Strand Segregation Research Articles

The Roles of Nucleoid-Associated Proteins and Topoisomerases in Chromosome Structure, Strand Segregation and the Generation of Phenotypic Heterogeneity in Bacteria.

How to adapt to a changing environment is a fundamental, recurrent problem confronting cells. One solution is for cells to organise their constituents into a limited number of spatially extended, functionally relevant, macromolecular assemblies or hyperstructures, and then to segregate these hyperstructures asymmetrically into daughter cells. This asymmetric segregation becomes a particularly powerful way of generating a coherent phenotypic diversity when the segregation of certain hyperstructures is with only one of the parental DNA strands and when this pattern of segregation continues over successive generations. Candidate hyperstructures for such asymmetric segregation in prokaryotes include those containing the Nucleoid-Associated Proteins (NAPs) and the topoisomerases. Another solution to the problem of creating a coherent phenotypic diversity is by creating a growth-environment-dependent gradient of supercoiling generated along the replication origin-to-terminus axis of the bacterial chromosome. This gradient is modulated by transcription, NAPs and topoisomerases. Here, we focus primarily on two topoisomerases, TopoIV and DNA gyrase in Escherichia coli, on three of its NAPs (H-NS, HU and IHF), and on the single-stranded binding protein, SSB. We propose that the combination of supercoiling-gradient-dependent and strand-segregation-dependent topoisomerase activities result in significant differences in the supercoiling of daughter chromosomes and hence in the phenotypes of daughter cells.

DNA template strand segregation in developing zebrafish

Asymmetric inheritance of sister chromatids has long been predicted to be linked to discordant fates of daughter cells and even hypothesized to minimize accumulation of mutations in stem cells. Here, we use (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine (F-ara-EdU), bromodeoxyuridine (BrdU), and light sheet microscopy to track embryonic DNA in whole zebrafish. Larval development results in rapid depletion of older DNA template strands from stem cell niches in the retina, brain, and intestine. Prolonged label retention occurs in quiescent progenitors that resume replication in later development. High-resolution microscopy reveals no evidence of asymmetric template strand segregation in >100 daughter cell pairs, making it improbable that asymmetric DNA segregation prevents mutational burden according to the immortal strand hypothesis in developing zebrafish.

A probabilistic framework for cellular lineage reconstruction using integrated single-cell 5-hydroxymethylcytosine and genomic DNA sequencing

SummaryLineage reconstruction is central to understanding tissue development and maintenance. To overcome the limitations of current techniques that typically reconstruct clonal trees using genetically encoded reporters, we report scPECLR, a probabilistic algorithm to endogenously infer lineage trees at a single-cell-division resolution by using 5-hydroxymethylcytosine (5hmC). When applied to 8-cell pre-implantation mouse embryos, scPECLR predicts the full lineage tree with greater than 95% accuracy. In addition, we developed scH&G-seq to sequence both 5hmC and genomic DNA from the same cell. Given that genomic DNA sequencing yields information on both copy number variations and single-nucleotide polymorphisms, when combined with scPECLR it enables more accurate lineage reconstruction of larger trees. Finally, we show that scPECLR can also be used to map chromosome strand segregation patterns during cell division, thereby providing a strategy to test the “immortal strand” hypothesis. Thus, scPECLR provides a generalized method to endogenously reconstruct lineage trees at an individual-cell-division resolution.

Generation of Bacterial Diversity by Segregation of DNA Strands.

The generation in a bacterial population of a diversity that is coherent with present and future environments is a fundamental problem. Here, we use modeling to investigate growth rate diversity. We show that the combination of (1) association of extended assemblies of macromolecules with the DNA strands and (2) the segregation of DNA strands during cell division allows cells to generate different patterns of growth rate diversity with little effect on the overall growth rate of the population and thereby constitutes an example of “order for free” on which evolution can act.

DNA Template Strand Segregation in Whole Zebrafish

Asymmetric inheritance of sister chromatids has long been predicted to be linked to discordant fates of daughter cells and even hypothesized to minimize accumulation of mutations in stem cells. Here, we use (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine (F-ara-EdU), bromodeoxyuridine (BrdU), and light-sheet microscopy to track embryonic DNA in whole zebrafish. Larval development resulted in rapid depletion of older, template DNA strands from stem cell niches in retina, brain and intestine. Prolonged label retention occurred in quiescent progenitors that resumed replication in later development. Quantifying >100 daughter cell pairs throughout the body showed no evidence of asymmetric template strand segregation, making it improbable that asymmetric DNA segregation prevents mutational burden.

Mutation-rate plasticity and the germline of unicellular organisms.

The mutation rate is a fundamental factor in evolutionary genetics. Recently, mutation rates were found to be strongly reduced at high density in a wide range of unicellular organisms, prokaryotic and eukaryotic. Independently, cell division was found to become more asymmetrical at increasing density in diverse organisms; some ‘mother’ cells continue dividing, while their ‘offspring’ cells do not divide further. Here, we investigate how this increased asymmetry in cell division at high density can be reconciled with reduced mutation-rate estimates. We calculated the expected number of mutant cells due to replication errors under various modes of segregation of template-DNA strands and copy-DNA strands, both under symmetrical (exponential) and asymmetrical (linear) growth. We show that the observed reduction in the mutation rate at high density can be explained if mother cells preferentially retain the template-DNA strands, since new mutations are then confined to non-dividing daughter cells, thus reducing the spread of mutant cells. Any other inheritance mode results in an increase in the number of mutant cells at higher density. The proposed hypothesis that patterns of DNA-strand segregation are density-dependent fundamentally challenges our current understanding of mutation-rate estimates and extends the distinction between germline and soma to unicellular organisms.

Levels of Coordination in Early Semantic Development

Abstract In this paper, we show that early interaction can be seen as comprising of strands of coordinated activity on multiple levels and timescales. In tracing the development of such multilayered organization from an embodied and situated perspective, we underscore the role of the reliable presence of the structured environment, an enacted niche, supporting the segregation and integration of participatory interaction strands. This perspective allows us to study the development of social coordination not only in terms of development of individual skills but, crucially, as a change of participatory emergent patterns, a transformation in engagement. We illustrate this approach with some results from the collaborative research project on Early Semantic Development (EASE). Using qualitative microanalysis combined with quantitative dynamical time series analyses, we were able to demonstrate several layers of such organization: from local forms of coordination, such as basic informational coupling within a modality, and the emergence of specific social affordances, to more global co-action structures such as affect imbued ‘action arcs’ – dynamic action contours with a beginning, build-up, climax and resolution, co-enacted by participants. Pointing to future work, we underscore the potential of these global structures to contribute to the emergence of more complex interactions, such as composite activities within ‘pragmatic frames’, narratives, or language.

Variation of mutational burden in healthy human tissues suggests non-random strand segregation and allows measuring somatic mutation rates.

The immortal strand hypothesis poses that stem cells could produce differentiated progeny while conserving the original template strand, thus avoiding accumulating somatic mutations. However, quantitating the extent of non-random DNA strand segregation in human stem cells remains difficult in vivo. Here we show that the change of the mean and variance of the mutational burden with age in healthy human tissues allows estimating strand segregation probabilities and somatic mutation rates. We analysed deep sequencing data from healthy human colon, small intestine, liver, skin and brain. We found highly effective non-random DNA strand segregation in all adult tissues (mean strand segregation probability: 0.98, standard error bounds (0.97,0.99)). In contrast, non-random strand segregation efficiency is reduced to 0.87 (0.78,0.88) in neural tissue during early development, suggesting stem cell pool expansions due to symmetric self-renewal. Healthy somatic mutation rates differed across tissue types, ranging from 3.5 × 10−9/bp/division in small intestine to 1.6 × 10−7/bp/division in skin.

Asymmetric stem-cell division ensures sustained keratinocyte hyperproliferation in psoriatic skin lesions

Excessive expansion of the transit-amplifying (TA) cell compartment is a distinct morphological characteristic of psoriatic epidermal hyperplasia. In order to examine the activation of basal stem cells and how they replenish such an enlarged compartment of TA cells in psoriatic epidermis, we utilized a BrdU labeling method to monitor mitotic stem cells in a mouse model of psoriasiform dermatitis, which was induced by imiquimod. Our results showed that perpendicular and parallel cell division characteristics of dividing stem cells existed in the inflamed epidermis. When we analyzed template-DNA strand segregation in trypsin-dissociated human psoriatic keratinocytes using BrdU pulse-chase labeling, we found that the percentage of asymmetric segregation of BrdU was significantly increased in the cell pairs of psoriatic epidermal cells compared with normal epidermal cells. Furthermore, we also examined the effects of both interleukin (IL)-17A and IL-22 cytokines on the differentiation status of cultured human keratinocytes. The results indicated that both cytokines had synergistic effects on passage-one epidermal cell sheets derived from skin explants and also on cultured keratinocytes, were involved in the maintenance of the undifferentiated stem cell phenotype, and these results suggest an efficient mechanism for preventing the premature loss of basal stem-cell pools in the pro-inflammatory cytokine-enriched milieu of the psoriatic epidermis. Our findings suggest that inhibition of hyperactive stem cells represents a potential therapeutic target to combat recalcitrant epidermal hyperplasia in psoriasis.

The (not so) immortal strand hypothesis

BackgroundNon-random segregation of DNA strands during stem cell replication has been proposed as a mechanism to minimize accumulated genetic errors in stem cells of rapidly dividing tissues. According to this hypothesis, an “immortal” DNA strand is passed to the stem cell daughter and not the more differentiated cell, keeping the stem cell lineage replication error-free. After it was introduced, experimental evidence both in favor and against the hypothesis has been presented. Principal findingsUsing a novel methodology that utilizes cancer sequencing data we are able to estimate the rate of accumulation of mutations in healthy stem cells of the colon, blood and head and neck tissues. We find that in these tissues mutations in stem cells accumulate at rates strikingly similar to those expected without the protection from the immortal strand mechanism. SignificanceUtilizing an approach that is fundamentally different from previous efforts to confirm or refute the immortal strand hypothesis, we provide evidence against non-random segregation of DNA during stem cell replication. Our results strongly suggest that parental DNA is passed randomly to stem cell daughters and provides new insight into the mechanism of DNA replication in stem cells.

Evaluating the immortal strand hypothesis in cancer stem cells: Symmetric/self-renewal as the relevant surrogate marker of tumorigenicity

Stem cells subserve repair functions for the lifetime of the organism but, as a consequence of this responsibility, are candidate cells for accumulating numerous genetic and/or epigenetic aberrations leading to malignant transformation. However, given the importance of this guardian role, stem cells likely harbor some process for maintaining their precious genetic code such as non-random segregation of chromatid strands as predicted by the Immortal Strand Hypothesis (ISH). Discerning such non-random chromosomal segregation and asymmetric cell division in normal or cancer stem cells has been complicated by methodological shortcomings but also by differing division kinetics amongst tissues and the likelihood that both asymmetric and symmetric cell divisions, dictated by local extrinsic factors, are operant in these cells. Recent data suggest that cancer stem cells demonstrate a higher incidence of symmetric versus asymmetric cell division with both daughter cells retaining self-renewal characteristics, a profile which may underlie poorly differentiated morphology and marked clonal diversity in tumors. Pathways and targets are beginning to emerge which may provide opportunities for preventing such a predilection in cancer stem cells and that will hopefully translate into new classes of chemotherapeutics in oncology. Thus, although the existence of the ISH remains controversial, the shift of cell division dynamics to symmetric random chromosome segregation/self-renewal, which would negate any likelihood of template strand retention, appears to be a surrogate marker for the presence of highly malignant tumorigenic cell populations.

Influence of polymer stabilization on the dielectric relaxations of an antiferroelectric liquid crystal

Dielectric spectroscopic investigations are reported on an antiferroelectric liquid crystal in its pristine form and when stabilized by a polymer network without and with application of a DC bias field. While the in-phase mode associated with the helix distortion is hardly affected, the high frequency mode connected with the antiphase fluctuations shows a large decrease with increasing concentration of the photoactive monomer used to generate the polymer stabilization. With the help of X-ray data, we rule out any inter-layer nanophase segregation of the polymer strands, and argue that this strong effect is due to the large elastic interaction between the liquid crystal molecules and the polymer strands. Employing expression from a Landau theory we extract several relevant parameters, including the viscosities associated with the different relaxation modes, the elastic constant, and importantly the antiferroelectric interaction, which exhibits a substantial decrease upon polymer stabilization.

Asymmetric segregation of template DNA strands in basal-like human breast cancer cell lines

Background and methodsStem or progenitor cells from healthy tissues have the capacity to co-segregate their template DNA strands during mitosis. Here, we set out to test whether breast cancer cell lines also possess the ability to asymmetrically segregate their template DNA strands via non-random chromosome co-segregation, and whether this ability correlates with certain properties attributed to breast cancer stem cells (CSCs). We quantified the frequency of asymmetric segregation of template DNA strands in 12 human breast cancer cell lines, and correlated the frequency to molecular subtype, CD44+/CD24-/lo phenotype, and invasion/migration ability. We tested if co-culture with human mesenchymal stem cells, which are known to increase self-renewal, can alter the frequency of asymmetric segregation of template DNA in breast cancer.ResultsWe found a positive correlation between asymmetric segregation of template DNA and the breast cancer basal-like and claudin-low subtypes. There was an inverse correlation between asymmetric segregation of template DNA and Her2 expression. Breast cancer samples with evidence of asymmetric segregation of template DNA had significantly increased invasion and borderline significantly increased migration abilities. Samples with high CD44+/CD24-/lo surface expression were more likely to harbor a consistent population of cells that asymmetrically segregated its template DNA; however, symmetric self-renewal was enriched in the CD44+/CD24-/lo population. Co-culturing breast cancer cells with human mesenchymal stem cells expanded the breast CSC pool and decreased the frequency of asymmetric segregation of template DNA.ConclusionsBreast cancer cells within the basal-like subtype can asymmetrically segregate their template DNA strands through non-random chromosome segregation. The frequency of asymmetric segregation of template DNA can be modulated by external factors that influence expansion or self-renewal of CSC populations. Future studies to uncover the underlying mechanisms driving asymmetric segregation of template DNA and dictating cell fate at the time of cell division may explain how CSCs are maintained in tumors.

WITHDRAWN: Symmetries and Asymmetries Associated with Non-Random Segregation of Sister DNA Strands in Escherichia coli

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.semcdb.2013.05.010. The duplicate article has therefore been withdrawn.

Selective tracking of template DNA strands after induction of mitosis with unreplicated genomes (MUGs) in Drosophila S2 cells

According to the "immortal" DNA strand hypothesis (Cairns Nature 255:197-200, 1975), stem cells would keep their template strands in order to prevent the accumulation of mutations, which could occur during DNA replication. Despite the growing number of studies that attempt to test this hypothesis, the conclusions remain highly controversial. In the base of this controversy lie the current limitations of available methodology to selectively and faithfully track the fate of template DNA strands throughout and upon cell division. Here, we developed a method that allows the unequivocal tracking of single chromatids containing template DNA strands in Drosophila S2 cells in culture. This method consists in the induction of mitosis with unreplicated genomes (MUGs) in which cells are allowed to enter mitosis without prior DNA replication. This is achieved by RNAi-mediated knockdown of Double parked, a conserved protein required for the initiation of DNA replication and post-replication checkpoint response. The advantages of this system when compared with MUGs generated in mammalian cells is the preservation of chromatid morphology, the ease of loss-of-function studies and the possibility of in vivo applications. Altogether, this approach allows for the readily visualization and tracking of template DNA strands by simply monitoring cells stably expressing GFP-fusions with either Histone H2B or the centromeric Histone variant CID/CENP-A by time-lapse fluorescence microscopy. This might be useful for the dissection of the molecular mechanism behind asymmetric DNA strand segregation.

Sorting DNA with asymmetry: a new player in gene regulation?

In recent years, our views on how DNA and genes are organised and regulated have evolved significantly. One example is provided by reports that single DNA strands in the double helix could carry distinct forms of information. That chromatids carrying old and nascently replicated DNA strands are recognised by the mitotic machinery, then segregated in a concerted way to distinct daughter cells after cell division is remarkable. Notably, this phenomenon in several cases has been associated with the cell fate choice of resulting daughter cells. Here, we review the evidence for asymmetric or template DNA strand segregation in mammals with a focus on skeletal muscle.

Differential Gene Expression in Nuclear Label-Retaining Cells in the Developing Mouse Mammary Gland

The immortal strand theory postulates stem cells protect themselves from DNA replication-associated mutations and subsequent cancer risk through selective segregation of template DNA strands. Stem cells self-renew by asymmetric cellular division. During asymmetric division, stem cells maintain their template DNA strands, while the newly synthesized DNA strands segregate to newly formed daughter cells. Previous studies have demonstrated that self-renewing mammary stem cells originate in the expanding mammary ducts during puberty-associated allometric growth. In this study, we labeled newly forming mammary stem cells with the thymidine analog 5-ethynl-2'-deoxyuridine for 2 weeks during allometric ductal expansion. Cells that incorporate and retain the nuclear label following extended chase periods are termed label-retaining cells (LRCs). A second nuclear label, 5-bromodeoxyuridine, was administered before euthanasia to identify cells traversing the cell cycle. Mammary cells collected following euthanasia were sorted based on nuclear label retention. Members of the Notch and Wnt signaling pathways were found differentially expressed by mammary LRCs. These pathways are involved in the regulation of stem cells in the mouse mammary gland. Upon further analysis, we found that in contrast to non-LRCs, Notch1 and Notch2 are expressed and localized in the nuclei of the LRCs. Expression of Notch-inducible genes, Hes1 and Hey2, was elevated in LRCs. Inhibition of Notch1 by shRNA reduced colony forming potential and label retention by mammary epithelial cells in vitro. These results indicate that genes are differentially regulated in the LRC population of mammary glands and Notch1 mediates asymmetric cell division of mammary progenitor cells.

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.

A pilot study of raltegravir and cisplatin in head and neck squamous cell carcinoma (HNSCC).

TPS5602 Background: Metnase is a recently characterized DNA repair component present in anthropoid primates. Metnase, the fusion of a SET histone methylase domain and a Transposase nuclease (Tn) domain, enhances DNA double-stranded break (DSB) repair. The Tn domain trims free DNA ends for optimal end-joining, and is required to re-start stalled replication forks. The SET domain di-methylates H3K36 at the DSB, recruiting non-homologous end-joining repair components. Cisplatin (CDDP), the central chemotherapy in HNSCC, covalently binds DNA leading to intra- and interstrand crosslinks (ICL). In addition to blocking strand transcription and segregation, the ICL stalls DNA replication fork progression leading to collapse and DSB. The role of Metnase in DNA repair, including overcoming the stalled replication fork, makes it a potential therapeutic target. We hypothesize that Metnase inhibition may be a useful adjunct to CDDP. 3D modeling of the Metnase Tn domain identified significant homology with human immunodeficiency virus 1 (HIV-1) integrase. A virtual screen of the Chem Div library identified the HIV-1 integrase inhibitor, raltegravir, as a lead compound for inhibiting the Metnase Tn domain. We designed a pilot study in HNSCC to evaluate potentiation of CDDP DNA damage by raltegravir. Methods: The primary objective of this window-of-opportunity study is to analyze biomarkers of DNA damage, fork arrest, and apoptosis in serial tumor biopsies from patients (pts) with HNSCC undergoing CDDP, with and without raltegravir. Eligible pts: 1) have HNSCC with tumor site amenable to repeat, awake biopsy; 2) are appropriate candidates for CDDP. Pts are treated with 2 doses of CDDP 30 mg/m2. One CDDP dose is administered with raltegravir 400 mg bid for 5 days, starting the day before CDDP. Pts undergo 3 research biopsies, at baseline and 48-72 hours after each CDDP. Serial biopsies will be evaluated for expression changes in γH2AX, Annexin V, pChk1, pChk2, and p53BP1 by immunohistochemistry. Numerical increase in biomarker expression in tumors exposed to CDDP-raltegravir vs. CDDP will justify development of a phase I/II study. Four of 12 pts have enrolled and completed all biopsies. Supported by a grant from the American Cancer Society.

Parameter estimation for an immortal model of colonic stem cell division using approximate Bayesian computation

In this paper we use approximate Bayesian computation to estimate the parameters in an immortal model of colonic stem cell division. We base the inferences on the observed DNA methylation patterns of cells sampled from the human colon. Utilising DNA methylation patterns as a form of molecular clock is an emerging area of research and has been used in several studies investigating colonic stem cell turnover. There is much debate concerning the two competing models of stem cell turnover: the symmetric (immortal) and asymmetric models. Early simulation studies concluded that the observed methylation data were not consistent with the immortal model. A later modified version of the immortal model that included preferential strand segregation was subsequently shown to be consistent with the same methylation data. Most of this earlier work assumes site independent methylation models that do not take account of the known processivity of methyltransferases whilst other work does not take into account the methylation errors that occur in differentiated cells. This paper addresses both of these issues for the immortal model and demonstrates that approximate Bayesian computation provides accurate estimates of the parameters in this neighbour-dependent model of methylation error rates. The results indicate that if colonic stem cells divide asymmetrically then colon stem cell niches are maintained by more than 8 stem cells. Results also indicate the possibility of preferential strand segregation and provide clear evidence against a site-independent model for methylation errors. In addition, algebraic expressions for some of the summary statistics used in the approximate Bayesian computation (that allow for the additional variation arising from cell division in differentiated cells) are derived and their utility discussed.