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.

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.

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.

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）.

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.

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.

cGAS guards against chromosome endto-end fusions during mitosis and facilitates replicative senescence

As a sensor of cytosolic DNA, the role of cyclic GMP-AMP synthase (cGAS) in innate immune response is well established, yet how its functions in different biological conditions remain to be elucidated. Here, we identify cGAS as an essential regulator in inhibiting mitotic DNA double-strand break (DSB) repair and protecting short telomeres from end-to-end fusion independent of the canonical cGAS-STING pathway. cGAS associates with telomeric/subtelomeric DNA during mitosis when TRF1/TRF2/POT1 are deficient on telomeres. Depletion of cGAS leads to mitotic chromosome end-to-end fusions predominantly occurring between short telomeres. Mechanistically, cGAS interacts with CDK1 and positions them to chromosome ends. Thus, CDK1 inhibits mitotic non-homologous end joining (NHEJ) by blocking the recruitment of RNF8. cGAS-deficient human primary cells are defective in entering replicative senescence and display chromosome end-to-end fusions, genome instability and prolonged growth arrest. Altogether, cGAS safeguards genome stability by controlling mitotic DSB repair to inhibit mitotic chromosome end-to-end fusions, thus facilitating replicative senescence.

Silicon-mediated genotoxic alterations in Brassica juncea under arsenic stress: A comparative study of biochemical and molecular markers

Arsenic(As),one of the most harmful toxicant at the global level,severely affects plant metabolism when taken up.Interestingly,the presence of silicon(Si)as a fertilizer in As-contaminated soil is an effective strategy to decrease As accumulation in plants.Brassica juncea(var.Varuna)were grown hydroponically to investigate the role of Si at biochemical and molecular levels under arsenite(As^3+)stress.Seedlings of B.juncea were exposed to As^3+,Si,and a combination of both elements.Our data demonstrated that seedlings exposed to As^3+showed an inhibition in shoot length,chlorophyll,carotenoid,and protein,while co-application of Si improved these growth parameters.Silicon supplementation reduced As accumulation in shoot.Increase/decrease was observed in stress-related parameters(cysteine and proline),antioxidant enzymes(superoxide dismutase,ascorbate peroxidase,and catalase),and oxidative stress markers(malondialdehyde and H2O2),which were improved upon co-application of Si as compared to As^3+alone treatment.Random amplified polymorphic DNA(RAPD)is a suitable biomarker assay for plants for assessing the genotoxicity.Seven RAPD primers produced a total of 39 and 48 bands in the leaves of the untreated and treated seedlings,respectively.The RAPD band-profiles and genomic template stability were consistent with other growth and physiological parameters.In conclusion,the genotoxic alterations along with the biochemical parameters indicate that the exposure to Si mitigates As^3+-induced oxidative stress by improving the stress-related parameters and antioxidant system in B.juncea.

DNA End Resection:Facts and Mechanisms

DNA double-strand breaks（DSBs）,which arise following exposure to a number of endogenous and exogenous agents,can be repaired by either the homologous recombination（HR）or non-homologous end-joining（NHEJ） pathways in eukaryotic cells.A vital step in HR repair is DNA end resection,which generates a long 30single-stranded DNA（ss DNA） tail that can invade the homologous DNA strand.The generation of 30 ss DNA is not only essential for HR repair,but also promotes activation of the ataxia telangiectasia and Rad3-related protein（ATR）.Multiple factors,including the MRN/X complex,C-terminal-binding protein interacting protein（Ct IP）/Sae2,exonuclease 1（EXO1）,Bloom syndrome protein（BLM）/Sgs1,DNA2 nuclease/helicase,and several chromatin remodelers,cooperate to complete the process of end resection.Here we review the basic machinery involved in DNA end resection in eukaryotic cells.

R-loop:The new genome regulatory element in plants

An R-loop is a three-stranded chromatin structure that consists of a displaced single strand of DNA and an RNA:DNA hybrid duplex,which was thought to be a rare by-product of transcription.However,recent genome-wide data have shown that R-loops are widespread and pervasive in a variety of genomes,and a growing body of experimental evidence indicates that R-loops have both beneficial and harmful effects on an organism.To maximize benefit and avoid harm,organisms have evolved several means by which they tightly regulate R-loop levels.Here,we summarize our current understanding of the biogenesis and effects of R-loops,the mechanisms that regulate them,and methods of R-loop profiling,reviewing recent research advances on R-loops in plants.Furthermore,we provide perspectives on future research directions for R-loop biology in plants,which might lead to a more comprehensive understanding of R-loop functions in plant genome regulation and contribute to future agricultural improvements.

RIP140 regulates POLK gene expression and the response to alkylating drugs in colon cancer cells

Aim:The transcription factor RIP140(receptor interacting protein of 140 kDa)is involved in intestinal tumorigenesis.It plays a role in the control of microsatellite instability(MSI),through the regulation of MSH2 and MSH6 gene expression.The aim of this study was to explore its effect on the expression of POLK,the gene encoding the specialized translesion synthesis(TLS)DNA polymeraseκknown to perform accurate DNA synthesis at microsatellites.Methods:Different mouse models and engineered human colorectal cancer(CRC)cell lines were used to analyze by RT-qPCR,while Western blotting and luciferase assays were used to elucidate the role of RIP140 on POLK gene expression.Published DNA microarray datasets were reanalyzed.The in vitro sensitivity of CRC cells to methyl methane sulfonate and cisplatin was determined.Results:RIP140 positively regulates,at the transcriptional level,the expression of the POLK gene,and this effect involves,at least partly,the p53 tumor suppressor.In different cohorts of CRC biopsies(with or without MSI),a strong positive correlation was observed between RIP140 and POLK gene expression.In connection with its effect on POLK levels and the TLS function of this polymerase,the cellular response to methyl methane sulfonate was increased in cells lacking the Rip140 gene.Finally,the association of RIP140 expression with better overall survival of CRC patients was observed only when the corresponding tumors exhibited low levels of POLK,thus strengthening the functional link between the two genes in human CRC.Conclusion:The regulation of POLK gene expression by RIP140 could thus contribute to the maintenance of microsatellite stability,and more generally to the control of genome integrity.