the control of differences in gene activity between sister chromatids—A possible factor in cell differentiation

Since genetic replication does not entail replication of the molecules mediating genetic repression, the set of “new” genes which become repressed may differ from the set of “old” genes which remain repressed. Therefore, the genetic activity of two sister chromatids may differ and these differences, through mitosis, may play a role in cellular differentiation. A newly synthesized gene will be, at least initially, derepressed. Which new genes become repressed, and therefore whether sister chromatids differ in activity, will be determined by at least three factors: the particular genetic feedback network; the set of repressor molecules synthesized by genes already active at the time of DNA replication; the particular sequence of repressor molecules synthesized by new, derepressed genes as they replicate in a particular temporal sequence. The model suggests that sister chromatids can be identical, but that a change in the set of genes active at replication, or in the order of replication, may cause the chromatids to differ, resulting in daughter cells which differ. Therefore, a genetic feedback network, by undergoing sequential change in gene activity, or by altering the order of replication, could itself control not only the occurrence of sister chromatid differences, but, at each replication period, the particular and possibly different way in which they differ. Since several sister chromatid pairs may differ simultaneously, a cell's capacity to create daughter cells which differ in specific ways would be aided by non-random segregation of sister chromatids at mitosis. Evidence from the literature is adduced to show that sister chromatids may differ, that newly synthesized genes are initially active, that the order of replication changes during embryogenesis and is associated with altered gene expression, that an altered order of replication may give rise to sister chromatids with different gene activities, and that mitosis separates sister chromatids non-randomly with respect to the age of their DNA.