scholarly journals Cell-Cycle—Dependent Chromatin Dynamics at Replication Origins

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1998
Author(s):  
Yulong Li ◽  
Alexander J. Hartemink ◽  
David M. MacAlpine
Keyword(s):  
Cell Cycle ◽  
Consensus Sequence ◽  
S Phase ◽  
Micrococcal Nuclease ◽  
Dependent Manner ◽  
Replication Origins ◽  
Chromatin Dynamics ◽  
Cell Cycles ◽  
Cycle Dependent ◽  
Replication Factors

Origins of DNA replication are specified by the ordered recruitment of replication factors in a cell-cycle—dependent manner. The assembly of the pre-replicative complex in G1 and the pre-initiation complex prior to activation in S phase are well characterized; however, the interplay between the assembly of these complexes and the local chromatin environment is less well understood. To investigate the dynamic changes in chromatin organization at and surrounding replication origins, we used micrococcal nuclease (MNase) to generate genome-wide chromatin occupancy profiles of nucleosomes, transcription factors, and replication proteins through consecutive cell cycles in Saccharomyces cerevisiae. During each G1 phase of two consecutive cell cycles, we observed the downstream repositioning of the origin-proximal +1 nucleosome and an increase in protected DNA fragments spanning the ARS consensus sequence (ACS) indicative of pre-RC assembly. We also found that the strongest correlation between chromatin occupancy at the ACS and origin efficiency occurred in early S phase, consistent with the rate-limiting formation of the Cdc45–Mcm2-7–GINS (CMG) complex being a determinant of origin activity. Finally, we observed nucleosome disruption and disorganization emanating from replication origins and traveling with the elongating replication forks across the genome in S phase, likely reflecting the disassembly and assembly of chromatin ahead of and behind the replication fork, respectively. These results provide insights into cell-cycle–regulated chromatin dynamics and how they relate to the regulation of origin activity.

scholarly journals Cell cycle-dependent chromatin dynamics at replication origins

2021 ◽  
Author(s):  
Yulong Li ◽  
Alexander J. Hartemink ◽  
David MacAlpine
Keyword(s):  
Cell Cycle ◽  
Consensus Sequence ◽  
S Phase ◽  
Micrococcal Nuclease ◽  
Dependent Manner ◽  
Replication Origins ◽  
Chromatin Dynamics ◽  
Cell Cycles ◽  
Cycle Dependent ◽  
Replication Factors

Origins of DNA replication are specified by the ordered recruitment of replication factors in a cell cycle dependent manner. The assembly of the pre-replicative complex in G1 and the pre-initiation complex prior to activation in S-phase are well characterized; however, the interplay between the assembly of these complexes and the local chromatin environment is less well understood. To investigate the dynamic changes in chromatin organization at and surrounding replication origins, we used micrococcal nuclease (MNase) to generate genome-wide chromatin occupancy profiles of nucleosomes, transcription factors and replication proteins through consecutive cell cycles in Saccharomyces cerevisiae. During each G1 phase of two consecutive cell cycles, we observed the downstream repositioning of the origin-proximal +1 nucleosome and an increase in protected DNA fragments spanning the ARS consensus sequence (ACS) indicative of pre-RC assembly. We also found that the strongest correlation between the chromatin occupancy at the ACS and origin efficiency occurred in early S-phase consistent with the rate limiting formation of the Cdc45-Mcm2-7-GINS (CMG) complex being a determinant of origin activity. Finally, we observed nucleosome disruption and disorganization emanating from replication origins and traveling with the elongating replication forks across the genome in S-phase, likely reflecting the disassembly and assembly of chromatin ahead of and behind the replication fork, respectively. These results provide insights into cell cycle-regulated chromatin dynamics and how they relate to the regulation of origin activity.

The efficiency and timing of plasmid DNA replication in Xenopus eggs: correlations to the extent of prior chromatin assembly

1992 ◽  
Vol 103 (4) ◽  
pp. 907-918 ◽  
Author(s):  
J.A. Sanchez ◽  
D. Marek ◽  
L.J. Wangh
Keyword(s):  
Cell Cycle ◽  
Enzymatic Digestion ◽  
S Phase ◽  
Chromatin Assembly ◽  
Type I ◽  
Dependent Manner ◽  
Cell Cycles ◽  
Gene Insertion ◽  
Cycle Dependent

Injection of the circular plasmid FV1 (derived from type I bovine papilloma virus) into Xenopus eggs before the start of the first cell cycle dramatically increases the efficiency of plasmid replication once eggs are chemically activated. We call this the preloading effect and report kinetic and quantitative characterization of this phenomenon here. The timing and the amount of FV1 synthesis were measured by both BrdUTP density labelling and an optimized method of selective enzymatic digestion of replicated and unreplicated molecules using the three methyladenosine-sensitive isoschizomers, DpnI, MboI and Sau3a. DpnI in 100 mM NaCl proved particularly useful for distinguishing and quantitating unreplicated, once-replicated, and repeatedly replicated molecules accumulated over several cell cycles. Our results reveal that both the amount of DNA replicated and the timing of synthesis during the first S-phase correlate with the length of the preloading period. Longer preloading leads to larger amounts of DNA being replicated sooner. In fact, up to 30–50% of 1 ng injected plasmid can replicate in a semiconservative cell cycle-dependent manner during the first S-phase. But such high levels of synthesis during the first cell cycle appear to limit the egg's ability to rereplicate this material in subsequent cell cycles. The preloading effect does not depend on synthesis of either viral or egg proteins, but does appear to correlate with the extent of plasmid assembly into chromatin before the start of the cell cycle. We postulate that each plasmid molecule must achieve a critical degree of chromatin assembly before it can proceed along the replication pathway. These observations illuminate some of the difficulties inherent in building a vector for gene insertion into Xenopus embryos, but also suggest an experimental strategy toward this aim.

scholarly journals Phosphorylation of ORC2 Protein Dissociates Origin Recognition Complex from Chromatin and Replication Origins

2012 ◽  
Vol 287 (15) ◽  
pp. 11891-11898 ◽  
Author(s):  
Kyung Yong Lee ◽  
Sung Woong Bang ◽  
Sang Wook Yoon ◽  
Seung-Hoon Lee ◽  
Jong-Bok Yoon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Origin Recognition Complex ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Replication Origins ◽  
Eukaryotic Chromosome ◽  
M Phase ◽  
Cycle Dependent ◽  
Prereplicative Complex ◽  
Origin Recognition

During the late M to the G1 phase of the cell cycle, the origin recognition complex (ORC) binds to the replication origin, leading to the assembly of the prereplicative complex for subsequent initiation of eukaryotic chromosome replication. We found that the cell cycle-dependent phosphorylation of human ORC2, one of the six subunits of ORC, dissociates ORC2, -3, -4, and -5 (ORC2–5) subunits from chromatin and replication origins. Phosphorylation at Thr-116 and Thr-226 of ORC2 occurs by cyclin-dependent kinase during the S phase and is maintained until the M phase. Phosphorylation of ORC2 at Thr-116 and Thr-226 dissociated the ORC2–5 from chromatin. Consistent with this, the phosphomimetic ORC2 protein exhibited defective binding to replication origins as well as to chromatin, whereas the phosphodefective protein persisted in binding throughout the cell cycle. These results suggest that the phosphorylation of ORC2 dissociates ORC from chromatin and replication origins and inhibits binding of ORC to newly replicated DNA.

scholarly journals EGT2 gene transcription is induced predominantly by Swi5 in early G1.

1996 ◽  
Vol 16 (7) ◽  
pp. 3264-3274 ◽  
Author(s):  
B Kovacech ◽  
K Nasmyth ◽  
T Schuster
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Cell Separation ◽  
S Phase ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Concentration Dependent Manner ◽  
A Cell ◽  
Cycle Dependent ◽  
Ho Gene

In a screen for cell cycle-regulated genes in the yeast Saccharomyces cerevisiae, we have identified a gene, EGT2, which is involved in cell separation in the G1 stage of the cell cycle. Transcription of EGT2 is tightly regulated in a cell cycle-dependent manner. Transcriptional levels peak at the boundary of mitosis and early G1 The transcription factors responsible for EGT2 expression in early G1 are Swi5 and, to a lesser extent, Ace2. Swi5 is involved in the transcriptional activation of the HO gene during late G1 and early S phase, and Ace2 induces CTS1 transcription during early and late G1 We show that Swi5 activates EGT2 transcription as soon as it enters the nucleus at the end of mitosis in a concentration-dependent manner. Since Swi5 is unstable in the nucleus, its level drops rapidly, causing termination of EGT2 transcription before cells are committed to the next cell cycle. However, Swi5 is still able to activate transcription of HO in late G1 in conjunction with additional activators such as Swi4 and Swi6.

scholarly journals Identification of Novel Saccharomyces cerevisiaeProteins with Nuclear Export Activity: Cell Cycle-Regulated Transcription Factor Ace2p Shows Cell Cycle-Independent Nucleocytoplasmic Shuttling

2000 ◽  
Vol 20 (21) ◽  
pp. 8047-8058 ◽  
Author(s):  
Torben Heick Jensen ◽  
Megan Neville ◽  
Jean Christophe Rain ◽  
Terri McCarthy ◽  
Pierre Legrain ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Nuclear Export ◽  
Consensus Sequence ◽  
Dependent Manner ◽  
Reading Frame ◽  
Export Activity ◽  
Amino Acid Region ◽  
Rna Triphosphatase ◽  
Cycle Dependent

ABSTRACT Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiaeproteins not previously known to have nuclear export activity. These proteins are the 5′ RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G1-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G1 but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner.

Dynamic cell cycle-dependent phosphorylation modulates CENP-L-CENP-N centromere recruitment

2021 ◽  
Author(s):  
Alexandra P Navarro ◽  
Iain M Cheeseman
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
S Phase ◽  
Dependent Manner ◽  
Dynamic Cell ◽  
A Cell ◽  
Cycle Dependent ◽  
Unique Cell ◽  
Localization Behavior ◽  
Constitutive Phosphorylation

The kinetochore is a macromolecular structure that is required to ensure proper chromosome segregation during each cell division. The kinetochore is assembled upon a platform of the 16-subunit Constitutive Centromere Associated Network (CCAN), which is present at centromeres throughout the cell cycle. The nature and regulation of CCAN assembly, interactions, and dynamics required to facilitate changing centromere properties and requirements remain to be fully elucidated. The CENP-LN CCAN sub-complex displays a unique cell cycle-dependent localization behavior, peaking in S phase. Here, we demonstrate that phosphorylation of CENP-L and CENP-N controls CENP-LN complex formation and localization in a cell cycle-dependent manner. Mimicking constitutive phosphorylation of either CENP-L or CENP-N or simultaneously preventing phosphorylation of both proteins prevents CENP-LN localization and disrupts chromosome segregation. Together, our work suggests that cycles of phosphorylation and dephosphorylation are critical for CENP-LN complex recruitment and dynamics at centromeres to enable cell cycle-dependent CCAN reorganization.

SF3B1 Interactions with Chromatin Are Dynamic and Regulated in a Cell Cycle-Dependent Manner

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1480-1480
Author(s):  
Tushar Murthy ◽  
Theresa Bluemn ◽  
Manoj M. Pillai ◽  
Alex C Minella
Keyword(s):  
Cell Cycle ◽  
Rna Splicing ◽  
Cell Cycle Progression ◽  
Conflicts Of Interest ◽  
S Phase ◽  
Chromatin Interaction ◽  
Dependent Manner ◽  
Direct Role ◽  
Cycle Progression ◽  
Cycle Dependent

Abstract Splicing factor 3B1 (SF3B1) is a member of the U2 snRNP complex that is a key regulator of RNA splicing. RNA splicing begins with the recognition of splice sites (ss) at the 5' and 3' ends of introns and ends with the removal of introns and joining of exons flanking them. SF3B1 plays an important role in this process by facilitating the recognition of the 3'ss. SF3B1 is frequently mutated in numerous cancers as well as the myelodysplastic syndromes (MDS). Mutations within the HEAT domain of the protein potentially contribute to disease pathogenesis. In addition to influencing splicing by binding to pre-mRNA, SF3B1 has been shown to affect splicing of exons by associating with them directly on chromatin via histone/nucleosome interactions. However, it is not understood if or how SF3B1 association with chromatin is regulated. Given that N-terminal serine and threonine residues on SF3B1 are known substrates of cyclin E-Cdk2, which phosphorylates histone subunits and other chromatin associated proteins, we hypothesized that CDK2 activity regulates SF3B1-nucleosome interactions. Although SF3B1 is phosphorylated during splicing catalysis, the function of this phosphorylation has remained unknown. We have now discovered, using nucleosome preparations and histone subunit co-immunoprecipitation assays in synchronized cells, that endogenous SF3B1 interacts with nucleosomes in a highly cell-cycle dependent manner, while total cellular abundance of SF3B1 remains invariant during cell cycle progression. In human and mouse cells, including hematopoietic cell lines, SF3B1 is excluded from chromatin during both G0 (quiescence) and G2/M phases of cell cycle. Notably, SF3B1 is enriched within chromatin maximally during S-phase. Unexpectedly, we found that the inhibition of Cdc2 (Cdk1) during G2/M enforces the SF3B1-chromatin interaction, pointing to a direct role for Cdc2 in restraining this interaction during mitosis. Further, SF3B1 loading onto chromatin during early cell cycle progression from G0 to S-phase is inhibited by Cdk2 inhibition. Thus, Cdk2 and Cdc2 appear to have antagonistic roles in controlling SF3B1-chromatin interactions during the cell cycle. Our findings suggest that Cdk activity may regulate the recruitment of the spliceosome machinery in order to coordinate splicing of particular transcripts with cell cycle progression. Current studies are focusing on how disease-associated mutations in the HEAT domain of SF3B1 affect the dynamics of its cell cycle-dependent interaction with nucleosomes and corresponding alterations to splicing outcomes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Topoisomerase IIα Prevents, But Does Not Resolve, Ultrafine Anaphase Bridges By Two Mechanisms

2019 ◽  
Author(s):  
Simon Gemble ◽  
Géraldine Buhagiar-Labarchède ◽  
Rosine Onclercq-Delic ◽  
Sarah Lambert ◽  
Mounira Amor-Guéret
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
S Phase ◽  
Cycle Phase ◽  
Dependent Manner ◽  
Topoisomerase Iiα ◽  
Anaphase Bridges ◽  
Double Stranded Dna ◽  
A Cell ◽  
Cycle Dependent

AbstractTopoisomerase IIα (Topo IIα), a well-conserved double-stranded DNA (dsDNA)-specific decatenase, processes dsDNA catenanes resulting from DNA replication during mitosis. Topo IIα defects lead to an accumulation of ultrafine anaphase bridges (UFBs), a type of chromosome non-disjunction. Topo IIα has been reported to resolve DNA anaphase threads, possibly accounting for the increase in UFB frequency upon Topo IIα inhibition. We hypothesized that the excess UFBs might also result, at least in part, from an impairment of the prevention of UFB formation by Topo IIα. We found that Topo IIα inhibition promotes UFB formation without affecting UFB resolution during anaphase. Moreover, we showed that Topo IIα inhibition promotes the formation of two types of UFBs depending on cell-cycle phase. Topo IIα inhibition during S-phase compromises complete DNA replication, leading to the formation of UFB-containing unreplicated DNA, whereas Topo IIα inhibition during mitosis impedes DNA decatenation at metaphase-anaphase transition, leading to the formation of UFB-containing DNA catenanes. Thus, Topo IIα activity is essential to prevent UFB formation in a cell-cycle dependent manner, but dispensable for UFB resolution during anaphase.

Nuclear accumulation of ZFP36L1 is cell cycle-dependent and determined by a C-terminal serine-rich cluster

2020 ◽  
Vol 168 (5) ◽  
pp. 477-489
Author(s):  
Yuki Matsuura ◽  
Aya Noguchi ◽  
Shunsuke Sakai ◽  
Naoto Yokota ◽  
Hiroyuki Kawahara
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Rna Binding ◽  
S Phase ◽  
Nuclear Accumulation ◽  
Protein Accumulation ◽  
Dependent Manner ◽  
Rich Cluster ◽  
G2 Phase ◽  
Cycle Dependent

Abstract ZFP36L1 is an RNA-binding protein responsible for mRNA decay in the cytoplasm. ZFP36L1 has also been suggested as a nuclear-cytoplasmic shuttling protein because it contains a potential nuclear localization signal and a nuclear export signal. However, it remains unclear how the nuclear localization of ZFP36L1 is controlled. In this study, we provide evidence that the nuclear accumulation of ZFP36L1 protein is modulated in a cell cycle-dependent manner. ZFP36L1 protein accumulation in fractionated nuclei was particularly prominent in cells arrested at G1-/S-phase boundary, while it was downregulated in S-phase cells, and eventually disappeared in G2-phase nuclei. Moreover, forced nuclear targeting of ZFP36L1 revealed marked downregulation of this protein in S- and G2-phase cells, suggesting that ZFP36L1 can be eliminated in the nucleus. The C-terminal serine-rich cluster of ZFP36L1 is critical for the regulation of its nuclear accumulation because truncation of this probable disordered region enhanced the nuclear localization of ZFP36L1, increased its stability and abolished its cell cycle-dependent fluctuations. These findings provide the first hints to the question of how ZFP36L1 nuclear accumulation is controlled during the course of the cell cycle.

ors12, a mammalian autonomously replicating DNA sequence, associates with the nuclear matrix in a cell cycle-dependent manner

1993 ◽  
Vol 105 (3) ◽  
pp. 807-818
Author(s):  
D.C. Mah ◽  
P.A. Dijkwel ◽  
A. Todd ◽  
V. Klein ◽  
G.B. Price ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Matrix ◽  
Pcr Amplification ◽  
S Phase ◽  
Dependent Manner ◽  
The Matrix ◽  
S Period ◽  
Cycle Dependent

Origin enriched sequence ors8 and ors12, have been isolated previously by extrusion of nascent CV-1 cell DNA from replication bubbles at the onset of S-phase. Both have been shown to direct autonomous DNA replication in vivo and in vitro. Here, we have examined the association of genomic ors8 and ors12 with the nuclear matrix in asynchronous and synchronized CV-1 cells. In asynchronously growing cells, ors8 was found to be randomly distributed, while ors12 was found to be enriched on the nuclear matrix. Using an in vitro binding assay, we determined that ors12 contains two attachment sites, each located in AT-rich domains. Surprisingly, in early and mid-S-phase cells, ors12 homologous sequences were recovered mainly from the DNA loops, while in late-S the majority had shifted to positions on the nuclear matrix. In contrast, the distribution of ors8 over the matrix and loop DNA fractions did not change during the cell cycle. By bromodeoxyuridine substitution of replicating DNA, followed by immunoprecipitation with anti-bromodeoxyuridine antibodies and PCR amplification, we demonstrated that ors12 replicates almost exclusively on the matrix in early and mid-S-phase; replicating ors8 was also found to be enriched on the matrix in early S-phase. Chase experiments showed that the ors12 sequences labelled with bromodeoxyuridine in the first 2 hours of S-phase remain attached to the nuclear matrix, resulting in an accumulation of ors12 on the nuclear matrix at the end of the S period.

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