scholarly journals Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points

2017 ◽  
Vol 216 (3) ◽  
pp. 607-621 ◽  
Author(s):  
Yael Nechemia-Arbely ◽  
Daniele Fachinetti ◽  
Karen H. Miga ◽  
Nikolina Sekulic ◽  
Gautam V. Soni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein A ◽  
Epigenetic Mark ◽  
Specific Cell ◽  
Entry And Exit ◽  
Satellite Repeat ◽  
Reference Models ◽  
Centromere Protein A ◽  
Cell Cycle Phases ◽  
Dna Unwrapping

Chromatin assembled with centromere protein A (CENP-A) is the epigenetic mark of centromere identity. Using new reference models, we now identify sites of CENP-A and histone H3.1 binding within the megabase, α-satellite repeat–containing centromeres of 23 human chromosomes. The overwhelming majority (97%) of α-satellite DNA is found to be assembled with histone H3.1–containing nucleosomes with wrapped DNA termini. In both G1 and G2 cell cycle phases, the 2–4% of α-satellite assembled with CENP-A protects DNA lengths centered on 133 bp, consistent with octameric nucleosomes with DNA unwrapping at entry and exit. CENP-A chromatin is shown to contain equimolar amounts of CENP-A and histones H2A, H2B, and H4, with no H3. Solid-state nanopore analyses show it to be nucleosomal in size. Thus, in contrast to models for hemisomes that briefly transition to octameric nucleosomes at specific cell cycle points or heterotypic nucleosomes containing both CENP-A and histone H3, human CENP-A chromatin complexes are octameric nucleosomes with two molecules of CENP-A at all cell cycle phases.

scholarly journals Propagation of centromeric chromatin requires exit from mitosis

2007 ◽  
Vol 176 (6) ◽  
pp. 795-805 ◽  
Author(s):  
Lars E.T. Jansen ◽  
Ben E. Black ◽  
Daniel R. Foltz ◽  
Don W. Cleveland
Keyword(s):  
Protein A ◽  
S Phase ◽  
Epigenetic Mark ◽  
Multiprotein Complex ◽  
Centromeric Chromatin ◽  
Microtubule Attachment ◽  
Centromere Protein A ◽  
Histone H3 Variant ◽  
Multiple Cell ◽  
Labeling Approach

Centromeres direct chromosomal inheritance by nucleating assembly of the kinetochore, a large multiprotein complex required for microtubule attachment during mitosis. Centromere identity in humans is epigenetically determined, with no DNA sequence either necessary or sufficient. A prime candidate for the epigenetic mark is assembly into centromeric chromatin of centromere protein A (CENP-A), a histone H3 variant found only at functional centromeres. A new covalent fluorescent pulse-chase labeling approach using SNAP tagging has now been developed and is used to demonstrate that CENP-A bound to a mature centromere is quantitatively and equally partitioned to sister centromeres generated during S phase, thereby remaining stably associated through multiple cell divisions. Loading of nascent CENP-A on the megabase domains of replicated centromere DNA is shown to require passage through mitosis but not microtubule attachment. Very surprisingly, assembly and stabilization of new CENP-A–containing nucleosomes is restricted exclusively to the subsequent G1 phase, demonstrating direct coupling between progression through mitosis and assembly/maturation of the next generation of centromeres.

scholarly journals Assembly in G1 phase and long-term stability are unique intrinsic features of CENP-A nucleosomes

2013 ◽  
Vol 24 (7) ◽  
pp. 923-932 ◽  
Author(s):  
Dani L. Bodor ◽  
Luis P. Valente ◽  
João F. Mata ◽  
Ben E. Black ◽  
Lars E. T. Jansen
Keyword(s):  
Cell Cycle ◽  
Dna Sequences ◽  
Protein A ◽  
G1 Phase ◽  
Term Stability ◽  
Nucleosome Core ◽  
Long Term Stability ◽  
Centromere Position ◽  
Centromere Protein A

Centromeres are the site of kinetochore formation during mitosis. Centromere protein A (CENP-A), the centromere-specific histone H3 variant, is essential for the epigenetic maintenance of centromere position. Previously we showed that newly synthesized CENP-A is targeted to centromeres exclusively during early G1 phase and is subsequently maintained across mitotic divisions. Using SNAP-based fluorescent pulse labeling, we now demonstrate that cell cycle–restricted chromatin assembly at centromeres is unique to CENP-A nucleosomes and does not involve assembly of other H3 variants. Strikingly, stable retention is restricted to the CENP-A/H4 core of the nucleosome, which we find to outlast general chromatin across several cell divisions. We further show that cell cycle timing of CENP-A assembly is independent of centromeric DNA sequences and instead is mediated by the CENP-A targeting domain. Unexpectedly, this domain also induces stable transmission of centromeric nucleosomes, independent of the CENP-A deposition factor HJURP. This demonstrates that intrinsic properties of the CENP-A protein direct its cell cycle–restricted assembly and induces quantitative mitotic transmission of the CENP-A/H4 nucleosome core, ensuring long-term stability and epigenetic maintenance of centromere position.

scholarly journals Forkhead Box M1 Regulates the Transcriptional Network of Genes Essential for Mitotic Progression and Genes Encoding the SCF (Skp2-Cks1) Ubiquitin Ligase

2005 ◽  
Vol 25 (24) ◽  
pp. 10875-10894 ◽  
Author(s):  
I-Ching Wang ◽  
Yi-Ju Chen ◽  
Douglas Hughes ◽  
Vladimir Petrovic ◽  
Michael L. Major ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ubiquitin Ligase ◽  
Kinase Inhibitor ◽  
Protein A ◽  
Mitotic Progression ◽  
Early Passage ◽  
Forkhead Box ◽  
Forkhead Box M1 ◽  
Centromere Protein A ◽  
Nuclear Levels

ABSTRACT The Forkhead box m1 (Foxm1) gene is critical for G1/S transition and essential for mitotic progression. However, the transcriptional mechanisms downstream of FoxM1 that control these cell cycle events remain to be determined. Here, we show that both early-passage Foxm1 − / − mouse embryonic fibroblasts (MEFs) and human osteosarcoma U2OS cells depleted of FoxM1 protein by small interfering RNA fail to grow in culture due to a mitotic block and accumulate nuclear levels of cyclin-dependent kinase inhibitor (CDKI) proteins p21Cip1 and p27Kip1. Using quantitative chromatin immunoprecipitation and expression assays, we show that FoxM1 is essential for transcription of the mitotic regulatory genes Cdc25B, Aurora B kinase, survivin, centromere protein A (CENPA), and CENPB. We also identify the mechanism by which FoxM1 deficiency causes elevated nuclear levels of the CDKI proteins p21Cip1 and p27Kip1. We provide evidence that FoxM1 is essential for transcription of Skp2 and Cks1, which are specificity subunits of the Skp1-Cullin 1-F-box (SCF) ubiquitin ligase complex that targets these CDKI proteins for degradation during the G1/S transition. Moreover, early-passage Foxm1 − / − MEFs display premature senescence as evidenced by high expression of the senescence-associated β-galactosidase, p19ARF, and p16INK4A proteins. Taken together, these results demonstrate that FoxM1 regulates transcription of cell cycle genes critical for progression into S-phase and mitosis.

Phosphatidylcholine catabolism in the MCF-7 cell cycle

2006 ◽  
Vol 84 (5) ◽  
pp. 737-744 ◽  
Author(s):  
Weiyang Lin ◽  
Gilbert Arthur
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Specific Cell ◽  
Synchronized Cells ◽  
M Phase ◽  
G2 Phase ◽  
Cell Cycle Phases ◽  
Kinetics Of ◽  
Mcf 7

The catabolism of phosphatidylcholine (PtdCho) appears to play a key role in regulating the net accumulation of the lipid in the cell cycle. Current protocols for measuring the degradation of PtdCho at specific cell-cycle phases require prolonged periods of incubation with radiolabelled choline. To measure the degradation of PtdCho at the S and G2 phases in the MCF-7 cell cycle, protocols were developed with radiolabelled lysophosphatidylcholine (lysoPtdCho), which reduces the labelling period and minimizes the recycling of labelled components. Although most of the incubated lysoPtdCho was hydrolyzed to glycerophosphocholine (GroPCho) in the medium, the kinetics of the incorporation of label into PtdCho suggests that the labelled GroPCho did not contribute significantly to cellular PtdCho formation. A protocol which involved exposing the cells twice to hydroxyurea, was also developed to produce highly synchronized MCF-7 cells with a profile of G1:S:G2/M of 90:5:5. An analysis of PtdCho catabolism in the synchronized cells following labelling with lysoPtdCho revealed that there was increased degradation of PtdCho in early to mid-S phase, which was attenuated in the G2/M phase. The results suggest that the net accumulation of PtdCho in MCF-7 cells may occur in the G2 phase of the cell cycle.

scholarly journals Functional Complementation of Human Centromere Protein A (CENP-A) by Cse4p from Saccharomyces cerevisiae

2004 ◽  
Vol 24 (15) ◽  
pp. 6620-6630 ◽  
Author(s):  
Gerhard Wieland ◽  
Sandra Orthaus ◽  
Sabine Ohndorf ◽  
Stephan Diekmann ◽  
Peter Hemmerich
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Budding Yeast ◽  
Protein A ◽  
Human Cells ◽  
Cycle Arrest ◽  
Centromere Protein ◽  
Centromere Protein A

ABSTRACT We have employed a novel in vivo approach to study the structure and function of the eukaryotic kinetochore multiprotein complex. RNA interference (RNAi) was used to block the synthesis of centromere protein A (CENP-A) and Clip-170 in human cells. By coexpression, homologous kinetochore proteins from Saccharomyces cerevisiae were then tested for the ability to complement the RNAi-induced phenotypes. Cse4p, the budding yeast CENP-A homolog, was specifically incorporated into kinetochore nucleosomes and was able to complement RNAi-induced cell cycle arrest in CENP-A-depleted human cells. Thus, Cse4p can structurally and functionally substitute for CENP-A, strongly suggesting that the basic features of centromeric chromatin are conserved between yeast and mammals. Bik1p, the budding yeast homolog of human CLIP-170, also specifically localized to kinetochores during mitosis, but Bik1p did not rescue CLIP-170 depletion-induced cell cycle arrest. Generally, the newly developed in vivo complementation assay provides a powerful new tool for studying the function and evolutionary conservation of multiprotein complexes from yeast to humans.

A Cyclin D2-derived peptide acts on specific cell cycle phases by activating ERK1/2 to cause the death of breast cancer cells

2017 ◽  
Vol 151 ◽  
pp. 24-32 ◽  
Author(s):  
Lilian C. Russo ◽  
Christiane B. Araujo ◽  
Leo K. Iwai ◽  
Emer S. Ferro ◽  
Fabio L. Forti
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Specific Cell ◽  
Cyclin D2 ◽  
Cell Cycle Phases

scholarly journals Dual excitation multi-fluorescence flow cytometry for detailed analyses of viability and apoptotic cell transition

10.4081/838 ◽  
2009 ◽  
Vol 47 (4) ◽  
pp. 289 ◽  
Author(s):  
G Mazzini ◽  
C Ferrari ◽  
E Erba
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Membrane ◽  
Apoptotic Cell ◽  
Intact Cell ◽  
Specific Cell ◽  
Apoptotic Cells ◽  
Apoptotic Pathway ◽  
Culture Models ◽  
Cell Cycle Phases

The discrimination of live/dead cells as well as the detection of apoptosis is a frequent need in many areas of experimental biology. Cell proliferation is linked to apoptosis and controlled by several genes. During the cell life, specific events can stimulate proliferation while others may trigger the apoptotic pathway. Very few methods (i.e. TUNEL) are now available for studies aimed at correlation between apoptosis and proliferation. Therefore, there is interest in developing new methodological approaches that are able to correlate apoptosis to the cell cycle phases. Recently new approaches have been proposed to detect and enumerate apoptotic cells by flow cytometry. Among these, the most established and applied are those based on the cell membrane modifications induced in the early phases of the apoptotic process. The dye pair Hoechst 33342 (HO) and Propidium Iodide (PI), thanks to their peculiar characteristics to be respectively permeable and impermeable to the intact cell membrane, seems to be very useful. Unfortunately the spectral interaction of these dyes generates a consistent “energy transfer” from HO to PI. The co-presence of the dyes in a nucleus results in a modification in the intensity of both the emitted fluorescences. In order to designate the damaged cells (red fluorescence) to the specific cell cycle phases (blue fluorescence), we have tested different staining protocols aimed to minimize the interference of these dyes as much as possible. In cell culture models, we are able to detect serum-starved apoptotic cells as well as to designate their exact location in the cell cycle phases using a very low PI concentration. Using a Partec PAS flow cytometer equipped with HBO lamp and argon ion laser, a double UV/blue excitation has been performed. This analytical approach is able to discriminate live blue cells from the damaged (blue-red) ones even at 0.05 ?g/mL PI. The same instrumental setting allows performing other multi-colour analyses including AnnexinV-FITC as well as the possibility to make a correlated analysis to phenotype markers.

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