Cell Cycle

Numerical abnormalities in chromosomal states, referred to as aneuploidy, is commonly observed in many cancer cells. Although numerous internal and external factors induce aneuploidy, the primary cause of aneuploidy in humans remains unclear. DNA damage is identified as a potential cause of aneuploidy by inducing chromosome segregation errors. However, a direct relationship between DNA damage and aneuploidy remains poorly understood. A major reason for this is the extremely low frequency of aneuploidy in cultured cells, making quantitative analyses challenging. In this study, we investigated the relationship between DNA damage and aneuploidy in cell lines containing minichromosomes. These chromosomes are more prone to loss than normal chromosomes, with the rate of loss substantially increased following exposure to various DNA-damaging agents. To determine whether damaged chromosomes were subjected to direct loss or whether chromosome loss occurred as an indirect consequence of a prolonged G2 phase or other factors, we used the CRISPR-Cas9 system to introduce a single DNA double-strand break (DSB) on a minichromosome. The rate of minichromosome loss increased by approximately seven-fold compared with that of the control. Furthermore, the loss rate was significantly elevated in the absence of KU70, a key factor in non-homologous end joining, and upon inhibition of ataxia telangiectasia mutated (ATM), a DNA damage checkpoint protein. Finally, two closely spaced nicks, believed to generate a 5-overhang, were also shown to induce minichromosome loss. These findings indicated that a single DSB or two closely spaced nicks can cause aneuploidy if improperly repaired in vertebrates.

Backgroundp53 is a transcription activator or repressor that acts mainly by having direct control over the expression of CDK inhibitor - p21 in response to DNA damage. MethodsWe used qPCR, Western Blot, protein co-immunoprecipitation, chromatin immunoprecipitation, RNA-seq, confocal microscopy, flow cytometry and resazurin assay to investigate how the p53 regulate gene expression upon sub-lethal doses of cisplatin. ResultsIn this study, molecular evidence was provided for the occurrence of p53 at the subset of E2F1-driven promoters and their suppression, despite the co-occurrence of p53 with p300. P53 repressed promoters were characterized by relatively high nucleosome density and demethylation of H3K4, followed by low H3K27 acetylation and trimethylation of H3K4. Induction of the ATM/ATR-Chek1/2-p53 pathway by sub-lethal doses of cisplatin caused the release of p53 from gene promoters, chromatin relaxation and the gain of transcription permissive histone marks. Mechanistically, p53 maintained the KDM5B that is associated with gene promoters, thereby conditioning the demethylation of H3K4me3. P53 formed an immunoprecipitable complex with KDM5B, E2F1, p300 and H3K4me2 in intact cells, which decomposed with cisplatin and substantially increased the level of H3K4me3 in the p300 interactome. The extrusion of KDM5B from the chromatin was triggered by cisplatin, transient p53 silencing or KDM5B inhibition, also enabled p300 enrichment and increased gene transcription. The molecular and functional interdependence between p53 and KDM5B was observed in distinct cancer cell types, and the co-expression of TP53 and KDM5B can be considered as a doxorubicin response biomarker. Conclusionsp53 directly suppressed the subset of E2F1-driven genes in proliferating cells by maintaining KDM5B associated with gene promoters and inhibiting p300-mediated transcription.

EWSR1 (Ewing sarcoma breakpoint region 1) was originally identified as a part of an aberrant EWSR1/FLI1 fusion gene in Ewing sarcoma, the second most common pediatric bone cancer. Due to formation of the EWSR1/FLI1 fusion gene in the tumor genome, the cell loses one wild type EWSR1 allele. Our previous study demonstrated that the loss of ewsr1a (homologue of human EWSR1) in zebrafish leads to the high incidence of mitotic dysfunction, of aneuploidy, and of tumorigenesis in the tp53 mutant background. To dissect the molecular function of EWSR1, we successfully established a stable DLD-1 cell line that enables a conditional knockdown of EWSR1 using Auxin Inducible Degron (AID) system. When both EWSR1 genes of DLD-1 cell were tagged with mini-AID at its 5-end using CRISPR/Cas9 system, treatment of the (AID-EWSR1/AID-EWSR1) DLD-1 cells with a plant-based Auxin (AUX) led to the significant levels of degradation of AID-EWSR1 proteins. During anaphase, the EWSR1 knockdown (AUX+) cells displayed higher incidence of lagging chromosomes compared to the control (AUX-) cells. This defect was proceeded by a lower incidence of the localization of Aurora B at inner centromeres, and by a higher incidence of the protein at kinetochores compared to the control cells during pro/metaphase. Despite these defects, the EWSR1 knockdown cells did not undergo mitotic arrest, suggesting that the cell lacks the error correction mechanism. Significantly, the EWSR1 knockdown (AUX+) cells induced higher incidence of aneuploidy compared to the control (AUX-) cells. Since our previous study demonstrated that EWSR1 interacts with the key mitotic kinase, Aurora B, we generated replacement lines of EWSR1-mCherry and EWSR1:R565A-mCherry (a mutant that has low affinity for Aurora B) in the (AID-EWSR1/AID-EWSR1) DLD-1 cells. The EWSR1-mCherry rescued the high incidence of aneuploidy of EWSR1 knockdown cells, whereas EWSR1-mCherry:R565A failed to rescue the phenotype. Together, we demonstrate that EWSR1 is essential to prevent aneuploidy through interaction with Aurora B, most likely by regulating the localization of Aurora B at centromere.

The centrosome cycle is a tightly regulated process to ensure proper segregation of chromosomes. Not surprisingly, centriole number is tightly controlled via multiple mechanisms, one of which involves PLK4, an upstream kinase facilitating centriole biogenesis and duplication. Aberrations in this process can result in supernumerary centrosomes, which are frequently observed in a variety of cancers due to high levels of PLK4. Interestingly, extra centrosomes induced by PLK4 over-expression go through unique intermediate structures called the centrosome rosettes (CRs), where the mother centriole is surrounded by numerous daughter centrioles. The maturation and molecular nature of these CRs have not been investigated in detail. Upon prolonged PLK4 over-expression, cells exhibited large centrosomes that were clustered and contained more than two CRs, which we defined as centrosome rosette clusters (CRCs). As expected, these structures required high PLK4 levels at two consecutive cell cycles and were still interconnected with canonical centrosomal linker proteins such as C-Nap1, Rootletin, and Cep68. Knockout of these linker proteins resulted in distancing of CRs and CRCs as observed by increased diameter of the CRCs in interphase. In contrast, Nek2 knockout inhibited the separation of CRCs in prometaphase, providing functional evidence for the binding of CRC structures with centrosomal linker proteins. These results suggest a cell cycle dependent model for PLK4 induced centrosome amplification, which occurs in two consecutive cell cycles: (i) CR state in the first cell cycle, and (ii) CRC state in the second cell cycle. Author summaryThe overexpression of PLK4 can lead to the formation of centrosome rosette structures, which harbor two centrioles around the mother centriole. Although the generation of centrosome rosettes by PLK4 overexpression has been previously investigated, little is known about the cell cycle-dependent maturation and linking of these structures. Here, we report that prolonged PLK4 overexpression results in amplification of centrosomes through the generation of centrosome rosette clusters (CRCs). These CRCs are interconnected via canonical centrosomal linker proteins such as C-Nap1, Rootletin, and CEP68 and are regulated by mechanisms controlling centrosome linking and separation. We also describe two different spatial binding types of amplified centrosomes following PLK4 induction: planar-oriented and circular-oriented. Since PLK4-associated centrosome amplification occurs naturally in both cancer and multiciliated cells, we believe that this research will contribute to a better understanding of the canonical mechanism of PLK4-induced centrosome amplification.

Poly(ADP-ribosyl)ation (PARylation) is a protein modification mostly synthesised and degraded by PARP1/2 and PARG enzymes, respectively. PARylation is involved in many covalent and non-covalent protein-protein interactions in the nucleus, making it a powerful form of molecular signaling in DNA metabolism. PARP inhibitors (PARPi) have shown efficacy in the treatment of Homologous Recombination (HR)-deficient cancers, yet the full range of molecular mechanisms underlying the activity of these drugs is not fully understood. Here, we decipher the direct consequences of PARPi-induced loss of PARylation on DNA replication. First, PARPi treatment during a single S phase induces replicative stress, delays S phase progression and causes genome-wide replication timing changes of disease-associated regions. These DNA replication alterations appear to be caused by an accumulation of SSBs in the DNA replicative template when PARylation is inhibited, which then seems to lead to one-ended DSBs and fork collapse during S phase. Second, PARPi deplete FANCD2-I, BRCA, monoubiquitinated PCNA and RAD18 proteins from the replisomes. The two PARPi tested then appear to modulate the choice of fork restart mechanisms and influence replisome dynamics in different ways. Taken together, our study highlights the common and unique primary effects of two PARPi on unperturbed DNA replication in human cells. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/639283v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@298f8dorg.highwire.dtl.DTLVardef@8f8cbcorg.highwire.dtl.DTLVardef@60952forg.highwire.dtl.DTLVardef@e2700a_HPS_FORMAT_FIGEXP M_FIG C_FIG

ADP-ribosyl-transferases (ADP-ribose polymerases) PARP1 and PARP2 are critical players in DNA damage response in the nucleus. Being activated by a genotoxic stress, these enzymes utilize NAD+ to attach ADP-ribose chains to wide variety of proteins; ribosomal proteins (RPs) have been identified among the major targets of the modification in different cell lines. However, little remained known concerning the peculiarities of the reaction of RPs ADP-ribosylation itself. Here, we study ADP-ribosylation of human RPs within the large (60S) and small (40S) ribosomal subunits and those isolated from the subunits, with PARP1 and PARP2 in vitro using radioactively labeled NAD+. We fail to detect the modification of ribosome-bound RPs but observed ADP-ribosylation of certain ribosome-free RPs when we use total protein isolated from the subunits. RPs from the 60S subunit were globally more modified than those from the 40S subunit, and ADP-ribosylation of several 60S RPs (but not 40S) was considerably enhanced in the presence of histone PARylation factor 1 (HPF1). With all kind RPs, HPF1 switches the modification preferentially to their serine/tyrosine residues. Major targets of the 60S RPs ADP-ribosylation were identified as RPL4 (uL4), RPL6 (eL6) and RPL13A/RPL15 (uL13/eL15). The modification levels of particular RPs differently depend on the concentration of total RP; the most selective HPF1-dependent ADP-ribosylation occurs in RPL6 (eL6). When present simultaneously with histones, RPs win linker histone H1 in the competition for both PARPs; in contrast, core histones strongly compete with RPs for ADP-ribosylation. Possible functional assignments of ADP-ribosylation of RPs are discussed. Bullet points- Free human ribosomal proteins are PARylated by PARP1 and PARP2; - PARylation of ribosomal 60S proteins but not 40S ones is mostly HPF1-dependent; - RPL4, RPL6 and RPL13A/RPL15 are the major targets of PARylation among 60S RPs; - Linker histone H1 is a poor competitor to ribosomal proteins for PARPs; - Core histones strongly competes with ribosomal proteins for PARPs.

Sister chromatid cohesion (SCC) is established during DNA replication by loading of the cohesin complex on newly replicated chromatids. Cohesin must then be maintained until mitosis to prevent segregation defects and aneuploidy. How SCC is established and maintained until mitosis remains incompletely understood and emerging evidence suggests that replication stress can lead to premature SCC loss. Here, we report that the single-stranded DNA-binding protein CTC1-STN1-TEN1 (CST) aids in SCC. CST primarily functions in telomere length regulation but also has known roles in replication restart and DNA repair. Following depletion of CST subunits, we observed an increase in the complete loss of SCC. Additionally, we determined that CST interacts with the cohesin complex. Unexpectedly, we did not find evidence of defective cohesion establishment or mitotic progression in the absence of CST. However, we did find that treatment with various replication inhibitors increased the association between CST and cohesin. Since replication stress was recently shown to induce SCC loss, we supposed that CST may be required to maintain SCC following fork stalling. In agreement with this idea, SCC loss was greatly increased in CST-depleted cells following exogenous replication stress. Based on our findings, we propose that CST aids in the maintenance of SCC at stalled replication forks to prevent premature cohesion loss.

Errors in mitosis can contribute to aneuploidy and CIN and play a pivotal role in cancer. So the identification of altered mitotic regulators can contribute to the understanding of the development and progression of breast cancer. In the present study we used an in vitro model of disease progression (the MCF10A series of BC continuum) and analyzed the errors of chromosome segregation that occur during the progression of the disease. Our findings indicated that the MCF10A series exhibited several abnormalities in chromosome segregation and its frequency increased with the disease progression. These errors included anaphase lagging chromosomes, micronuclei, nuclear buds, nucleoplasmic bridges, errors of chromosome alignment, and centrosome loss/amplification. Moreover, the presence of centrosome amplification disrupted the proper orientation of the mitotic spindle, resulting in the generation asymmetrical cell lines and aneuploidy in the MCF10A series. Hyper stable kinetochore-microtubule (kt-MT) attachment was also found in premalignant, preinvasive, and invasive cell lines, which can also explain the presence of errors of chromosome alignment. The human transcriptome array also determined possible negative regulators of ciliogenesis that can explain the mechanism of chromosome missegregation that lead to CIN found in the MCF10A series. Collectively, these findings highlight the importance of mitotic defects in the progression of breast cancer.

Recycling of parental histones is an important step in epigenetic inheritance. During DNA replication, DNA polymerase epsilon subunit DPB3/DPB4 and DNA replication helicase subunit MCM2 are involved in the transfer of parental histones to the leading and lagging DNA strands, respectively. Single Dpb3 deletion (dpb3{Delta}) or Mcm2 mutation (mcm2-3A), which each disrupt one parental histone transfer pathway, leads to the others predominance. However, the impact of the two histone transfer pathways on chromatin structure and DNA repair remains elusive. In this study, we used budding yeast Saccharomyces cerevisiae to determine the genetic and epigenetic outcomes from disruption of parental histone H3-H4 tetramer transfer. We found that a dpb3{Delta}/mcm2-3A double mutant did not exhibit the single dpb3{Delta} and mcm2-3A mutants asymmetric parental histone patterns, suggesting that the processes by which parental histones are transferred to the leading and lagging strands are independent. Surprisingly, the frequency of homologous recombination was significantly lower in dpb3{Delta}, mcm2-3A, and dpb3{Delta}/mcm2-3A mutants relative to the wild-type strain, likely due to the elevated levels of free histones detected in the mutant cells. Together, these findings indicate that proper transfer of parental histones to the leading and lagging strands during DNA replication is essential for maintaining chromatin structure and that high levels of free histones due to parental histone transfer defects are detrimental to cells.

DNA polymerase kappa (Pol{kappa}) has multiple cellular roles such as translesion DNA synthesis, replication of repetitive sequences and nucleotide excision repair. However, the mechanisms regulating Pol{kappa}s cellular activities are unknown. Since all polymerases insert the canonical deoxynucleotide triphosphates, it is difficult to determine which polymerase is active at a particular genomic site. To counter this difficulty, we utilized the selective Pol{kappa} substrate, N2-(4-ethynylbenyl)-2-deoxyguanosine (EBndG), as bait to capture proteins associated with Pol{kappa} synthesized DNA. Here we show that, Pol{kappa} is active in the nucleolus and Pol{kappa} synthesized DNA are enriched with nucleolar proteins. Exposure of cells to benzo[a]pyrene diol epoxide (BPDE) induced hallmarks of nucleolar stress, increased Pol{kappa} stability and nucleolar activity. Other agents that induce nucleolar stress such as mitomycin C, cisplatin and actinomycin D also increased Pol{kappa}s nucleolar activity. The Pol{kappa} activity was cell-cycle independent and dependent on PCNA ubiquitination. In addition, we identified that the expression and activity of Pol{kappa} is regulated by the polycomb complex protein Ring Finger Protein 2 (RNF2) and by poly(ADP)-ribose polymerase 1 (PARP1) catalyzed PARylation. This study provides insight into the novel role of Pol{kappa} in ribosomal RNA synthesis, in maintaining ribosomal DNA integrity after DNA damage thus protecting the cells from nucleolar stress.

DNA topoisomerase II (Top2) is a nuclear protein that resolves DNA topological problems and plays critical roles in multiple nuclear processes. Human cells have two Top2 proteins, Top2A and Top2B, that are localized in both the nucleoplasm and nucleolus. Previously, ATP depletion was shown to augment the nucleolar localization of Top2B, but the molecular details of subnuclear distributions, particularly of Top2A, remained to be fully elucidated in relation to the status of cellular ATP. Here, we analyzed the nuclear dynamics of human Top2A and Top2B in ATP-depleted cells. Both proteins rapidly translocated from the nucleoplasm to the nucleolus in response to ATP depletion. FRAP analysis demonstrated that they were highly mobile in the nucleoplasm and nucleolus. The nucleolar retention of both proteins was sensitive to the RNA polymerase I inhibitor BMH-21, and the Top2 proteins in the nucleolus were immediately dispersed into the nucleoplasm by BMH-21. Under ATP-depleted conditions, the Top2 poison etoposide was less effective, indicating the therapeutic relevance of Top2 subnuclear distributions. These results give novel insights into the subnuclear dynamics of Top2 in relation to cellular ATP levels and also provide discussions about its possible mechanisms and biological significance.

In the event of DNA damage, the cell cycle can be slowed or halted to allow for DNA repair. The mechanisms by which this occurs are well-characterised in interphase, although the mechanisms underpinning mitosis slowing in response to damage are unclear. Canonical checkpoints and DNA repair pathways are largely repressed in mitosis, and whilst there is some level of mitotic DNA synthesis and repair, the bulk of DNA damage is processed for post-mitotic repair. How the decision is made between mitotic DNA repair and post-mitotic DNA repair is not known. We have identified the antioxidant enzyme Superoxide Dismutase 1 (SOD1) as an essential factor mediating delayed mitotic progression in response to DNA damage and replication stress. Cells depleted of SOD1 no longer exhibit DNA damage dependent mitotic delay, and display increased levels of damaged centromeres and mitotic defects. Whilst reactive oxygen species (ROS)-inducing agents also lead to SOD1-dependent mitotic delay, intracellular ROS levels do not correlate with mitotic arrest. SOD1 appears to play an important role in DNA repair in interphase and is recruited to the nucleus in response to DNA damage. In addition to control of mitotic progression in response to genotoxic stress, SOD1 also plays a major role in mitotic DNA synthesis. SOD- depleted cells show reduced levels of mitotic EdU incorporation in response to either replication stress or DNA breaks, seemingly in tandem with Rad51 andSOD1-depletion induced mitotic progression in the presence of DNA breaks is Rad52-dependent. We suggest that there are two responses to DNA breaks in mitosis; either arrest and mitotic repair or progression and post-mitotic repair; and these two pathways exist in a fine balance, controlled by a signaling cascade involving SOD1.

The cell cycle is an ordered process in which cells replicate their DNA in S-phase and divide them into two identical daughter cells in mitosis. DNA replication takes place only once per cell cycle to preserve genome integrity, which is tightly regulated by Cyclin Dependent Kinase (CDK). Formation of the pre-replicative complex, a platform for origin licensing, is inhibited through CDK-dependent phosphorylation. Failure of this control leads to re-licensing, re-replication and DNA damage. Eukaryotic cells have evolved surveillance mechanisms to maintain genome integrity, termed cell cycle checkpoints. It has been shown that the DNA damage checkpoint is activated upon the induction of DNA re-replication and arrests cell cycle in mitosis in S. cerevisiae. In this study, we show that PP2A-Cdc55 is responsible for the metaphase arrest induced by DNA re-replication, leading to dephosphorylation of APC component, Exclusion of Cdc55 from the nucleus bypassed the mitotic arrest and resulted in enhanced cell lethality in re-replicating cells. The metaphase arrest in re-replication cells was retained in the absence of Mad2, a key component of the spindle assembly checkpoint. Moreover, re-replicating cells showed the same rate of DNA damage induction in the presence or absence of Cdc55. These results indicate that PP2A-Cdc55 maintains metaphase arrest upon DNA re-replication and DNA damage through APC inhibition.

Lamins are emerging as major regulators in the maintenance of nuclear architecture and genome organization. Extensive research for the last two decades has enormously contributed to understanding the roles of lamins in various signaling mechanisms which are drastically modified in neoplasia. It is interesting to record that alteration in lamin A/C expression and distribution drives tumorigenesis of almost all tissues of human bodies. One of the important signatures of a cancer cell is its inability to repair DNA damage which befalls several genomic events that transform the cells to be sensitive to chemotherapeutic agents. This genomic and chromosomal instability is the most common feature found in cases of high-grade ovarian serous carcinoma. Here, we report elevated levels of lamins in OVCAR3 cells (High grade ovarian serous carcinoma cell line) in comparison to IOSE (Immortalised ovarian surface epithelial cells) and consequently altered damage repair machinery in OVCAR3. We have analyzed the changes in global gene expression as a sequel to DNA damage induced by etoposide in ovarian carcinoma where lamin A is particularly elevated in expression and reported some differentially expressed genes associated with pathways conferring cellular proliferation and chemoresistance. We highlight new avenues unraveling the role of upregulated lamin A in confronting chemically induced genomic instability in the context of high grade ovarian serous cancer through a combination of HR and NHEJ mechanisms.

Apoptosis inhibitor 5 (Api5) is an inhibitor of apoptosis, which is found to be upregulated in several cancers and promotes invasion as well as metastasis. Over-expression of Api5 is positively co-related with poor survival of cancers and inhibition of DNA damage induced apoptosis in cancerous cells. Acetylation at lysine 251 (K251) on Api5 facilitates the stability of the protein and thus functionally provides resistance to cancer cells against chemotherapeutic or anti-cancerous agents. However, the regulation of Api5 upon DNA damage is not yet known. In this study, we demonstrate that Api5 undergoes degradation following DNA damage via the ubiquitin-proteasome system. Upon DNA damage, ATR was observed to phosphorylate Api5 at serine 138 which led to the cytoplasmic localisation of Api5. The E3-ubiquitin ligase, SCF-FBXW2 ubiquitinates Api5 leading to its proteasomal degradation.

Cell cycle progression in the presence of damaged DNA can lead to accumulation of mutations and pose a risk for tumour development. In response to DNA damage in G2 phase, human cells can be forced to exit the cell cycle in a p53-p21- and APC/CCdh1-dependent manner. Cells that exit the cell cycle in G2 phase become senescent, but it is unclear what determines this commitment and whether other cell fates occur. We find that a subset of immortalised RPE-1 cells and primary human fibroblasts spontaneously initiate DNA re-replication several days after forced cell cycle exit in G2 phase. By combining single cell tracking for more than a week with quantitative immunofluorescence, we find that the resulting polyploid cells contain increased levels of damaged DNA and frequently exit the cell cycle again in the next G2 phase. Subsequently, these cells either enter senescence or commit to another round of DNA re-replication, further increasing the ploidy. At least a subset of the polyploid cells show abnormal centrosome numbers or localisation. In conclusion, cells that are forced to exit the cell cycle in G2 phase face multiple choices that lead to various phenotypes, including propagation of cells with different ploidies. Our findings suggest a mechanism by which p53-positive cells can evade senescence that risks genome integrity. Main points-Cell cycle exit from G2 phase does not necessarily lead to senescence -Resumption of proliferation after G2 phase cell cycle exit starts with DNA replication -Successive cell cycle exits lead to propagation of cells with different ploidies -A p53-dependent mechanism allows eventual proliferation after DNA damage

The Restriction Point (R) in the mammalian cell cycle is regarded as a critical transition in G1 when cells become committed to enter S phase even in the absence of further growth factor stimulation. Classic time-lapse studies by Zetterberg and Larsson suggested that the acquisition of growth factor independence (i.e. passage of R) occurred very abruptly 3-4 hours after mitosis, with most cell cycle variability arising between R and entry into S phase. However, the cycle times of the post-R cells that continued on to mitosis after serum step-down without perturbation were far less variable than the control cells with which they were compared. A re-analysis of the data, presented here, shows that when the timing of R and entry in mitosis are compared for the same experiments, the curves are superimposable and statistically indistinguishable. This indicates that the data are compatible with the timing of R contributing to much of the overall variability in the cell cycle, contrary to the conclusions of Zetterberg and colleagues.

Inositol is an essential metabolite that serves as a precursor for structural and signaling molecules. Although perturbation of inositol homeostasis has been implicated in numerous human disorders, surprisingly little is known about how inositol levels are regulated in mammalian cells. A recent study in mouse embryonic fibroblasts (MEFs) demonstrated that nuclear translocation of inositol hexakisphosphate kinase 1 (IP6K1) mediates repression of myo-3-P synthase (MIPS), the rate-limiting inositol biosynthetic enzyme. Binding of IP6K1 to phosphatidic acid (PA) is required for this repression. The current study was carried out to elucidate the role of PA in IP6K1 repression. The results indicate that increasing PA levels through pharmacological stimulation of phospholipase D (PLD) or direct supplementation of 18:1 PA induces nuclear translocation of IP6K1 and represses expression of MIPS protein. This effect was specific to PA synthesized in the plasma membrane, as ER-derived PA did not induce IP6K1 translocation. PLD-mediated PA synthesis can be stimulated by the master metabolic regulator 5 AMP-activated protein kinase (AMPK). Activation of AMPK by glucose deprivation or by treatment with the mood stabilizing drugs valproate (VPA) or lithium recapitulated IP6K1 nuclear translocation and decreased MIPS expression. This study demonstrates for the first time that modulation of PA levels through the AMPK-PLD pathway regulates IP6K1-mediated repression of MIPS.

Members of the conserved subfamily, JADE1S and JADE1L isoforms, are expressed in epithelial cells, fibroblasts, and epithelial cell lining in vivo. JADE1 proteins interact with histone acetyl transferase HBO1 complex. The two consecutive PHD zinc fingers of JADE1 bind chromatin. We recently reported novel effects of JADE1S on cytokinesis progression. JADE1S depletion facilitated G2/M-to-G1 transition and increased polyploidy and aneuploidy. JADE1S over-expression arrested cells in late cytokinesis, an effect reversed by AURKB inhibitor. In late cytokinesis cells JADE1S protein localized to the midbody. Results suggested a JADE1S role in final abscission delay. Here we investigated the expression of JADE1 in the central spindle, interactions with HBO1, and the role of PHD fingers in late cytokinesis arrest. The midzone begins to assemble in anaphase and forms into a midbody in cytokinesis. The midbody structure connects two daughter cells and is thought to bear factors controlling the final abscission. We questioned whether, similar to established factors, JADE1S is targeted to the central spindle structures in anaphase. Indeed, in cells transitioning from mitosis to cytokinesis, JADE1S was sequentially targeted to early midzone, midbody flanking zone, and midbody. The step-wise increase of JADE1S expression in midzone and midbody of synchronously dividing cells suggested protein recruitment. The increase of late cytokinesis arrest caused by recombinant JADE1S correlated with increased expression in midbody. Spatial analysis of the members of the chromatin passenger complex, microtubule associated proteins, and centralspindlin, revealed transient co-localization with JADE1S and mapped JADE1S within the cytokinesis bridge. Deletion of the two PHD zinc fingers inactivated JADE1S ability to arrest cells in late cytokinesis but did not affect its midbody localization. Thus, PHD zinc fingers are required for JADE1S cytokinesis delay but not for midbody targeting. Recombinant HBO1 protein decreased the proportion of late cytokinesis cells, prevented late cytokinesis arrest by JADE1S as well as its midbody localization. Enzyme inactive HBO1 mutant recapitulated the wild type phenotype. The results demonstrate antagonistic relationship and suggest HBO1-mediated midbody dislocation of JADE1S. Our study supports the role of JADE1S in cytokinesis delay and implicates protein partners.

C9ORF78 is a poorly characterized protein found in diverse eukaryotes. Previous work indicated overexpression of hC9ORF78 (aka HCA59) in malignant tissues indicating a possible involvement in growth regulatory pathways. Additional studies in fission yeast and humans uncover a potential function in regulating the spliceosome. In studies of GFP-tagged hC9ORF78 we observed a dramatic reduction in protein abundance in cells grown to confluence and/or deprived of serum growth factors. Serum stimulation induced synchronous re-expression of the protein in HeLa cells. This effect was also observed with the endogenous protein. Overexpressing either E2F1 or N-Myc resulted in elevated hC9ORF78 expression potentially explaining the serum-dependent upregulation of the protein. Immunofluorescence analysis indicates that hC9ORF78 localizes to nuclei in interphase but does not appear to concentrate in speckles as would be expected for a splicing protein. Surprisingly, a subpopulation of hC9ORF78 co-localizes with ACA, Mad1 and Hec1 in mitotic cells suggesting that this protein may associate with kinetochores or centromeres. Furthermore, knocking-down hC9ORF78 caused mis-alignment of chromosomes in mitosis. These studies uncover novel mitotic function and subcellular localization of cancer antigen hC9ORF78. SUMMARY STATEMENThC9ORF78 regulates chromosome segregation.