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RAD51 is critical to the homologous recombination (HR) pathway that repairs DNA double strand breaks (DSBs) and protects replication forks (RFs). Previously, we showed that the S181P (SP) mutation in RAD51causes defective RF maintenance but is proficient for DSB repair. Here we report that SP/SP female mice exhibit a shortened lifespan compared to +/+ females but not males. Histological analysis found that most mice in this study died from lymphoma, independent of genotype and sex. We propose that a potential cause for shortened lifespan in SP/SP females is due to the RF defect.

Heat shock protein 90 (Hsp90) family proteins are molecular chaperones that modulate the functions of various substrate proteins (clients) implicated in pro-tumorigenic pathways. In this study, the mitochondria-targeted antioxidant mitoquinone (MitoQ) was identified as a potent inhibitor of mitochondrial Hsp90, known as a tumor necrosis factor receptor-associated protein 1 (TRAP1). Structural analyses revealed an asymmetric bipartite interaction between MitoQ and the previously unrecognized drug binding sites located in the middle domain of TRAP1, believed to be a client binding region. MitoQ effectively competed with TRAP1 clients, and MitoQ treatment facilitated the identification of 103 TRAP1-interacting mitochondrial proteins in cancer cells. MitoQ and its redox-crippled SB-U014/SB-U015 exhibited more potent anticancer activity in vitroand in vivothan previously reported mitochondria-targeted TRAP1 inhibitors. The findings indicate that targeting the client binding site of Hsp90 family proteins offers a novel strategy for the development of potent anticancer drugs.

Haematopoietic stem and progenitor cells (HSPCs) have been the focus of developmental and regenerative studies, yet our understanding of the signalling events regulating their specification remains incomplete. We demonstrate that supt16h, a component of the Facilitates chromatin transcription (FACT) complex, is required for HSPC formation. Zebrafish supt16hmutants express reduced levels of Notch-signalling components, genes essential for HSPC development, due to abrogated transcription. Whereas global chromatin accessibility in supt16hmutants is not substantially altered, we observe a specific increase in p53accessibility, causing an accumulation of p53. We further demonstrate that p53 influences expression of the Polycomb-group protein PHC1, which functions as a transcriptional repressor of Notch genes. Suppression of phc1or its upstream regulator, p53, rescues the loss of both Notch and HSPC phenotypes in supt16hmutants. Our results highlight a relationship between supt16h, p53and phc1to specify HSPCs via modulation of Notch signalling.

ABSTRACTCentrosomes are the primary microtubule-organizing centers that are important for mitotic spindle assembly. Centrosome amplification is commonly observed in human cancer cells and contributes to genomic instability. However, it is not clear how centrosome duplication is dysregulated in cancer cells. Here, we report that ATAD5, a replisome protein that unloads PCNA from chromatin as a replication factor C-like complex (RLC), plays an important role in regulating centrosome duplication. ATAD5 is present at the centrosome, specifically at the base of the mother and daughter centrioles that undergo duplication. UAF1, which interacts with ATAD5 and regulates PCNA deubiquitination as a complex with ubiquitin-specific protease 1, is also localized at the centrosome. Depletion of ATAD5 or UAF1 increases cells with over-duplicated centrosome whereas ATAD5 overexpression reduces such cells. Consistently, the proportion of cells showing the multipolar mode of chromosome segregation is increased among ATAD5-depleted cells. The localization and function of ATAD5 at the centrosomes do not require other RLC subunits. UAF1 interacts and co-localizes with ID1, a protein that increases centrosome amplification upon overexpression. ATAD5 depletion reduces interactions between UAF1 and ID1 and increases ID1 signal at the centrosome, providing a mechanistic framework for understanding the role of ATAD5 in centrosome duplication.

Motile cilia of multiciliated epithelial cells have important roles in animal development and cell homeostasis. Although several studies have identified and reported proteins localized in this complex organelle and the related immotile primary cilia from various cell types, it is still challenging to isolate high quantities of ciliary proteins for proteomic analysis. In this study, African clawed frog (Xenopus laevis) embryos, which have many multiciliated cells in the epidermis, were treated with a simple ionic buffer to identify 1009 proteins conserved across vertebrates; these proteins were putatively localized in motile cilia. Using two ciliary proteome databases, we confirmed that previously validated cilia-associated proteins are highly enriched in our ciliary proteome. Proteins localized at the transition zone and Ellis-van Creveld zone, which are distinct regions at the base of cilia, near the junction with the apical cell surface, were isolated using our method. Among the newly identified ciliary proteins, we report that KRT17 may have an unrecognized function in motile cilia. Hence, the method developed in this study would be useful for understanding the ciliary proteome.

The higher-order structural organization and dynamics of the chromosomes play a central role in gene regulation. To explore this structure–function relationship, it is necessary to directly visualize genomic elements in living cells. Genome imaging based on the CRISPR system is a powerful approach but has limited applicability due to background signals and nonspecific aggregation of fluorophores within nuclei. To address this issue, we developed a novel visualization scheme combining tripartite fluorescent proteins with the SunTag system and demonstrated that it strongly suppressed background fluorescence and amplified locus-specific signals, allowing long-term tracking of genomic loci. We integrated the multicomponent CRISPR system into stable cell lines to allow quantitative and reliable analysis of dynamic behaviors of genomic loci. Due to the greatly elevated signal-to-background ratio, target loci with only small numbers of sequence repeats could be successfully tracked, even under a conventional fluorescence microscope. This feature enables the application of CRISPR-based imaging to loci throughout the genome and opens up new possibilities for the study of nuclear processes in living cells.

ABSTRACTImatinib is the first molecularly targeted compound for chronic myeloid leukemia (CML) capable to inhibit BCR-ABL kinase activity. However, recent clinical evidence indicates that a substantial proportion of CML patients exhibit BCR-ABL-dependent or independent resistance to imatinib. Despite the importance of imatinib resistance in CML, the underlying molecular mechanisms of this resistance are largely unknown. Here, we identified GCA (grancalcin) as a critical regulator of imatinib resistance in chronic phase CML via activation of autophagy. Mechanistically, we demonstrated that GCA activates TRAF6 ubiquitin ligase activity to induce Lys63 ubiquitination of ULK1, a crucial regulator of autophagy, resulting in its stabilization and activation. We also highlighted the role of GCA-TRAF6-ULK1 autophagy regulatory axis in imatinib resistance. Our findings represent the basis for novel therapeutic strategies against CML.Abbreviation:ACTB/β-actin: actin beta; ADM: adrenomedullin; AMBRA1: autophagy and beclin 1 regulator 1; AMPK: AMP-activated protein kinase; ANXA5: annexin A5; CP: cytogenetic response; CML: chronic myeloid leukemia; CUL3: cullin 3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCA: grancalcin; Dx: at diagnosis; E-64-d: (2S,3S)-trans-Epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester; IMres: Imatinib resistance; KLHL20: Kelch-like protein 20; LRMP: lymphoid-restricted membrane protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MMR: major molecular response; NH4Cl: ammonium chloride; PBMCs: peripheral blood mononuclear cells; PTPRC: protein tyrosine phosphatase, receptor type, C; SQSTM1/p62: sequestosome 1; SYK: spleen associated tyrosine kinase; TAP1: transporter 1, ATP binding cassette subfamily B member; TKIs: ABL-specific tyrosine kinase inhibitors; TLR9: toll- like receptor 9; TRAF6: TNF receptor associated factor 6; ULK1: unc-51 like autophagy activating kinase 1

Background: Ubiquitin-related rare diseases are generally characterized by developmental delays and mental retardation, but the exact incidence or prevalence is not yet fully understood. The clinical application of next-generation sequencing for pediatric seizures and developmental delay of unknown causes has become common in studies aimed at identification of a causal gene in patients with ubiquitin-related rare diseases that cannot be diagnosed using conventional fluorescence in situ hybridization or chromosome microarray tests. Our study aimed to investigate the effects of ubiquitin–proteasome system on ultra-rare neurodevelopmental diseases, through functional identification of candidate genes and variants. Methods: In our present work, we carried out genome analysis of a patient with clinical phenotypes of developmental delay and intractable convulsion, to identify causal mutations. Further characterization of the candidate gene was performed using zebrafish, through gene knockdown approaches. Transcriptomic analysis using whole embryos of zebrafish knockdown morphants and additional functional studies identified downstream pathways of the candidate gene affecting neurogenesis. Results: Through trio-based whole-genome sequencing analysis, we identified a de novo missense variant of the ubiquitin system-related gene UBE2H(c.449C>T; p.Thr150Met) in the proband. Using zebrafish, we found that Ube2h is required for normal brain development. Differential gene expression analysis revealed activation of the ATM-p53 signaling pathway in the absence of Ube2h. Moreover, depletion of ube2hled to induction of apoptosis, specifically in the differentiated neural cells. Finally, we found that a missense mutation in zebrafish, ube2h(c.449C>T; p.Thr150Met), which mimics a variant identified in a patient with neurodevelopmental defects, causes aberrant Ube2h function in zebrafish embryos. Conclusion: A de novo heterozygous variant in the UBE2Hc.449C>T (p.Thr150Met) has been identified in a pediatric patient with global developmental delay and UBE2H is essential for normal neurogenesis in the brain.

Herein, we report chemoselective trifluoroethylation routes of unmasked 2-arylquinazolin-4(3H)-ones using mesityl(2,2,2-trifluoroethyl)iodonium triflate at room temperature. Homologous C-, O-, and N-functionalized subclasses are accessed in a straightforward manner with a wide substrate scope. These chemoselective branching events are driven by Pd-catalyzed ortho-selective C–H activation at the pendant aryl ring and base-promoted reactivity modulation of the amide group, leveraging the intrinsic directing capability and competing pronucleophilicity of the quinazolin-4(3H)-one framework. Furthermore, outstanding photostability of the quinazolin-4(3H)-one family associated with nonradiative decay is presented.

ObjectivesHepatocellular carcinoma (HCC) is a common cancer with high rate of recurrence and mortality. Diverse aetiological agents and wide heterogeneity in individual tumours impede effective and personalised treatment. Tonicity-responsive enhancer-binding protein (TonEBP) is a transcriptional cofactor for the expression of proinflammatory genes. Although inflammation is intimately associated with the pathogenesis of HCC, the role of TonEBP is unknown. We aimed to identify function of TonEBP in HCC.DesignTumours with surrounding hepatic tissues were obtained from 296 patients with HCC who received completion resection. TonEBP expression was analysed by quantitative reverse transcription–quantitative real-time PCR (RT-PCR) and immunohfistochemical analyses of tissue microarrays. Mice with TonEBP haplodeficiency, and hepatocyte-specific and myeloid-specific TonEBP deletion were used along with HCC and hepatocyte cell lines.ResultsTonEBP expression is higher in tumours than in adjacent non-tumour tissues in 92.6% of patients with HCC regardless of aetiology associated. The TonEBP expression in tumours and adjacent non-tumour tissues predicts recurrence, metastasis and death in multivariate analyses. TonEBP drives the expression of cyclo-oxygenase-2 (COX-2) by stimulating the promoter. In mouse models of HCC, three common sites of TonEBP action in response to diverse aetiological agents leading to tumourigenesis and tumour growth were found: cell injury and inflammation, induction by oxidative stress and stimulation of the COX-2 promoter.ConclusionsTonEBP is a key component of the common pathway in tumourigenesis and tumour progression of HCC in response to diverse aetiological insults. TonEBP is involved in multiple steps along the pathway, rendering it an attractive therapeutic target as well as a prognostic biomarker.

Dopamine synapses play a crucial role in volitional movement and reward-related behaviors, while dysfunction of dopamine synapses causes various psychiatric and neurological disorders. Despite this significance, the true biological nature of dopamine synapses remains poorly understood. Here, we show that dopamine transmission is strongly correlated with GABA co-transmission across the brain and dopamine synapses are structured and function like GABAergic synapses with marked regional heterogeneity. In addition, GABAergic-like dopamine synapses are clustered on the dendrites, and GABA transmission at dopamine synapses has distinct physiological properties. Interestingly, the knockdown of neuroligin-2, a key postsynaptic protein at GABAergic synapses, unexpectedly does not weaken GABA co-transmission but instead facilitates it at dopamine synapses in the striatal neurons. More importantly, the attenuation of GABA co-transmission precedes deficits in dopaminergic transmission in animal models of Parkinson’s disease. Our findings reveal the spatial and functional nature of GABAergic-like dopamine synapses in health and disease.

ABSTRACTThere are hundreds of copies of rDNA repeats in mammalian chromosomes and the ratio of active, poised, or inactive rDNA is regulated in epigenetic manners. Recent studies demonstrated that a post-DNA replication repair enzyme, SHPRH affects rRNA transcription by recognizing epigenetic markers on rDNA promoters and unveiled potential links between DNA repair and ribosome biogenesis. This study suggests that SHPRH could be a link between mTOR-mediated epigenetic regulations and rRNA transcription, while concomitantly affecting genomic integrity.

Genome duplication is an essential process to preserve genetic information between generations. The eukaryotic cell cycle is composed of functionally distinct phases: G1, S, G2, and M. One of the key replicative proteins that participate at every stage of DNA replication is the Mcm2-7 complex, a replicative helicase. In the G1 phase, inactive Mcm2-7 complexes are loaded on the replication origins by replication-initiator proteins, ORC and Cdc6. Two kinases, S-CDK and DDK, convert the inactive origin-loaded Mcm2-7 complex to an active helicase, the CMG complex in the S phase. The activated CMG complex begins DNA unwinding and recruits enzymes essential for DNA synthesis to assemble a replisome at the replication fork. After completion of DNA synthesis, the inactive CMG complex on the replicated DNA is removed from chromatin to terminate DNA replication. In this review, we will discuss the structure, function, and regulation of the molecular machines involved in each step of DNA replication.

Maintaining genome stability is crucial for all cells. The budding yeast Elg1 protein, the major subunit of a replication factor C-like complex, is important for genome stability, since cells lacking Elg1 exhibit increased recombination and chromosomal rearrangements. This genome maintenance function of Elg1 seems to be conserved in higher eukaryotes, since removal of the human Elg1 homolog, encoded by the ATAD5gene, also causes genome instability leading to tumorigenesis. The fundamental molecular function of the Elg1/ATAD5-replication factor C-like complex (RLC) was, until recently, elusive, although Elg1/ATAD5-RLC was known to interact with the replication sliding clamp PCNA. Two papers have now reported that following DNA replication, the Elg1/ATAD5-RLC is required to remove PCNA from chromatin in yeast and human cells. In this Review, we summarize the evidence that Elg1/ATAD5-RLC acts as a PCNA unloader and discuss the still enigmatic relationship between the function of Elg1/ATAD5-RLC in PCNA unloading and the role of Elg1/ATAD5 in maintaining genomic stability.

Genomic integrity depends on DNA replication, recombination, and repair, particularly in S phase. We demonstrate that a human homologue of yeast Elg1 plays an important role in S phase to preserve genomic stability. The level of ELG1 is induced during recovery from a variety of DNA damage. In response to DNA damage, ELG1 forms distinct foci at stalled DNA replication forks that are different from DNA double strand break foci. Targeted gene knockdown of ELG1 resulted in spontaneous foci formation of γ-H2AX, 53BP1, and phosphorylated-ATM that mark chromosomal breaks.  Abnormal chromosomes including fusions, inversions and hypersensitivity to DNA damaging agents were also observed in cells expressing low level of ELG1 by targeted gene knockdown. Knockdown of ELG1 by siRNA reduced homologous recombination frequency in the I-SceI induced double strand break-dependent assay. In contrast, spontaneous homologous recombination frequency and sister chromatin exchange rate were up-regulated when ELG1 was silenced by shRNA. Taken together, we propose that ELG1 would be a new member of proteins involved in maintenance of genomic integrity.

ABSTRACTGross chromosomal rearrangements (GCRs) are frequently observed in cancer cells. Abnormalities in different DNA metabolism including DNA replication, cell cycle checkpoints, chromatin remodeling, telomere maintenance, and DNA recombination and repair cause GCRs in Saccharomyces cerevisiae. Recently, we used genome-wide screening to identify several genes the deletion of which increases GCRs in S. cerevisiae. Elg1, which was discovered during this screening, functions in DNA replication by participating in an alternative replication factor complex. Here we further characterize the GCR suppression mechanisms observed in the elg1? mutant strain in conjunction with the telomere maintenance role of Elg1. The elg1? mutation enhanced spontaneous DNA damage and resulted in GCR formation. However, DNA damage due to inactivation of Elg1 activates the intra-S checkpoints, which suppress further GCR formation. The intra-S checkpoints activated by the elg1? mutation also suppress GCR formation in strains defective in the DNA replication checkpoint. Lastly, the elg1? mutation increases telomere size independently of other previously known telomere maintenance proteins such as the telomerase inhibitor Pif1 or the telomere size regulator Rif1. The increase in telomere length caused by the elg1? mutation was suppressed by a defect in the DNA replication checkpoint, which suggests that DNA replication surveillance by Dpb11-Mec1/Tel1-Dun1 also has an important role in telomere length regulation.

The Ku autoantigen is a heterodimer of 70 (p70) and ∼80 kDa (p80) subunits that is the DNA-binding component of the DNA-dependent protein kinase (DNA-PK) complex involved in DNA repair and V(D)J recombination. Binding to DNA ends is critical to the function of DNA-PK, but how Ku interacts with DNA is not completely understood. To define the role of p70 and p80 and their dimerization in DNA binding, heterodimers were assembled by co-expressing the subunits using recombinant baculoviruses. Two p70 dimerization sites, amino acids 1–115 and 430–482, respectively, were identified. Binding of p70 to linear double-stranded DNA could be demonstrated by an immunoprecipitation assay, and required the C-terminal portion (amino acids 430–609), but not interaction with p80. The p70 mutants 1–600, 1–542, 1–115, and 430–600 did not bind DNA efficiently. However, DNA binding of 1–600, 1–542, and 1–115, but not 430–600, was restored by dimerization with p80, indicating that p70 has two DNA binding sites, each partially overlapping one of the dimerization sites. The C-terminal domain can bind DNA by itself, but the N-terminal domain requires dimerization with p80. These observations could be relevant to the multiple functional activities of Ku and explain controversies regarding the role of dimerization in DNA binding.

Background: Diabetic peripheral neuropathy (DPN), the most common microvascular complication of type-2 diabetes mellitus (T2DM), results in nontraumatic lower-limb amputations. When DPN is not detected early, disease progression is irreversible. Thus, biomarkers for diagnosing DPN are needed.Methods: We analyzed three data sets of T2DM DPN: two for mouse models (GSE70852 and GSE34889) and one for a human model (GSE24290). We found common differentially expressed genes (DEGs) in the two mouse data sets and validated them in the human data set. To identify the phenotypic function of the DEGs, we overexpressed them in zebrafish embryos. Clinical information and serum samples of T2DM patients with and without DPN were obtained from the Korea Biobank Network. To assess the plausibility of DEGs as biomarkers of DPN, we performed an enzyme-linked immunosorbent assay.Results: Among the DEGs, only NPYand SLPIwere validated in the human data set. As npyis conserved in zebrafish, its mRNA was injected into zebrafish embryos, and it was observed that the branches of the central nervous system became thicker and the number of dendritic branches increased. Baseline characteristics between T2DM patients with and without DPN did not differ, except for the sex ratio. The mean serum NPY level was higher in T2DM patients with DPN than in those without DPN (p= 0.0328), whereas serum SLPI levels did not differ (p= 0.9651).Conclusion: In the pathogenesis of DPN, NPYmay play a protective role in the peripheral nervous system and may be useful as a biomarker for detecting T2DM DPN.

Proliferating cell nuclear antigen (PCNA) is a DNA clamp essential for DNA replication. During DNA synthesis, PCNA is continuously loaded onto and unloaded from DNA. PCNA recruits various proteins to nascent DNA to facilitate chromosome duplication. Therefore, timely PCNA unloading is crucial for high-fidelity DNA replication. The ATAD5-RFC-like complex (ATAD5-RLC) unloads PCNA from replicated DNA. It is unclear how ATAD5-RLC activity is regulated to prevent premature PCNA unloading. Here, we find that BRD4, an acetyl-histone-binding chromatin reader, inhibits the PCNA-unloading activity of ATAD5-RLC. The BRD4 ET domain interacts with a region upstream of the ATAD5 PCNA-unloading domain. BRD4-ATAD5 binds to acetyl-histones in nascent chromatin. BRD4 release from chromatin correlates with PCNA unloading. Disruption of the interaction between BRD4 and acetyl-histones or between BRD4 and ATAD5 reduces the PCNA amount on chromatin. In contrast, the overexpression of BRD4 increases the amount of chromatin-bound PCNA. Thus, acetyl-histone-bound BRD4 fine-tunes PCNA unloading from nascent DNA.

ITGBL1 promotes chondrogenesis and protects joint cartilage from osteoarthritis by inhibiting integrin-extracellular matrix interaction.

Comment on: Parnas O, et al. Cell Cycle 2011; 10:2894-903

Comment on: Maradeo ME, et al. Cell Cycle 2010; 9:4370–8.

Genome instability is characteristic of cancer cells. Although it frequently occurs during carcinogenesis, the mechanism underlying genome instability is not clearly understood. Recent extensive genetic analyses from different organisms have begun to reveal mechanisms for the suppression of genome instability in general DNA metabolisms including DNA replication, recombination, DNA repair, and signal transduction. One DNA repair pathway called postreplication repair (also known as DNA damage bypass) has been highlighted for its role in genome stability. Central to DNA damage bypass, proliferating cell nuclear antigen (PCNA) directs different pathways through its mono- or polyubiquitination and sumoylation. In this review, we will discuss template switching dictated by the PCNA polyubiquitination and its roles in the suppression of genome instabilities.