H2B oncohistones cause homologous recombination defect and genomic instability through reducing H2B monoubiquitination in Schizosaccharomyces pombe.

Canonical oncohistones are histone H3 mutations in the N-terminal tail associated with tumors and affect gene expression by altering H3 post-translational modifications (PTMs) and the epigenetic landscape. Noncanonical oncohistone mutations occur in both tails and globular domains of all 4 core histones and alter gene expression by perturbing chromatin remodeling. However, the effects and mechanisms of noncanonical oncohistones remain largely unknown. Here we characterized 16 noncanonical H2B oncohistones in the fission yeast Schizosaccharomyces pombe. We found that 7 of them exhibited temperature sensitivities and 11 exhibited genotoxic sensitivities. A detailed study of 2 of these onco-mutants H2BG52D and H2BP102L revealed that they were defective in homologous recombination (HR) repair with compromised histone eviction and Rad51 recruitment. Interestingly, their genotoxic sensitivities and HR defects were rescued by inactivation of the H2BK119 deubiquitination function of Ubp8 in the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. The levels of H2BK119 monoubiquitination (H2Bub) in the H2BG52D and H2BP102L mutants are reduced in global and local DNA break sites presumably due to enhanced recruitment of Ubp8 onto nucleosomes, and are recovered upon loss of H2B deubiquitination function of the SAGA complex. Moreover, H2BG52D and H2BP102L heterozygotes exhibit genotoxic sensitivities and reduced H2Bub in cis. We therefore conclude that H2BG52D and H2BP102L oncohistones affect HR repair and genome stability via reduction of H2Bub and propose that other noncanonical oncohistones may also affect histone PTMs to cause diseases.

The deletion of ppr2 interferes iron sensing and leads to oxidative stress response in Schizosaccharomyces pombe.

Pentatricopeptide repeat proteins are involved in mitochondrial both transcriptional and posttranscriptional regulation. Schizosaccharomyces pombe Ppr2 is a general mitochondrial translation factor that plays a critical role in the synthesis of all mitochondrial DNA-encoded oxidative phosphorylation subunits, which are essential for mitochondrial respiration. Our previous analysis showed that ppr2 deletion resulted in increased expression of iron uptake genes and caused ferroptosis-like cell death in S. pombe. In the present work, we showed that deletion of ppr2 reduced viability on glycerol- and galactose-containing media.Php4 is a transcription repressor that regulates iron homeostasis in fission yeast. We found that in the ppr2 deletion strain, Php4 was constitutively active and accumulated in the nucleus in the stationary phase. We also found that deletion of ppr2 decreased the ferroptosis-related protein Gpx1 in the mitochondria. Overexpression of Gpx1 improves the viability of Δppr2 cells. We showed that the deletion of ppr2 increased the production of ROS, downregulated heme synthesis and iron-sulfur cluster proteins, and induced stress proteins. Finally, we observed the nuclear accumulation of Pap1-GFP and Sty1-GFP, suggesting that Sty1 and Pap1 in response to cellular stress in the ppr2 deletion strain. These results suggest thatppr2 deletion may cause mitochondrial dysfunction, which is likely to lead to iron-sensing defect and iron starvation response, resulting in perturbation of iron homeostasis and increased hydroxyl radical production. The increased hydroxyl radical production triggers cellular responses in theppr2 deletion strain.

Mrz1, a Novel Mitochondrial Outer Membrane RING Finger Protein, is Degraded Through the Ubiquitin-Proteasome Pathway in Schizosaccharomyces pombe.

The RING (Really Interesting New Gene) finger proteins are a large diverse group of Zinc finger proteins. Many determined RING finger proteins are ubiquitin-protein E3 ligases and RING E3s are the most abundant type of ubiquitin ligase. RING finger and RING finger E3s have been discovered in many organisms where they play various functions, including DNA repair, ubiquitination and mitochondrial protein quality control. In this study, we identified a novel mitochondrial protein (SPBC16G5.03) with predicted RING finger domain within an N-terminal 21-60 amino acids and named it Mrz1 (mitochondrial RING finger protein). Our results showed that Mrz1 is localized in the mitochondrial outer membrane. Deletion of mrz1 did not affect cell growth in an unstressed state, but increases sensitivity to selenite. We showed that Mrz1 was degraded during the stationary phase and blocked by addition proteasome inhibitor MG132. We further showed that the E2 enzyme Ubc13 was identified among 8 candidate proteins as the ubiquitin-conjugating enzyme in this system. These data suggested that the Mrz1 was degraded likely through the ubiquitin-proteasome system.

Clinical implications and biological features of a novel postoperative recurrent HCC classification: A multi-centre study.

BACKGROUND & AIMS: The multiplicity of hepatocellular carcinoma (HCC) recurrence patterns is the most important determinant of patients' postsurgical survival. A systematic HCC recurrence classification is needed to help prevent and treat postoperative HCC recurrence in the era of precision medicine. METHODS: A total of 1319 patients with recurrent HCC from four hospitals were enrolled and divided into a development cohort (n = 916), internal validation cohort (n = 225) and external validation cohort (n = 178). A comprehensive study of patients' clinicopathological factors and biological features was conducted. RESULTS: Four subtypes of recurrence were identified, which integrated recurrence features, survival, effects on systemic and liver function and potential therapeutics after recurrence: type I (solitary-intrahepatic oligorecurrence); type II (multi-intrahepatic oligorecurrence); type III (progression recurrence) and type IV (hyper-progression recurrence). Type III~IV recurrence indicated exceptionally poor prognosis. Subsequently, two nomogram models were established for type III~IV recurrence prediction, and both demonstrated excellent predictive performance and applicability of pre and postoperative strategy formulation. Multiple biological analyses revealed that HCC cases with type III~IV recurrence were characterized by enrichment in p53 mutations, CCND1 amplification, high proliferation/metastasis potential, inactive metabolism and immune exhaustion features. Over-expression of high mobility group protein 2 (HMGA2) enhanced the highly malignant behaviour of HCC through multiple molecular pathways, making it a potential prognostic predictor and therapeutic target. CONCLUSIONS: This 'recurrent HCC classification' has important potential value in identifying patients with surgical benefit, predicting postsurgical survival and guiding treatment strategies. Multidimensional biological insights also increased knowledge of factors associated with HCC recurrence.

Global view of dynamic expression and precise mapping of mitochondrial tRNAs-derived fragments during stressed conditions in S. pombe.

In this study, we provide a global view of population and processing of mitochondrial tRNAs-derived fragments (mt-tRFs) in fission yeast Schizosaccharomyces pombe. Here, mt-tRFs of 15-30 nucleotides were retrieved from S. pombe small RNA libraries obtained from unstressed, stress, and during stationary phase conditions. We demonstrate that production of these fragments increase during heat stress and stationary phase conditions in S. pombe, especially (most notably) in stationary phase. Analysis of data also reveals depending on the tRNA, either 5'-mt-tRF or 3'-mt-tRF was found and major mt-tRNA processing sites have been precisely identified. Furthermore, RNA-seq reveals that inactivation of trz2 encoding S. pombe mitochondrial tRNase ZL globally impairs mt-tRF processing. Finally, our result showed mt-tRFs were predicted to target mitochondrial genome mapping mtDNA-encoded protein gene. These observations suggest that mitochondrial tRFs may play an important regulatory role in response to stress and development.

S100P as a novel biomarker of microvascular invasion and portal vein tumor thrombus in hepatocellular carcinoma.

BACKGROUND: Portal vein tumor thrombus (PVTT) and microvascular invasion (MVI) are types of intrahepatic vascular metastasis of hepatocellular carcinoma (HCC) and are highly correlated with poor prognosis. However, the underlying biomarkers of PVTT and MVI are unclear. METHODS: We identified a PVTT/MVI-associated gene S100P by cDNA microarray analysis, and assess the potential value of serum S100P measurement in the differential diagnosis of HCC and prediction of MVI status with large retrospective and perspective cohort studies. RESULTS: The mRNA and protein of S100P was increased in HCCs with PVTT or MVI. High S100P immunostaining in tumors was correlated with inferior tumor-free survival. Serum S100P values discriminated patients with HCCs from those with benign liver tumors, and it showed predictive potential of MVI status in both retrospective and perspective cohorts. S100P may regulate HCC tumorigenicity and invasive ability; S100P also was associated with up-regulation of CD44, which may mediate HCC cell adhesion to form PVTT/MVI. CONCLUSIONS: Serum S100P may be a novel differential diagnostic marker for HCC and a potential predictor of MVI status pre-surgery for HCC patients. S100P overexpression in HCC is highly correlated with the formation of PVTT and MVI, which may make S100P as a potential therapeutic target for HCC metastasis.

dnm1 deletion blocks mitochondrial fragmentation in Δfzo1 cells.

Mitochondrial division and fusion play critical roles in maintaining functional mitochondria. Fzo1 is an outer mitochondrial membrane GTPase that played an essential role in mitochondrial fusion in budding yeast Saccharomyces cerevisiae. Here, we report the characterization of the Schizosaccharomyces pombe homologue of S. cerevisiae Fzo1p, Fzo1. Disruption of the fzo1 gene in S. pombe results in a fragmented mitochondrial morphology and a dramatically reduced growth on glycerol medium phenotype, indicating that deletion of fzo1 compromises respiratory function. Fluorescence microscopy shows that Fzo1p is located in the mitochondria. Overexpressing Fzo1 from a heterologous promoter induces mitochondrial aggregation. We also find that dnm1 mutations could both block mitochondrial fragmentation and rescue respiration growth defect in Δfzo1 single mutant cells. Our results proposed that a genetic interaction between fzo1 and a balance between division- and fusion-controlled mitochondrial shape and function in S. pombe. This study represents the first report of Fzo1 mediator of mitochondrial fusion in S. pombe.

CpATG8, a Homolog of Yeast Autophagy Protein ATG8, Is Required for Pathogenesis and Hypovirus Accumulation in the Chest Blight Fungus.

Autophagy is a degradation system in the cell, involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. Autophagy is induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. In this study, by using electron microscopy, we observed that hypovirus CHV1-EP713 infection of Cryphonectria parasitica, the causative agent of chestnut blight disease, caused proliferation of autophagic-like vesicles. This phenomenon could be mimicked by treating the wild-type strain of the fungus EP155 with the autophagy induction drug rapamycin. Some of the hypovirulence-associated traits, including reduced pigmentation and conidiation, were also observed in the rapamycin-treated EP155. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) revealed that genes involved in autophagy were up-regulated in expression. Deletion of cpatg8, a gene encoding a homolog of ATG8 in Saccharomyces cerevisiae, resulted in attenuation of virulence and reduction in sporulation, as well as accumulation of the double-stranded viral RNA. Furthermore, virus-encoded p29 protein was found to co-localize with CpATG8, implying that the viral protein may interfere with the function of CpATG8. Taken together, these findings show that cpatg8 can be regulated by the hypovirus and is required for virulence and development of the fungus and accumulation of viral dsRNA in chestnut blight fungus.

Overexpression of Schizosaccharomyces pombe tRNA 3'-end processing enzyme Trz2 leads to an increased cellular iron level and apoptotic cell death.

The fission yeast Schizosaccharomyces pombe has two tRNase ZL genes (trz1 and trz2) involved in nuclear and mitochondrial tRNA 3'-end processing, respectively. Overexpression of trz2 but not trz1 is toxic to cells. In the present work, we showed that trz2 overexpression led to apoptotic cell death, as revealed by DAPI and Annexin V-FITC staining. Overexpression of trz2 also caused a loss of mitochondrial membrane potential and an increased reactive oxygen species (ROS) formation. These effects required mitochondrial localization but not its catalytic activity. RNA sequencing (RNA-seq) analysis revealed increased expression levels of genes involved in iron uptake and/or iron homeostasis, suggesting an elevated level of intracellular iron in the trz2-overexpressing cells. Indeed, we showed that overexpressing trz2 increased the level of intracellular iron by ∼2-fold. We further showed that the iron chelator, bathophenanthroline disulfonic acid (BPS) nearly restored the viability of trz2-overexpression cells and reduced ROS levels in the cells. These results suggest that trz2 overexpression may cause mitochondrial dysfunction, which is likely to lead to perturbation of iron homeostasis, ROS accumulation and induction of apoptotic cell death in S. pombe.

Circulating Tumor Cells Undergoing EMT Provide a Metric for Diagnosis and Prognosis of Patients with Hepatocellular Carcinoma.

To clarify the significance of circulating tumor cells (CTC) undergoing epithelial-mesenchymal transition (EMT) in patients with hepatocellular carcinoma (HCC), we used an advanced CanPatrol CTC-enrichment technique and in situ hybridization to enrich and classify CTC from blood samples. One hundred and one of 112 (90.18%) patients with HCC were CTC positive, even with early-stage disease. CTCs were also detected in 2 of 12 patients with hepatitis B virus (HBV), both of whom had small HCC tumors detected within 5 months. CTC count ≥16 and mesenchymal-CTC (M-CTC) percentage ≥2% prior to resection were significantly associated with early recurrence, multi-intrahepatic recurrence, and lung metastasis. Postoperative CTC monitoring in 10 patients found that most had an increased CTC count and M-CTC percentage before clinically detectable recurrence nodules appeared. Analysis of HCC with high CTC count and high M-CTC percentage identified 67 differentially expressed cancer-related genes involved in cancer-related biological pathways (e.g., cell adhesion and migration, tumor angiogenesis, and apoptosis). One of the identified genes, BCAT1, was significantly upregulated, and knockdown in Hepg2, Hep3B, and Huh7 cells reduced cell proliferation, migration, and invasion while promoting apoptosis. A concomitant increase in epithelial marker expression (EpCAM and E-cadherin) and reduced mesenchymal marker expression (vimentin and Twist) suggest that BCAT1 may trigger the EMT process. Overall, CTCs were highly correlated with HCC characteristics, representing a novel marker for early diagnosis and a prognostic factor for early recurrence. BCAT1 overexpression may induce CTC release by triggering EMT and may be an important biomarker of HCC metastasis.Significance: In liver cancer, CTC examination may represent an important "liquid biopsy" tool to detect both early disease and recurrent or metastatic disease, providing cues for early intervention or adjuvant therapy. Cancer Res; 78(16); 4731-44. ©2018 AACR.

The S. pombe mitochondrial transcriptome.

Mitochondrial gene expression is largely controlled through post-transcriptional processes including mitochondrial RNA (mt-RNA) processing, modification, decay, and quality control. Defective mitochondrial gene expression results in mitochondrial oxidative phosphorylation (OXPHOS) deficiency and has been implicated in human disease. To fully understand mitochondrial transcription and RNA processing, we performed RNA-seq analyses of mt-RNAs from the fission yeast Schizosaccharomyces pombe RNA-seq analyses show that the abundance of mt-RNAs vary greatly. Analysis of data also reveals mt-RNA processing sites including an unusual RNA cleavage event by mitochondrial tRNA (mt-tRNA) 5'-end processing enzyme RNase P. Additionally, this analysis reveals previously unknown mitochondrial transcripts including the rnpB-derived fragment, mitochondrial small RNAs (mitosRNAs) such as mt-tRNA-derived fragments (mt-tRFs) and mt-tRNA halves, and mt-tRNAs marked with 3'-CCACCA/CCACC in S. pombe Finally, RNA-seq reveals that inactivation of trz2 encoding S. pombe mitochondrial tRNA 3'-end processing enzyme globally impairs mt-tRNA 3'-end processing, inhibits mt-mRNA 5'-end processing, and causes accumulation of unprocessed transcripts, demonstrating the feasibility of using RNA-seq to examine the protein known or predicted to be involved in mt-RNA processing in S. pombe Our work uncovers the complexity of a fungal mitochondrial transcriptome and provides a framework for future studies of mitochondrial gene expression using S. pombe as a model system.

Complexity of roles and regulation of the PMK1-MAPK pathway in mycelium development, conidiation and appressorium formation in Magnaporthe oryzae.

MST50, MST11, MST7, PMK1 and GAS1/GAS2 genes are the important components in the PMK1-MAPK signal transduction pathway in fungi. Mutants with deletion of these five genes of Magnaporthe oryzae, a pathogen of the rice blast, were constructed. A cDNA array containing 4108 unique genes of M. oryzae was developed and used to analyze the gene expression profiles of these mutants against the wild type to dissect the gene expression regulation networks responsible for conidiation and appressorium formation. With this approach, differentially regulated genes by these five components were identified. The vast majority of the regulated genes were mutant-specific, while only a small proportion were in common for all of the mutants, suggesting that each of these genes has its own regulon. Functional groups and expression patterns of the regulated genes showed that (1) gene members in the PMK1-MAPK pathway are associated with multiple signaling pathways; (2) the regulation of PMK1-mediated signaling pathways is very complex and likely involved in other signaling networks; (3) glucose metabolism and signals are required in mycelium development; and (4) appressorium formation likely shares the mechanisms responsible for sexual conjugation and meiosis, which is affected by carbohydrate metabolism.

Comparative vesicle proteomics reveals selective regulation of protein expression in chestnut blight fungus by a hypovirus.

The chestnut blight fungus (Cryphonectria parasitica) and hypovirus constitute a model system to study fungal pathogenesis and mycovirus-host interaction. Knowledge in this field has been gained largely from investigations at gene transcription level so far. Here we report a systematic analysis of the vesicle proteins of the host fungus with/without hypovirus infection. Thirty-three differentially expressed protein spots were identified in the purified vesicle protein samples by two-dimensional electrophoresis and mass spectrometry. Down-regulated proteins were mostly cargo proteins involved in primary metabolism and energy generation and up-regulated proteins were mostly vesicle associated proteins and ABC transporter. A virus-encoded protein p48 was found to have four forms with different molecular mass in vesicles from the virus-infected strain. While a few of the randomly selected differentially expressed proteins were in accordance with their transcription profiles, majority were not in agreement with their mRNA accumulation patterns, suggesting that an extensive post-transcriptional regulation may have occurred in the host fungus upon a hypovirus infection.

Functional analysis of an S-adenosylhomocysteine hydrolase homolog of chestnut blight fungus.

S-adenosylhomocysteine (SAH), formed after donation of the methyl group of S-adenosylmethionine (SAM) to a methyl acceptor, is reversibly hydrolyzed to adenosine (ADO) and homocysteine (HCY) by S-adenosylhomocysteine hydrolase (SAHH). In chestnut blight fungus (Cryphonectria parasitica), sahh, a hypovirus-regulated gene that encodes a deduced SAHH protein was shown to have an SAHH enzymatic activity in vitro. Deletion of sahh resulted in the increased accumulation of intracellular SAH and SAM but decreased ADO, and a remarkably increased accumulation of transcripts that encode adenosine kinase, methionine adenosyltransferase, and an O-methyltransferase, key components of the methylation pathway. The Δsahh knockout mutants showed a phenotype of slower growth rate, fewer aerial hyphae, loss of orange pigment, absence of asexual fruiting bodies and conidia, and a significant reduction in virulence. Deletion of sahh significantly reduced the accumulation level of transcripts of the cyp1 that encodes cyclophilin A as well as genes of the heterotrimeric G-protein signaling pathways including cpga1, cpgb1, and cpgc1 and ste12, a target activated by the MAP kinase cascade. Taken together, we demonstrated that SAHH is required for virulence and multiple traits of phenotype in C. parasitica, by regulation of the expression of genes involved in key process of the cell.

CYP1, a hypovirus-regulated cyclophilin, is required for virulence in the chestnut blight fungus.

Cyclophilins are peptidyl-prolyl cis-trans isomerases that are highly conserved throughout eukaryotes and are the cellular target of the immunosuppressive drug cyclosporin A (CsA). We cloned cyp1, a cyclophilin A-encoding gene in the phytopathogenic fungus Cryphonectria parasitica, and showed that this gene was downregulated following infection by a virulence-attenuating hypovirus. The function of cyp1 was further investigated by construction of a cyp1 deletion mutant. Although the wild-type C. parasitica strain EP155 was sensitive to CsA, the Δcyp1 strain was highly tolerant to CsA, indicating that CYP1 was the target of CsA. Deletion of cyp1 resulted in reduced virulence when inoculated to chestnut stems. Transcriptional analysis revealed that deletion of cyp1 also reduced transcript levels for genes encoding key components of the heterotrimeric guanosine triphosphate-binding protein signalling pathway that are essential for sensing environmental cues and are involved in C. parasitica development and virulence.

Large-scale expressed sequence tag analysis for the chestnut blight fungus Cryphonectria parasitica.

Cryphonectria parasitica is the causal fungal agent responsible for the chestnut blight disease. We report the generation of 14,263 expressed sequence tags representing 6318 unisequences for the fungus. Functional annotation of these unisequences revealed different gene expression patterns for wild-type and hypovirus-infected cultures at the sporulation stage and allowed the reconstruction of key C. parasitica signal transduction pathways conserved in the Sorbidaryomycetes. A list of homologous genes implicated in pathogenicity, sporulation, and virus replication in other fungi were also identified. A large-scale gene comparison indicated that C. parasitica is most closely related to the plant pathogen Fusarium graminearum but more closely related to the non-pathogen Neurospora crassa than to the rice pathogen Magnaporthe grisea. This transcriptional information lays a new and solid ground for further investigation of both the biology of the fungus and interaction between a hypovirus and the host fungus.

Deletion of the cpku80 gene in the chestnut blight fungus, Cryphonectria parasitica, enhances gene disruption efficiency.

The chestnut blight fungus, Cryphonectria parasitica, and associated virulence-attenuating hypoviruses have emerged as an important model system for studying molecular mechanisms underlying fungal-plant pathogenic interactions. As more gene sequence information becomes available as a result of C. parasitica express sequence tags (ESTs) and ongoing whole genome sequencing projects, the development of an efficient gene disruption system has become an urgent need for functional genomics studies of this important forestry pathogen. Here, we report the cloning of the C. parasitica gene cpku80 that encodes a key component of the nonhomologous end joining DNA repair pathway and the construction of a corresponding deletion mutant strain. The cpku80 mutant was indistinguishable from the parental wild-type strain EP155 in colony morphology, ability to support hypovirus replication, conidiation and virulence. As predicted, the Deltacpku80 strain did exhibit an increased sensitivity to the mutagen methyl methanesulfonate. A test with three selected genes resulted in a gene disruption efficiency of about 80% for the Deltacpku80 strain, a significant increase over the 2-5% levels of homologous recombination generally observed for the wild-type strain EP155. This efficient homologous recombination system provides a powerful tool for large-scale analysis of gene functions in C. parasitica.