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Replication stress has been suggested to be an ultimate trigger of carcinogenesis. Oncogenic signal, such as overexpression of CyclinE, has been shown to induce replication stress. Here, we show that various biological stresses, including heat, oxidative stress, osmotic stress, LPS, hypoxia, and arsenate induce activation of Chk1, a key effector kinase for replication checkpoint. Some of these stresses indeed reduce the fork rate, inhibiting DNA replication. Analyses of Chk1 activation in the cell population with western analyses showed that Chk1 activation by these stresses is largely dependent on Claspin. On the other hand, single cell analyses with Fucci cells indicated that while Chk1 activation during S phase is dependent on Claspin, that in G1 is mostly independent of Claspin. We propose that various biological stresses activate Chk1 either directly by stalling DNA replication fork or by some other mechanism that does not involve replication inhibition. The former pathway predominantly occurs in S phase and depends on Claspin, while the latter pathway, which may occur throughout the cell cycle, is largely independent of Claspin.Our findings provide evidence for novel links between replication stress checkpoint and other biological stresses and points to the presence of unknown mechanisms of Chk1 activation in mammalian cells.

Human cells need to cope with the stalling of DNA replication to complete replication of the entire genome to minimize genome instability. They respond to “replication stress” by activating the conserved ATR-Claspin-Chk1 replication checkpoint pathway. The stalled replication fork is detected and stabilized by the checkpoint proteins to prevent disintegration of the replication fork, to remove the lesion or problems that are causing fork block, and to facilitate the continuation of fork progression. Claspin, a factor conserved from yeasts to human, plays a crucial role as a mediator that transmits the replication fork arrest signal from the sensor kinase, ataxia telangiectasia and Rad3-related (ATR), to the effector kinase, Checkpoint kinase 1 (Chk1). Claspin interacts with multiple kinases and replication factors and facilitates efficient replication fork progression and initiation during the normal course of DNA replication as well. It interacts with Cdc7 kinase through the acidic patch segment near the C-terminus and this interaction is critical for efficient phosphorylation of Mcm in non-cancer cells and also for checkpoint activation. Phosphorylation of Claspin by Cdc7, recruited to the acidic patch, regulates the conformation of Claspin through affecting the intramolecular interaction between the N- and C-terminal segments of Claspin. Abundance of Claspin is regulated at both mRNA and protein levels (post-transcriptional regulation and protein stability) and affects the extent of replication checkpoint. In this article, we will discuss how the ATR-Claspin-Chk1 regulates normal and stressed DNA replication and provide insight into the therapeutic potential of targeting replication checkpoint for efficient cancer cell death.

Nearly 70 years after the proposal of semiconservative replication of generic material by Watson and Crick, we now understand many of the proteins involved in the replication of host chromosomes and how they operate. The initiator and replicator, proposed in the replicon hypothesis, are now well defined in both prokaryotes and eukaryotes. On the other hand, studies in prokaryotes and Archaea indicate alternative modes of initiation, which may not depend on an initiator. Here I summarize recent progress in the field of DNA replication and discuss the evolution of replication systems.

Posttranslational modifications of histone are intimately related to chromatin/chromosome-mediated cellular events. Among all, the roles of histone modifications including acetylation, methylation, ubiquitination, and SUMOylation of lysine or arginine residue of nucleosome core histones in gene expression have been intensively studied. Genome-wide profiles of histone modification marks revealed their combinatorial organization in the functional features of chromatin. Analysis of histone modification by chromatin immunoprecipitation (ChIP) is one of the standard assays to examine chromatin states. Although high-throughput sequencing analysis (ChIP-seq) is now widely conducted, classical ChIP-qPCR analysis has advantages in investigation of multiple histone modification marks at a target site simply through the use of relatively small numbers of cells. Since ChIP-qPCR is devoid of biases caused by overamplification and inaccurate mapping of sequencing reads, it is a more reliable quantification method than genome-wide ChIP-seq especially for analyses of the low-mappability regions, which harbor many repetitive sequences and/or highly homologous segmental multiplications as found in gene clusters. We have recently analyzed histone H3 and H4 modifications of the Zscan4 family gene loci in an 880 kb gene cluster and found that the atypical enhancer-like structure is formed upon derepression of Zscan4. In this chapter, we describe the detailed protocols for histone modification ChIP-assay of repeat-enriched gene cluster regions. The protocol here we applied to mouse ES cells, but the protocol is perfectly applicable to human cultured cells and specimens.

UTX/KDM6A encodes a major histone H3 lysine 27 (H3K27) demethylase, and is frequently mutated in various types of human cancers. Although UTX appears to play a crucial role in oncogenesis, the mechanisms involved are still largely unknown. Here we show that a specific pharmacological inhibitor of H3K27 demethylases, GSK-J4, induces the expression of transcription activating factor 4 (ATF4) protein as well as the ATF4 target genes (e.g. PCK2, CHOP, REDD1, CHAC1 and TRIB3). ATF4 induction by GSK-J4 was due to neither transcriptional nor post-translational regulation. In support of this view, the ATF4 induction was almost exclusively dependent on the heme-regulated eIF2α kinase (HRI) in mouse embryonic fibroblasts (MEFs). Gene expression profiles with UTX disruption by CRISPR-Cas9 editing and the following stable re-expression of UTX showed that UTX specifically suppresses the expression of the ATF4 target genes, suggesting that UTX inhibition is at least partially responsible for the ATF4 induction. Apoptosis induction by GSK-J4 was partially and cell-type specifically correlated with the activation of ATF4-CHOP. These findings highlight that the anti-cancer drug candidate GSK-J4 strongly induces ATF4 and its target genes via HRI activation and raise a possibility that UTX might modulate cancer formation by regulating the HRI-ATF4 axis.

MSCs (mesenchymal stem cells), responsible for tissue repair, rarely undergo cell fusion with somatic cells. Here, we show that approximately 5% of bladder cancer cells (UMUC-3) fuses with bone marrow-derived MSC (BM-MSC) in co-culture and exhibits increased tumorigenicity. Eleven fusion cell clones are established, and 116 genes are identified whose expression is specifically altered in the fusion cells. Many of them are interferon-stimulated genes (ISG), but are activated in a manner independent of interferon. Among them, we show that PD-L1 is induced in fusion cells, and its knockout decreases tumorigenesis in a xenograft model. PD-L1 is induced in a manner independent of STAT1 known to regulate PD-L1 expression, but is regulated by histone modification, and is likely to inhibit phagocytosis by PD1-expressing macrophages, thus protecting cancer cells from immunological attacks. The fusion cells overexpress multiple cytokines including CCL2 that causes tumor progression by converting infiltrating macrophages to tumor-associated-macrophage (TAM). The results present mechanisms of how cell fusion promotes tumorigenesis, revealing a novel link between cell fusion and PD-L1, and underscores the efficacy of cancer immunotherapy.

Chromatin is compartmentalized in nuclei and its architecture and nuclear location may have impacts on chromatin events. Rif1, identified as a potent suppressor of hsk1-null mutation (defective in initiation of DNA replication) in fission yeast, recognizes G-quadruplex structures and inhibits origin firing in their 50∼100-kb vicinity, leading us to postulate that Rif1 may generate chromatin higher-order structures inhibitory for initiation. However, effects of Rif1 on chromatin localization in nuclei have not been known. We show here that overexpression of Rif1 causes growth inhibition and eventually cell death in fission yeast. Chromatin binding activities of Rif1, but not recruitment of phosphatase PP1, are required for growth inhibitory effect. Overexpression of a PP1 binding site mutant of Rif1 does not delay S-phase, but still causes cell death, indicating that cell death is caused not by S-phase problems but by issues in other phases of cell cycle, most likely M-phase. Indeed, Rif1 overexpression generates cells with unequally segregated chromosomes. Rif1 overexpression relocates chromatin near nuclear periphery in a manner dependent on its chromatin-binding ability, and this correlates with growth inhibition and cell death induction. Thus, regulated Rif1-mediated chromatin association with nuclear periphery is important for coordinated progression of S- and M-phases.

DNA replication is spatially and temporally regulated during S phase to execute efficient and coordinated duplication of entire genome. Various epigenomic mechanisms operate to regulate the timing and locations of replication. Among them, Rif1 plays a major role to shape the ‘replication domains’ that dictate which segments of the genome are replicated when and where in the nuclei. Rif1 achieves this task by generating higher-order chromatin architecture near nuclear membrane and by recruiting a protein phosphatase. Rif1 is a G4 binding protein, and G4 binding activity of Rif1 is essential for replication timing regulation in fission yeast. In this article, we first summarize strategies by which cells regulate their replication timing and then describe how Rif1 and its interaction with G4 contribute to regulation of chromatin architecture and replication timing.

G-quadrupex is now known to play crucial roles in various biological reactions. However, direct evidence for its presence in cells has been limited, due to the lack of versatile and non-biased methodology. We use Rif1 binding sites on the fission yeast genome, which has been shown to adopt G4 structures, as a model to prove that Rif1 BS indeed adopt G4 structure in cells. We take advantage of the presence of a single-stranded loop in the G4 structure. Rif1BS is unique in that they contain unusually long loop sequences, and we replace them with a 18 bp I-SceI restriction site. We show in vitro that I-SceI in the loop is not cleaved when G4 is formed on duplex Rif1BS DNA, but is cleaved when G4 is not formed due to a mutation in the G-tracts. This is observed both heat-induced and transcription-induced G4 structure, and gives proof of evidence for this procedure. We apply this strategy for detection of a G4 structure at the same Rif1BS in fission yeast cells. We present evidence that in vivo cleavage of I-SceI can be a measure for the presence of G4 at the target sequence in cells as well. The method described here gives a platform strategy for genome-wide analyses of cellular G4 and their dynamic formation and disruption.

G-quadruplex is one of the best-studied non-B type DNA that is now known to be prevalently present in the genomes of almost all the biological species. Recent studies reveal roles of G-quadruplex (G4) structures in various nucleic acids and chromosome transactions. In this short article, we will first describe recent findings on the roles of G4 in regulation of DNA replication. G4 is involved in regulation of spatio-temporal regulation of DNA replication through interaction with a specific binding protein, Rif1. This regulation is at least partially mediated by generation of specific chromatin architecture through Rif1-G4 interactions. We will also describe recent studies showing the potential roles of G4 in initiation of DNA replication. Next, we will present showcases of highly diversified roles of DNA G4 and RNA G4 in regulation of nucleic acid and chromosome functions. Finally, we will discuss how the formation of cellular G4 could be regulated.

The ubiquitously transcribed tetratricopeptide repeat on X chromosome (UTX) is a major histone H3 lysine 27 (H3K27) demethylase and the mixed-lineage leukemia (MLL) proteins are the H3K4 methyltransferases. UTX is one of the major components of MLL3- and MLL4-containing (MlLL3/4) complexes and likely has functions within the complexes. Although UTX is frequently mutated in various types of cancer and is thought to play a crucial role as a tumor suppressor, the importance of UTX interaction with MLL3/4 complexes in cancer formation is poorly understood. Here, we analyzed the ability of cancer-derived UTX mutant proteins to interact with ASH2L, which is a common core component of all the MLL complexes, and MLL3/4-specific components PTIP and PA1, and found that several single-amino acid substitution mutations in the tetratricopeptide repeat (TPR) affect UTX interaction with these components. Interaction-compromised mutants G137V and D336G and a TPR-deleted mutant Δ80-397 were preferentially localized to the cytoplasm, suggesting that UTX is retained in the nucleus by MLL3/4 complexes through their interaction with the TPR. Intriguingly, WT UTX suppressed colony formation in soft agar, whereas G137V failed. This suggests that interaction of UTX with MLL3/4 complex plays a crucial role in their tumor suppressor function. Preferential cytoplasmic localization was also observed for endogenous proteins of G137V and another mutant G137VΔ138 in HCT116 created by CRISPR-Cas9 gene editing. Interestingly, expression levels of these mutants were low and MG312 stabilized both endogenous as well as exogenous G137V proteins. These results reveal a novel mechanism of UTX regulation and reinforce the importance of UTX interaction with MLL3/4 complexes in cancer formation.

Stalled replication forks need to be swiftly detected and protected from collapse and the cause for fork stall be removed to restore the active replication fork. In bacteria, stalled forks are recognized and stabilized by PriA, a DEXH-type helicase, which also facilitates reassembly of an active replication fork. A TT-pocket (three-prime terminus binding pocket) present in the N-terminal segment of PriA plays a crucial role in stabilization of the stalled forks by specifically binding to the 3$^\prime$-terminus of the nascent leading strand. Eukaryotic proteins, Rad5/HLTF, contain a TT-pocket related domain, HIRAN, that specifically binds to 3′-terminus of DNA and play a role in stalled fork processing. While the TT-pocket of PriA facilitates the formation of an apparently stable and immobile complex on a fork with a 3′-terminus at the fork junction, HIRAN of Rad5/HLTF facilitates fork regression by itself. A recent report shows that HIRAN can displace 3 nucleotides at the end of the duplex DNA, providing mechanistic insight into how stalled forks are reversed in eukaryotes. In this article, I will compare the roles of 3′-terminus binding domains in stalled fork processing in prokaryotes and in eukaryotes.

Upregulation of Notch3 expression has been reported in many cancers and is considered a marker for poor prognosis. Hypoxia is a driving factor of the Notch3 signaling pathway; however, the induction mechanism and role of hypoxia-inducible factor-1α (HIF-1α) in the Notch3 response are still unclear. In this study, we found that HIF-1α and poly [ADP-ribose] polymerase 1 (PARP-1) regulate Notch3 induction under hypoxia via a noncanonical mechanism. In the analyzed cancer cell lines, Notch3 expression was increased during hypoxia at both the mRNA and protein levels. HIF-1α knockdown and Notch3 promoter reporter analyses indicated that the induction of Notch3 by hypoxia requires HIF-1α and also another molecule that binds the Notch3 promoter's guanine-rich region, which lacks the canonical hypoxia response element. Therefore, using mass spectrometry analysis to identify the binding proteins of the Notch3 promoter, we found that PARP-1 specifically binds to the Notch3 promoter. Interestingly, analyses of the Notch3 promoter reporter and knockdown of PARP-1 revealed that PARP-1 plays an important role in Notch3 regulation. Furthermore, we demonstrate that PARP inhibitors, including an inhibitor specific for PARP-1, attenuated the induction of Notch3 by hypoxia. These results uncover a novel mechanism in which HIF-1α associates with PARP-1 on the Notch3 promoter in a hypoxia response element-independent manner, thereby inducing Notch3 expression during hypoxia. Further studies on this mechanism could facilitate a better understanding of the broader functions of HIF-1α, the roles of Notch3 in cancer formation, and the insights into novel therapeutic strategies.

Inflammation is implicated in the onset and progression of various diseases, including cerebral pathologies. Here, we report that DJ-1, which plays a role within cells as an antioxidant protein, functions as a damage-associated molecular pattern (DAMP) and triggers inflammation if released from dead cells into the extracellular space. We first found that recombinant DJ-1 protein induces the production of various inflammatory cytokines in bone marrow–derived macrophages (BMMs) and dendritic cells (BMDCs). We further identified a unique peptide sequence in the αG and αH helices of DJ-1 that activates Toll-like receptor 2 (TLR2) and TLR4. In the ischemic brain, DJ-1 is released into the extracellular space from necrotic neurons within 24 h after stroke onset and makes direct contact with TLR2 and TLR4 in infiltrating myeloid cells. Although DJ-1 deficiency in a murine model of middle cerebral artery occlusion did not attenuate neuronal injury, the inflammatory cytokine expression in infiltrating immune cells was significantly decreased. Next, we found that the administration of an antibody to neutralize extracellular DJ-1 suppressed cerebral post-ischemic inflammation and attenuated ischemic neuronal damage. Our results demonstrate a previously unknown function of DJ-1 as a DAMP and suggest that extracellular DJ-1 could be a therapeutic target to prevent inflammation in tissue injuries and neurodegenerative diseases., Intracellular expression of the antioxidant protein DJ-1 has previously been shown to be neuroprotective. This study reveals that extracellularly released DJ-1 from necrotic neurons is a trigger of sterile inflammation that promotes neuronal injury and neurological deficits after ischemic stroke.

HP1 (heterochromatin protein 1), a key factor for the formation of heterochromatin, binds to the methylated lysine 9 of histone H3 (H3K9me) and represses transcription. While the H3K9me mark and HP1 binding are thought to be faithfully propagated to daughter cells, the heterochromatin structure could be dynamically regulated during cell cycle. As evidenced by the well-known phenomenon called position effect variegation (PEV), heterochromatin structure is dynamically and stochastically altered during developmental processes, and thus the expression of genes within or in the vicinity of heterochromatin could be affected by mutations in factors regulating DNA replication as well as by other epigenetic factors. Recent reports show that HP1 also plays an important role in the maintenance and transmission of chromosomes. Like many other factors ensuring faithful chromosome segregation, HP1 family proteins are subjected to posttranslational modifications, most notably phosphorylation, in a cell cycle-dependent manner. Recent studies identified a conserved phosphorylation site that profoundly affects the functions of HP1 during mitotic phase. In this commentary, we discuss dynamic regulation of HP1 protein by phosphorylation during transcriptional repression and cell cycle.

Low-density lipoprotein (LDL)-mimetic lipid nanoparticles (LNPs), decorated with MRI contrast agents and fluorescent dyes, were prepared by the covalent attachment of apolipoprotein-mimetic peptide (P), Gd(iii)-chelate (Gd), and sulforhodamine B (R) moieties on the LNP surface. The functionalized LNPs were prepared using the amide-forming potassium acyltrifluoroborate (KAT) ligation reaction. The KAT groups on the surface of LNPs were allowed to react with the corresponding hydroxylamine (HA) derivatives of P and Gd to provide bi-functionalized LNPs (PGd-LNP). The reaction proceeded with excellent yields, as observed by ICP-MS (for B and Gd amounts) and MALDI-TOF-MS data, and did not alter the morphology of the LNPs (mean diameter: ca. 50 nm), as shown by DLS and cryoTEM analyses. With the help of the efficient KAT ligation, a high payload of Gd(iii)-chelate on the PGd-LNP surface (ca. 2800 Gd atoms per LNP) was successfully achieved and provided a high r1 relaxivity (r1 = 22.0 s−1 mM−1 at 1.4 T/60 MHz and 25 °C; r1 = 8.2 s−1 mM−1 at 9.4 T/400 MHz and 37 °C). This bi-functionalized PGd-LNP was administered to three atherosclerotic apoE−/− mice to reveal the clear enhancement of atherosclerotic plaques in the brachiocephalic artery (BA) by MRI, in good agreement with the high accumulation of Gd in the aortic arch as shown by ICP-MS. The parallel in vivo MRI and ex vivo studies of whole mouse cryo-imaging were performed using triply functionalized LNPs with P, Gd, and R (PGdR-LNP). The clear presence of atherosclerotic plaques in BA was observed by ex vivo bright field cryo-imaging, and they were also observed by high emission fluorescent imaging. These directly corresponded to the enhanced tissue in the in vivo MRI of the identical mouse., LDL-mimetic lipid nanoparticles, decorated with MRI contrast agents and fluorescent dyes, were prepared by the covalent attachments of an apoB100-mimetic peptide, Gd(iii)-chelate, and rhodamine to enhance atherosclerosis in the in vivo imaging.

Inflammation is implicated in the onset and progression of various diseases, including cerebral pathologies. Here we report that DJ-1, which plays a role within cells as an antioxidant protein, functions as a damage-associated molecular pattern (DAMP), and triggers inflammation if released from dead cells into the extracellular space. We first found that recombinant DJ-1 protein induces the production of various inflammatory cytokines in bone marrow-derived macrophages (BMMs). We further identified a unique peptide sequence in the αG and αH helices of DJ-1 that activates Toll-like receptor 2 (TLR2) and TLR4. In the ischemic brain, DJ-1 is released into the extracellular space from necrotic neurons within 24 hours after stroke onset and makes direct contact with the surfaces of infiltrating myeloid cells. Administration of an antibody against DJ-1 suppresses the expression of inflammatory cytokines in infiltrating immune cells and attenuates ischemic neuronal damage. Our results demonstrate a previously unknown function of DJ-1 as a DAMP and suggest that extracellular DJ-1 could be a therapeutic target to prevent inflammation in tissue injuries and neurodegenerative diseases.Significance statementDJ-1 has been thoroughly investigated as a cytoprotective antioxidant protein in neurons. However, here we demonstrate that extracellularly released DJ-1 triggers neurotoxic inflammation after ischemic stroke. Intracellular DJ-1 increases in response to oxidative stress in ischemic neurons, but if ischemic stresses result in necrotic cell death, DJ-1 is released extracellularly. Released DJ-1 interacts with TLR2 and TLR4 on the surface of infiltrating myeloid cells and triggers post-ischemic inflammation, leading to the exacerbated pathologies of ischemic stroke. Thus, extracellular DJ-1 is a previously unknown inflammatogenic DAMP, and may be a putative target for therapeutic intervention to prevent progression of inflammatory and neurodegenerative diseases.

G-quadruplexes (G4s) are four-stranded DNA structures comprising stacks of four guanines, are prevalent in genomes, and have diverse biological functions in various chromosomal structures. A conserved protein, Rap1-interacting factor 1 (Rif1) from fission yeast (Schizosaccharomyces pombe), binds to Rif1-binding sequence (Rif1BS) and regulates DNA replication timing. Rif1BS is characterized by the presence of multiple G-tracts, often on both strands, and their unusual spacing. Although previous studies have suggested generation of G4-like structures on duplex Rif1BS, its precise molecular architecture remains unknown. Using gel-shift DNA binding assays and DNA footprinting with various nuclease probes, we show here that both of the Rif1BS strands adopt specific higher-order structures upon heat denaturation. We observed that the structure generated on the G-strand is consistent with a G4 having unusually long loop segments and that the structure on the complementary C-strand does not have an intercalated motif (i-motif). Instead, we found that the formation of the C-strand structure depends on the G4 formation on the G-strand. Thus, the higher-order structure generated at Rif1BS involved both DNA strands, and in some cases, G4s may form on both of these strands. The presence of multiple G-tracts permitted the formation of alternative structures when some G-tracts were mutated or disrupted by deazaguanine replacement, indicating the robust nature of DNA higher-order structures generated at Rif1BS. Our results provide general insights into DNA structures generated at G4-forming sequences on duplex DNA.

Rap1-interacting protein 1 (Rif1) regulates telomere length in budding yeast. We previously reported that, in metazoans and fission yeast, Rif1 also plays pivotal roles in controlling genome-wide DNA replication timing. We proposed that Rif1 may assemble chromatin compartments that contain specific replication-timing domains by promoting chromatin loop formation. Rif1 also is involved in DNA lesion repair, restart after replication fork collapse, anti-apoptosis activities, replicative senescence, and transcriptional regulation. Although multiple physiological functions of Rif1 have been characterized, biochemical and structural information on mammalian Rif1 is limited, mainly because of difficulties in purifying the full-length protein. Here, we expressed and purified the 2418-amino-acid-long, full-length murine Rif1 as well as its partially truncated variants in human 293T cells. Hydrodynamic analyses indicated that Rif1 forms elongated or extended homo-oligomers in solution, consistent with the presence of a HEAT-type helical repeat segment known to adopt an elongated shape. We also observed that the purified murine Rif1 bound G-quadruplex (G4) DNA with high specificity and affinity, as was previously shown for Rif1 from fission yeast. Both the N-terminal (HEAT-repeat) and C-terminal segments were involved in oligomer formation and specifically bound G4 DNA, and the central intrinsically disordered polypeptide segment increased the affinity for G4. Of note, pulldown assays revealed that Rif1 simultaneously binds multiple G4 molecules. Our findings support a model in which Rif1 modulates chromatin loop structures through binding to multiple G4 assemblies and by holding chromatin fibers together.

Rif1 regulates DNA replication timing and double-strand break repair, and its depletion induces transcriptional bursting of 2-cell (2C) zygote-specific genes in mouse ES cells. However, how Rif1 regulates zygotic transcription is unclear. We show here that Rif1 depletion promotes the formation of a unique Zscan4 enhancer structure harboring both histone H3 lysine 27 acetylation (H3K27ac) and moderate levels of silencing chromatin mark H3K9me3. Curiously, another enhancer mark H3K4me1 is missing whereas DNA methylation is still maintained in the structure, which spreads across gene bodies and neighboring regions within the Zscan4 gene cluster. We also found by function analyses of Rif1 domains in ES cells that ectopic expression of Rif1 lacking N-terminal domain results in upregulation of 2C transcripts. This appears to be caused by dominant negative inhibition of endogenous Rif1 protein localization at the nuclear periphery through formation of hetero-oligomers between the N-terminally truncated and endogenous forms. Strikingly, in murine 2-cell embryos, most of Rif1-derived polypeptides are expressed as truncated forms in soluble nuclear or cytosolic fraction, and are likely non-functional. Toward the morula stage, the full-length form of Rif1 gradually increased. Our results suggest that the absence of the functional full-length Rif1 due to its instability or alternative splicing and potential inactivation of Rif1 through dominant inhibition by N-terminally truncated Rif1 polypeptides, may be involved in 2C-specific transcription program.

Mrc1 and its vertebrate homologue Claspin serve as a mediator for replication stress checkpoint signaling, receiving the signal from Mec1/Rad3/ATR sensor kinase and transmitting it to the effector Rad53/Cds1/Chk1 kinase. They are likely to be a part of the replisome and facilitate the S-phase progression by promoting replication fork progression. Recent reports on Mrc1/Claspin indicate their new role in regulating the replication initiation through interaction with Cdc7, a key conserved serine-threonine kinase that triggers firing at each replication origin. Mrc1/Claspin has a specific domain that specifically interacts with Cdc7, and this domain is involved also in intramolecular interaction with its N-terminal segment. Mechanisms for novel regulation of origin firing and its timing through recruitment of Cdc7 to Mrc1/Claspin will be discussed.

Replication checkpoint is essential for maintaining genome integrity in response to various replication stresses as well as during the normal growth. The evolutionally conserved ATR-Claspin-Chk1 pathway is induced during replication checkpoint activation. Cdc7 kinase, required for initiation of DNA replication at replication origins, has been implicated in checkpoint activation but how it is involved in this pathway has not been known. Here, we show that Cdc7 is required for Claspin-Chk1 interaction in human cancer cells by phosphorylating CKBD (Chk1-binding-domain) of Claspin. The residual Chk1 activation in Cdc7-depleted cells is lost upon further depletion of casein kinase1 (CK1γ1), previously reported to phosphorylate CKBD. Thus, Cdc7, in conjunction with CK1γ1, facilitates the interaction between Claspin and Chk1 through phosphorylating CKBD. We also show that, whereas Cdc7 is predominantly responsible for CKBD phosphorylation in cancer cells, CK1γ1 plays a major role in non-cancer cells, providing rationale for targeting Cdc7 for cancer cell-specific cell killing., eLife digest It takes a human cell between six and eight hours to copy all three billion letters of its genome. During this time, any interruption to the process can lead to genetic errors, putting the cell in danger of developing disease. To guard against this, cells use a checkpoint system, testing their own health before, during and after DNA replication to make sure that they are ready for the next step. If a cell detects a problem while copying its DNA, it responds by activating proteins called checkpoint kinases. These stop the cell from continuing until the problem is resolved. One of these checkpoint kinases is a protein called Chk1, which switches on if the cell gets stuck part way through copying its DNA. To switch Chk1 on, the cell first needs to activate a protein called Claspin. Activating Claspin involves adding a chemical phosphate group to part of the Claspin protein. A third protein takes on this role, but its identity is controversial. Recent research points to a protein called casein kinase 1, but it was also possible that another protein, Cdc7 kinase, might be involved. To find out, Yang et al. used gene editing to lower the levels of Cdc7 in human cancer cells. The cells were able to copy their DNA under normal conditions, but they struggled to activate Chk1 when DNA replication stopped. Biochemical tests revealed that this was because, without Cdc7, Claspin was not receiving the phosphate group it needed. Even so, the cancer cells still had some Chk1 activation, which meant that they must be able to activate some of their Claspin. So, Yang et al. tried getting rid of both Cdc7 and the other candidate protein, casein kinase 1. This stopped Chk1 activation completely, revealing that although the cancer cells mainly used Cdc7 to activate Claspin, they also used casein kinase 1. In tests on non-cancerous cells, the results were the other way around; healthy cells mainly used casein kinase 1 and relied less heavily on Cdc7. These differences could prove useful for drug design. One of the challenges in cancer treatment is producing drugs that target cancer cells while leaving healthy cells unharmed. Future research could explore whether blocking Cdc7 could stop Chk1 activation in cancer cells only. This could stop the diseased cells fixing problems with their DNA replication, making it harder for them to survive.

Rif1 is a conserved protein regulating replication timing and binds preferentially to the vicinity of late-firing/dormant origins in fission yeast. The Rif1 binding sites on the fission yeast genome have an intrinsic potential to generate G-quadruplex (G4) structures to which purified Rif1 preferentially binds. We previously proposed that Rif1 generates chromatin architecture that may determine replication timing by facilitating the chromatin loop formation. Here, we conducted detailed biochemical analyses on Rif1 and its G4 binding. Rif1 prefers sequences containing long stretches of guanines and binds preferentially to the multimeric G4 of parallel or hybrid/mix topology. Rif1 forms oligomers and binds simultaneously to multiple G4. We present a model on how Rif1 may facilitate the formation of chromatin architecture through its G4 binding and oligomerization properties.

Rif1 is a key factor for spatiotemporal regulation of DNA replication. Rif1 suppresses origin firing in the mid-late replication domains by generating replication-suppressive chromatin architecture and by recruiting a protein phosphatase., Rif1 is a key factor for spatiotemporal regulation of DNA replication. Rif1 suppresses origin firing in the mid-late replication domains by generating replication-suppressive chromatin architecture and by recruiting a protein phosphatase. In fission yeast, the function of Hsk1, a kinase important for origin firing, can be bypassed by rif1Δ due to the loss of origin suppression. Rif1 specifically binds to G-quadruplex (G4) in vitro. Here, we show both conserved N-terminal HEAT repeats and C-terminal nonconserved segments are required for origin suppression. The N-terminal 444 amino acids and the C-terminal 229 amino acids can each mediate specific G4 binding, although high-affinity G4 binding requires the presence of both N- and C-terminal segments. The C-terminal 91 amino acids, although not able to bind to G4, can form a multimer. Furthermore, genetic screening led to identification of two classes of rif1 point mutations that can bypass Hsk1, one that fails to bind to chromatin and one that binds to chromatin. These results illustrate functional domains of Rif1 and indicate importance of both the N-terminal HEAT repeat segment and C-terminal G4 binding/oligomerization domain as well as other functionally unassigned segments of Rif1 in regulation of origin firing.

ATF4 is a pro-oncogenic transcription factor whose translation is activated by eIF2 phosphorylation through delayed re-initiation involving two uORFs in the mRNA leader. However, in yeast, the effect of eIF2 phosphorylation can be mimicked by eIF5 overexpression, which turns eIF5 into translational inhibitor, thereby promoting translation of GCN4, the yeast ATF4 equivalent. Furthermore, regulatory protein termed eIF5-mimic protein (5MP) can bind eIF2 and inhibit general translation. Here, we show that 5MP1 overexpression in human cells leads to strong formation of 5MP1:eIF2 complex, nearly comparable to that of eIF5:eIF2 complex produced by eIF5 overexpression. Overexpression of eIF5, 5MP1 and 5MP2, the second human paralog, promotes ATF4 expression in certain types of human cells including fibrosarcoma. 5MP overexpression also induces ATF4 expression in Drosophila. The knockdown of 5MP1 in fibrosarcoma attenuates ATF4 expression and its tumor formation on nude mice. Since 5MP2 is overproduced in salivary mucoepidermoid carcinoma, we propose that overexpression of eIF5 and 5MP induces translation of ATF4 and potentially other genes with uORFs in their mRNA leaders through delayed re-initiation, thereby enhancing the survival of normal and cancer cells under stress conditions.

B lymphocyte-induced maturation protein 1 (Blimp-1) encoded by Prdm1 is a master regulator of plasma cell differentiation. The transcription factor Bach2 represses Blimp-1 expression in B cells to stall terminal differentiation, by which it supports reactions such as class switch recombination of the antibody genes. We found that histones H3 and H4 around the Prdm1 intron 5 Maf recognition element were acetylated at higher levels in X63/0 plasma cells expressing Blimp-1 than in BAL17 mature B cells lacking its expression. Conversely, methylation of H3-K9 was lower in X63/0 cells than BAL17 cells. Purification of the Bach2 complex in BAL17 cells revealed its interaction with histone deacetylase 3 (HDAC3), nuclear co-repressors NCoR1 and NCoR2, transducin β-like 1X-linked (Tbl1x), and RAP1-interacting factor homolog (Rif1). Chromatin immunoprecipitation confirmed the binding of HDAC3 and Rif1 to the Prdm1 locus. Reduction of HDAC3 or NCoR1 expression by RNA interference in B cells resulted in an increased Prdm1 mRNA expression. Bach2 is suggested to cooperate with HDAC3-containing co-repressor complexes in B cells to regulate the stage-specific expression of Prdm1 by writing epigenetic modifications at the Prdm1 locus.

Arrest of replication fork progression is one of the most common causes for increasing the genomic instability. In bacteria, PriA, a conserved DEXH-type helicase, plays a major role in recognition of the stalled forks and restart of DNA replication. We took advantage of PriA's ability to specifically recognize stalled replication forks to determine the genomic loci where replication forks are prone to stall on the Escherichia coli genome. We found that PriA binds around oriC upon thymine starvation which reduces the nucleotide supply and causes replication fork stalling. PriA binding quickly disappeared upon readdition of thymine. Furthermore, BrdU was incorporated at around oriC upon release from thymine starvation. Our results indicate that reduced supply of DNA replication precursors causes replication fork stalling preferentially in the 600 kb segment centered at oriC. This suggests that replication of the vicinity of oriC requires higher level of nucleotide precursors. The results also point to a possibility of slow fork movement and/or the presence of multiple fork arrest signals within this segment. Indeed, we have identified rather strong fork stall/pausing signals symmetrically located at ∼50 kb away from oriC. We speculate that replication pausing and fork-slow-down shortly after initiation may represent a novel checkpoint that ensures the presence of sufficient nucleotide supply prior to commitment to duplication of the entire genome.