Immortal DNA Strands Research Articles

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.

Decreased H3K27 and H3K4 trimethylation on mortal chromosomes in distributed stem cells.

The role of immortal DNA strands that co-segregate during mitosis of asymmetrically self-renewing distributed stem cells (DSCs) is unknown. Previously, investigation of immortal DNA strand function and molecular mechanisms responsible for their nonrandom co-segregation was precluded by difficulty in identifying DSCs and immortal DNA strands. Here, we report the use of two technological innovations, selective DSC expansion and establishment of H2A.Z chromosomal asymmetry as a specific marker of ‘immortal chromosomes,' to investigate molecular properties of immortal chromosomes and opposing ‘mortal chromosomes' in cultured mouse hair follicle DSCs. Although detection of the respective suppressive and activating H3K27me3 and H3K4me3 epigenetic marks on immortal chromosomes was similar to randomly segregated chromosomes, detection of both was lower on mortal chromosomes destined for lineage-committed sister cells. This global epigenomic feature of nonrandom co-segregation may reveal a mechanism that maintains an epigenome-wide ‘poised' transcription state, which preserves DSC identity, while simultaneously activating sister chromosomes for differentiation.

Higher 5-hydroxymethylcytosine identifies immortal DNA strand chromosomes in asymmetrically self-renewing distributed stem cells

Immortal strands are the targeted chromosomal DNA strands of nonrandom sister chromatid segregation, a mitotic chromosome segregation pattern unique to asymmetrically self-renewing distributed stem cells (DSCs). By nonrandom segregation, immortal DNA strands become the oldest DNA strands in asymmetrically self-renewing DSCs. Nonrandom segregation of immortal DNA strands may limit DSC mutagenesis, preserve DSC fate, and contribute to DSC aging. The mechanisms responsible for specification and maintenance of immortal DNA strands are unknown. To discover clues to these mechanisms, we investigated the 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) content on chromosomes in mouse hair follicle DSCs during nonrandom segregation. Although 5-methylcytosine content did not differ significantly, the relative content of 5hmC was significantly higher in chromosomes containing immortal DNA strands than in opposed mitotic chromosomes containing younger mortal DNA strands. The difference in relative 5hmC content was caused by the loss of 5hmC from mortal chromosomes. These findings implicate higher 5hmC as a specific molecular determinant of immortal DNA strand chromosomes. Because 5hmC is an intermediate during DNA demethylation, we propose a ten-eleven translocase enzyme mechanism for both the specification and maintenance of nonrandomly segregated immortal DNA strands. The proposed mechanism reveals a means by which DSCs "know" the generational age of immortal DNA strands. The mechanism is supported by molecular expression data and accounts for the selection of newly replicated DNA strands when nonrandom segregation is initiated. These mechanistic insights also provide a possible basis for another characteristic property of immortal DNA strands, their guanine ribonucleotide dependency.

Asymmetric Chromatid Segregation in Cardiac Progenitor Cells Is Enhanced by Pim-1 Kinase

Cardiac progenitor cells (CPCs) in the adult heart are used for cell-based treatment of myocardial damage, but factors determining stemness, self-renewal, and lineage commitment are poorly understood. Immortal DNA strands inherited through asymmetric chromatid segregation correlate with self-renewal of adult stem cells, but the capacity of CPCs for asymmetric segregation to retain immortal strands is unknown. Cardioprotective kinase Pim-1 increases asymmetric cell division in vivo, but the ability of Pim-1 to enhance asymmetric chromatid segregation is unknown. We aimed to demonstrate immortal strand segregation in CPCs and the enhancement of asymmetric chromatid distribution by Pim-1 kinase. Asymmetric segregation is tracked by incorporation of bromodeoxyuridine. The CPC DNA was labeled for several generations and then blocked in second cytokinesis during chase to determine distribution of immortal versus newly synthesized strands. Intensity ratios of binucleated CPCs with bromodeoxyuridine of ≥70:30 between daughter nuclei indicative of asymmetric chromatid segregation occur with a frequency of 4.57, and asymmetric chromatid segregation is demonstrated at late mitotic phases. Asymmetric chromatid segregation is significantly enhanced by Pim-1 overexpression in CPCs (9.19 versus 4.79 in eGFP-expressing cells; P=0.006). Asymmetric segregation of chromatids in CPCs is increased nearly two-fold with Pim-1 kinase overexpression, indicating that Pim-1 promotes self-renewal of stem cells.

A New Look at an Immortal DNA Hypothesis for Stem Cell Self-Renewal

Copyright: © 2012 Mull JL, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Thirty years ago Cairns and Potten first noted that intestinal epithelial cells fail to produce carcinomas at a rate proportionate to the number of divisions they undergo throughout their lifetimes [1,2]. This observation led to the formation of the immortal DNA strand hypothesis: the suggestion that somatic stem cells segregate their DNA asymmetrically, retaining an “immortal” DNA template while passing on newly formed chromatids to daughter cells. A distinct, yet related, idea posits that somatic stem cells divide infrequently, maintaining a state of relative quiescence. In tandem, mitotic quiescence and asymmetric DNA segregation are thought to spare stem cells from accumulating mutations as they divide to replenish their respective tissues [3].

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.

Semiconservative quasispecies equations for polysomic genomes: The general case

This paper develops a formulation of the quasispecies equations appropriate for polysomic, semiconservatively replicating genomes. This paper is an extension of previous work on the subject, which considered the case of haploid genomes. Here, we develop a more general formulation of the quasispecies equations that is applicable to diploid and even polyploid genomes. Interestingly, with an appropriate classification of population fractions, we obtain a system of equations that is formally identical to the haploid case. As with the work for haploid genomes, we consider both random and immortal DNA strand chromosome segregation mechanisms. However, in contrast to the haploid case, we have found that an analytical solution for the mean fitness is considerably more difficult to obtain for the polyploid case. Accordingly, whereas for the haploid case we obtained expressions for the mean fitness for the case of an analog of the single-fitness-peak landscape for arbitrary lesion repair probabilities (thereby allowing for noncomplementary genomes), here we solve for the mean fitness for the restricted case of perfect lesion repair.

Nonselective Sister Chromatid Segregation in Mouse Embryonic Neocortical Precursor Cells

We have investigated whether the precursor cells that give rise to the neurons of the neocortex during mouse embryonic development segregate sister chromatids nonrandomly upon mitosis, as would be predicted by the immortal strand hypothesis. Using various protocols of 5-bromo-2-deoxyuridine (BrdU) labeling and chase, we were unable to detect BrdU label-retaining neocortical precursor cells at any of the embryonic stages analyzed, even when the entire brain was analyzed by serial sectioning. Analysis of mitotic neuroepithelial and radial glial cells revealed BrdU-labeled sister chromatid segregation to both nascent daughter cells, which showed a mirror-symmetrical pattern in the first and a non-mirror-symmetrical pattern in the second division after BrdU labeling. Taken together, our data are incompatible with embryonic neocortical precursor cells segregating the sister chromatids selectively to one daughter cell upon mitosis and hence argue against the existence of immortal DNA strands in these cells. In light of the previously reported existence of immortal DNA strands in adult neural stem cells, we discuss that either 1) embryonic and adult neural stem cells in the cortex are distinct or 2) that most, if not all, of the embryonic precursor cells to neocortical neurons are progenitor cells rather than true neural stem cells.

A New Mechanism for Aging: Chemical “Age Spots” in Immortal DNA Strands in Distributed Stem Cells

The existence of immortal DNA strands (IDSs) in distributed stem cells (DSCs) of adult human tissues was first inferred by Cairns. Cairns deduced the existence of IDSs by connecting two seemingly disparate observations - one his own and the other belonging to Lark. Cairns noted a mathematical discrepancy between predicted human tissue cell mutation rates and human cancer incidence. He integrated this insight with Lark's earlier discovery of non-random mitotic chromosome segregation in both plant root tip cells and mouse fetal fibroblast cultures to predict the existence of IDSs as the essential elements of a mutation-defense mechanism in DSCs. Since Cairns' seminal prediction, several laboratories have identified IDSs in diverse mammalian cells with DSC properties. Past studies focused on the potential roles of IDSs as originally envisioned in DSC genetic fidelity or in the maintenance of the DSC phenotype. Another possible consequence of IDSs, aging, has received little attention. Herein, the potential for cumulative chemical modifications and decompositions (i.e., "age spots") of IDSs in DSCs to act as a major determinant of human aging is considered. If accrued chemical alterations of IDSs prove to be essential determinants of aging, then a means to restore IDSs may yield new strategies for tissue rejuvenation.

Abstract 3422: Asymmetric Division of Human Cardiac Progenitor Cells Involves Immortal DNA Strand Cosegregation

The objective of this study was to determine whether asymmetric division of human cardiac progenitor cells (hCPCs) occurs by: random DNA template segregation; selective retention of the old template DNA strand; or a combination of both processes. Myocardial samples were enzymatically dissociated and hCPCs were sorted for the stem cell antigen c-kit. During in vitro expansion, hCPCs were exposed to BrdU for 36 hours to reach a 90% degree of labeling. BrdU-tagged hCPCs were plated at limiting dilution to obtain single cell-derived clones. Sixty clones comprising 10 –125 c-kit-positive hCPCs developed in 7–10 days. In four cases, one single BrdU-bright hCPC was identified while the remaining clonogenic cells were negative for the halogenated nucleotide. In these clones, the number of BrdU-negative hCPCs was 25, 85, 95, and 111. Conversely, in 56 clones hCPCs were uniformly labeled and showed very low levels of BrdU. In a second group of experiments, hCPCs in late-anaphase initial-telophase were identified and the distribution of BrdU in the two clusters of chromosomes was analyzed. In 5% of mitotic cells, three-dimensional reconstruction by confocal microscopy documented that BrdU-labeling was restricted at one pole only of the dividing hCPCs. PCNA which is highly expressed in newly synthesized DNA was restricted to the BrdU-negative chromosomes. In a third set of studies, hCPCs were loaded with quantum dots, cultured for 36 hours in the presence of BrdU and examined 96 hours later. Quantum dots are progressively diluted by cell division independently from the modality of DNA template segregation. Thus, hCPCs with minimal levels of quantum dots and bright BrdU localization were interpreted as replicating cells which retained the old DNA strand. By this approach, 5% hCPCs displayed these two critical properties. The uneven distribution of the cell fate determinants Numb and α-adaptin confirmed that hCPCs underwent asymmetric division. In conclusion, these data support the hypothesis that immortal DNA strand cosegregation participates in asymmetric kinetics of hCPCs although random-segregation of DNA template is the prevailing mechanism of hCPC growth.

Quantitative proliferation dynamics and random chromosome segregation of hair follicle stem cells

Regulation of stem cell (SC) proliferation is central to tissue homoeostasis, injury repair, and cancer development. Accumulation of replication errors in SCs is limited by either infrequent division and/or by chromosome sorting to retain preferentially the oldest 'immortal' DNA strand. The frequency of SC divisions and the chromosome-sorting phenomenon are difficult to examine accurately with existing methods. To address this question, we developed a strategy to count divisions of hair follicle (HF) SCs over time, and provide the first quantitative proliferation history of a tissue SC during its normal homoeostasis. We uncovered an unexpectedly high cellular turnover in the SC compartment in one round of activation. Our study provides quantitative data in support of the long-standing infrequent SC division model, and shows that HF SCs do not retain the older DNA strands or sort their chromosome. This new ability to count divisions in vivo has relevance for obtaining basic knowledge of tissue kinetics.

Does 'Immortal DNA strand' exist in 'immortal' stem cells?

Stem cells function to generate differentiated cells, and, at the same time, are maintained as 'immortal' cells through self-renewal. Accumulated evidence indicates that adult stem cells may be the most common precursor for cancers in adult mammalian tissue 1, and this concept is gaining increasing support 2, 3. Meanwhile, this also raises the question of how stem cells, which have a much longer lifespan compared to other progenitor and mature cells, can avoid accumulating mutations from DNA replication errors, thereby reducing the risk of tumorigenesis. In 1975, J Cairns proposed an intriguing hypothesis from an evolutionary point of view to explain this phenomenon in organisms with long life spans. According to this 'immortal DNA strand' model, adult stem cells might be able to retain template (old) DNA strands in each asymmetric division to avoid accumulation of mutations during the process of DNA replication 4.

Evolutionary dynamics of adult stem cells: Comparison of random and immortal-strand segregation mechanisms

This paper develops a point-mutation model describing the evolutionary dynamics of a population of adult stem cells. Such a model may prove useful for quantitative studies of tissue aging and the emergence of cancer. We consider two modes of chromosome segregation: (1) random segregation, where the daughter chromosomes of a given parent chromosome segregate randomly into the stem cell and its differentiating sister cell and (2) "immortal DNA strand" co-segregation, for which the stem cell retains the daughter chromosomes with the oldest parent strands. Immortal strand co-segregation is a mechanism, originally proposed by [Cairns Nature (London) 255, 197 (1975)], by which stem cells preserve the integrity of their genomes. For random segregation, we develop an ordered strand pair formulation of the dynamics, analogous to the ordered strand pair formalism developed for quasispecies dynamics involving semiconservative replication with imperfect lesion repair (in this context, lesion repair is taken to mean repair of postreplication base-pair mismatches). Interestingly, a similar formulation is possible with immortal strand co-segregation, despite the fact that this segregation mechanism is age dependent. From our model we are able to mathematically show that, when lesion repair is imperfect, then immortal strand co-segregation leads to better preservation of the stem cell lineage than random chromosome segregation. Furthermore, our model allows us to estimate the optimal lesion repair efficiency for preserving an adult stem cell population for a given period of time. For human stem cells, we obtain that mispaired bases still present after replication and cell division should be left untouched, to avoid potentially fixing a mutation in both DNA strands.

Immortal DNA Strand Cosegregation Requires p53/IMPDH–Dependent Asymmetric Self-renewal Associated with Adult Stem Cells

Because they are long-lived and cycle continuously, adult stem cells (ASCs) are predicted as the most common precursor for cancers in adult mammalian tissues. Two unique attributes have been proposed to restrict the carcinogenic potential of ASCs. These are asymmetric self-renewal that limits their number and immortal DNA strand cosegregation that limits their accumulation of mutations due to DNA replication errors. Until recently, the molecular basis and regulation of these important ASC-specific functions were unknown. We developed engineered cultured cells that exhibit asymmetric self-renewal and immortal DNA strand cosegregation. These model cells were used to show that both ASC-specific functions are regulated by the p53 cancer gene. Previously, we proposed that IMP dehydrogenase (IMPDH) was an essential factor for p53-dependent asymmetric self-renewal. We now confirm this proposal and provide quantitative evidence that asymmetric self-renewal is acutely sensitive to even modest changes in IMPDH expression. These analyses reveal that immortal DNA strand cosegregation is also regulated by IMPDH and confirm the original implicit precept that immortal DNA strand cosegregation is specific to cells undergoing asymmetric self-renewal (i.e., ASCs). With IMPDH being the rate-determining enzyme for guanine ribonucleotide (rGNP) biosynthesis, its requirement implicates rGNPs as important regulators of ASC asymmetric self-renewal and immortal DNA strand cosegregation. An in silico analysis of global gene expression data from human cancer cell lines underscored the importance of p53-IMPDH-rGNP regulation for normal tissue cell kinetics, providing further support for the concept that ASCs are key targets for adult tissue carcinogenesis.

Parental DNA strands segregate randomly during embryonic development of Caenorhabditis elegans

The fate of gamete DNA was followed in the next generation embryos of the nematode C. elegans. Either male worms or spermless hermaphrodites were grown on bromodeoxyuridine-containing E. coli in order to label germ-line DNA. Matings then produced embryos in which only the DNA strands provided by the gametes contained label. This original gamete DNA could be detected during embryonic development by using a fluorescently labeled monoclonal antibody specific to bromodeoxyuridine. Both the number and position of fluorescent spots in the embryo indicate that gamete DNA strands segregate randomly during development. Random segregation of parental DNA strands rules out models of development that invoke chromosome imprinting or immortal DNA strands.