End Of S-phase Research Articles

Visualizing Active Replication Regions in S-Phase Chromosomes.

A basic question of cell biology is how DNA folds to chromosome. A number of recently accumulated evidences have suggested that folding of chromosome proceeds tightly coupled with DNA replication progresses. Drug-induced PCC is a useful tool for visualization of the interphase nuclei, in particular, S-phase, as S-phase prematurely condensed chromosomes (S-phase PCC). Active replicating DNA is labeled directly with Cy3-dUTP by bead loading method, and then S-phase nuclei is immediately condensed prematurely by calyculin A to obtain S-phase PCC. Active replicating regions on S-PCC are observed under a scanning confocal microscope. Cy3-dUTP-labeled S-phase PCCs clearly reveal the drastic transitional change of chromosome formation through S-phase, starting from a "cloudy nebula" to numerous numbers of "beads on a string" and finally to "striped arrays of banding structured chromosome" known as G- or R-banding pattern. The number, distribution, and shape of replication foci were also measured in individual subphase of S-phase; maximally ~1400 foci of 0.35 μm average radius size were scored at the beginning of S-phase, and the number is reduced to ~100 at the end of S-phase. Drug-induced PCC clearly provided the new insight that eukaryote DNA replication is tightly coupled with the chromosome condensation/compaction for construction of eukaryote higher-ordered chromosome structure.

Knockouts of TUT7 and 3'hExo show that they cooperate in histone mRNA maintenance and degradation.

Metazoan histone mRNAs are the only cellular eukaryotic mRNAs that are not polyadenylated, ending instead in a conserved stem-loop. SLBP is bound to the 3' end of histone mRNAs and is required for translation of histone mRNA. The expression of histone mRNAs is tightly cell-cycle regulated. A major regulatory step is rapid degradation of histone mRNA at the end of S-phase or when DNA synthesis is inhibited in S-phase. 3'hExo, a 3' to 5' exonuclease, binds to the SLBP/SL complex and trims histone mRNA to 3 nt after the stem-loop. Together with a terminal uridyl transferase, 3'hExo maintains the length of the histone mRNA during S-phase. 3'hExo is essential for initiating histone mRNA degradation on polyribosomes, initiating degradation into the 3' side of the stem-loop. There is extensive uridylation of degradation intermediates in the 3' side of the stem when histone mRNA is degraded. Here, we knocked out TUT7 and 3'hExo and we show that both modification of histone mRNA during S-phase and degradation of histone mRNA involve the interaction of 3'hExo, and a specific TUTase, TENT3B (TUT7, ZCCHC6). Knockout of 3'hExo prevents the initiation of 3' to 5' degradation, stabilizing histone mRNA, whereas knockout of TUT7 prevents uridylation of the mRNA degradation intermediates, slowing the rate of degradation. In synchronized 3'hExo KO cells, histone mRNA degradation is delayed, but the histone mRNA is degraded prior to mitosis by a different pathway.

Centriole growth is limited by the Cdk/Cyclin-dependent phosphorylation of Ana2/STIL.

Centrioles duplicate once per cell cycle, but it is unclear how daughter centrioles assemble at the right time and place and grow to the right size. Here, we show that in Drosophila embryos the cytoplasmic concentrations of the key centriole assembly proteins Asl, Plk4, Ana2, Sas-6, and Sas-4 are low, but remain constant throughout the assembly process-indicating that none of them are limiting for centriole assembly. The cytoplasmic diffusion rate of Ana2/STIL, however, increased significantly toward the end of S-phase as Cdk/Cyclin activity in the embryo increased. A mutant form of Ana2 that cannot be phosphorylated by Cdk/Cyclins did not exhibit this diffusion change and allowed daughter centrioles to grow for an extended period. Thus, the Cdk/Cyclin-dependent phosphorylation of Ana2 seems to reduce the efficiency of daughter centriole assembly toward the end of S-phase. This helps to ensure that daughter centrioles stop growing at the correct time, and presumably also helps to explain why centrioles cannot duplicate during mitosis.

Role of oligouridylation in normal metabolism and regulated degradation of mammalian histone mRNAs.

Metazoan replication-dependent histone mRNAs are the only known cellular mRNAs that are not polyadenylated. Histone mRNAs are present in large amounts only in S-phase cells, and their levels are coordinately regulated with the rate of DNA replication. In mammals, the stemloop at the 3' end of histone mRNA is bound to stemloop binding protein, a protein required for both synthesis and degradation of histone mRNA, and an exonuclease, 3'hExo (ERI1). Histone mRNAs are rapidly degraded when DNA synthesis is inhibited in S-phase cells and at the end of S-phase. Upf1 is also required for rapid degradation of histone mRNA as is the S-phase checkpoint. We report that Smg1 is required for histone mRNA degradation when DNA replication is inhibited, suggesting it is the PI-like kinase that activates Upf1 for histone mRNA degradation. We also show that some mutant Upf1 proteins are recruited to histone mRNAs when DNA replication is inhibited and act as dominant negative factors in histone mRNA degradation. We report that the pathway of rapid histone mRNA degradation when DNA replication is inhibited in S-phase cells that are activating the S-phase checkpoint is similar to the pathway of rapid degradation of histone mRNA at the end of S-phase.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'.

Carcinogen susceptibility is regulated by genome architecture and predicts cancer mutagenesis.

The development of many sporadic cancers is directly initiated by carcinogen exposure. Carcinogens induce malignancies by creating DNA lesions (i.e., adducts) that can result in mutations if left unrepaired. Despite this knowledge, there has been remarkably little investigation into the regulation of susceptibility to acquire DNA lesions. In this study, we present the first quantitative human genome-wide map of DNA lesions induced by ultraviolet (UV) radiation, the ubiquitous carcinogen in sunlight that causes skin cancer. Remarkably, the pattern of carcinogen susceptibility across the genome of primary cells significantly reflects mutation frequency in malignant melanoma. Surprisingly, DNase-accessible euchromatin is protected from UV, while lamina-associated heterochromatin at the nuclear periphery is vulnerable. Many cancer driver genes have an intrinsic increase in carcinogen susceptibility, including the BRAF oncogene that has the highest mutation frequency in melanoma. These findings provide a genome-wide snapshot of DNA injuries at the earliest stage of carcinogenesis. Furthermore, they identify carcinogen susceptibility as an origin of genome instability that is regulated by nuclear architecture and mirrors mutagenesis in cancer.

Mec1ATR is needed for extensive telomere elongation in response to ethanol in yeast.

Telomere length homeostasis is essential for cell survival. In humans, telomeres shorten as a function of age. Short telomeres are known determinants of cell senescence and longevity. The yeast Saccharomyces cerevisiae expresses telomerase and maintains a strict telomere length homeostasis during vegetative growth. We have previously reported that different environmental signals promote changes in telomere length in S. cerevisiae. In particular, exposure to ethanol induces an extensive telomere elongation response due to a reduction in RAP1 mRNA and protein levels. Here we show that the reduction in Rap1 protein levels disrupts the physical interaction between Rap1 and Rif1, which in turn reduces the recruitment of these two proteins to telomeres during G2-phase. Although elongation of the shortest telomeres has been shown to depend on the Rif2 telomeric protein and on the Tel1(ATM) protein kinase, we show here that the extensive telomere elongation in response to ethanol exposure is Rif1 and Mec1 (ATR)-dependent. Our results fit a model in which Rif1 and Rap1 form a complex that is loaded onto telomeres at the end of S-phase. Reduced levels of the Rap1-Rif1 complex in ethanol lead to continuous telomere elongation in a Mec1-dependent process.

MP83-13 ONCOLYTIC VACCINIA VIRUS HAS SIGNIFICANT ACTIVITY AND INDUCES ANTI-TUMOR IMMUNITY IN BLADDER CANCER

INTRODUCTION AND OBJECTIVES: Most Bladder Cancers (BCs) are diagnosed as non-muscle invasive, however with a recurrence rate of up to 80%, many patients require multiple treatment strategies that often fail. In particular, Bacillus Calmette-Guerin (BCG) carries the risk of infection and can be dangerous for immunocompromised (IC) patients. 30% of patients fail BCG therapy and cystectomy remains the standard treatment in these cases. Oncolytic viruses preferentially replicate in and lyse cancer cells while sparing normal cells. Virus replication requires an abundant supply of dNTPs, but because cellular enzymes that synthesize dNTPs are degraded at the end of S-phase, vaccinia virus (VAC) expresses its own biosynthetic enzymes including both I4L (large, R1) and F4L (small, R2) subunits of the heterodimeric ribonucleotide reductase. We have shown that deleting the F4L gene inhibits virus replication in normal cells and reduces virulence while retaining replication proficiency in cancer cells. METHODS: We developed preclinical and clinical grade VACs with deletions in F4L, J2R (viral thymidine kinase) or both. Oncolytic activities of these VACs were evaluated in a panel of human and rodent BC or normal cell lines in vitro. Two BC models were used to assess the VACs in vivo: Syngeneic AY-27 in immunocompetent rats and human RT112-luc xenografts in IC mice. RESULTS: Cytotoxicity assays showed a high degree of cell killing in infections with VACs. Highly efficient VAC replication was seen in BC cells with significantly less replication seen in normal cells (p<0.01). VACs selectively replicated in both AY-27 immunocompetent and RT112-luc IC BC models causing significant tumor regression (p<0.001) or complete ablation with no toxicity. Rats treated with the VACs developed a protective CD8+ T-cell mediated anti-tumor immunity, evident by tumor rejection upon challenge and in vitro assays. Finally, VAC replicated in BC cell lines made BCGrefractory. CONCLUSIONS: Our data indicate that DF4L VACs retain their replication proficiency and cytotoxicity in BC cells despite deletion of these critical viral genes required in non-transformed cells. Significant replication in BC cells that are resistant to the uptake of BCG was observed. Treatment with DF4L VACs in vivo is selective for BC cells, elicits an anti-tumor immunity, and shows no signs of toxicity. Further mechanistic investigations are being conducted on the antitumor effects of DF4L VACs in a BCG-resistant animal model. If successful VAC could provide an essential line of therapy for patients, particularly those refractory to BCG treatment.

Analysis of the Mechanism of DNA Damage and Replication Arrest‐induced Histone mRNA Decay Pornpen Panomwan*and Carl Smythe

Histone mRNA decay (HD) is the process which ensures that histone mRNA is rapidly degraded following completion of DNA replication at the end of S-phase. Strict coordination between histone protein production and DNA replication is essential for the correct packaging of newly replicated DNA, as imbalances can lead to deleterious effects such as genomic instability. Interestingly, histone mRNA stability is controlled by the presence of a stem-loop structure at the 3' end of histone mRNA, and a protein, HBP/SLBP (hairpin/stem loop binding protein) which specifically binds to this structure, plays a major role in histone mRNA metabolism. Importantly, previous studies have shown HD is also one functional target of an intra S-phase checkpoint activated when DNA synthesis is inhibited, ensuring that histone mRNA is rapidly destroyed when global DNA replication is blocked. Consistent with this, replication stress-induced histone mRNA decay is blocked in the presence of inhibitors of the PIKK family of checkpoint protein kinases, implicating PIKK family members in the regulation of this process. Therefore, we aim to utilise a proteomic approach to to identify HBP/SLBP-associated proteins and post-translational status in order to elucidate the detailed mechanism of checkpoint activated HD. We have established isogenic cell lines stably expressing functional, tagged HBP/SLBP by using the Flp-InTM T-RexTM Expression system. Our results indicate that isogenic Flp-In HeLa cell lines inducibly expressing tagged forms of SLBP under the control of a doxycycline promoter are a useful model system for the molecular analysis of SLBP function during replication stress.

The spatial and temporal organization of origin firing during the S-phase of fission yeast.

Eukaryotes duplicate their genomes using multiple replication origins, but the organization of origin firing along chromosomes and during S-phase is not well understood. Using fission yeast, we report the first genome-wide analysis of the spatial and temporal organization of replication origin firing, analyzed using single DNA molecules that can approach the full length of chromosomes. At S-phase onset, origins fire randomly and sparsely throughout the chromosomes. Later in S-phase, clusters of fired origins appear embedded in the sparser regions, which form the basis of nuclear replication foci. The formation of clusters requires proper histone methylation and acetylation, and their locations are not inherited between cell cycles. The rate of origin firing increases gradually, peaking just before mid S-phase. Toward the end of S-phase, nearly all the available origins within the unreplicated regions are fired, contributing to the timely completion of genome replication. We propose that the majority of origins do not fire as a part of a deterministic program. Instead, origin firing, both individually and as clusters, should be viewed as being mostly stochastic.

Epigenetic regulation of the human genome: coherence between promoter activity and large-scale chromatin environment

Increasing knowledge of chromatin structure in various cell types raises the challenge of deciphering the contribution of epigenetic modifications to the regulation of nuclear functions in mammals. In a recent study, we have analysed the genome-wide distributions of thirteen epigenetic marks in the human cell line K562 at 100 kb resolution of Mean Replication Timing (MRT) data. Using classical clustering techniques, we have shown that the combinatorial complexity of these epigenetic data can be reduced to four predominant chromatin states that replicate at different periods of the S-phase. C1 is an early replicating transcriptionally active euchromatin state, C2 a mid-S repressive type of chromatin associated with Polycomb complexes, C3 a silent chromatin with lack of chromatin marks that replicates later than C2 but before C4, a HP1-associated heterochromatin state that replicates at the end of S-phase. These four chromatin states display remarkable similarities with those recently reported in fly, worm and plants at higher ∼ 1 kb resolution of gene expression data. Here, we extend our integrative analysis of epigenetic data in the K562 human cell line to this smaller scale by focusing on gene promoters (±3 kb around transcription start sites). We show that these promoters can similarly be classified into four main chromatin states: P1 regroups all the marks of transcriptionally active chromatin and corresponds to CpG rich promoters of highly expressed genes; P2 is notably associated with the histone modification H3K27me3 that is the mark of a polycomb repressed chromatin state; P3 corresponds to promoters that are not enriched for any available marks as the signature of a ‘null’ or ‘black’ silent heterochromatin state and P4 characterizes the few gene promoters that contain only the constitutive heterochromatin histone modification H3K9me3. When investigating the coherence between promoter activity (P1, P2, P3 or P4) and the large-scale chromatin environment (C1, C2, C3 or C4), we find that the higher the gene density in a considered 100 kb-window, the higher (resp. the lower) the probability of a P1 active promoter (resp. silent P2, P3 and P4 promoters) to be surrounded by an open euchromatin C1 (resp. facultative C2, black C3 or HP1-associated C4 heterochromatin) environment. From large to small scales, it is mainly C4 and to a lesser extent C3 heterochromatin environments both corresponding to gene poor regions, that strongly conditions promoters to belong to the inactive P3 and P4 classes. If C1 (resp. C2) environment surrounds a majority of corresponding active P1 (resp. P2) promoters, it also contains a non-negligible proportion of inactive P2 and P3 (resp. active P1 and inactive P3) promoters. When further investigating the large-scale organization of human genes with respect to ‘master’ replication origins that were shown to border megabase-sized U-shaped MRT domains, we reveal some significant enrichment of highly expressed P1 genes in a closed neighbourhood of these early initiation zones consistently with the gradient of chromatin states observed from C1 at U-domain borders followed by C2, C3 and C4 at U-domain centers. On the contrary to P2 promoters that are mainly found in the C2 environment at finite distance (∼200–300 kb) from U-domain borders, the inactive P3 and P4 promoters are distributed rather homogeneously inside U-domains. The generalization of our study to different cell types including ES, somatic and cancer cells is likely to provide new insight on the global reorganization of replication domains during differentiation (or disease) in relation to coordinated changes in chromatin environment and gene expression.

DNA Binding of the Cell Cycle Transcriptional Regulator GcrA Depends on N6-Adenosine Methylation in Caulobacter crescentus and Other Alphaproteobacteria

Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer) cell and a replicative S-phase (stalked) cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation), biochemical and biophysical experimentation, and genetics to show that GcrA is a dimeric DNA–binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits the RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable) epigenetic switch that regulates gene expression during the cell cycle.

Prevalent coordination of mitochondrial DNA transcription and initiation of replication with the cell cycle

Nuclear (nDNA) and mitochondrial DNA (mtDNA) communication is essential for cell function, but it remains unclear whether the replication of these genomes is linked. We inspected human cells with a novel fluorescence in situ hybridization protocol (mitochondrial Transcription and Replication Imaging Protocol) that identifies mitochondrial structures engaged in initiation of mtDNA replication and unique transcript profiles, and reconstruct the temporal series of mitochondrial and nuclear events in single cells during the cell cycle. We show that mtDNA transcription and initiation of replication are prevalently coordinated with the cell cycle, preceding nuclear DNA synthesis, and being reactivated towards the end of S-phase. This coordination is achieved by modulating the fraction of mitochondrial structures that intiate mtDNA synthesis and/or contain transcript at a given time. Thus, although replication of the mitochondrial genome is active through the entire cell cycle, but in a limited fraction of mitochondrial structures, peaks of these activities are synchronized with nDNA synthesis. After release from blockage of mtDNA replication with either nocodazole or double thymidine treatment, prevalent mtDNA and nDNA synthesis occurred simultaneously, indicating that mitochondrial coordination with the nuclear phase can be adjusted in response to physiological alterations. These findings will help redefine other nuclear–mitochondrial links in cell function.

Epigenetic Silencing of Core Histone Genes by HERS in Drosophila

Cell cycle-dependent expression of canonical histone proteins enables newly synthesized DNA to be integrated into chromatin in replicating cells. However, the molecular basis of cell cycle-dependency in the switching of histone gene regulation remains to be uncovered. Here, we report the identification and biochemical characterization of a molecular switcher, HERS (histone gene-specific epigenetic repressor in late S phase), for nucleosomal core histone gene inactivation in Drosophila. HERS protein is phosphorylated by a cyclin-dependent kinase (Cdk) at the end of S-phase. Phosphorylated HERS binds to histone gene regulatory regions and anchors HP1 and Su(var)3-9 to induce chromatin inactivation through histone H3 lysine 9 methylation. These findings illustrate a salient molecular switch linking epigenetic gene silencing to cell cycle-dependent histone production.

Cells and polyamines do it cyclically

Cell-cycle progression is a one-way journey where the cell grows in size to be able to divide into two equally sized daughter cells. The cell cycle is divided into distinct consecutive phases defined as G(1) (first gap), S (synthesis), G(2) (second gap) and M (mitosis). A non-proliferating cell, which has retained the ability to enter the cell cycle when it receives appropriate signals, is in G(0) phase, and cycling cells that do not receive proper signals leave the cell cycle from G(1) into G(0). One of the major events of the cell cycle is the duplication of DNA during S-phase. A group of molecules that are important for proper cell-cycle progression is the polyamines. Polyamine biosynthesis occurs cyclically during the cell cycle with peaks in activity in conjunction with the G(1)/S transition and at the end of S-phase and during G(2)-phase. The negative regulator of polyamine biosynthesis, antizyme, shows an inverse activity compared with the polyamine biosynthetic activity. The levels of the polyamines, putrescine, spermidine and spermine, double during the cell cycle and show a certain degree of cyclic variation in accordance with the biosynthetic activity. When cells in G(0)/G(1) -phase are seeded in the presence of compounds that prevent the cell-cycle-related increases in the polyamine pools, the S-phase of the first cell cycle is prolonged, whereas the other phases are initially unaffected. The results point to an important role for polyamines with regard to the ability of the cell to attain optimal rates of DNA replication.

Generation and management of excess histones during the cell cycle

Histones are essential proteins that package the DNA in all eukaryotes into chromosomes. However, histones can accumulate upon a decrease in DNA synthesis that occurs at the end of S-phase or following replication arrest. These positively charged histones can associate non-specifically with the negatively charged DNA and other cellular biomolecules, impairing their normal function. Hence, cells have evolved numerous strategies to limit the generation of excess histones and prevent deleterious effects due to their accumulation. Such strategies for histone regulation are discussed here, with particular emphasis on recent studies that implicate the DNA damage checkpoint kinases in the regulation of histone levels, especially in response to replication inhibition. We have also focused upon the recently discovered regulatory mechanism involving histone proteolysis in the budding yeast. Additionally, we speculate that cells may possess a surveillance mechanism for sensing histone levels, particularly in the G1 and S-phases of the cell cycle. Proper regulation of histone levels has major implications for the maintenance of epigenetic marks on chromatin, genomic stability and the packaging of sperm DNA.

Aβ40 promotes neuronal cell fate in neural progenitor cells

Sequential cleavage of the amyloid precursor protein (APP) by beta- and then gamma- secretase gives rise to Abeta(1-40) (Abeta40), a major species of Abeta (beta-amyloid) produced by neurons under physiological conditions. Abeta(1-42) (Abeta42), a minor species of Abeta, is also produced by a similar but less understood mechanism of the gamma-secretase. The physiological functions of these Abeta species remain to be defined. In this report, we demonstrate that freshly prepared soluble Abeta40 significantly promotes neurogenesis in primary neural progenitor cells (NPCs). First, Abeta40 increases neuronal markers as determined by NeuN expression and Tuj1 promoter activity, differing from Abeta42, which induces astrocyte markers in NPCs. Second, Abeta40 induces neuronal differentiation at the end of S-phase in the cell cycle. Third, Abeta40 promotes NPC entry into S-phase, playing a role in NPC self-renewal. Interestingly, Abeta40 does not significantly increase apoptotic indexes such as DNA condensation and DNA fragmentation. In addition, Abeta40 does not augment caspase-3 activation in NeuN(+) or nestin(+) cells. Collectively, this report provides strong evidence that Abeta40 is a neurogenic factor and suggests that the debilitated function of Abeta40 in neurogenesis may account for the shortage of neurons in Alzheimer's disease.

Global Profiling of DNA Replication Timing and Efficiency Reveals that Efficient Replication/Firing Occurs Late during S-Phase in S. pombe

BackgroundDuring S. pombe S-phase, initiation of DNA replication occurs at multiple sites (origins) that are enriched with AT-rich sequences, at various times. Current studies of genome-wide DNA replication profiles have focused on the DNA replication timing and origin location. However, the replication and/or firing efficiency of the individual origins on the genomic scale remain unclear.Methodology/Principal FindingsUsing the genome-wide ORF-specific DNA microarray analysis, we show that in S. pombe, individual origins fire with varying efficiencies and at different times during S-phase. The increase in DNA copy number plotted as a function of time is approximated to the near-sigmoidal model, when considering the replication start and end timings at individual loci in cells released from HU-arrest. Replication efficiencies differ from origin to origin, depending on the origin's firing efficiency. We have found that DNA replication is inefficient early in S-phase, due to inefficient firing at origins. Efficient replication occurs later, attributed to efficient but late-firing origins. Furthermore, profiles of replication timing in cds1Δ cells are abnormal, due to the failure in resuming replication at the collapsed forks. The majority of the inefficient origins, but not the efficient ones, are found to fire in cds1Δ cells after HU removal, owing to the firing at the remaining unused (inefficient) origins during HU treatment.Conclusions/SignificanceTaken together, our results indicate that efficient DNA replication/firing occurs late in S-phase progression in cells after HU removal, due to efficient late-firing origins. Additionally, checkpoint kinase Cds1p is required for maintaining the efficient replication/firing late in S-phase. We further propose that efficient late-firing origins are essential for ensuring completion of DNA duplication by the end of S-phase.

Heterochromatin and ND10 are cell-cycle regulated and phosphorylation-dependent alternate nuclear sites of the transcription repressor Daxx and SWI/SNF protein ATRX.

Placing regulatory proteins into different multiprotein complexes should modify key cellular processes. Here, we show that the transcription repressor Daxx and the SWI/SNF protein ATRX are both associated with two intranuclear domains: ND10/PML bodies and heterochromatin. The accumulation of ATRX at nuclear domain 10 (ND10) was mediated by its interaction with the N-terminus of Daxx. Binding of this complex to ND10 was facilitated by the interaction of the Daxx C-terminus with SUMOylated promyelocytic leukemia protein (PML). Although ATRX was present at heterochromatin during the entire cell cycle, Daxx was actively recruited to this domain at the end of S-phase. The FACT-complex member structure-specific recognition protein 1 (SSRP1) accumulated at heterochromatin simultaneously with Daxx and accumulation of both proteins depended on ATRX phosphorylation. Both Daxx and SSRP1 were released from heterochromatin early in G(2) phase and Daxx was recruited back to ND10, indicating that both proteins localize to heterochromatin during a very short temporal window of the cell cycle. ATRX seems to assemble a repression multiprotein complex including Daxx and SSRP1 at heterochromatin during a specific stage of the cell cycle, whereas Daxx functions as an adapter for ATRX accumulation at ND10. A potential functional consequence of Daxx accumulation at heterochromatin was found in the S- to G(2)-phase transition. In Daxx(-/-) cells, S-phase was accelerated and the propensity to form double nuclei was increased, functional changes that could be rescued by Daxx reconstitution and that might be the basis for the developmental problems observed in Daxx knockout animals.

The extracellular matrix, p53 and estrogen compete to regulate cell-surface Fas/Apo-1 suicide receptor expression in proliferating embryonic cerebral cortical precursors, and reciprocally, Fas-ligand modifies estrogen control of cell-cycle proteins

BackgroundApoptosis is important for normal cerebral cortical development. We previously showed that the Fas suicide receptor was expressed within the developing cerebral cortex, and that in vitro Fas activation resulted in caspase-dependent death. Alterations in cell-surface Fas expression may significantly influence cortical development. Therefore, in the following studies, we sought to identify developmentally relevant cell biological processes that regulate cell-surface Fas expression and reciprocal consequences of Fas receptor activation.ResultsFlow-cytometric analyses identified two distinct neural sub-populations that expressed Fas on their cell surface at high (FasHi) or moderate (FasMod) levels. The anti-apoptotic protein FLIP further delineated a subset of Fas-expressing cells with potential apoptosis-resistance. FasMod precursors were mainly in G0, while FasHi precursors were largely apoptotic. However, birth-date analysis indicated that neuroblasts express the highest levels of cell-surface Fas at the end of S-phase, or after their final round of mitosis, suggesting that Fas expression is induced at cell cycle checkpoints or during interkinetic nuclear movements. FasHi expression was associated with loss of cell-matrix adhesion and anoikis. Activation of the transcription factor p53 was associated with induction of Fas expression, while the gonadal hormone estrogen antagonistically suppressed cell-surface Fas expression. Estrogen also induced entry into S-phase and decreased the number of Fas-expressing neuroblasts that were apoptotic. Concurrent exposure to estrogen and to soluble Fas-ligand (sFasL) suppressed p21/waf-1 and PCNA. In contrast, estrogen and sFasL, individually and together, induced cyclin-A expression, suggesting activation of compensatory survival mechanisms.ConclusionsEmbryonic cortical neuronal precursors are intrinsically heterogeneous with respect to Fas suicide-sensitivity. Competing intrinsic (p53, cell cycle, FLIP expression), proximal (extra-cellular matrix) and extrinsic factors (gonadal hormones) collectively regulate Fas suicide-sensitivity either during neurogenesis, or possibly during neuronal migration, and may ultimately determine which neuroblasts successfully contribute neurons to the differentiating cortical plate.

Requirement for phosphatidylinositol-3 kinase activity during progression through S-phase and entry into mitosis

Phosphatidylinositol-3 kinase (PI3K) proteins are important regulators of cell survival and proliferation. PI3K-dependent signalling regulates cell proliferation by promoting G1- to S-phase progression during the cell cycle. However, a definitive role for PI3K at other times during the cell cycle is less clear. In these studies, we provide evidence that PI3K activity is required during DNA synthesis (S-phase) and G2-phase of the cell cycle. Inhibition of PI3K with LY294002 at the onset of S-phase caused a 4- to 5-h delay in progression through G2/M. LY294002 treatment at the end of S-phase caused an approximate 2-h delay in progression through G2/M, indicating that PI3K activity functions for both S- and G2-phase progression. The expression of constitutively activated Akt partially reversed the inhibitory effects of LY294002 on mitotic entry, which demonstrated that Akt was one PI3K target that was required during G2/M transitions. Inhibition of PI3K resulted in enhanced susceptibility of G2/M synchronized cells to undergo apoptosis in response to DNA damage as compared to asynchronous cells. Thus, similar to its role in promoting cell survival and cell cycle transitions from G1 to S phase, PI3K activity appears to promote entry into mitosis and protect against cell death during S- and G2-phase progression.