iPSC lines that do not silence the expression of the ectopic reprogramming factors may display enhanced propensity to genomic instability

Here, we provide data suggesting that the absence of silencing of the ectopic reprogramming factors used to reprogram somatic cells to induced pluripotent stem cells （iPSCs） may predispose iPSCs to genomic instability. We encourage stem cell scientists to undertake an extensive characterization and standardization of much larger cohorts of iPSC lines in order to set up rigorous criteria to define safe and stable bonafide iPSCs.

Lower genomic stability of induced pluripotent stem cells reflects increased non-homologous end joining

Background:Induced pluripotent stem cells(iPSCs)and embryonic stem cells(ESCs)share many common features,including similar morphology,gene expression and in vitro differentiation profiles.However,genomic stability is much lower in iPSCs than in ESCs.In the current study,we examined whether changes in DNA damage repair in iPSCs are responsible for their greater tendency towards mutagenesis.Methods:Mouse iPSCs,ESCs and embryonic fibroblasts were exposed to ionizing radiation(4 Gy)to introduce dou-ble-strand DNA breaks.At 4 h later,fidelity of DNA damage repair was assessed using whole-genome re-sequencing.We also analyzed genomic stability in mice derived from iPSCs versus ESCs.Results:In comparison to ESCs and embryonic fibroblasts,iPSCs had lower DNA damage repair capacity,more somatic mutations and short indels after irradiation.iPSCs showed greater non-homologous end joining DNA repair and less homologous recombination DNA repair.Mice derived from iPSCs had lower DNA damage repair capacity than ESC-derived mice as well as C57 control mice.Conclusions:The relatively low genomic stability of iPSCs and their high rate of tumorigenesis in vivo appear to be due,at least in part,to low fidelity of DNA damage repair.

A Deleted Deletion Site in a New Vector Strain and Exceptional Genomic Stability of Plaque-Purified Modified Vaccinia Ankara(MVA)

Vectored vaccines based on highly attenuated modified vaccinia Ankara(MVA) are reported to be immunogenic, tolerant to pre-existing immunity, and able to accommodate and stably maintain very large transgenes. MVA is usually produced on primary chicken embryo fibroblasts, but production processes based on continuous cell lines emerge as increasingly robust and cost-effective alternatives. An isolate of a hitherto undescribed genotype was recovered by passage of a nonplaque-purified preparation of MVA in a continuous anatine suspension cell line(CR.pIX) in chemically defined medium.The novel isolate(MVA-CR19) replicated to higher infectious titers in the extracellular volume of suspension cultures and induced fewer syncytia in adherent cultures. We now extend previous studies with the investigation of the point mutations in structural genes of MVA-CR19 and describe an additional point mutation in a regulatory gene. We furthermore map and discuss an extensive rearrangement of the left telomer of MVA-CR19 that appears to have occurred by duplication of the right telomer. This event caused deletions and duplications of genes that may modulate immunologic properties of MVACR19 as a vaccine vector. Our characterizations also highlight the exceptional genetic stability of plaque-purified MVA:although the phenotype of MVA-CR19 appears to be advantageous for replication, we found that all genetic markers that differentiate wildtype and MVA-CR19 are stably maintained in passages of recombinant viruses based on either wildtype or MVA-CR.

Fertility,genome stability,and homozygosity in a diverse set of resynthesized rapeseed lines

Rapeseed(Brassica napus,AACC)was formed by hybridization between progenitor species Brassica rapa(AA)and Brassica oleracea(CC).As a result of a limited number of hybridization events between specific progenitor genotypes and strong breeding selection for oil quality traits,rapeseed has limited genetic diversity.The production of resynthesized B.napus lines via interspecific hybridization of the diploid progenitor species B.rapa and B.oleracea is one possible way to increase genetic variation in rapeseed.However,most resynthesized lines produced so far have been reported to be meiotically unstable and infertile,in contrast to established B.napus cultivars.This hinders both maintenance and use of this germplasm in breeding programs.We characterized a large set of 140 resynthesized lines produced by crosses between B.rapa and B.oleracea,as well as between B.rapa and wild C genome species(B.incana,B.hilarionis,B.montana,B.Bourgeaui,B.villosa and B.cretica)for purity(homozygosity),fertility,and genome stability.Self-pollinated seed set,seeds per ten pods as well as percentage pollen viability were used to estimate fertility.SNP genotyping was performed using the Illumina Infinium Brassica 60K array for 116 genotypes,with at least three individuals per line.Most of the material which had been advanced through multiple generations was no longer pure,with heterozygosity detected corresponding to unknown parental contributions via outcrossing.Fertility and genome stability were both genotypedependent.Most lines had high numbers of copy number variants(CNVs),indicative of meiotic instability,and high numbers of CNVs were significantly associated with reduced fertility.Eight putatively stable resynthesized B.napus lines were observed.Further investigation of these lines may reveal the mechanisms underlying this effect.Our results suggest that selection of stable resynthesized lines for breeding purposes is possible.

Expressions of genes related to genome stability and DNA repair in nasopharyngeal carcinoma clustering families

Objective： The aim of the study was to observe the expressions of genes related to genome stability and DNA repair in the members of nasopharyngeal carcinoma （NPC） clustedng families. Methods： In the Zhongshan City where there is highly incidence rate of NPC, we chose the members of the NPC clustering families as objects, and the patients of nasopharyngitis and NPC as the control group. We isolated the RNA from the nasopharyngeal tissue, and synthesized its cRNA, the genome stability and DNA repair genes chip technique, chemiluminescent detection and real-time fluorescence quantita- tive technique were used to examine the genome stability and DNA repair genes in the nasopharyngeal tissue. Results： More genome stability and DNA repair genes were up-regulated in the members of the NPC clustering families than the NPC patients, and the range of up-regulated was high, with the over up-regulated 100 times genes including TEP1, MSH4, PMS2LI. Fewer genome stability and DNA repair genes were down-regulated in the members of the NPC clustering families than the NPC patients, the ubiquitin genes almost were down-regulated, the results also could be confirmed by real-time fluorescence quantitative PCR. Conclusion： There are specially expression character of genome stability and DNA repair genes in the members of NPC clustering families.

Genomic analysis of Nypa fruticans elucidates its intertidal adaptations and early palm evolution

Nypa fruticans(Wurmb),a mangrove palm species with origins dating back to the Late Cretaceous period,is a unique species for investigating long-term adaptation strategies to intertidal environments and the early evolution of palms.Here,we present a chromosome-level genome sequence and assembly for N.fruticans.We integrated the genomes of N.fruticans and other palm family members for a comparative genomic analysis,which confirmed that the common ancestor of all palms experienced a whole-genome duplication event around 89 million years ago,shaping the distinctive characteristics observed in this clade.We also inferred a low mutation rate for the N.fruticans genome,which underwent strong purifying selection and evolved slowly,thus contributing to its stability over a long evolutionary period.Moreover,ancient duplicates were preferentially retained,with critical genes having experienced positive selection,enhancing waterlogging tolerance in N.fruticans.Furthermore,we discovered that the pseudogenization of Early Methionine-labelled 1(EM1)and EM6 in N.fruticans underly its crypto-vivipary characteristics,reflecting its intertidal adaptation.Our study provides valuable genomic insights into the evolutionary history,genome stability,and adaptive evolution of the mangrove palm.Our results also shed light on the long-term adaptation of this species and contribute to our understanding of the evolutionary dynamics in the palm family.

Dual role of lipids for genome stability and pluripotency facilitates full potency of mouse embryonic stem cells

While Mek1/2 and Gsk3βinhibition("2i")supports the maintenance of murine embryonic stem cells(EsCs)in a homogenous naive state,prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential.Additionally,2i fails to support derivation and culture of fully potent female ESCs.Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin(AlbuMAx)undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture.Mechanisticaily,lipids in AlbuMAx impact intracellular metabolism including nucleotide biosynthesis,lipid biogenesis,and TCA cycle intermediates,with enhanced expression of DNMT3s that prevent DNA hypomethylation.Lipids induce a formative-like pluripotent state through direct stimulation of Erk2 phosphorylation,which also alleviates X chromosome loss in female ESCs.Importantly,both male and female"all-ESc"mice can be generated from de novo derived ESCs using AlbuMAXbased media.Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.

Regulation of DNA double-strand break repair pathway choice

DNA double-strand breaks （DSBs） are critical lesions that can result in cell death or a wide variety of genetic alterations including largeor small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining （NHEJ） and homologous recombination （HR）, and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources including reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1（XRS2） complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit （DNA-PKcs）, and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.

Homologous recombination in DNA repair and DNA damage tolerance

Homologous recombination （HR） comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks （DSBs） and interstrand crosslinks （ICLs）. In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR： homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.

The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases

In their seminal publication describing the structure of the DNA double helix , Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that ＂It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.＂ Half a century later, we more fully appreciate what a huge challenge it is to replicate six billion nucleotides with the accuracy needed to stably maintain the human genome over many generations. This challenge is perhaps greater than was realized 50 years ago, because subsequent studies have revealed that the genome can be destabilized not only by environmental stresses that generate a large number and variety of potentially cytotoxic and mutagenic lesions in DNA but also by various sequence motifs of normal DNA that present challenges to replication. Towards a better understanding of the many determinants of genome stability, this chapter reviews the fidelity with which undamaged and damaged DNA is copied, with a focus on the eukaryotic B- and Y-family DNA polymerases, and considers how this fidelity is achieved.

Physiological,Biochemical and Molecular Responses of Barley Seedlings to Aluminum Stress

Barley(Hordeum vulgare L.)is one of the most Aluminum(Al)sensitive cereal species.In this study,the physiological,biochemical,and molecular response of barley seedlings to Al treatment was examined to gain insight into Al response and tolerance mechanisms.The results showed that superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)activity were inhibited to different degrees following Al exposure.The MDA content also significantly increased with increasing Al concentrations.SRAP results indicated significant differences between Al treatments and controls in terms of SRAP profile,and the genomic template stability(GTS)decreased with increasing Al concentration and duration.These integrative results help to elucidate the underlying mechanisms that the barley response to Al toxicity.

Analysis of 12 variants in the development of gastric and colorectal cancers

AIM To evaluate the relation between 12 polymorphisms and the development of gastric cancer(GC) and colorectal cancer(CRC).METHODS In this study,we included 125 individuals with GC diagnosis,66 individuals with CRC diagnosis and 475 cancer-free individuals. All participants resided in the North region of Brazil and authorized the use of their samples. The 12 polymorphisms(in CASP8,CYP2 E1,CYP19 A1,IL1 A,IL4,MDM2,NFKB1,PAR1,TP53,TYMS,UGT1 A1 and XRCC1 genes) were genotyped in a single PCR for each individual,followed by fragment analysis. To avoid misinterpretation due to population substructure,we applied a previously developed set of 61 ancestryinformative markers that can also be genotyped by multiplex PCR. The statistical analyses were performed in Structure v.2.3.4,R environment and SPSS v.20.RESULTS After statistical analyses with the control of confounding factors,such as genetic ancestry,three markers(rs79071878 in IL4,rs3730485 in MDM2 and rs28362491 in NFKB1) were positively associated with the development of GC. One of these markers(rs28362491) and the marker in the UGT1 A1 gene(rs8175347) were positively associated with the development of CRC. Therefore,we investigated whether the joint presence of the deleterious alleles of each marker could affect the development of cancer and we obtained positive results in all analyses. Carriers of the combination of alleles RP1 + DEL(rs79071878 and rs28361491,respectively) are at 10-times greater risk of developing GC than carriers of other combinations. Similarly,carriers of the combination of DEL + RARE(rs283628 and rs8175347) are at about 12-times greater risk of developing CRC than carriers of other combinations.CONCLUSION These findings are important for the comprehension of gastric and CRC development,particularly in highly admixed populations,such as the Brazilian population.

The genomics of desmoplastic small round cell tumor reveals the deregulation of genes related to DNA damage response, epithelial-mesenchymal transition, and immune response

Background:Desmoplastic small round cell tumor(DSRCT)is a rare,aggressive,and poorly investigated simple sarcoma with a low frequency of genetic deregulation other than an Ewing sarcoma RNA binding protein 1(EWSR1)-Wilm’s tumor suppressor(WT1)translocation.We used whole-exome sequencing to interrogate six consecutive pretreated DSRCTs whose gene expression was previously investigated.Methods:DNA libraries were prepared from formalin-fixed,paraffin-embedded archival tissue specimens following the Agilent SureSelectXT2 target enrichment protocol and sequenced on Illumina NextSeq 500.Raw sequence data were aligned to the reference genome with Burrows-Wheeler Aligner algorithm.Somatic mutations and copy number alterations(CNAs)were identified using MuTect2 and EXCAVATOR2,respectively.Biological functions associated with altered genes were investigated through Ingenuity Pathway Analysis(IPA)software.Results:A total of 137 unique somatic mutations were identified:133 mutated genes were case-specific,and 2 were mutated in two cases but in different positions.Among the 135 mutated genes,27%were related to two biological categories:DNA damage-response(DDR)network that was also identified through IPA and mesenchymal-epithelial reverse transition(MErT)/epithelial-mesenchymal transition(EMT)already demonstrated to be relevant in DSRCT.The mutated genes in the DDR network were involved in various steps of transcription and particularly affected pre-mRNA.Half of these genes encoded RNA-binding proteins or DNA/RNA-binding proteins,which were recently rec-ognized as a new class of DDR players.CNAs in genes/gene families,involved in MErT/EMT and DDR,were recurrent across patients and mostly segregated in the MErT/EMT category.In addition,recurrent gains of regions in chromosome 1 involving many MErT/EMT gene families and loss of one arm or the entire chromosome 6 affecting relevant immune-regulatory genes were recorded.Conclusions:The emerging picture is an extreme inter-tumor heterogeneity,characterized by the concurrent deregulation of the DDR and MErT/EMT dynamic and plastic programs that could favour genomic instability and explain the refractory DSRCT profile.

RNase P:Beyond Precursor tRNA Processing

Ribonuclease P(RNase P)was first described in the 1970’s as an endoribonuclease acting in the maturation of precursor transfer RNAs(tRNAs).More recent studies,however,have uncovered non-canonical roles for RNase P and its components.Here,we review the recent progress of its involvement in chromatin assembly,DNA damage response,and maintenance of genome stability with implications in tumorigenesis.The possibility of RNase P as a therapeutic target in cancer is also discussed.

Special Issue on "Genome Stability"

We are very pleased to announce a special issue, to be published in June, 2016, on ＂Genome Stability＂ in the journal Genomics, Proteomies ＆ Bioinformaties （GPB）. This special issue aims to provide a platform for specialists or experts in the field to report their results and share their opinions on the topic. We are inviting you to submit high-quality papers to this special issue. The guest editors for this special issue are Dr. Zhao-Qi Wang （The Leibniz Institute on Aging - Fritz Lipmann Institute （FLI）, Germany）, Dr. Xingzbi Xu （Capital Normal University, China）, and Dr. Daochun Kong （Peking University, China）.

CHK2 kinase in the DNA damage response and beyond

The serine/threonine kinase CHK2 is a keycomponent of the DNA damage response. In human cells, following genotoxic stress, CHK2 is activated and phosphorylates 〉20 proteins to induce the appropriate cellular response, which, depending on the extent of damage, the cell type, and other factors, could be cell cycle checkpoint activation, induction of apoptosis or senescence, DNA repair, or tolerance of the damage. Recently, CHK2 has also been found to have cellular functions independent of the presence of nuclear DNA lesions. In par- ticular, CHK2 participates in several molecular processes involved in DNA structure modification and cell cycle progression. In this review, we discuss the activity of CHK2 in response to DNA damage and in the maintenance of the biological functions in unstressed cells. These activities are also considered in relation to a possible role of CHK2 in tumorigenesis and, as a consequence, as a target of cancer therapy.

Abscisic Acid Suppresses the Highly Occurred Somatic Homologous Recombination in Arabidopsis rfc1 Mutant

The phytohormone abscisic acid （ABA） regulates many aspects of division. Previous study indicates that ABA treatment increases DNA plant growth, including seed germination, root growth and cell damage and somatic homologous recombination （HR） in Arabi- dopsis abo4/pol ε （aba overly-sensitive 4/DNA polymerase ε） mutants. DNA replication factor C （RFC） complex is required for loading PCNA （Proliferating Cell Nuclear Antigen） during DNA replication. The defect in RFC1, the largest subunit of RFC, causes the high HR and DNA damage sensitivity in Arabidopsis. Here we found that like pol e/abo4, rfcl is sensitive to ABA in both ABA-inhibiting seed germination and root growth. However, ABA treatment greatly reduces HR and also reduces the expression of the DNA-damaged marker genes in ,rfcl. These results suggest that RFCI plays critical roles in ABA-mediated HR in Arabidopsis.

Telomerase as a “stemness” enzyme

Pluripotent or multipotent stem cells are involved in development and tissue homeostasis;they have the ability to self-renew and differentiate into various types of functional cells.To maintain these properties,stem cells must undergo sustained or unlimited proliferation that requires the stabilization of telomeres,which are essential for chromosome end protection.Telomerase,an RNA-dependent DNA polymerase,synthesizes telomeric DNA.Through the lengthening of telomeres the lifespans of cells are extended,or indefinite proliferation is conferred;this is intimately associated with stem cell phenotype.This review highlights our current understanding of telomerase as a"stemness"enzyme and discusses the underlying implications.

Genetic and Epigenetic Effects of Plant-Pathogen Interactions： An Evolutionary Perspective

Recent reports suggest that exposure to stress is capable of influencing the frequency and pattern of inherited changes in various parts of the genome. In this review, we will discuss the influence of viral pathogens on somatic and meiotic genome stability of Nicotiana tabacum and Arabidopsis thaliana. Plants infected with a compatible pathogen generate a systemic recombination signal that precedes the spread of pathogens and results in changes in the somatic and meiotic recombination frequency. The progeny of infected plants exhibit changes in global and locusspecific DNA methylation patterns, genomic rearrangements at transgenic reporter loci and resistance gene-like-loci, and even tolerance to pathogen infection and abiotic stress. Here, we will discuss the contribution of environmental stresses to genome evolution and will focus on the role of heritable epigenetic changes in response to pathogen infection.

Prevention of DNA re-replication in eukaryotic cells

DNA replication is a highly regulated process involving a number of licensing and replication factors that function in a carefully orchestrated manner to faithfully replicate DNA during every cell cycle.Loss of proper licensing control leads to deregulated DNA replication including DNA re-replication,which can cause genome instability and tumorigenesis.Eukaryotic organisms have established several conserved mechanisms to prevent DNA re-replication and to counteract its potentially harmful effects.These mechanisms include tightly controlled regulation of licensing factors and activation of cell cycle and DNA damage checkpoints.Deregulated licensing control and its associated compromised checkpoints have both been observed in tumor cells,indicating that proper functioning of these pathways is essential for maintaining genome stability.In this review,we discuss the regulatory mechanisms of licensing control,the deleterious consequences when both licensing and checkpoints are compromised,and present possible mechanisms to prevent re-replication in order to maintain genome stability.