Recruitment of the cell cycle checkpoint kinase ATR to chromatin during S-phase

The ataxia telangiectasia-mutated (ATM) and Rad3-related kinase (ATR) is a central component of the cell cycle checkpoint machinery required to induce cell cycle arrest in response to DNA damage. Accumulating evidence suggests a role for ATR in signaling DNA damage during S-phase. Here we show that ATR is recruited to nuclear foci induced by replication fork stalling in a manner that is dependent on the single stranded binding protein replication protein A (RPA). ATR associates with chromatin in asynchronous cell cultures, and we use a variety of approaches to examine the association of ATR with chromatin in the absence of agents that cause genotoxic stress. Under our experimental conditions, ATR exhibits a decreased affinity for chromatin in quiescent cells and cells synchronized at mitosis but an increased affinity for chromatin as cells re-enter the cell cycle. Using centrifugal elutriation to obtain cells enriched at various stages of the cell cycle, we show that ATR associates with chromatin in a cell cycle-dependent manner, specifically during S-phase. Cell cycle association of ATR with chromatin mirrors that of RPA in addition to claspin, a cell cycle checkpoint protein previously shown to be a component of the replication machinery. Furthermore, association of ATR with chromatin occurs in the absence of detectable DNA damage and cell cycle checkpoint activation. These data are consistent with a model whereby ATR is recruited to chromatin during the unperturbed cell cycle and points to a role of ATR in monitoring genome integrity during normal S-phase progression.     

Polyamines may regulate S-phase progression but not the dynamic changes of chromatin during the cell cycle

Several studies suggest that polyamines may stabilize chromatin and play a role in its structural alterations. In line with this idea, we found here by chromatin precipitation and micrococcal nuclease (MNase) digestion analyses, that spermidine and spermine stabilize or condense the nucleosomal organization of chromatin in vitro. We then investigated the possible physiological role of polyamines in the nucleosomal organization of chromatin during the cell cycle in Chinese hamster ovary (CHO) cells deficient in ornithine decarboxylase (ODC) activity. An extended polyamine deprivation (for 4 days) was found to arrest 70% of the odc⁻ cells in S phase. MNase digestion analyses revealed that these cells have a highly loosened and destabilized nucleosomal organization. However, no marked difference in the chromatin structure was detected between the control and polyamine-depleted cells following the synchronization of the cells at the S-phase. We also show in synchronized cells that polyamine deprivation retards the traverse of the cells through the S phase already in the first cell cycle. Depletion of polyamines had no significant effect on the nucleosomal organization of chromatin in G₁–early S. The polyamine-deprived cells were also capable of condensing the nucleosomal organization of chromatin in the S/G₂ phase of the cell cycle. These data indicate that polyamines do not regulate the chromatin condensation state during the cell cycle, although they might have some stabilizing effect on the chromatin structure. Polyamines may, however, play an important role in the control of S-phase progression. J. Cell Biochem. 68:200–212, 1998. © 1998 Wiley-Liss, Inc.     

Chromatin remodeling by cell cycle stage-specific extracts from Physarum polycephalum

Remodeling of chromatin is an essential process allowing the establishment of specific genetic programs. The slime mold Physarum polycephalum presents the attractive advantage of natural synchrony of the cell cycle in several million nuclei. Whole-cell extracts prepared at precise stages during the cell cycle were tested for the ability to induce remodeling in erythrocyte nuclei as monitored by microscopy, protamine competition assays, micrococcal nuclease digestions, and release of histone H5. Extracts derived from two specific cell cycle stages caused opposite types of changes in erythrocyte nuclei. An increase in chromatin compaction was imparted by extracts prepared during S-phase while extracts harvested at the end of G2-phase caused increases in nuclear volume, DNA accessibility, and release of linker histone. We also found that late G2 extracts had the ability to alter the DNase I digestion profile of mononucleosomes reconstituted in vitro in a classical nucleosomes remodeling assay. The relevance of these finding to the Physarum cell cycle is discussed.     

Proteolysis of DNA replication licensing factor Cdt1 in S-phase is performed independently of geminin through its N-terminal region

Licensing of replication origins is carefully regulated in a cell cycle to maintain genome integrity. Using an in vivo ubiquitination assay and temperature-sensitive cell lines we demonstrate that Cdt1 in mammalian cells is degraded through ubiquitin-dependent proteolysis in S-phase. siRNA experiments for Geminin indicate that Cdt1 is degraded in the absence of Geminin. The N terminus of Cdt1 is required for its nuclear localization, associates with cyclin A, but is dispensable for the association of Cdt1 with Geminin in cells. This region is responsible for proteolysis of Cdt1 in S-phase. On the other hand, the N terminus-truncated Cdt1 is stable in S-phase, and associates with the licensing inhibitor, Geminin. High level expression of this form of Cdt1 brings about cells having higher DNA content. Proteasome inhibitors stabilize Cdt1 in S-phase, and the protein is detected in the nucleus in a complex with Geminin. This form of Cdt1 associates with chromatin as tightly as that of G1-cells, when no Geminin is detected. Our data show that proteolysis and Geminin binding independently inactivate Cdt1 after the onset of S-phase to prevent re-replication.     

Nek1 interacts with Ku80 to assist chromatin loading of replication factors and S-phase progression

NIMA-related kinases (Neks) play divergent roles in mammalian cells. While several Neks regulate mitosis, Nek1 was reported to regulate DNA damage response, centrosome duplication and primary cilium formation. Whether Nek1 participates in cell cycle regulation is not known. Here we report that loss of Nek1 results in severe proliferation defect due to a delay in S-phase of the cell cycle. Nek1-deficient cells show replication stress and checkpoint activation under normal growth conditions. Nek1 accumulates on the chromatin during normal DNA replication. In response to replication stress, Nek1 is further activated for chromatin localization. Nek1 interacts with Ku80 and, in Nek1-deficient cells chromatin localization of Ku80 and several other DNA replication factors is significantly reduced. Thus, Nek1 may facilitate S-phase progression by interacting with Ku80 and regulating chromatin loading of replication factors.     

Retinoblastoma tumor suppressor protein signals through inhibition of cyclin-dependent kinase 2 activity to disrupt PCNA function in S phase

The retinoblastoma tumor suppressor protein (RB) is a negative regulator of the cell cycle that inhibits both G(1) and S-phase progression. While RB-mediated G(1) inhibition has been extensively studied, the mechanism utilized for S-phase inhibition is unknown. To delineate the mechanism through which RB inhibits DNA replication, we generated cells which inducibly express a constitutively active allele of RB (PSM-RB). We show that RB-mediated S-phase inhibition does not inhibit the chromatin binding function of MCM2 or RPA, suggesting that RB does not regulate the prereplication complex or disrupt early initiation events. However, activation of RB in S-phase cells disrupts the chromatin tethering of PCNA, a requisite component of the DNA replication machinery. The action of RB was S phase specific and did not inhibit the DNA damage-mediated association of PCNA with chromatin. We also show that RB-mediated PCNA inhibition was dependent on downregulation of CDK2 activity, which was achieved through the downregulation of cyclin A. Importantly, restoration of cyclin-dependent kinase 2 (CDK2)-cyclin A and thus PCNA activity partially restored S-phase progression in the presence of active RB. Therefore, the data presented identify RB-mediated regulation of PCNA activity via CDK2 attenuation as a mechanism through which RB regulates S-phase progression. Together, these findings identify a novel pathway of RB-mediated replication inhibition.     

Cell-cycle-regulated control of VSG expression site silencing by histones and histone chaperones ASF1A and CAF-1b in Trypanosoma brucei

Antigenic variation in African trypanosomes involves monoallelic expression and reversible silencing of variant surface glycoprotein (VSG) genes found adjacent to telomeres in polycistronic expression sites (ESs). We assessed the impact on ES silencing of five candidate essential chromatin-associated factors that emerged from a genome-wide RNA interference viability screen. Using this approach, we demonstrate roles in VSG ES silencing for two histone chaperones. Defects in S-phase progression in cells depleted for histone H3, or either chaperone, highlight in particular the link between chromatin assembly and DNA replication control. S-phase checkpoint arrest was incomplete, however, allowing G₂/M-specific VSG ES derepression following knockdown of histone H3. In striking contrast, knockdown of anti-silencing factor 1A (ASF1A) allowed for derepression at all cell cycle stages, whereas knockdown of chromatin assembly factor 1b (CAF-1b) revealed derepression predominantly in S-phase and G₂/M. Our results support a central role for chromatin in maintaining VSG ES silencing. ASF1A and CAF-1b appear to play constitutive and DNA replication-dependent roles, respectively, in the recycling and assembly of chromatin. Defects in these functions typically lead to arrest in S-phase but defective cells can also progress through the cell cycle leading to nucleosome depletion and derepression of telomeric VSG ESs.     

Developmental differences in genome replication program and origin activation

To ensure error-free duplication of all (epi)genetic information once per cell cycle, DNA replication follows a cell type and developmental stage specific spatio-temporal program. Here, we analyze the spatio-temporal DNA replication progression in (un)differentiated mouse embryonic stem (mES) cells. Whereas telomeres replicate throughout S-phase, we observe mid S-phase replication of (peri)centromeric heterochromatin in mES cells, which switches to late S-phase replication upon differentiation. This replication timing reversal correlates with and depends on an increase in condensation and a decrease in acetylation of chromatin. We further find synchronous duplication of the Y chromosome, marking the end of S-phase, irrespectively of the pluripotency state. Using a combination of single-molecule and super-resolution microscopy, we measure molecular properties of the mES cell replicon, the number of replication foci active in parallel and their spatial clustering. We conclude that each replication nanofocus in mES cells corresponds to an individual replicon, with up to one quarter representing unidirectional forks. Furthermore, with molecular combing and genome-wide origin mapping analyses, we find that mES cells activate twice as many origins spaced at half the distance than somatic cells. Altogether, our results highlight fundamental developmental differences on progression of genome replication and origin activation in pluripotent cells.     

3D chromatin conformation correlates with replication timing and is conserved in resting cells

Although chromatin folding is known to be of functional importance to control the gene expression program, less is known regarding its interplay with DNA replication. Here, using Circular Chromatin Conformation Capture combined with high-throughput sequencing, we identified megabase-sized self-interacting domains in the nucleus of a human lymphoblastoid cell line, as well as in cycling and resting peripheral blood mononuclear cells (PBMC). Strikingly, the boundaries of those domains coincide with early-initiation zones in every cell types. Preferential interactions have been observed between the consecutive early-initiation zones, but also between those separated by several tens of megabases. Thus, the 3D conformation of chromatin is strongly correlated with the replication timing along the whole chromosome. We furthermore provide direct clues that, in addition to the timing value per se, the shape of the timing profile at a given locus defines its set of genomic contacts. As this timing-related scheme of chromatin organization exists in lymphoblastoid cells, resting and cycling PBMC, this indicates that it is maintained several weeks or months after the previous S-phase. Lastly, our work highlights that the major chromatin changes accompanying PBMC entry into cell cycle occur while keeping largely unchanged the long-range chromatin contacts.     

Stabilization and Localization of Xist RNA are Controlled by Separate Mechanisms and are Not Sufficient for X Inactivation

We examined the effect of cell cycle progression on various levels of chromosome organization in Drosophila. Using bromodeoxyuridine incorporation and DNA quantitation in combination with fluorescence in situ hybridization, we detected gross chromosomal movements in diploid interphase nuclei of larvae. At the onset of S-phase, an increased separation was seen between proximal and distal positions of a long chromsome arm. Progression through S-phase disrupted heterochromatic associations that have been correlated with gene silencing. Additionally, we have found that large-scale G1 nuclear architecture is continually dynamic. Nuclei display a Rabl configuration for only ∼2 h after mitosis, and with further progression of G1-phase can establish heterochromatic interactions between distal and proximal parts of the chromosome arm. We also find evidence that somatic pairing of homologous chromosomes is disrupted during S-phase more rapidly for a euchromatic than for a heterochromatic region. Such interphase chromosome movements suggest a possible mechanism that links gene regulation via nuclear positioning to the cell cycle: delayed maturation of heterochromatin during G1-phase delays establishment of a silent chromatin state.     

DNAase I and cellular factors that affect chromatin structure

The use of DNAase I as a probe of chromatin structure is frequently fraught with problems of irreproducibility. We have recently evaluated this procedure, documented the sources of the problems, and standardized the method for reproducible results (Prentice and Gurley (1983) Biochim. Biophys. Acta 740, 134-144). We have now used this probe to detect differences in chromatin structure between cells blocked (1) in G1 phase by isoleucine deprivation, or (2) in early S phase by sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea. The cells blocked in G1 phase have easily-digestible chromatin, while cells blocked in early S phase have chromatin which is much more resistant to DNAase I. These differences were found to be the result of diffusible factors found in the cytoplasm and nuclei of G1- and S-phase cells, respectively. The G1 cells contained a cytoplasmic factor which modulates the chromatin structure of S-phase nuclei to a more easily digestible state, while cells blocked in S phase contain a nuclear factor which modulates the chromatin structure of G1 nuclei to a state more resistant to digestion. DNAase I is much more sensitive to these cell cycle-specific chromatin changes than is micrococcal nuclease. The results indicate that, under controlled conditions, DNAase I should be a valuable probe for detecting chromatin structural changes associated with cell cycle traverse, differentiation, development, hormone action and chemical toxicity.     

Use of a double-label method to detect rapid changes in the rate of cell proliferation.

We developed a double-label method to directly measure the rate at which cells enter S-phase of the cell cycle. All cells in S-phase were first labeled with a short pulse of [3H]-thymidine. This was followed by a longer incubation in bromodeoxyuridine (BrdU), a thymidine analogue. Nuclei labeled with [3H]-thymidine were detected by autoradiography and those labeled with BrdU by immunocytochemistry. Cells labeled only with BrdU must have entered S-phase at some time after the end of the [3H]-thymidine pulse. Thus, the rate of entry of cells into S-phase could be determined. This method was shown to be more accurate and more sensitive than determining changes in the rate at which cells entered S-phase with a continuous labeling protocol. It was possible to detect changes in proliferative activity that occurred in less than 1 hr. We used this double-label technique to study changes in the cell cycle during the terminal differentiation of chicken embryo lens fiber cells. These studies revealed differences in the effects of several treatments known to stimulate fiber cell differentiation. They also demonstrated the presence in the embryonic eye of factors that stimulate and prevent lens cell proliferation and differentiation.     

Drosophila SUUR protein associates with PCNA and binds chromatin in a cell cycle-dependent manner

Drosophila SUUR (Suppressor of UnderReplication) protein was shown to regulate the DNA replication elongation process in endocycling cells. This protein is also known to be the component of silent chromatin in polyploid and diploid cells. To mark the different cell cycle stages, we used immunostaining patterns of PCNA, the main structural component of replication fork. We demonstrate that SUUR chromatin binding is dynamic throughout the endocyle in Drosophila salivary glands. We observed that SUUR chromosomal localization changed along with PCNA pattern and these proteins largely co-localized during the late S-phase in salivary glands. The hypothesized interaction between SUUR and PCNA was confirmed by co-immunoprecipitation from embryonic nuclear extracts. Our findings support the idea that the effect of SUUR on replication elongation depends on the cell cycle stage and can be mediated through its physical interaction with replication fork.