Sumario:
The International Journal of Developmental Biology
Linking Development, Stem Cells and Cancer Research
Euskal Herriko Unibertsitateko Argitalpen Zerbitzua / Servicio Editorial de la Universidad del País Vasco / University of the Basque Country Press
Volume 60 - Numbers 7-8-9 (2016) / Pages 189-320 Editor-in-Chief: Juan Aréchaga
MORE INFORMATION [Abstract - FullText / Full Text Open Access]
ISSN: 0214-6282 / ISSN-e: 1696-3547 www.intjdevbiol.com
----------------------------------------------------------------------------------------------------------------------
Special Issue: Cell-free Extracts in Development & Cancer Research
Guest Editors: Jacek Z. Kubiak
CONTENTS
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 191 (2016)
doi: 10.1387/ijdb.160222jk / © UBC Press ( www.a360grados.net )
Preface: Cell-free extracts in Development and Cancer Research for over 40 years
Jacek Z. Kubiak
CNRS, UMR6290 and Université de Rennes 1, Institut de Génétique et Développement de Rennes, Rennes, France and Department of Regenerative Medicine, Military Institute of Hygiene and Epidemiology, Warsaw, Poland
Abstract: Analysis of cell-free extracts has allowed us to understand many of the fundamental processes of cell physiology and pathology, including those involved in embryo development and cancer. This methodology is being continuously modified and improved. Papers selected for this Special Issue will show readers the plethora of systems and applications of this methodology.
--------------------------------------------------------------------------------------------------
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 193-200 (2016)
doi: 10.1387/ijdb.160348jk / © UBC Press ( www.a360grados.net )
MPF, starfish oocyte and cell-free extract in the background - an interview with Takeo Kishimoto
Jacek Z. Kubiak 1,2 and Takeo Kishimoto 3
1. CNRS, UMR 6290, Institute of Genetics and Development of Rennes, Cell Cycle Group, Rennes, France
2. University Rennes 1, UEB, IFR 140, Faculty of Medicine, Rennes, France
3. Ochanomizu University, Science & Education Center, Bunkyo-ku, Tokyo, Japan
Abstract: Professor Takeo Kishimoto’s research has an enormous impact on the cell cycle field. Although his favorite model has always been a starfish oocyte, he has used many other model organisms in his research. Cell-free extracts have been wildly used in his laboratory as a very useful tool to answer cell cycle research questions. Recently, professor Kishimoto discovered the identity of the M-phase promoting factor (MPF) that was thought for years to be cyclin-dependent kinase 1 (CDK1). However, Takeo Kishimoto found that MPF consists in fact of two kinases: CDK1 and Greatwall kinase. While CDK1 phosphorylates mitotic substrates, Greatwall kinase allows these substrates to persist in their phosphorylated state because it regulates phosphatase PP2A, which dephosphorylates the majority of CDK1 substrates. When I started to interview Prof. Kishimoto, I was mostly interested in his experiences with cell-free extracts. However, as you will see below we almost immediately turned to the problem of the identity of MPF. This is fully understandable because the identity of MPF seems to be a major interest in Takeo’s scientific career. I hope readers will enjoy this interview and will be able to learn about many aspects of scientific research, which do not usually appear in regular research papers.
Keywords: CDK1, Cdc2, p34, Greatwall kinase, PP2A, cell cycle, cyclin B, oocyte maturation, MPF
Introductory papers ---------------------------------------------------
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 201-207 (2016)
doi: 10.1387/ijdb.160142jb / © UBC Press ( www.a360grados.net )
Xenopus cell-free extracts and their contribution to the study of DNA replication and other complex biological processes
J. Julian Blow 1 and Ronald A. Laskey 2
1. School of Life Sciences, University of Dundee, Dundee
2. Department of Zoology, University of Cambridge, Cambridge, UK
Abstract: Here we discuss the important contributions that cell-free extracts have made to the study of complex biological processes. We provide a brief history of how cell-free extracts of frog eggs were developed to avoid many of the problems that can arise from the dilution and mixing of cellular components that typically occur when cell-free extracts are prepared. We briefly describe how Xenopus egg extracts have been fundamental to the study of many important cellular processes including DNA replication, cell cycle progression, nuclear protein import, nuclear assembly and chromosome organisation. We describe how, in particular, Xenopus egg extracts have made a major contributions to the study of DNA replication, by permitting the direct manipulation of proteins in a system that is extraordinarily faithful to the way that DNA replication occurs in the living embryo. Finally we consider how results obtained using Xenopus egg extracts are being translated to produce diagnostic reagents for cancer screening and diagnosis.
Keywords: Xenopus, frog, egg, cell-free extract, in vitro, DNA replication
--------------------------------------------------------------------------------------------------
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 209-220 (2016)
doi: 10.1387/ijdb.160077dp / © UBC Press ( www.a360grados.net )
A sea urchin cell-free system to study male pronuclear assembly and activation
Dominic Poccia
Department of Biology, Amherst College, Amherst, MA, USA
Abstract: Typically sperm nuclei are genetically inert and contain extremely compacted chromatin. Following fertilization, the first steps in their conversion to somatic nuclei (male pronuclei) which will support further development involve chromatin decondensation and the formation of a new nuclear envelope. We have studied the reactivation of sea urchin sperm nuclei in a cell-free system derived from homogenates of activated sea urchin egg cytoplasm. The cell-free system has provided several novel insights including requirements for sperm-specific histone phosphorylation on N- and C-terminal extensions and disassembly of the sperm nuclear lamina for decondensation, the utilization of remnant regions of the sperm nuclear envelope to direct polarized binding and fusion of egg membranes to form the new nuclear envelope, and a role for phosphoinositide metabolism in initiation of membrane fusion through binding of a minor membrane fraction enriched in PtdIns (4,5)P2, PLCγ and SFK1 which locally produces a fusigenic lipid, diacylglycerol.
Keywords: male pronucleus, chromatin, nuclear envelope, membrane fusion, sea urchin
--------------------------------------------------------------------------------------------------
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 221-227 (2016)
doi: 10.1387/ijdb.160103vc / © UBC Press ( www.a360grados.net )
Studying essential DNA metabolism proteins in Xenopus egg extract
Vincenzo Sannino, Arun M. Kolinjivadi, Giorgio Baldi and Vincenzo Costanzo
DNA metabolism Laboratory, IFOM, The FIRC institute of Molecular Oncology, Milan, Italy
Abstract: The correct duplication of genetic information is essential to maintain genome stability, which is lost in cancer cells. Replication fork integrity is ensured by a number of DNA metabolism proteins that assist replication of chromatin regions difficult to replicate due to their intrinsic DNA sequence composition, coordinate repair of DNA molecules resulting from aberrant replication events or protect replication forks in the presence of lesions impairing their progression. Some DNA metabolism genes involved in DNA repair are essential in higher eukaryotes even in unchallenged conditions, suggesting the existence of biological processes requiring these specialized functions in organisms with complex genomes. The impact on cell survival of null mutants of many DNA metabolism genes has precluded complete in depth analysis of their function. Cell free extracts represent a fundamental tool to overcome survival issues. The Xenopus laevis egg cell free extract is an ideal system to study replication-associated functions of essential genes. We are taking advantage of this system together with innovative imaging and proteomic based experimental approaches to characterize the molecular function of essential DNA metabolism proteins. Using this approach we have uncovered the role of some essential homologous recombination and fork protection proteins in chromosomal DNA replication and we have characterized some of the factors required for faithful replication of specific vertebrate genomic regions. This approach will be instrumental to study the molecular mechanisms underlying the function of a number of essential DNA metabolism proteins involved in the maintenance of genome stability in complex genomes.
Keywords: Xenopus laevis, immuno-depletion, DNA replication, DNA recombination and reversed forks
Review articles ------------------------------------------------------------
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 229-236 (2016)
doi: 10.1387/ijdb.160113sy / © UBC Press ( www.a360grados.net )
Cell-free Xenopus egg extracts for studying DNA damage response pathways
Steven Cupello, Christine Richardson and Shan Yan
Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC, USA
Abstract: In response to a variety of DNA replication stress or DNA damaging agents, the DNA damage response (DDR) pathways are triggered for cells to coordinate DNA repair, cell cycle checkpoints, apoptosis, and senescence. Cell-free Xenopus egg extracts, derived from the eggs of African clawed frogs (Xenopus laevis), have been widely used for studies concerning DDR pathways. In this review, we focus on how different experimental systems have been established using Xenopus egg extracts to investigate the DDR pathways that are activated in response to DNA replication stress, double-strand breaks (DSBs), inter-strand crosslinks (ICLs), and oxidative stress. We summarize how molecular details of DDR pathways are dissected by the mechanistic studies with Xenopus egg extracts. We also provide an update on the regulation of translesion DNA synthesis (TLS) polymerases (Pol ĸ and REV1) in the DDR pathways. A better understanding of DDR pathways using Xenopus egg extracts has opened new avenues for future cancer therapeutics. Finally, we offer our perspectives of future directions for studies of DDR pathways with Xenopus egg extracts.
Keywords: Xenopus egg extracts, DNA damage response, ATR-Chk1, ATM-Chk2, TLS
--------------------------------------------------------------------------------------------------
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 237-244 (2016)
doi: 10.1387/ijdb.160125bb / © UBC Press ( www.a360grados.net )
Escherichia coli-based cell-free extract development for protein-based cancer therapeutic production
Amin S.M. Salehi, Conner C. Earl, Christina Muhlestein and Bradley C. Bundy
Department of Chemical Engineering, Brigham Young University, Provo, UT, USA
Abstract: Cell-free protein synthesis has been around for decades but it has never been close to becoming a robust tool for the production of biotherapeutic agents. In this review, we focus on how Escherichia coli-based cell-free protein synthesis can be modified in various ways to produce challenging, complex anticancer biotherapeutics. Here we report progress in extract preparation and its relation to cell-free cancer research. The future prospects of cell-free technology and its potential in various areas of cancer therapeutics production are also highlighted.
Keywords: cell-free protein synthesis, cell extract, cancer research, anticancer biotherapeutics
--------------------------------------------------------------------------------------------------
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 245-254 (2016)
doi: 10.1387/ijdb.160155tl / © UBC Press ( www.a360grados.net )
The master Greatwall kinase, a critical regulator of mitosis and meiosis
Suzanne Vigneron, Perle Robert, Khaled Hached, Lena Sundermann, Sophie Charrasse, Jean-Claude Labbé, Anna Castro and Thierry Lorca
Equipe Labellisée Ligue Contre le Cancer. Université de Montpellier, Centre de Recherche de Biologie Cellulaire de Montpellier, CNRS UMR 5237, France
Abstract: Entry into mitosis requires the coordinated activation of various protein kinases and phosphatases that together activate sequential signaling pathways allowing entry, progression and exit of mitosis. The limiting step is thought to be the activation of the mitotic Cdk1-cyclin B kinase. However, this model has recently evolved with new data showing that in addition to the Cdk1-cyclin B complex, Greatwall (Gwl) kinase is also required to enter into and maintain mitosis. This new concept proposes that entry into mitosis is now based on the combined activation of both kinases Cdk1-cyclin B and Gwl, the former promoting massive phosphorylation of mitotic substrates and the latter inhibiting PP2A-B55 phosphatase responsible for dephosphorylation of these substrates. Activated Gwl phosphorylates both Arpp19 and ENSA, which associate and inhibit PP2A-B55. This pathway seems relatively well conserved from yeast to humans, although some differences appear based on models or techniques used. While Gwl is activated by phosphorylation, its inactivation requires dephosphorylation of critical residues. Several phosphatases such as PP1, PP2A-B55 and FCP1 are required to control the dephosphorylation and inactivation of Gwl and a properly regulated mitotic exit. Gwl has also been reported to be involved in cancer processes and DNA damage recovery. These new findings support the idea that the Gwl-Arpp19/ENSA-PP2A-B55 pathway is essential to achieve an efficient division of cells and to maintain genomic stability.
Keywords: Greatwall, Arpp19, ENSA, mitosis, meiosis
--------------------------------------------------------------------------------------------------
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 255-262 (2016)
doi: 10.1387/ijdb.160189jk / © UBC Press ( www.a360grados.net )
Aurora-A: an expedition to the pole of the spindle in Xenopus egg extracts
Jacek Z. Kubiak 1,2,3 and Claude Prigent 1,2
1. CNRS, Unité Mixte de Recherche UMR6290
2. Université de Rennes 1, Institut de Génétique et Développement de Rennes, UEB, 35000 Rennes, France
3. Department of Hygiene and Physiology, Military Institute of Hygiene and Epidemiology, Warsaw, Poland
Abstract: The aim of this short review is to describe the contribution of Xenopus laevis egg extracts to the discovery and understanding of the regulation and function of the serine/threonine kinase Aurora-A. The power of these extracts to recapitulate cell cycle events makes them a precious tool to decipher complex biological processes at the molecular level, including the mechanisms that affect Aurora-A (post-translational modifications) and mechanisms in which Aurora-A plays a crucial role (bipolar spindle assembly). We focus on the results obtained in cell-free extracts, but we also give an updated overview of Aurora A functions found in other systems.
Keywords: Aurora-A, Eg2, Ipl1, cell-free extract, Xenopus laevis, TPX2, spindle assembly
--------------------------------------------------------------------------------------------------
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 263-270 (2016)
doi: 10.1387/ijdb.160186gm / © UBC Press ( www.a360grados.net )
Ubiquitin-mediated proteolysis in Xenopus extract
Gary S. McDowell 1,2,3 and Anna Philpott 4,5
1. Center for Regenerative and Developmental Biology, Department of Biology, Tufts University, Medford, MA, USA
2. The Future of Research (www.futureofresearch.org), Abington, MA
3. Manylabs (www.manylabs.org), San Francisco, CA, USA
4. Department of Oncology, MRC/Hutchison Research Centre, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK
5. Wellcome Trust – Medical Research Council, Cambridge Stem Cell Institute, Cambridge, UK
Abstract: The small protein modifier, ubiquitin, can be covalently attached to proteins in the process of ubiquitylation, resulting in a variety of functional outcomes. In particular, the most commonly-associated and well-studied fate for proteins modified with ubiquitin is their ultimate destruction: degradation by the 26S proteasome via the ubiquitin-proteasome system, or digestion in lysosomes by proteolytic enzymes. From the earliest days of ubiquitylation research, a reliable and versatile “cell-in-a-test-tube” system has been employed in the form of cytoplasmic extracts from the eggs and embryos of the frog Xenopus laevis. Biochemical studies of ubiquitin and protein degradation using this system have led to significant advances particularly in the study of ubiquitin-mediated proteolysis, while the versatility of Xenopus as a developmental model has allowed investigation of the in vivo consequences of ubiquitylation. Here we describe the use and history of Xenopus extract in the study of ubiquitin-mediated protein degradation, and highlight the versatility of this system that has been exploited to uncover mechanisms and consequences of ubiquitylation and proteolysis.
Keywords: extract system, ubiquitin, degron, protein degradation, 26S proteasome, Xenopus
--------------------------------------------------------------------------------------------------
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 271-276 (2016)
doi: 10.1387/ijdb.160188ds / © UBC Press ( www.a360grados.net )
Chaperone-mediated chromatin assembly and transcriptional regulation in Xenopus laevis
Takashi Onikubo and David Shechter
Department of Biochemistry. Albert Einstein College of Medicine, Bronx, NY, USA
Abstract: Chromatin is the complex of DNA and histone proteins that is the physiological form of the eukaryotic genome. Chromatin is generally repressive for transcription, especially so during early metazoan development when maternal factors are explicitly in control of new zygotic gene expression. In the important model organism Xenopus laevis, maturing oocytes are transcriptionally active with reduced rates of chromatin assembly, while laid eggs and fertilized embryos have robust rates of chromatin assembly and are transcriptionally repressed. As the DNA-to-cytoplasmic ratio decreases approaching the mid-blastula transition (MBT) and the onset of zygotic genome activation (ZGA), the chromatin assembly process changes with the concomitant reduction in maternal chromatin components. Chromatin assembly is mediated in part by histone chaperones that store maternal histones and release them into new zygotic chromatin. Here, we review literature on chromatin and transcription in frog embryos and cell-free extracts and highlight key insights demonstrating the roles of maternal and zygotic histone deposition and their relationship with transcriptional regulation. We explore the central historical and recent literature on the use of Xenopus embryos and the key contributions provided by experiments in cell-free oocyte and egg extracts for the interplay between histone chaperones, chromatin assembly, and transcriptional regulation. Ongoing and future studies in Xenopus cell free extracts will likely contribute essential new insights into the interplay between chromatin assembly and transcriptional regulation.
Keywords: transcription, histone chaperone, H2A-H2B, H3-H4
--------------------------------------------------------------------------------------------------
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 277-288 (2016)
doi: 10.1387/ijdb.160158dl / © UBC Press ( www.a360grados.net )
Use of Xenopus cell-free extracts to study size regulation of subcellular structures
Predrag Jevtić, Ana Milunović-Jevtić, Matthew R. Dilsaver, Jesse C. Gatlin and Daniel L. Levy
Department of Molecular Biology, University of Wyoming, Laramie, WY, USA
Abstract: Striking size variations are prominent throughout biology, at the organismal, cellular, and subcellular levels. Important fundamental questions concern organelle size regulation and how organelle size is regulated relative to cell size, also known as scaling. Uncovering mechanisms of organelle size regulation will inform the functional significance of size as well as the implications of misregulated size, for instance in the case of nuclear enlargement in cancer. Xenopus egg and embryo extracts are powerful cell-free systems that have been utilized extensively for mechanistic and functional studies of various organelles and subcellular structures. The open biochemical nature of the extract permits facile manipulation of its composition, and in recent years extract approaches have illuminated mechanisms of organelle size regulation. This review largely focuses on in vitro Xenopus studies that have identified regulators of nuclear and spindle size. We also discuss potential relationships between size scaling of the nucleus and spindle, size regulation of other subcellular structures, and extract experiments that have clarified developmental timing mechanisms. We conclude by offering some future prospects, notably the integration of Xenopus extract with microfluidic technology.
Keywords: nuclear size, spindle size, developmental size scaling, midblastula transition, cancer, microfluidics
--------------------------------------------------------------------------------------------------
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 289-296 (2016)
doi: 10.1387/ijdb.160163at / © UBC Press ( www.a360grados.net )
Reprogramming of somatic cells and nuclei by Xenopus oocyte and egg extracts
Alexander A. Tokmakov 1,2, Tetsushi Iwasaki 2, Ken-Ichi Sato 1 and Shinji Kamada 2
1. Department of Molecular Biosciences, Kyoto Sangyo University, Kyoto
2. Research Center for Environmental Genomics, Kobe University, Kobe, Japan
Abstract: Differentiated somatic cells and nuclei can be reprogrammed to a pluripotent undifferentiated state in the cytoplasm of oocytes and eggs. The ability of the gamete cells to induce reprogramming is not species-specific, so the extracts prepared from the oocytes and eggs of the African clawed frog Xenopus laevis can reprogram somatic mammalian cells. Thus, Xenopus egg extract-mediated reprogramming may constitute an alternative or complement other experimental reprogramming approaches, such as nuclear transfer, cell fusion, and transcription factor transduction. Here, we discuss the major reprogramming events induced by the extracts in somatic nuclei and cells, including remodeling of nuclear structure, replacement of somatic proteins with their embryonic counterparts, epigenetic modification of DNA and histones, transcriptional reprogramming, and initiation of DNA replication. We also address the advantages and limitations of the extract-based reprogramming approach.
Keywords: Xenopus laevis, oocyte, egg, cell extract, somatic cells, reprogramming
Original Articles -------------------------------------------------
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 297-304 (2016)
doi: 10.1387/ijdb.160139oh / © UBC Press ( www.a360grados.net )
Single-molecule, antibody-free fluorescent visualisation of replication tracts along barcoded DNA molecules
Francesco De Carli 1,2, Vincent Gaggioli 1,3, Gaël A. Millot 4,5 and Olivier Hyrien 1
1. Institut de Biologie de l'Ecole Normale Supérieure (IBENS), CNRS UMR 8197, Inserm U1024, Ecole Normale Supérieure, PSL Research University, Paris, France
2. Sorbonne Universités, UPMC Univ Paris 06, IFD, Paris, France
3. Welcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, England, UK
4. Institut Curie, PSL Research University, CNRS, UMR 3244, Paris, France
5. Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR 3244, Paris, France.
Abstract: DNA combing is a standard technique to map DNA replication at the single molecule level. Typically, replicating DNA is metabolically labelled with nucleoside or nucleotide analogs, purified, stretched on coverslips and treated with fluorescent antibodies to reveal tracts of newly synthesized DNA. Fibres containing a locus of interest can then be identified by fluorescent in situ hybridization (FISH) with DNA probes. These steps are complex and the throughput is low. Here, we describe a simpler, antibody-free method to reveal replication tracts and identify the locus of origin of combed DNA replication intermediates. DNA was replicated in Xenopus egg extracts in the presence of a fluorescent dUTP. Purified DNA was barcoded by nicking with Nt.BspQI, a site-specific nicking endonuclease (NE), followed by limited nick-translation in the presence of another fluorescent dUTP. DNA was then stained with YOYO-1, a fluorescent DNA intercalator, and combed. Direct epifluorescence revealed the DNA molecules, their replication tracts and their Nt.BspQI sites in three distinct colours. Replication intermediates could thus be aligned to a reference genome map. In addition, replicated DNA segments showed a stronger YOYO-1 fluorescence than unreplicated segments. The entire length, replication tracts, and NE sites of combed DNA molecules can be simultaneously visualized in three distinct colours by standard epifluorescence microscopy, with no need for antibody staining and/or FISH detection. Furthermore, replication bubbles can be detected by quantitative YOYO-1 staining, eliminating the need for metabolic labelling. These results provide a starting point for genome-wide, single-molecule mapping of DNA replication in any organism.
Keywords: DNA replication, DNA combing, DNA barcoding, single-molecule analysis, Xenopus egg extract
--------------------------------------------------------------------------------------------------
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 305-314 (2016)
doi: 10.1387/ijdb.160134jk / © UBC Press ( www.a360grados.net )
Flexibility vs. robustness in cell cycle regulation of timing of M-phase entry in Xenopus laevis embryo cell-free extract
Mateusz Debowski 1, Mohammed El Dika 2,3, Jacek Malejczyk 4, Robert Zdanowski 5, Claude Prigent 2,3, Jean-Pierre Tassan 2,3, Malgorzata Kloc 6, Miroslaw Lachowicz 1 and Jacek Z. Kubiak 2,3,5
1. Institute of Applied Mathematics and Mechanics, Faculty of Mathematics, Informatics and Mechanics, University of Warsaw, Warsaw, Poland
2. CNRS, UMR 6290, Institute of Genetics and Development of Rennes, Cell Cycle Group, Rennes, France
3. University Rennes 1, UEB, IFR 140, Faculty of Medicine, Rennes, France
4. Medical University of Warsaw, Department of Histology & Embryology, Warsaw, Poland
5. Department of Regenerative Medicine, Military Institute of Hygiene and Epidemiology, Warsaw, Poland
6. The Methodist Hospital Research Institute, Bertner Ave, Houston, TX, USA
Abstract: During the cell cycle, cyclin dependent kinase 1 (CDK1) and protein phosphatase 2A (PP2A) play major roles in the regulation of mitosis. CDK1 phosphorylates a series of substrates triggering M-phase entry. Most of these substrates are dephosphorylated by PP2A. To allow phosphorylation of CDK1 substrates, PP2A is progressively inactivated upon M-phase entry. We have shown previously that the interplay between these two activities determines the timing of M-phase entry. Slight diminution of CDK1 activity by the RO3306 inhibitor delays M-phase entry in a dose-dependent manner in Xenopus embryo cell-free extract, while reduction of PP2A activity by OA inhibitor accelerates this process also in a dose-dependent manner. However, when a mixture of RO3306 and OA is added to the extract, an intermediate timing of M-phase entry is observed. Here we use a mathematical model to describe and understand this interplay. Simulations showing acceleration and delay in M-phase entry match previously described experimental data. CDC25 phosphatase is a major activator of CDK1 and acts through CDK1 Tyr15 and Thr14 dephosphorylation. Addition of CDC25 activity to our mathematical model was also consistent with our experimental results. To verify whether our assumption that the dynamics of CDC25 activation used in this model are the same in all experimental variants, we analyzed the dynamics of CDC25 phosphorylation, which reflect its activation. We confirm that these dynamics are indeed very similar in control extracts and when RO3306 and OA are present separately. However, when RO3306 and OA are added simultaneously to the extract, activation of CDC25 is slightly delayed. Integration of this parameter allowed us to improve our model. Furthermore, the pattern of CDK1 dephosphorylation on Tyr15 showed that the real dynamics of CDK1 activation are very similar in all experimental variants. The model presented here accurately describes, in mathematical terms, how the interplay between CDK1, PP2A and CDC25 controls the flexible timing of M-phase entry.
Keywords: cell cycle regulation, M-phase entry, CDK, PP2A, CDC25, mathematical model
--------------------------------------------------------------------------------------------------
EHU/UPV/UBC - The International Journal of Developmental Biology 60: 315-320 (2016)
doi: 10.1387/ijdb.160161ds / © UBC Press ( www.a360grados.net )
Chromatin assembly and transcriptional cross-talk in Xenopus laevis oocyte and egg extracts
Wei-Lin Wang and David Shechter
Department of Biochemistry. Albert Einstein College of Medicine, Bronx, NY, USA
Abstract: Chromatin, primarily a complex of DNA and histone proteins, is the physiological form of the genome. Chromatin is generally repressive for transcription and other information transactions that occur on DNA. A wealth of post-translational modifications on canonical histones and histone variants encode regulatory information to recruit or repel effector proteins on chromatin, promoting and further repressing transcription and thereby form the basis of epigenetic information. During metazoan oogenesis, large quantities of histone proteins are synthesized and stored in preparation for the rapid early cell cycles of development and to elicit maternal control of chromatin assembly pathways. Oocyte and egg cell-free extracts of the frog Xenopus laevis are a compelling model system for the study of chromatin assembly and transcription, precisely because they exist in an extreme state primed for rapid chromatin assembly or for transcriptional activity. We show that chromatin assembly rates are slower in the X. laevis oocyte than in egg extracts, while conversely, only oocyte extracts transcribe template plasmids. We demonstrate that rapid chromatin assembly in egg extracts represses RNA Polymerase II dependent transcription, while pre-binding of TATA-Binding Protein (TBP) to a template plasmid promotes transcription. Our experimental evidence presented here supports a model in which chromatin assembly and transcription are in competition and that the onset of zygotic genomic activation may be in part due to stable transcriptional complex assembly.
Keywords: histone, RNA polymerase II, TATA-binding protein, transcriptional complex
|
