DHX36 maintains genomic integrity by unwinding G-quadruplexes.

The guanine-rich stretch of single-stranded DNA (ssDNA) forms a G-quadruplex (G4) in a fraction of genic and intergenic chromosomal regions. The probability of G4 formation increases during events causing ssDNA generation, such as transcription and replication. In turn, G4 abrogates these events, leading to DNA damage. DHX36 unwinds G4-DNA in vitro and in human cells. However, its spatial correlation with G4-DNA in vivo and its role in genome maintenance remain unclear. Here, we demonstrate a connection between DHX36 and G4-DNA and its implications for genomic integrity. The nuclear localization of DHX36 overlapped with that of G4-DNA, RNA polymerase II, and a splicing-related factor. Depletion of DHX36 resulted in accumulated DNA damage, slower cell growth, and enhanced cell growth inhibition upon treatment with a G4-stabilizing compound; DHX36 expression reversed these defects. In contrast, the reversal upon expression of DHX36 mutants that could not bind G4 was imperfect. Thus, DHX36 may suppress DNA damage by promoting the clearance of G4-DNA for cell growth and survival. Our findings deepen the understanding of G4 resolution in the maintenance of genomic integrity.

The CST complex facilitates cell survival under oxidative genotoxic stress.

Genomic DNA is constantly exposed to a variety of genotoxic stresses, and it is crucial for organisms to be equipped with mechanisms for repairing the damaged genome. Previously, it was demonstrated that the mammalian CST (CTC1-STN1-TEN1) complex, which was originally identified as a single-stranded DNA-binding trimeric protein complex essential for telomere maintenance, is required for survival in response to hydroxyurea (HU), which induces DNA replication fork stalling. It is still unclear, however, how the CST complex is involved in the repair of diverse types of DNA damage induced by oxidizing agents such as H2O2. STN1 knockdown (KD) sensitized HeLa cells to high doses of H2O2. While H2O2 induced DNA strand breaks throughout the cell cycle, STN1 KD cells were as resistant as control cells to H2O2 treatment when challenged in the G1 phase of the cell cycle, but they were sensitive when exposed to H2O2 in S/G2/M phase. STN1 KD cells showed a failure of DNA synthesis and RAD51 foci formation upon H2O2 treatment. Chemical inhibition of RAD51 in shSTN1 cells did not exacerbate the sensitivity to H2O2, implying that the CST complex and RAD51 act in the same pathway. Collectively, our results suggest that the CST complex is required for maintaining genomic stability in response to oxidative DNA damage, possibly through RAD51-dependent DNA repair/protection mechanisms.

The interferon stimulated gene-encoded protein HELZ2 inhibits human LINE-1 retrotransposition and LINE-1 RNA-mediated type I interferon induction.

Some interferon stimulated genes (ISGs) encode proteins that inhibit LINE-1 (L1) retrotransposition. Here, we use immunoprecipitation followed by liquid chromatography-tandem mass spectrometry to identify proteins that associate with the L1 ORF1-encoded protein (ORF1p) in ribonucleoprotein particles. Three ISG proteins that interact with ORF1p inhibit retrotransposition: HECT and RLD domain containing E3 ubiquitin-protein ligase 5 (HERC5); 2'-5'-oligoadenylate synthetase-like (OASL); and helicase with zinc finger 2 (HELZ2). HERC5 destabilizes ORF1p, but does not affect its cellular localization. OASL impairs ORF1p cytoplasmic foci formation. HELZ2 recognizes sequences and/or structures within the L1 5'UTR to reduce L1 RNA, ORF1p, and ORF1p cytoplasmic foci levels. Overexpression of WT or reverse transcriptase-deficient L1s lead to a modest induction of IFN-α expression, which is abrogated upon HELZ2 overexpression. Notably, IFN-α expression is enhanced upon overexpression of an ORF1p RNA binding mutant, suggesting ORF1p binding might protect L1 RNA from "triggering" IFN-α induction. Thus, ISG proteins can inhibit retrotransposition by different mechanisms.

Fission yeast Stn1 maintains stability of repetitive DNA at subtelomere and ribosomal DNA regions.

Telomere binding protein Stn1 forms the CST (Cdc13/CTC1-STN1-TEN1) complex in budding yeast and mammals. Likewise, fission yeast Stn1 and Ten1 form a complex indispensable for telomere protection. We have previously reported that stn1-1, a high-temperature sensitive mutant, rapidly loses telomere DNA at the restrictive temperature due to frequent failure of replication fork progression at telomeres and subtelomeres, both containing repetitive sequences. It is unclear, however, whether Stn1 is required for maintaining other repetitive DNAs such as ribosomal DNA. In this study, we have demonstrated that stn1-1 cells, even when grown at the permissive temperature, exhibited dynamic rearrangements in the telomere-proximal regions of subtelomere and ribosomal DNA repeats. Furthermore, Rad52 and γH2A accumulation was observed at ribosomal DNA repeats in the stn1-1 mutant. The phenotypes exhibited by the stn1-1 allele were largely suppressed in the absence of Reb1, a replication fork barrier-forming protein, suggesting that Stn1 is involved in the maintenance of the arrested replication forks. Collectively, we propose that Stn1 maintains the stability of repetitive DNAs at subtelomeres and rDNA regions.

Chromosome instability induced by a single defined sister chromatid fusion.

Chromosome fusion is a frequent intermediate in oncogenic chromosome rearrangements and has been proposed to cause multiple tumor-driving abnormalities. In conventional experimental systems, however, these abnormalities were often induced by randomly induced chromosome fusions involving multiple different chromosomes. It was therefore not well understood whether a single defined type of chromosome fusion, which is reminiscent of a sporadic fusion in tumor cells, has the potential to cause chromosome instabilities. Here, we developed a human cell-based sister chromatid fusion visualization system (FuVis), in which a single defined sister chromatid fusion is induced by CRISPR/Cas9 concomitantly with mCitrine expression. The fused chromosome subsequently developed extra-acentric chromosomes, including chromosome scattering, indicative of chromothripsis. Live-cell imaging and statistical modeling indicated that sister chromatid fusion generated micronuclei (MN) in the first few cell cycles and that cells with MN tend to display cell cycle abnormalities. The powerful FuVis system thus demonstrates that even a single sporadic sister chromatid fusion can induce chromosome instability and destabilize the cell cycle through MN formation.

Proteostasis failure and cellular senescence in long-term cultured postmitotic rat neurons.

Cellular senescence, a stress-induced irreversible cell cycle arrest, has been defined for mitotic cells and is implicated in aging of replicative tissues. Age-related functional decline in the brain is often attributed to a failure of protein homeostasis (proteostasis), largely in postmitotic neurons, which accordingly is a process distinct by definition from senescence. It is nevertheless possible that proteostasis failure and cellular senescence have overlapping molecular mechanisms. Here, we identify postmitotic cellular senescence as an adaptive stress response to proteostasis failure. Primary rat hippocampal neurons in long-term cultures show molecular changes indicative of both senescence (senescence-associated ß-galactosidase, p16, and loss of lamin B1) and proteostasis failure relevant to Alzheimer's disease. In addition, we demonstrate that the senescent neurons exhibit resistance to stress. Importantly, treatment of the cultures with an mTOR antagonist, protein synthesis inhibitor, or chemical compound that reduces the amount of protein aggregates relieved the proteotoxic stresses as well as the appearance of senescence markers. Our data propose mechanistic insights into the pathophysiological brain aging by establishing senescence as a primary cell-autonomous neuroprotective response.

Telomere-binding proteins Taz1 and Rap1 regulate DSB repair and suppress gross chromosomal rearrangements in fission yeast.

Genomic rearrangements (gross chromosomal rearrangements, GCRs) threatens genome integrity and cause cell death or tumor formation. At the terminus of linear chromosomes, a telomere-binding protein complex, called shelterin, ensures chromosome stability by preventing chromosome end-to-end fusions and regulating telomere length homeostasis. As such, shelterin-mediated telomere functions play a pivotal role in suppressing GCR formation. However, it remains unclear whether the shelterin proteins play any direct role in inhibiting GCR at non-telomeric regions. Here, we have established a GCR assay for the first time in fission yeast and measured GCR rates in various mutants. We found that fission yeast cells lacking shelterin components Taz1 or Rap1 (mammalian TRF1/2 or RAP1 homologues, respectively) showed higher GCR rates compared to wild-type, accumulating large chromosome deletions. Genetic dissection of Rap1 revealed that Rap1 contributes to inhibiting GCRs via two independent pathways. The N-terminal BRCT-domain promotes faithful DSB repair, as determined by I-SceI-mediated DSB-induction experiments; moreover, association with Poz1 mediated by the central Poz1-binding domain regulates telomerase accessibility to DSBs, leading to suppression of de novo telomere additions. Our data highlight unappreciated functions of the shelterin components Taz1 and Rap1 in maintaining genome stability, specifically by preventing non-telomeric GCRs.

Time-Series Analysis of Tumorigenesis in a Murine Skin Carcinogenesis Model.

Recent years have witnessed substantial progress in understanding tumor heterogeneity and the process of tumor progression; however, the entire process of the transition of tumors from a benign to metastatic state remains poorly understood. In the present study, we performed a prospective cancer genome-sequencing analysis by employing an experimental carcinogenesis mouse model of squamous cell carcinoma to systematically understand the evolutionary process of tumors. We surgically collected a part of a lesion of each tumor and followed the progression of these tumors in vivo over time. Comparative time-series analysis of the genomes of tumors with different fates, i.e., those that eventually metastasized and regressed, suggested that these tumors acquired and inherited different mutations. These findings suggest that despite the occurrence of an intra-tumor selection event for malignant alteration during the transformation from early- to late-stage papilloma, the fate determination of tumors might be determined at an even earlier stage.

ATF7 mediates TNF-α-induced telomere shortening.

Telomeres maintain the integrity of chromosome ends and telomere length is an important marker of aging. The epidemiological studies suggested that many types of stress including psychosocial stress decrease telomere length. However, it remains unknown how various stresses induce telomere shortening. Here, we report that the stress-responsive transcription factor ATF7 mediates TNF-α-induced telomere shortening. ATF7 and telomerase, an enzyme that elongates telomeres, are localized on telomeres via interactions with the Ku complex. In response to TNF-α, which is induced by various stresses including psychological stress, ATF7 was phosphorylated by p38, leading to the release of ATF7 and telomerase from telomeres. Thus, a decrease of ATF7 and telomerase on telomeres in response to stress causes telomere shortening, as observed in ATF7-deficient mice. These findings give credence to the idea that various types of stress might shorten telomere.

Structure of the Dnmt1 Reader Module Complexed with a Unique Two-Mono-Ubiquitin Mark on Histone H3 Reveals the Basis for DNA Methylation Maintenance.

The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.

Hippocampal TERT Regulates Spatial Memory Formation through Modulation of Neural Development.

The molecular mechanism of memory formation remains a mystery. Here, we show that TERT, the catalytic subunit of telomerase, gene knockout (Tert-/-) causes extremely poor ability in spatial memory formation. Knockdown of TERT in the dentate gyrus of adult hippocampus impairs spatial memory processes, while overexpression facilitates it. We find that TERT plays a critical role in neural development including dendritic development and neuritogenesis of hippocampal newborn neurons. A monosynaptic pseudotyped rabies virus retrograde tracing method shows that TERT is required for neural circuit integration of hippocampal newborn neurons. Interestingly, TERT regulated neural development and spatial memory formation in a reverse transcription activity-independent manner. Using X-ray irradiation, we find that hippocampal newborn neurons mediate the modulation of spatial memory processes by TERT. These observations reveal an important function of TERT through a non-canonical pathway and encourage the development of a TERT-based strategy to treat neurological disease-associated memory impairment.

Three-Dimensional Localization of an Individual Fluorescent Molecule with Angstrom Precision.

Among imaging techniques, fluorescence microscopy is a unique method to noninvasively image individual molecules in whole cells. If the three-dimensional spatial precision is improved to the angstrom level, various molecular arrangements in the cell can be visualized on an individual basis. We have developed a cryogenic reflecting microscope with a numerical aperture of 0.99 and an imaging stability of 0.05 nm in standard deviation at a temperature of 1.8 K. The key optics to realize the cryogenic performances is the reflecting objective developed by our laboratory. With this cryogenic microscope, an individual fluorescent molecule (ATTO647N) at 1.8 K was localized with standard errors of 0.53 nm (x), 0.31 nm (y), and 0.90 nm (z) when 106 fluorescence photons from the molecule were accumulated in 5 min.

Fission yeast Stn1 is crucial for semi-conservative replication at telomeres and subtelomeres.

The CST complex is a phylogenetically conserved protein complex consisting of CTC1/Cdc13, Stn1 and Ten1 that protects telomeres on linear chromosomes. Deletion of the fission yeast homologs stn1 and ten1 results in complete telomere loss; however, the precise function of Stn1 is still largely unknown. Here, we have isolated a high-temperature sensitive stn1 allele (termed stn1-1). stn1-1 cells abruptly lost telomeric sequence almost completely at the restrictive temperature. The loss of chromosomal DNA happened without gradual telomere shortening, and extended to 30 kb from the ends of chromosomes. We found transient and modest single-stranded G-strand exposure, but did not find any evidence of checkpoint activation in stn1-1 at the restrictive temperature. When we probed neutral-neutral 2D gels for subtelomere regions, we found no Y-arc-shaped replication intermediates in cycling cells. We conclude that the loss of telomere and subtelomere DNAs in stn1-1 cells at the restrictive temperature is caused by very frequent replication fork collapses specifically in subtelomere regions. Furthermore, we identified two independent suppressor mutants of the high-temperature sensitivity of stn1-1: a multi-copy form of pmt3 and a deletion of rif1. Collectively, we propose that fission yeast Stn1 primarily safeguards the semi-conservative DNA replication at telomeres and subtelomeres.

NEK6-mediated phosphorylation of human TPP1 regulates telomere length through telomerase recruitment.

Shelterin component TPP1 plays critical roles in chromosome end protection and telomere length regulation. Specifically, TPP1 contains an OB-fold domain that provides an interface to recruit telomerase. However, it remains largely unknown how telomerase recruitment is regulated by cell cycle regulators. We show that TPP1 interacts with the cell cycle regulator kinase NEK6 in human cells. We found that NEK6-mediated phosphorylation of TPP1 Ser255 in G2/M phase regulates the association between telomerase activity and TPP1. Furthermore, we found evidence that POT1 negatively regulates TPP1 phosphorylation because the level of Ser255 phosphorylation was elevated when telomeres were elongated by a POT1 mutant lacking its OB-fold domains. Ser255 is located in the intervening region between the telomerase-recruiting OB-fold and the POT1 recruitment domains. Ser255 and the surrounding amino acids are conserved among vertebrates. These observations suggest that a region adjacent to the OB-fold domain of TPP1 is involved in telomere length regulation via telomerase recruitment.

A human artificial chromosome recapitulates the metabolism of native telomeres in mammalian cells.

Telomeric and subtelomeric regions of human chromosomes largely consist of highly repetitive and redundant DNA sequences, resulting in a paucity of unique DNA sequences specific to individual telomeres. Accordingly, it is difficult to analyze telomere metabolism on a single-telomere basis. To circumvent this problem, we have exploited a human artificial chromosome (HAC#21) derived from human chromosome 21 (hChr21). HAC#21 was generated through truncation of the long arm of native hChr21 by the targeted telomere seeding technique. The newly established telomere of HAC#21 lacks canonical subtelomere structures but possesses unique sequences derived from the target vector backbone and the internal region of hChr21 used for telomere targeting, which enabled us to molecularly characterize the single HAC telomere. We established HeLa and NIH-3T3 sub-lines containing a single copy of HAC#21, where it was robustly maintained. The seeded telomere is associated with telomeric proteins over a length similar to that reported in native telomeres, and is faithfully replicated in mid-S phase in HeLa cells. We found that the seeded telomere on HAC#21 is transcribed from the newly juxtaposed site. The transcript, HAC-telRNA, shares several features with TERRA (telomeric repeat-containing RNA): it is a short-lived RNA polymerase II transcript, rarely contains a poly(A) tail, and associates with chromatin. Interestingly, HAC-telRNA undergoes splicing. These results suggest that transcription into TERRA is locally influenced by the subtelomeric context. Taken together, we have established human and mouse cell lines that will be useful for analyzing the behavior of a uniquely identifiable, functional telomere.

Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication.

Faithful propagation of DNA methylation patterns during DNA replication is critical for maintaining cellular phenotypes of individual differentiated cells. Although it is well established that Uhrf1 (ubiquitin-like with PHD and ring finger domains 1; also known as Np95 and ICBP90) specifically binds to hemi-methylated DNA through its SRA (SET and RING finger associated) domain and has an essential role in maintenance of DNA methylation by recruiting Dnmt1 to hemi-methylated DNA sites, the mechanism by which Uhrf1 coordinates the maintenance of DNA methylation and DNA replication is largely unknown. Here we show that Uhrf1-dependent histone H3 ubiquitylation has a prerequisite role in the maintenance DNA methylation. Using Xenopus egg extracts, we successfully reproduce maintenance DNA methylation in vitro. Dnmt1 depletion results in a marked accumulation of Uhrf1-dependent ubiquitylation of histone H3 at lysine 23. Dnmt1 preferentially associates with ubiquitylated H3 in vitro though a region previously identified as a replication foci targeting sequence. The RING finger mutant of Uhrf1 fails to recruit Dnmt1 to DNA replication sites and maintain DNA methylation in mammalian cultured cells. Our findings represent the first evidence, to our knowledge, of the mechanistic link between DNA methylation and DNA replication through histone H3 ubiquitylation.

Receptor for activated C-kinase (RACK1) homolog Cpc2 facilitates the general amino acid control response through Gcn2 kinase in fission yeast.

General amino acid control (GAAC) is crucial for sensing and adaptation to nutrient availability. Amino acid starvation activates protein kinase Gcn2, which plays a central role in the GAAC response by phosphorylating the α-subunit of eukaryotic initiation factor 2 (eIF2α), leading to the translational switch to stimulate selective expression of stress-responsive genes. We report here that in fission yeast Schizosaccharomyces pombe, Cpc2, a homolog of mammalian receptor for activated C-kinase (RACK1), is important for the GAAC response. Deletion of S. pombe cpc2 impairs the amino acid starvation-induced phosphorylation of eIF2α and the expression of amino acid biosynthesis genes, thereby rendering cells severely sensitive to amino acid limitation. Unlike the Saccharomyces cerevisiae Cpc2 ortholog, which normally suppresses the GAAC response, our findings suggest that S. pombe Cpc2 promotes the GAAC response. We also found that S. pombe Cpc2 is required for starvation-induced Gcn2 autophosphorylation, which is essential for Gcn2 function. These results indicate that S. pombe Cpc2 facilitates the GAAC response through the regulation of Gcn2 activation and provide a novel insight for the regulatory function of RACK1 on Gcn2-mediated GAAC response.

Portrait of replication stress viewed from telomeres.

Genetic instability is the driving force of the malignant progression of cancer cells. Recently, replication stress has attracted much attention as a source of genetic instability that gives rise to an accumulation of mutations during the lifespan of individuals. However, the molecular details of the process are not fully understood. Here, recent progress in understanding how genetic alterations accumulate at telomeres will be reviewed. In particular, two aspects of telomere replication will be discussed in this context, covering conventional semi-conservative replication, and DNA synthesis by telomerase plus the C-strand fill-in reactions. Although these processes are seemingly telomere-specific, I will emphasize the possibility that the molecular understanding of the telomere events may shed light on genetic instability at other genetic loci in general.