scholarly journals Quiescent cells actively replenish CENP-A nucleosomes to maintain centromere identity and proliferative potential

2018 ◽  
Author(s):  
S. Zachary Swartz ◽  
Liliana S. McKay ◽  
Kuan-Chung Su ◽  
Abbas Padeganeh ◽  
Paul S. Maddox ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Epigenetic Inheritance ◽  
Proliferative Potential ◽  
Quiescent Cells ◽  
Differentiated Cells ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Robust Model ◽  
Histone H3 Variant ◽  
Cycle Dependent

SummaryCentromeres provide a robust model for epigenetic inheritance as they are specified by sequence-independent mechanisms involving the histone H3-variant CENP-A. Prevailing models indicate that the high intrinsic stability of CENP-A nucleosomes maintains centromere identity indefinitely. Here, we demonstrate that CENP-A is not stable at centromeres, but is instead gradually and continuously incorporated in quiescent cells including G0-arrested tissue culture cells and prophase I-arrested oocytes. Quiescent CENP-A incorporation involves the canonical CENP-A deposition machinery, but displays distinct requirements from cell cycle-dependent deposition. We demonstrate that Plk1 is required specifically for G1 CENP-A deposition, whereas transcription promotes CENP-A incorporation in quiescent oocytes. Preventing CENP-A deposition during quiescence results in significantly reduced CENP-A levels and perturbs chromosome segregation following the resumption of cell division. In contrast to quiescent cells, terminally differentiated cells fail to maintain CENP-A levels. Our work reveals that quiescent cells actively maintain centromere identity providing an indicator of proliferative potential.

scholarly journals Centromere transcription allows CENP-A to transit from chromatin association to stable incorporation

2018 ◽  
Vol 217 (6) ◽  
pp. 1957-1972 ◽  
Author(s):  
Georg O.M. Bobkov ◽  
Nick Gilbert ◽  
Patrick Heun
Keyword(s):  
Rna Polymerase Ii ◽  
Chromatin Remodeling ◽  
Chromosome Segregation ◽  
De Novo ◽  
Histone H3 ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Histone H3 Variant ◽  
Loading System ◽  
Stable Incorporation

Centromeres are essential for chromosome segregation and are specified epigenetically by the presence of the histone H3 variant CENP-A. In flies and humans, replenishment of the centromeric mark is uncoupled from DNA replication and requires the removal of H3 “placeholder” nucleosomes. Although transcription at centromeres has been previously linked to the loading of new CENP-A, the underlying molecular mechanism remains poorly understood. Here, we used Drosophila melanogaster tissue culture cells to show that centromeric presence of actively transcribing RNA polymerase II temporally coincides with de novo deposition of dCENP-A. Using a newly developed dCENP-A loading system that is independent of acute transcription, we found that short inhibition of transcription impaired dCENP-A incorporation into chromatin. Interestingly, initial targeting of dCENP-A to centromeres was unaffected, revealing two stability states of newly loaded dCENP-A: a salt-sensitive association with the centromere and a salt-resistant chromatin-incorporated form. This suggests that transcription-mediated chromatin remodeling is required for the transition of dCENP-A to fully incorporated nucleosomes at the centromere.

scholarly journals Epigenetic inheritance of centromere identity in a heterologous system

2019 ◽  
Author(s):  
Virginie Roure ◽  
Bethan Medina-Pritchard ◽  
Eduard Anselm ◽  
A. Arockia Jeyaprakash ◽  
Patrick Heun
Keyword(s):  
Chromosome Segregation ◽  
Chromosomal Region ◽  
Histone H3 ◽  
Epigenetic Inheritance ◽  
Centromeric Chromatin ◽  
Centromere Proteins ◽  
Heterologous System ◽  
Histone H3 Variant ◽  
Self Association

SUMMARYThe centromere is an essential chromosomal region required for accurate chromosome segregation. Most eukaryotic centromeres are defined epigenetically by the histone H3 variant, CENP-A, yet how its self-propagation is achieved remains poorly understood. Here we developed a heterologous system to reconstitute epigenetic inheritance of centromeric chromatin by ectopically targeting the Drosophila centromere proteins dCENP-A, dCENP-C and CAL1 to LacO arrays in human cells. Dissecting the function of these three components uncovers the key role of self-association of dCENP-C and CAL1 for their mutual interaction and dCENP-A deposition. Importantly, we identify the components required for dCENP-C loading onto chromatin, involving a cooperation between CAL1 and dCENP-A nucleosomes, thus closing the epigenetic loop to ensure dCENP-C and dCENP-A replenishment during the cell division cycle. Finally, we show that all three Drosophila factors are sufficient for dCENP-A propagation and propose a model for the epigenetic inheritance of centromere identity.

scholarly journals Cell cycle–dependent association of polo kinase Cdc5 with CENP-A contributes to faithful chromosome segregation in budding yeast

2019 ◽  
Vol 30 (8) ◽  
pp. 1020-1036 ◽  
Author(s):  
Prashant K. Mishra ◽  
Gudjon Olafsson ◽  
Lars Boeckmann ◽  
Timothy J. Westlake ◽  
Ziad M. Jowhar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Growth Defects ◽  
Evolutionarily Conserved ◽  
Histone H3 Variant ◽  
Cycle Dependent

Evolutionarily conserved polo-like kinase, Cdc5 (Plk1 in humans), associates with kinetochores during mitosis; however, the role of cell cycle–dependent centromeric ( CEN) association of Cdc5 and its substrates that exclusively localize to the kinetochore have not been characterized. Here we report that evolutionarily conserved CEN histone H3 variant, Cse4 (CENP-A in humans), is a substrate of Cdc5, and that the cell cycle–regulated association of Cse4 with Cdc5 is required for cell growth. Cdc5 contributes to Cse4 phosphorylation in vivo and interacts with Cse4 in mitotic cells. Mass spectrometry analysis of in vitro kinase assays showed that Cdc5 phosphorylates nine serine residues clustered within the N-terminus of Cse4. Strains with cse4-9SA exhibit increased errors in chromosome segregation, reduced levels of CEN-associated Mif2 and Mcd1/Scc1 when combined with a deletion of MCM21. Moreover, the loss of Cdc5 from the CEN chromatin contributes to defects in kinetochore integrity and reduction in CEN-associated Cse4. The cell cycle–regulated association of Cdc5 with Cse4 is essential for cell viability as constitutive association of Cdc5 with Cse4 at the kinetochore leads to growth defects. In summary, our results have defined a role for Cdc5-mediated Cse4 phosphorylation in faithful chromosome segregation.

scholarly journals Cell Cycle-Regulated Transcription of CENP-A by the MBF Complex Ensures Optimal Level of CENP-A for Centromere Formation

Genetics ◽  
2019 ◽  
Vol 211 (3) ◽  
pp. 861-875 ◽  
Author(s):  
David Aristizabal-Corrales ◽  
Jinpu Yang ◽  
Fei Li
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Optimal Level ◽  
S Phase ◽  
Temporal Control ◽  
Centromere Function ◽  
Histone H3 Variant ◽  
Cycle Dependent ◽  
Regulation Of Cell Cycle ◽  
Cycle Regulation

The centromere plays an essential role in chromosome segregation. In most eukaryotes, centromeres are epigenetically defined by the conserved histone H3 variant CENP-A. Proper centromere assembly is dependent upon the tight regulation of CENP-A level. Cell cycle regulation of CENP-A transcription appears to be a universal feature across eukaryotes, but the molecular mechanism underlying the temporal control of CENP-A transcription and how such regulation contributes to centromere function remains elusive. CENP-A in fission yeast has been shown to be transcribed before S phase. Using various synchronization methods, we confirmed that CENP-A transcription occurs at G1, leading to an almost twofold increase of the protein during S phase. Through a genetic screen, we identified the MBF (MluI box-binding factors) complex as a key regulator of temporal control of CENP-A transcription. The periodic transcription of CENP-A is lost in MBF mutants, resulting in CENP-A mislocalization and chromosome segregation defects. We identified the MCB (MluI cell cycle box) motif in the CENP-A promoter, and further showed that the MBF complex binds to the motif to restrict CENP-A transcription to G1. Mutations of the MCB motif cause constitutive CENP-A expression and deleterious effects on cell survival. Using promoters driving transcription to different cell cycle stages, we found that timing of CENP-A transcription is dispensable for its centromeric localization. Our data instead indicate that cell cycle-regulated CENP-A transcription is a key step to ensure that a proper amount of CENP-A is generated across generations. This study provides mechanistic insights into the regulation of cell cycle-dependent CENP-A transcription, as well as its importance on centromere function.

Cytoplasmic Tubules in Cells Infected with Laryngotracheitis Virus

1969 ◽  
Vol 27 ◽  
pp. 376-377
Author(s):  
A. M. Watrach
Keyword(s):  
Tissue Culture ◽  
Small Proportion ◽  
Chicken Embryo ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Morphologic Characteristics ◽  
Infectious Laryngotracheitis ◽  
Laryngotracheitis Virus ◽  
In Cells

During a study of the development of infectious laryngotracheitis (LT) virus in tissue culture cells, unusual tubular formations were found in the cytoplasm of a small proportion of the affected cells. It is the purpose of this report to describe the morphologic characteristics of the tubules and to discuss their possible association with the development of virus.The source and maintenance of the strain of LT virus have been described. Prior to this study, the virus was passed several times in chicken embryo kidney (CEK) tissue culture cells.

Sulfhydryl bond formation is a prerequisite for proper cycling of centrosomes and chromosomes in mammalian tissue culture cells

1993 ◽  
Vol 51 ◽  
pp. 342-343
Author(s):  
Heide Schatten ◽  
Neidhard Paweletz ◽  
Ron Balczon
Keyword(s):  
Tissue Culture ◽  
Cell Cycle Progression ◽  
Group Formation ◽  
Sulfhydryl Group ◽  
Mammalian Tissue ◽  
Mitotic Chromosomes ◽  
Microtubule Network ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Transmission Electron

To study the role of sulfhydryl group formation during cell cycle progression, mammalian tissue culture cells (PTK2) were exposed to 100¼M 2-mercaptoethanol for 2 to 6 h during their exponential phase of growth. The effects of 2-mercaptoethanol on centrosomes, chromosomes, microtubules, membranes and intermediate filaments were analyzed by transmission electron microscopy (TEM) and by immunofluorescence microscopy (IFM) methods using a human autoimmune antibody directed against centrosomes (SPJ), and a mouse monoclonal antibody directed against tubulin (E7). Chromosomes were affected most by this treatment: premature chromosome condensation was detected in interphase nuclei, and the structure in mitotic chromosomes was altered compared to control cells. This would support previous findings in dividing sea urchin cells in which chromosomes are arrested at metaphase while the centrosome splitting cycle continues. It might also support findings that certairt-sulfhydryl-blocking agents block cyclin destruction. The organization of the microtubule network was scattered probably due to a looser organization of centrosomal material at the interphase centers and at the mitotic poles.

UDP-D-glucose 4-epimerase activity of the tissue culture of white poplars (Populus alba L. var. pyramidalis)

1982 ◽  
Vol 47 (5) ◽  
pp. 1530-1536 ◽  
Author(s):  
Ladislav Bilisics ◽  
Štefan Karácsonyi ◽  
Marta Kubačková
Keyword(s):  
Tissue Culture ◽  
Cell Walls ◽  
Enzyme Preparation ◽  
Uridine Diphosphate ◽  
Populus Alba ◽  
Activity Change ◽  
White Poplar ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Galactose Content

The presence of UDP-D-glucose 4-epimerase (EC 5.1.3.2) in the culture tissue of white poplar was evidenced. As found, the partially purified enzyme preparation contained UDP-D-glucose glucosyltransferase, UDP-D-galactose galactosyltransferase and non-specific enzymes able to cleave the uridine-diphosphate saccharides into the appropriate hexose monophosphates. The activity change of UDP-D-glucose 4-epimerase in tissue culture cells during the growth was in accord with changes in D-galactose content in cell walls and indicated the possibility to regulate the formation of polysaccharides containing D-galactose at the level of production of UDP-D-galactose in cells.

Sign in / Sign up

Export Citation Format

Share Document