Browsing by Subject "Cell Cycle"

Now showing 1 - 12 of 12
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    A genetic memory initiates the epigenetic loop necessary to preserve centromere position.
    (The EMBO journal, 2020-10) Hoffmann, Sebastian; Izquierdo, Helena M; Gamba, Riccardo; Chardon, Florian; Dumont, Marie; Keizer, Veer; Hervé, Solène; McNulty, Shannon M; Sullivan, Beth A; Manel, Nicolas; Fachinetti, Daniele
    Centromeres are built on repetitive DNA sequences (CenDNA) and a specific chromatin enriched with the histone H3 variant CENP-A, the epigenetic mark that identifies centromere position. Here, we interrogate the importance of CenDNA in centromere specification by developing a system to rapidly remove and reactivate CENP-A (CENP-AOFF/ON ). Using this system, we define the temporal cascade of events necessary to maintain centromere position. We unveil that CENP-B bound to CenDNA provides memory for maintenance on human centromeres by promoting de novo CENP-A deposition. Indeed, lack of CENP-B favors neocentromere formation under selective pressure. Occasionally, CENP-B triggers centromere re-activation initiated by CENP-C, but not CENP-A, recruitment at both ectopic and native centromeres. This is then sufficient to initiate the CENP-A-based epigenetic loop. Finally, we identify a population of CENP-A-negative, CENP-B/C-positive resting CD4+ T cells capable to re-express and reassembles CENP-A upon cell cycle entry, demonstrating the physiological importance of the genetic memory.
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    c-Myc is required for maintenance of glioma cancer stem cells.
    (PLoS One, 2008) Wang, J; Wang, H; Li, Z; Wu, Q; Lathia, JD; McLendon, RE; Hjelmeland, AB; Rich, JN
    BACKGROUND: Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G(0)/G(1) phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice. CONCLUSIONS/SIGNIFICANCE: These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.
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    Cell cycle Start is coupled to entry into the yeast metabolic cycle across diverse strains and growth rates.
    (Mol Biol Cell, 2016-01-01) Burnetti, Anthony J; Aydin, Mert; Buchler, Nicolas E
    Cells have evolved oscillators with different frequencies to coordinate periodic processes. Here we studied the interaction of two oscillators, the cell division cycle (CDC) and the yeast metabolic cycle (YMC), in budding yeast. Previous work suggested that the CDC and YMC interact to separate high oxygen consumption (HOC) from DNA replication to prevent genetic damage. To test this hypothesis, we grew diverse strains in chemostat and measured DNA replication and oxygen consumption with high temporal resolution at different growth rates. Our data showed that HOC is not strictly separated from DNA replication; rather, cell cycle Start is coupled with the initiation of HOC and catabolism of storage carbohydrates. The logic of this YMC-CDC coupling may be to ensure that DNA replication and cell division occur only when sufficient cellular energy reserves have accumulated. Our results also uncovered a quantitative relationship between CDC period and YMC period across different strains. More generally, our approach shows how studies in genetically diverse strains efficiently identify robust phenotypes and steer the experimentalist away from strain-specific idiosyncrasies.
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    Comparison of pattern detection methods in microarray time series of the segmentation clock.
    (PLoS One, 2008-08-06) Dequéant, Mary-Lee; Ahnert, Sebastian; Edelsbrunner, Herbert; Fink, Thomas MA; Glynn, Earl F; Hattem, Gaye; Kudlicki, Andrzej; Mileyko, Yuriy; Morton, Jason; Mushegian, Arcady R; Pachter, Lior; Rowicka, Maga; Shiu, Anne; Sturmfels, Bernd; Pourquié, Olivier
    While genome-wide gene expression data are generated at an increasing rate, the repertoire of approaches for pattern discovery in these data is still limited. Identifying subtle patterns of interest in large amounts of data (tens of thousands of profiles) associated with a certain level of noise remains a challenge. A microarray time series was recently generated to study the transcriptional program of the mouse segmentation clock, a biological oscillator associated with the periodic formation of the segments of the body axis. A method related to Fourier analysis, the Lomb-Scargle periodogram, was used to detect periodic profiles in the dataset, leading to the identification of a novel set of cyclic genes associated with the segmentation clock. Here, we applied to the same microarray time series dataset four distinct mathematical methods to identify significant patterns in gene expression profiles. These methods are called: Phase consistency, Address reduction, Cyclohedron test and Stable persistence, and are based on different conceptual frameworks that are either hypothesis- or data-driven. Some of the methods, unlike Fourier transforms, are not dependent on the assumption of periodicity of the pattern of interest. Remarkably, these methods identified blindly the expression profiles of known cyclic genes as the most significant patterns in the dataset. Many candidate genes predicted by more than one approach appeared to be true positive cyclic genes and will be of particular interest for future research. In addition, these methods predicted novel candidate cyclic genes that were consistent with previous biological knowledge and experimental validation in mouse embryos. Our results demonstrate the utility of these novel pattern detection strategies, notably for detection of periodic profiles, and suggest that combining several distinct mathematical approaches to analyze microarray datasets is a valuable strategy for identifying genes that exhibit novel, interesting transcriptional patterns.
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    Detecting separate time scales in genetic expression data.
    (BMC Genomics, 2010-06-16) Orlando, David A; Brady, Siobhan M; Fink, Thomas MA; Benfey, Philip N; Ahnert, Sebastian E
    BACKGROUND: Biological processes occur on a vast range of time scales, and many of them occur concurrently. As a result, system-wide measurements of gene expression have the potential to capture many of these processes simultaneously. The challenge however, is to separate these processes and time scales in the data. In many cases the number of processes and their time scales is unknown. This issue is particularly relevant to developmental biologists, who are interested in processes such as growth, segmentation and differentiation, which can all take place simultaneously, but on different time scales. RESULTS: We introduce a flexible and statistically rigorous method for detecting different time scales in time-series gene expression data, by identifying expression patterns that are temporally shifted between replicate datasets. We apply our approach to a Saccharomyces cerevisiae cell-cycle dataset and an Arabidopsis thaliana root developmental dataset. In both datasets our method successfully detects processes operating on several different time scales. Furthermore we show that many of these time scales can be associated with particular biological functions. CONCLUSIONS: The spatiotemporal modules identified by our method suggest the presence of multiple biological processes, acting at distinct time scales in both the Arabidopsis root and yeast. Using similar large-scale expression datasets, the identification of biological processes acting at multiple time scales in many organisms is now possible.
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    Human-chimpanzee differences in a FZD8 enhancer alter cell-cycle dynamics in the developing neocortex.
    (Curr Biol, 2015-03-16) Boyd, J Lomax; Skove, Stephanie L; Rouanet, Jeremy P; Pilaz, Louis-Jan; Bepler, Tristan; Gordân, Raluca; Wray, Gregory A; Silver, Debra L
    The human neocortex differs from that of other great apes in several notable regards, including altered cell cycle, prolonged corticogenesis, and increased size [1-5]. Although these evolutionary changes most likely contributed to the origin of distinctively human cognitive faculties, their genetic basis remains almost entirely unknown. Highly conserved non-coding regions showing rapid sequence changes along the human lineage are candidate loci for the development and evolution of uniquely human traits. Several studies have identified human-accelerated enhancers [6-14], but none have linked an expression difference to a specific organismal trait. Here we report the discovery of a human-accelerated regulatory enhancer (HARE5) of FZD8, a receptor of the Wnt pathway implicated in brain development and size [15, 16]. Using transgenic mice, we demonstrate dramatic differences in human and chimpanzee HARE5 activity, with human HARE5 driving early and robust expression at the onset of corticogenesis. Similar to HARE5 activity, FZD8 is expressed in neural progenitors of the developing neocortex [17-19]. Chromosome conformation capture assays reveal that HARE5 physically and specifically contacts the core Fzd8 promoter in the mouse embryonic neocortex. To assess the phenotypic consequences of HARE5 activity, we generated transgenic mice in which Fzd8 expression is under control of orthologous enhancers (Pt-HARE5::Fzd8 and Hs-HARE5::Fzd8). In comparison to Pt-HARE5::Fzd8, Hs-HARE5::Fzd8 mice showed marked acceleration of neural progenitor cell cycle and increased brain size. Changes in HARE5 function unique to humans thus alter the cell-cycle dynamics of a critical population of stem cells during corticogenesis and may underlie some distinctive anatomical features of the human brain.
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    Mating in wild yeast: delayed interest in sex after spore germination.
    (Molecular biology of the cell, 2018-12) McClure, Allison W; Jacobs, Katherine C; Zyla, Trevin R; Lew, Daniel J
    Studies of laboratory strains of Saccharomyces cerevisiae have uncovered signaling pathways involved in mating, including information-processing strategies to optimize decisions to mate or to bud. However, lab strains are heterothallic (unable to self-mate), while wild yeast are homothallic. And while mating of lab strains is studied using cycling haploid cells, mating of wild yeast is thought to involve germinating spores. Thus, it was unclear whether lab strategies would be appropriate in the wild. Here, we have investigated the behavior of several yeast strains derived from wild isolates. Following germination, these strains displayed large differences in their propensity to mate or to enter the cell cycle. The variable interest in sex following germination was correlated with differences in pheromone production, which were due to both cis- and trans-acting factors. Our findings suggest that yeast spores germinating in the wild may often enter the cell cycle and form microcolonies before engaging in mating.
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    Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.
    (Mol Biol Cell, 2014-11-05) Mazo-Vargas, Anyimilehidi; Park, Heungwon; Aydin, Mert; Buchler, Nicolas E
    Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15-20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.
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    Stochastic E2F activation and reconciliation of phenomenological cell-cycle models.
    (PLoS Biol, 2010-09-21) Lee, Tae J; Yao, Guang; Bennett, Dorothy C; Nevins, Joseph R; You, Lingchong
    The transition of the mammalian cell from quiescence to proliferation is a highly variable process. Over the last four decades, two lines of apparently contradictory, phenomenological models have been proposed to account for such temporal variability. These include various forms of the transition probability (TP) model and the growth control (GC) model, which lack mechanistic details. The GC model was further proposed as an alternative explanation for the concept of the restriction point, which we recently demonstrated as being controlled by a bistable Rb-E2F switch. Here, through a combination of modeling and experiments, we show that these different lines of models in essence reflect different aspects of stochastic dynamics in cell cycle entry. In particular, we show that the variable activation of E2F can be described by stochastic activation of the bistable Rb-E2F switch, which in turn may account for the temporal variability in cell cycle entry. Moreover, we show that temporal dynamics of E2F activation can be recast into the frameworks of both the TP model and the GC model via parameter mapping. This mapping suggests that the two lines of phenomenological models can be reconciled through the stochastic dynamics of the Rb-E2F switch. It also suggests a potential utility of the TP or GC models in defining concise, quantitative phenotypes of cell physiology. This may have implications in classifying cell types or states.
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    Survivin as a therapeutic target in Sonic hedgehog-driven medulloblastoma.
    (Oncogene, 2015-07) Brun, SN; Markant, SL; Esparza, LA; Garcia, G; Terry, D; Huang, J-M; Pavlyukov, MS; Li, X-N; Grant, GA; Crawford, JR; Levy, ML; Conway, EM; Smith, LH; Nakano, I; Berezov, A; Greene, MI; Wang, Q; Wechsler-Reya, RJ
    Medulloblastoma (MB) is a highly malignant brain tumor that occurs primarily in children. Although surgery, radiation and high-dose chemotherapy have led to increased survival, many MB patients still die from their disease, and patients who survive suffer severe long-term side effects as a consequence of treatment. Thus, more effective and less toxic therapies for MB are critically important. Development of such therapies depends in part on identification of genes that are necessary for growth and survival of tumor cells. Survivin is an inhibitor of apoptosis protein that regulates cell cycle progression and resistance to apoptosis, is frequently expressed in human MB and when expressed at high levels predicts poor clinical outcome. Therefore, we hypothesized that Survivin may have a critical role in growth and survival of MB cells and that targeting it may enhance MB therapy. Here we show that Survivin is overexpressed in tumors from patched (Ptch) mutant mice, a model of Sonic hedgehog (SHH)-driven MB. Genetic deletion of survivin in Ptch mutant tumor cells significantly inhibits proliferation and causes cell cycle arrest. Treatment with small-molecule antagonists of Survivin impairs proliferation and survival of both murine and human MB cells. Finally, Survivin antagonists impede growth of MB cells in vivo. These studies highlight the importance of Survivin in SHH-driven MB, and suggest that it may represent a novel therapeutic target in patients with this disease.
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    Ubiquitylation of p53 by the APC/C inhibitor Trim39.
    (Proc Natl Acad Sci U S A, 2012-12-18) Zhang, Liguo; Huang, Nai-Jia; Chen, Chen; Tang, Wanli; Kornbluth, Sally
    Tripartite motif 39 (Trim39) is a RING domain-containing E3 ubiquitin ligase able to inhibit the anaphase-promoting complex (APC/C) directly. Through analysis of Trim39 function in p53-positive and p53-negative cells, we have found, surprisingly, that p53-positive cells lacking Trim39 could not traverse the G1/S transition. This effect did not result from disinhibition of the APC/C. Moreover, although Trim39 loss inhibited etoposide-induced apoptosis in p53-negative cells, apoptosis was enhanced by Trim39 knockdown in p53-positive cells. Furthermore, we show here that the Trim39 can directly bind and ubiquitylate p53 in vitro and in vivo, leading to p53 degradation. Depletion of Trim39 significantly increased p53 protein levels and cell growth retardation in multiple cell lines. We found that the relative importance of Trim39 and the well-characterized p53-directed E3 ligase, murine double minute 2 (MDM2), varied between cell types. In cells that were relatively insensitive to the MDM2 inhibitor, nutlin-3a, apoptosis could be markedly enhanced by siRNA directed against Trim39. As such, Trim39 may serve as a potential therapeutic target in tumors with WT p53 when MDM2 inhibition is insufficient to elevate p53 levels and apoptosis.
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    Yeast screens identify the RNA polymerase II CTD and SPT5 as relevant targets of BRCA1 interaction.
    (PLoS One, 2008-01-16) Bennett, Craig B; Westmoreland, Tammy J; Verrier, Carmel S; Blanchette, Carrie AB; Sabin, Tiffany L; Phatnani, Hemali P; Mishina, Yuliya V; Huper, Gudrun; Selim, Alice L; Madison, Ernest R; Bailey, Dominique D; Falae, Adebola I; Galli, Alvaro; Olson, John A; Greenleaf, Arno L; Marks, Jeffrey R
    BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1) to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34) and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1). Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII) carboxy terminal domain (P-CTD), phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1 mutant breast cells. These results extend the mechanistic links between BRCA1 and transcriptional consequences in response to DNA damage and suggest an important role for RNAPII P-CTD cleavage in BRCA1-mediated cancer suppression.