Showing papers on "Nuclear DNA published in 2019"

Nuclear hnRNPA2B1 initiates and amplifies the innate immune response to DNA viruses

Abstract: INTRODUCTION Recognition of pathogen-derived nucleic acids by host cells is an evolutionarily conserved mechanism that induces immune defense responses to microbial infections. Most DNA viruses direct their genomic DNA into host cell nuclei, which can serve as an important molecular signature of DNA virus infection. However, little is known about the nuclear surveillance mechanisms for viral nucleic acids. RATIONALE Virus-induced type I interferon (IFN-I) expression depends on the TANK-binding kinase 1–interferon regulatory factor 3 (TBK1–IRF3) activation. We reasoned that nuclear DNA sensors may translocate to the cytoplasm to activate the TBK1–IRF3 pathway after recognizing viral DNA in the nucleus. Thus, we screened nuclear proteins that bound viral DNA and translocated from the nucleus to the cytoplasm after viral infection. Heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) was identified as a potential DNA sensor. We then conducted a series of in vivo and in vitro experiments to probe the biological importance and activation mechanisms of hnRNPA2B1. Additionally, we explored its relationship with known cytosolic stimulator of interferon genes (STING)–dependent DNA sensors such as cyclic GAMP synthase (cGAS). RESULTS hnRNPA2B1 was found to bind viral DNA in the cell nucleus during herpes simplex virus–1 (HSV-1) infection. It then translocated to the cytoplasm and activated TBK1 through the tyrosine kinase Src. Accordingly, hnRNPA2B1 knockdowns and deficiency resulted in impaired DNA virus– but not RNA virus–induced IFN-I production and prolonged viral replication. The production of proinflammatory cytokines such as tumor necrosis factor–α (TNF-α) and interleukin-6 (IL-6) was unaffected. hnRNPA2B1 became dimerized after HSV-1 infection. Mutation of the dimer interface abrogated its nucleocytoplasmic translocation upon HSV-1 infection. Thus, hnRNPA2B1 dimerization is required for its nucleocytoplasmic translocation. Additionally, hnRNPA2B1 was demethylated at Arg226 after HSV-1 infection, which led to its activation and the subsequent initiation of IFN-β expression. This demethylation was catalyzed by the arginine demethylase JMJD6. hnRNPA2B1 with dimer interface mutation was unable to associate with JMJD6 after HSV-1 infection and showed increased amounts of arginine methylation compared to full-length hnRNPA2B1, indicating that dimerization was required for its demethylation. To probe the relationship between hnRNPA2B1 and the recognized DNA sensor pathways, we found that the overexpression of hnRNPA2B1 increased HSV-1–induced TBK1 activation and Ifnb1 expression in Cgas–/– L929 cells. Thus, hnRNPA2B1 could induce IFN-I in a cGAS-independent manner at least in part. This is consistent with earlier evidence suggesting the existence of other IFN-I–initiating molecules in the innate response against DNA virus. Wild-type macrophages showed higher and more sustained Ifnb1 expression than Hnrnpa2b1–/– macrophages in response to DNA viruses. Thus, hnRNPA2B1 was required for fully activating type I interferon production against DNA viruses mediated by cGAS, interferon-γ–inducible protein 16 (IFI16), and STING pathways. Mechanistically, hnRNPA2B1 bound CGAS, IFI16,and STING mRNAs and promoted their nucleocytoplasmic trafficking to amplify cytoplasmic innate sensor signaling. The translation of these mRNAs was impaired in the absence of hnRNPA2B1 after HSV-1 infection. hnRNPA2B1 was constitutively associated with fat mass and obesity-associated protein (FTO). This association was abrogated after HSV-1 infection. By this means, hnRNPA2B1 promoted the N6-methyladenosine (m6A) modification and nucleocytoplasmic trafficking of CGAS, IFI16,and STING mRNAs. Thus, hnRNPA2B1 facilitates the efficient induction of antiviral IFN-I production mediated by cGAS, IFI16, and STING. CONCLUSION We identified hnRNPA2B1 as an innate sensor that initiates type I IFN production upon DNA virus infection in the nucleus. hnRNPA2B1 also amplifies type I IFN responses by directly enhancing STING-dependent cytosolic DNA sensing pathways.

Untangling "NETosis" from NETs.

Abstract: Neutrophil extracellular trap (NET) formation is a cellular function of neutrophils that facilitates the immobilization and killing of invading microorganisms in the extracellular milieu. To form NETs, neutrophils release a DNA scaffold consisting of mitochondrial DNA binding granule proteins. This process does not depend on cell death, but requires glycolytic ATP production for rearrangements in the microtubule network and F-actin. Such cytoskeletal rearrangements are essential for both mitochondrial DNA release and degranulation. However, the formation of NETs has also been described as a distinct form of programed, necrotic cell death, a process designated "NETosis." Necrotic cell death of neutrophils is associated with the permeabilization of both plasma and nuclear membranes resulting in a kind of extracellular cloud of nuclear DNA. The molecular mechanisms eliciting necrotic neutrophil death have been investigated and appear to be different from those responsible for NET formation following mitochondrial DNA release. Here, we discriminate between the mechanisms responsible for the release of mitochondrial versus nuclear DNA and address their respective functions. Our aim is to clarify existing differences of opinion in the fields of NET formation and neutrophil death.

Chemoptogenetic damage to mitochondria causes rapid telomere dysfunction.

Abstract: Reactive oxygen species (ROS) play important roles in aging, inflammation, and cancer. Mitochondria are an important source of ROS; however, the spatiotemporal ROS events underlying oxidative cellular damage from dysfunctional mitochondria remain unresolved. To this end, we have developed and validated a chemoptogenetic approach that uses a mitochondrially targeted fluorogen-activating peptide (Mito-FAP) to deliver a photosensitizer MG-2I dye exclusively to this organelle. Light-mediated activation (660 nm) of the Mito-FAP–MG-2I complex led to a rapid loss of mitochondrial respiration, decreased electron transport chain complex activity, and mitochondrial fragmentation. Importantly, one round of singlet oxygen produced a persistent secondary wave of mitochondrial superoxide and hydrogen peroxide lasting for over 48 h after the initial insult. By following ROS intermediates, we were able to detect hydrogen peroxide in the nucleus through ratiometric analysis of the oxidation of nuclear cysteine residues. Despite mitochondrial DNA (mtDNA) damage and nuclear oxidative stress induced by dysfunctional mitochondria, there was a lack of gross nuclear DNA strand breaks and apoptosis. Targeted telomere analysis revealed fragile telomeres and telomere loss as well as 53BP1-positive telomere dysfunction-induced foci (TIFs), indicating that DNA double-strand breaks occurred exclusively in telomeres as a direct consequence of mitochondrial dysfunction. These telomere defects activated ataxia-telangiectasia mutated (ATM)-mediated DNA damage repair signaling. Furthermore, ATM inhibition exacerbated the Mito-FAP–induced mitochondrial dysfunction and sensitized cells to apoptotic cell death. This profound sensitivity of telomeres through hydrogen peroxide induced by dysregulated mitochondria reveals a crucial mechanism of telomere–mitochondria communication underlying the pathophysiological role of mitochondrial ROS in human diseases.

Extranuclear DNA accumulates in aged cells and contributes to senescence and inflammation.

Abstract: Systemic inflammation is central to aging-related conditions. However, the intrinsic factors that induce inflammation are not well understood. We previously identified a cell-autonomous pathway through which damaged nuclear DNA is trafficked to the cytosol where it activates innate cytosolic DNA sensors that trigger inflammation. These results led us to hypothesize that DNA released after cumulative damage contributes to persistent inflammation in aging cells through a similar mechanism. Consistent with this notion, we found that older cells harbored higher levels of extranuclear DNA compared to younger cells. Extranuclear DNA was exported by a leptomycin B-sensitive process, degraded through the autophagosome-lysosomal pathway and triggered innate immune responses through the DNA-sensing cGAS-STING pathway. Patient cells from the aging diseases ataxia and progeria also displayed extranuclear DNA accumulation, increased pIRF3 and pTBK1, and STING-dependent p16 expression. Removing extranuclear DNA in old cells using DNASE2A reduced innate immune responses and senescence-associated (SA) β-gal enzyme activity. Cells and tissues of Dnase2a- / - mice with defective DNA degradation exhibited slower growth, higher activity of β-gal, or increased expression of HP-1β and p16 proteins, while Dnase2a- / - ;Sting- / - cells and tissues were rescued from these phenotypes, supporting a role for extranuclear DNA in senescence. We hypothesize a direct role for excess DNA in aging-related inflammation and in replicative senescence, and propose DNA degradation as a therapeutic approach to remove intrinsic DNA and revert inflammation associated with aging.

Mitochondrial DNA Stress Signalling Protects the Nuclear Genome.

Abstract: The mammalian genome comprises nuclear DNA (nDNA) derived from both parents and mitochondrial DNA (mtDNA) that is maternally inherited and encodes essential proteins required for oxidative phosphorylation. Thousands of copies of the circular mtDNA are present in most cell types that are packaged by TFAM into higher-order structures called nucleoids1. Mitochondria are also platforms for antiviral signalling2 and, due to their bacterial origin, mtDNA and other mitochondrial components trigger innate immune responses and inflammatory pathology2,3. We showed previously that cytoplasmic release of mtDNA activates the cGAS–STING–TBK1 pathway resulting in interferon-stimulated gene (ISG) expression that promotes antiviral immunity4. Here, we find that persistent mtDNA stress is not associated with basally activated NF-κB signalling or interferon gene expression typical of an acute antiviral response. Instead, a specific subset of ISGs that includes Parp9 remains activated by the unphosphorylated form of ISGF3 that enhances nDNA damage and repair responses. In cultured primary fibroblasts and cancer cells, the chemotherapeutic drug doxorubicin causes mtDNA damage and release, which leads to cGAS–STING–dependent ISG activation. In addition, mtDNA stress in TFAM-deficient mouse melanoma cells produces tumours that are more resistant to doxorubicin in vivo. Finally, Tfam+/− mice exposed to ionizing radiation exhibit enhanced nDNA repair responses in spleen. Therefore, we propose that damage to and subsequent release of mtDNA elicits a protective signalling response that enhances nDNA repair in cells and tissues, suggesting that mtDNA is a genotoxic stress sentinel. Persistent mitochondrial DNA stress is shown to upregulate nuclear DNA damage and repair responses via activation of the cGAS–STING pathway and a subset of interferon-stimulated genes.

Mitochondrial DNA Variants and Common Diseases: A Mathematical Model for the Diversity of Age-Related mtDNA Mutations.

Abstract: The mitochondrion is the only organelle in the human cell, besides the nucleus, with its own DNA (mtDNA). Since the mitochondrion is critical to the energy metabolism of the eukaryotic cell, it should be unsurprising, then, that a primary driver of cellular aging and related diseases is mtDNA instability over the life of an individual. The mutation rate of mammalian mtDNA is significantly higher than the mutation rate observed for nuclear DNA, due to the poor fidelity of DNA polymerase and the ROS-saturated environment present within the mitochondrion. In this review, we will discuss the current literature showing that mitochondrial dysfunction can contribute to age-related common diseases such as cancer, diabetes, and other commonly occurring diseases. We will then turn our attention to the likely role that mtDNA mutation plays in aging and senescence. Finally, we will use this context to develop a mathematical formula for estimating for the accumulation of somatic mtDNA mutations with age. This resulting model shows that almost 90% of non-proliferating cells would be expected to have at least 100 mutations per cell by the age of 70, and almost no cells would have fewer than 10 mutations, suggesting that mtDNA mutations may contribute significantly to many adult onset diseases.

G-quadruplex dynamics contribute to regulation of mitochondrial gene expression

Abstract: Single-stranded DNA or RNA sequences rich in guanine (G) can adopt non-canonical structures known as G-quadruplexes (G4). Mitochondrial DNA (mtDNA) sequences that are predicted to form G4 are enriched on the heavy-strand and have been associated with formation of deletion breakpoints. Increasing evidence supports the ability of mtDNA to form G4 in cancer cells; however, the functional roles of G4 structures in regulating mitochondrial nucleic acid homeostasis in non-cancerous cells remain unclear. Here, we demonstrate by live cell imaging that the G4-ligand RHPS4 localizes primarily to mitochondria at low doses. We find that low doses of RHPS4 do not induce a nuclear DNA damage response but do cause an acute inhibition of mitochondrial transcript elongation, leading to respiratory complex depletion. We also observe that RHPS4 interferes with mtDNA levels or synthesis both in cells and isolated mitochondria. Importantly, a mtDNA variant that increases G4 stability and anti-parallel G4-forming character shows a stronger respiratory defect in response to RHPS4, supporting the conclusion that mitochondrial sensitivity to RHPS4 is G4-mediated. Taken together, our results indicate a direct role for G4 perturbation in mitochondrial genome replication, transcription processivity, and respiratory function in normal cells.

Defects in mtDNA replication challenge nuclear genome stability through nucleotide depletion and provide a unifying mechanism for mouse progerias.

Abstract: Mitochondrial DNA (mtDNA) mutagenesis and nuclear DNA repair defects are considered cellular mechanisms of ageing. mtDNA mutator mice with increased mtDNA mutagenesis show signs of premature ageing. However, why patients with mitochondrial diseases, or mice with other forms of mitochondrial dysfunction, do not age prematurely remains unknown. Here, we show that cells from mutator mice display challenged nuclear genome maintenance similar to that observed in progeric cells with defects in nuclear DNA repair. Cells from mutator mice show slow nuclear DNA replication fork progression, cell cycle stalling and chronic DNA replication stress, leading to double-strand DNA breaks in proliferating progenitor or stem cells. The underlying mechanism involves increased mtDNA replication frequency, sequestering of nucleotides to mitochondria, depletion of total cellular nucleotide pools, decreased deoxynucleoside 5'-triphosphate (dNTP) availability for nuclear genome replication and compromised nuclear genome maintenance. Our data indicate that defects in mtDNA replication can challenge nuclear genome stability. We suggest that defects in nuclear genome maintenance, particularly in the stem cell compartment, represent a unified mechanism for mouse progerias. Therefore, through their destabilizing effects on the nuclear genome, mtDNA mutations are indirect contributors to organismal ageing, suggesting that the direct role of mtDNA mutations in driving ageing-like symptoms might need to be revisited.

Knock-In Strategy for Editing Human and Zebrafish Mitochondrial DNA Using Mito-CRISPR/Cas9 System.

Abstract: The mitochondria DNA (mtDNA) editing tool, zinc finger nucleases (ZFNs), transcription activator-like effector nuclease (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system, is a promising approach for the treatment of mtDNA diseases by eliminating mutant mitochondrial genomes. However, there have been no reports of repairing the mutant mtDNA with homologous recombination strategy to date. Here, we show a mito-CRISPR/Cas9 system that mito-Cas9 protein can specifically target mtDNA and reduce mtDNA copy number in both human cells and zebrafish. An exogenous single-stranded DNA with short homologous arm was knocked into the targeting loci accurately, and this mutagenesis could be steadily transmitted to F1 generation of zebrafish. Moreover, we found some major factors involved in nuclear DNA repair were upregulated significantly by the mito-CRISPR/Cas9 system. Taken together, our data suggested that the mito-CRISPR/Cas9 system could be a us...

G4-Interacting DNA Helicases and Polymerases: Potential Therapeutic Targets

Abstract: Background Guanine-rich DNA can fold into highly stable four-stranded DNA structures called G-quadruplexes (G4). In recent years, the G-quadruplex field has blossomed as new evidence strongly suggests that such alternately folded DNA structures are likely to exist in vivo. G4 DNA presents obstacles for the replication machinery, and both eukaryotic DNA helicases and polymerases have evolved to resolve and copy G4 DNA in vivo. In addition, G4-forming sequences are prevalent in gene promoters, suggesting that G4-resolving helicases act to modulate transcription. Methods We have searched the PubMed database to compile an up-to-date and comprehensive assessment of the field's current knowledge to provide an overview of the molecular interactions of Gquadruplexes with DNA helicases and polymerases implicated in their resolution. Results Novel computational tools and alternative strategies have emerged to detect G4-forming sequences and assess their biological consequences. Specialized DNA helicases and polymerases catalytically act upon G4-forming sequences to maintain normal replication and genomic stability as well as appropriate gene regulation and cellular homeostasis. G4 helicases also resolve telomeric repeats to maintain chromosomal DNA ends. Bypass of many G4-forming sequences is achieved by the action of translesion DNS polymerases or the PrimPol DNA polymerase. While the collective work has supported a role of G4 in nuclear DNA metabolism, an emerging field centers on G4 abundance in the mitochondrial genome. Conclusion Discovery of small molecules that specifically bind and modulate DNA helicases and polymerases or interact with the G4 DNA structure itself may be useful for the development of anticancer regimes.

Nuclear accumulation of UBC9 contributes to SUMOylation of lamin A/C and nucleophagy in response to DNA damage

Abstract: Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular mechanism for lysosomal degradation of damaged cellular components. The specific degradation of nuclear components by the autophagy pathway is called nucleophagy. Most studies have focused on autophagic turnover of cytoplasmic materials, and little is known about the role of autophagy in the degradation of nuclear components. Human MDA-MB-231 and MCF-7 breast cancer cell lines were used as model systems in vitro. Induction of nucleophagy by nuclear DNA leakage was determined by western blot and immunofluorescence analyses. The interaction and colocalization of LC3 and lamin A/C was determined by immunoprecipitation and immunofluorescence. The role of the SUMO E2 ligase, UBC9, on the regulation of SUMOylation of lamin A/C and nucleophagy was determined by siRNA silencing of UBC9, and analyzed by immunoprecipitation and immunofluorescence. DNA damage induced nuclear accumulation of UBC9 ligase which resulted in SUMOylation of lamin A/C and that SUMOylation of this protein was required for the interaction between the autophagy protein LC3 and lamin A/C, which was required for nucleophagy. Knockdown of UBC9 prevented SUMOylation of lamin A/C and LC3-lamin A/C interaction. This attenuated nucleophagy which degraded nuclear components lamin A/C and leaked nuclear DNA mediated by DNA damage. Our findings suggest that nuclear DNA leakage activates nucleophagy through UBC9-mediated SUMOylation of lamin A/C, leading to degradation of nuclear components including lamin A/C and leaked nuclear DNA.

Early Nuclear Events after Herpesviral Infection.

Abstract: Herpesviruses are important pathogens that can cause significant morbidity and mortality in the human population. Herpesviruses have a double-stranded DNA genome, and viral genome replication takes place inside the nucleus. Upon entering the nucleus, herpesviruses have to overcome the obstacle of cellular proteins in order to enable viral gene expression and genome replication. In this review, we want to highlight cellular proteins that sense incoming viral genomes of the DNA-damage repair (DDR) pathway and of PML-nuclear bodies (PML-NBs) that all can act as antiviral restriction factors within the first hours after the viral genome is released into the nucleus. We show the function and significance of both nuclear DNA sensors, the DDR and PML-NBs, and demonstrate for three human herpesviruses of the alpha-, beta- and gamma-subfamilies, HSV-1, HCMV and KSHV respectively, how viral tegument proteins antagonize these pathways.

Mitochondrial DNA methylation and copy number predict body composition in a young female population

Abstract: Since both genomic and environmental factors are involved in obesity etiology, several studies about the influence of adiposity on both nuclear DNA and mitochondrial DNA methylation patterns have been carried out. Nevertheless, few evidences exploring the usage of buccal swab samples to study mitochondrial DNA epigenetics can be found in literature. In this study, mitochondrial DNA from buccal swabs collected from a young Caucasian population (n = 69) have been used to examine potential correlation between mitochondrial DNA copy number and methylation with body composition (BMI, WHtR and bioimpedance measurements). A negative correlation between mitochondrial DNA copy number and BMI was measured in females (p = 0.028), but not in males. The mean percentage of D-loop methylation is significantly higher in overweight than in lean female subjects (p = 0.003), and a specific CpG located in the D-loop shows per se an association with impaired body composition (p = 0.004). Body composition impairment is predicted by a combined variable including mtDNA copy number and the D-loop methylation (AUC = 0.785; p = 0.009). This study corroborates the hypothesis that mitochondrial DNA carries relevant information about body composition. However, wider investigations able to validate the usage of mtDNA methylation from buccal swabs as a biomarker are warranted.

Intra- and Inter-Element Variability in Mitochondrial and Nuclear DNA from Fresh and Environmentally Exposed Skeletal Remains.

Abstract: Successful identification of skeletonized remains often relies upon DNA analyses, frequently focusing on the mid-diaphysis of weight-bearing long bones. This study explored intra-bone DNA variability using bovine and porcine femora, along with calcanei and tali. DNA from fresh and short-term environmentally exposed bone was extracted utilizing demineralization and standard lysis buffer protocols, and DNA quantity and quality were measured. Overall, femoral epiphyses, metaphyses, and the tarsals had more nuclear and mitochondrial DNA than did the femoral diaphyses. DNA loss was much more rapid in buried bones than in surface exposed bones, while DNA quality differed based on environment, but not bone region/element. The demineralization protocol generated more DNA in some bone regions, while the standard lysis was more effective in others, and neither significantly affected DNA quality. Taken together, these findings reinforce the importance of considering inter- and intra-bone heterogeneity when sampling skeletal material for forensic DNA-based identifications.

Plant DNA Polymerases.

Abstract: Maintenance of genome integrity is a key process in all organisms. DNA polymerases (Pols) are central players in this process as they are in charge of the faithful reproduction of the genetic information, as well as of DNA repair. Interestingly, all eukaryotes possess a large repertoire of polymerases. Three protein complexes, DNA Pol α, δ, and e, are in charge of nuclear DNA replication. These enzymes have the fidelity and processivity required to replicate long DNA sequences, but DNA lesions can block their progression. Consequently, eukaryotic genomes also encode a variable number of specialized polymerases (between five and 16 depending on the organism) that are involved in the replication of damaged DNA, DNA repair, and organellar DNA replication. This diversity of enzymes likely stems from their ability to bypass specific types of lesions. In the past 10–15 years, our knowledge regarding plant DNA polymerases dramatically increased. In this review, we discuss these recent findings and compare acquired knowledge in plants to data obtained in other eukaryotes. We also discuss the emerging links between genome and epigenome replication.

SD quants-Sensitive detection tetraplex-system for nuclear and mitochondrial DNA quantification and degradation inference

Abstract: Measuring the quantity of DNA present in a forensic sample is relevant in a number of ways. First, it informs the analyst about the general DNA content to adjust the volume of DNA extract used for the genotyping assay to the optimal conditions (when possible). Second, quantification values can serve as plausibility checks for the performance of the DNA extraction method used as extraction positive and negative controls demand expected values. Third and relevant to highly compromised specimens, DNA quantification can inform about the degradation state of the DNA extracted from the unknown biological sample and aid the choice of downstream genotyping assays. While there are different, commercial products for the quantification of nuclear DNA available, commercial mitochondrial DNA (mtDNA) quantification systems are rare. Even more so, the simultaneous quantification of nuclear and mtDNA that is of relevance in highly degraded forensic specimens has rarely been described. We present here a novel real-time qPCR based tetraplex system termed SD quants that targets two different-sized mtDNA and a nuclear DNA region and includes an internal positive control to monitor potential inhibition. SD quants was compared to other existing quantification systems and subjected to analysis of severely degraded DNA present in ancient DNA and aged forensic specimens. This study complies with the MIQE (Bustin et al., 2009) guidelines (when applicable).

Copy numbers of mitochondrial genes change during melon leaf development and are lower than the numbers of mitochondria.

Abstract: Melon is a useful plant species for studying mitochondrial genetics because it contains one of the largest and structurally diverse mitochondrial genomes among all plant species and undergoes paternal transmission of mitochondria. We used droplet digital (dd) PCR in combination with flow cytometric determination of nuclear DNA quantities to determine the absolute per-cell copy numbers of four mitochondrial genes (nad9, rps1, matR, and atp6) across four stages of melon leaf development. The copy numbers of these mitochondrial genes not only varied during leaf development but also differed among each other, and there was no correlation between the copy numbers of the mitochondrial genes and their transcript levels. The gene copy numbers varied from approximately 36.8 ± 4.5 (atp6 copies in the 15th leaf) to approximately 82.9 ± 5.7 (nad9 copies in the 9th leaf), while the mean number of mitochondria was approximately 416.6 ± 182.7 in the 15th leaf and 459.1 ± 228.2 in the 9th leaf. These observations indicate that the leaf cells of melon do not contain sufficient copies of mitochondrial genes to ensure that every mitochondrion possesses the entire mitochondrial genome. Given this cytological evidence, our results indicate that mtDNA in melon exists as a sub-genomic molecule rather than as a single-master circle and that the copy numbers of individual mitochondrial genes may vary greatly. An improved understanding of the molecular mechanism(s) controlling the relative prevalence and transmission of sub-genomic mtDNA molecules should provide insights into the continuity of the mitochondrial genome across generations.

Circulating Cell Free Nuclear DNA, Mitochondrial DNA and Global DNA Methylation: Potential Noninvasive Biomarkers for Breast Cancer Diagnosis.

Abstract: Eighty seven women with benign breast lesion, 120 patients with breast cancer (BC) and one hundred controls were included in the study. Quantification of mtDNA and nDNA was done by qPCR. Global DNA methylation was measured using ELISA. Circulating cell-free nDNA and mtDNA were significantly elevated in BC and benign breast lesions patients. Global methylation was significantly low in BC patients. Combining the studied parameters in one panel, nDNA/mtDNA/hypomethylation, improved their sensitivity in detecting BC to reach 92.5%. Circulating cell-free nDNA, mtDNA and global DNA hypomethylation can be used as diagnostic and prognostic markers for BC.

Change in nuclear DNA content and pollen size with polyploidisation in the sweet potato (Ipomoea batatas, Convolvulaceae) complex

Abstract: Genome size evolution, and its relationships with pollen grain size, has been investigated in the sweet potato (Ipomoea batatas), an economically important crop, and closely related diploid and tetraploid species, assessing the nuclear DNA content of 22 accessions from five Ipomoea species, 10 sweet potato varieties and two outgroup taxa. Nuclear DNA amounts were determined by flow cytometry. Pollen grains have been studied at scanning and transmission electron microscopy. 2C DNA content of hexaploid I. batatas ranged over 3.12-3.29 pg, mean monoploid genome size being 0.539 pg (527 Mbp) much as for the related diploid accessions. In tetraploid species I. trifida and I. tabascana, 2C DNA content was respectively 2.07 and 2.03 pg. In the diploid species closely related to sweet potato e.g. I. ×leucantha, I. tiliacea, I. trifida, I. triloba, 2C DNA content was 1.01-1.12 pg. However, two diploid outgroup species, I. setosa and I. purpurea, were clearly different from the other diploid species with 2C of 1.47-1.49 pg; they also have larger chromosomes. The I. batatas genome presents 60.0% of AT bases. DNA content and ploidy level were positively correlated within this complex. In I. batatas and the more closely related species I. trifida, genome size and ploidy levels were correlated with pollen size. Our results allow us proposing alternative or complementary hypotheses to the one currently proposed for the formation of hexaploid Ipomoea batatas. This article is protected by copyright. All rights reserved.

Impact of DNA degradation on massively parallel sequencing-based autosomal STR, iiSNP, and mitochondrial DNA typing systems.

Abstract: Biological samples, including skeletal remains exposed to environmental insults for extended periods of time, exhibit increasing levels of DNA damage and fragmentation. Human forensic identification methods typically use a combination of mitochondrial (mt) DNA sequencing and short tandem repeat (STR) analysis, which target segments of DNA ranging from 80 to 500 base pairs (bps). Larger templates are often unavailable as skeletal samples age and the associated DNA degrades. Single-nucleotide polymorphism (SNP) loci target shorter templates and may serve as a solution to the problem. Recently developed assays for STR and SNP analysis using a massively parallel sequencing approach, such as the ForenSeq kit (Verogen, San Diego, CA), offer a means for generating results from degraded samples as they target templates down to 60 to 170 bps. We performed a modeling study that demonstrates that SNPs can increase the significance of an identification when analyzing DNA down to an average size of 100 bps for input amounts between 0.375 and 1 ng of nuclear DNA. Observations from this study were then compared with human skeletal material results (n = 14, ninth to eighteenth centuries), which further demonstrated the utility of the ForenSeq kit for degraded samples. The robustness of the Promega PowerSeq™ Mito System was also tested with human skeletal remains (n = 70, ninth to eighteenth centuries), resulting in successful coverage of 99.29% of the mtDNA control region at 50× coverage or more. This was accompanied by modifications to a mainstream DNA extraction technique for skeletal remains that improved recovery of shorter templates.

Day/Night Separation of Oxygenic Energy Metabolism and Nuclear DNA Replication in the Unicellular Red Alga Cyanidioschyzon merolae

Abstract: The transition from G1 to S phase and subsequent nuclear DNA replication in the cells of many species of eukaryotic algae occur predominantly during the evening and night in the absence of photosynthesis; however, little is known about how day/night changes in energy metabolism and cell cycle progression are coordinated and about the advantage conferred by the restriction of S phase to the night. Using a synchronous culture of the unicellular red alga Cyanidioschyzon merolae, we found that the levels of photosynthetic and respiratory activities peak during the morning and then decrease toward the evening and night, whereas the pathways for anaerobic consumption of pyruvate, produced by glycolysis, are upregulated during the evening and night as reported recently in the green alga Chlamydomonas reinhardtii. Inhibition of photosynthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) largely reduced respiratory activity and the amplitude of the day/night rhythm of respiration, suggesting that the respiratory rhythm depends largely on photosynthetic activity. Even when the timing of G1/S-phase transition was uncoupled from the day/night rhythm by depletion of retinoblastoma-related (RBR) protein, the same patterns of photosynthesis and respiration were observed, suggesting that cell cycle progression and energy metabolism are regulated independently. Progression of the S phase under conditions of photosynthesis elevated the frequency of nuclear DNA double-strand breaks (DSB). These results suggest that the temporal separation of oxygenic energy metabolism, which causes oxidative stress, from nuclear DNA replication reduces the risk of DSB during cell proliferation in C. merolae. IMPORTANCE Eukaryotes acquired chloroplasts through an endosymbiotic event in which a cyanobacterium or a unicellular eukaryotic alga was integrated into a previously nonphotosynthetic eukaryotic cell. Photosynthesis by chloroplasts enabled algae to expand their habitats and led to further evolution of land plants. However, photosynthesis causes greater oxidative stress than mitochondrion-based respiration. In seed plants, cell division is restricted to nonphotosynthetic meristematic tissues and populations of photosynthetic cells expand without cell division. Thus, seemingly, photosynthesis is spatially sequestrated from cell proliferation. In contrast, eukaryotic algae possess photosynthetic chloroplasts throughout their life cycle. Here we show that oxygenic energy conversion (daytime) and nuclear DNA replication (night time) are temporally sequestrated in C. merolae. This sequestration enables “safe” proliferation of cells and allows coexistence of chloroplasts and the eukaryotic host cell, as shown in yeast, where mitochondrial respiration and nuclear DNA replication are temporally sequestrated to reduce the mutation rate.

Estimation of nuclear DNA content and its variation among Indian Tea accessions by flow cytometry

Abstract: Nuclear DNA content and genome size variation among 36 Indian tea accessions were analyzed by flow cytometry. Initial standardization of protocols for isolation of nuclei, DNA staining and selection of an internal standard for tea accessions which have significantly high amount of phenolic secondary metabolites in their cytosol was carried out. Results obtained revealed that 2C DNA content of Indian tea is 7.46 pg which corresponds to 1C genome size of 3673 Mb. Inter accession variation in 2C DNA content was also observed among 35 diploid taxa ranging from 7.23 to 7.73 pg which was significant at 1% probability level. The 2C DNA content of triploid (UPASI 3) was observed to be 11.47 pg which is concurrent with the expected value. Results obtained showed that Assam and Cambod type tea accession have higher 2C DNA content of 7.73 pg whereas Assam Cambod hybrids and Assam China hybrids have reduction in DNA content with 2C amounts, 7.23 and 7.32 pg DNA respectively. The present study suggests that the species involved in origin of Indian tea must have differed in their genome sizes owing to significant inter accession variation in nuclear DNA content.

Intergenomic gene transfer in diploid and allopolyploid Gossypium

Abstract: Intergenomic gene transfer (IGT) between nuclear and organellar genomes is a common phenomenon during plant evolution. Gossypium is a useful model to evaluate the genomic consequences of IGT for both diploid and polyploid species. Here, we explore IGT among nuclear, mitochondrial, and plastid genomes of four cotton species, including two allopolyploids and their model diploid progenitors (genome donors, G. arboreum: A2 and G. raimondii: D5). Extensive IGT events exist for both diploid and allotetraploid cotton (Gossypium) species, with the nuclear genome being the predominant recipient of transferred DNA followed by the mitochondrial genome. The nuclear genome has integrated 100 times more foreign sequences than the mitochondrial genome has in total length. In the nucleus, the integrated length of chloroplast DNA (cpDNA) was between 1.87 times (in diploids) to nearly four times (in allopolyploids) greater than that of mitochondrial DNA (mtDNA). In the mitochondrion, the length of nuclear DNA (nuDNA) was typically three times than that of cpDNA. Gossypium mitochondrial genomes integrated three nuclear retrotransposons and eight chloroplast tRNA genes, and incorporated chloroplast DNA prior to divergence between the diploids and allopolyploid formation. For mitochondrial chloroplast-tRNA genes, there were 2-6 bp conserved microhomologies flanking their insertion sites across distantly related genera, which increased to 10 bp microhomologies for the four cotton species studied. For organellar DNA sequences, there are source hotspots, e.g., the atp6-trnW intergenic region in the mitochondrion and the inverted repeat region in the chloroplast. Organellar DNAs in the nucleus were rarely expressed, and at low levels. Surprisingly, there was asymmetry in the survivorship of ancestral insertions following allopolyploidy, with most numts (nuclear mitochondrial insertions) decaying or being lost whereas most nupts (nuclear plastidial insertions) were retained. This study characterized and compared intracellular transfer among nuclear and organellar genomes within two cultivated allopolyploids and their ancestral diploid cotton species. A striking asymmetry in the fate of IGTs in allopolyploid cotton was discovered, with numts being preferentially lost relative to nupts. Our results connect intergenomic gene transfer with allotetraploidy and provide new insight into intracellular genome evolution.

hnRNPA2B1: a nuclear DNA sensor in antiviral immunity

Abstract: Pattern recognition receptors (PRRs) have critical roles in mediating innate immune responses. In a recent study published in Science, Cao and colleagues identified hnRNPA2B1 as a new nuclear DNA sensor that initiated type I interferon (IFN-I) production upon DNA virus infection and amplified IFN-I responses by directly enhancing STINGdependent cytosolic DNA sensing pathways. The innate immune system is activated by the recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs), which leads to the production of proinflammatory cytokines and type I interferons (IFN-I). PRRs that recognize viral DNA in cells include TLR9 in endosomes and several cytosolic sensors such as cyclic GMP-AMP synthase (cGAS), DNA-dependent activator of IFN-regulatory factors (DAI), DEAH box protein 9 (DHX9), DEAH box protein 36 (DHX36), absent in melanoma 2 (AIM2) and IFNγ-inducible protein 16 (IFI16). Viral DNA replication mostly occurs in the nucleus of infected cells, where the recognition of viral DNA could potentially happen. However, innate immune response sensors in the nucleus to recognize nuclear pathogen-derived DNA remain unclear. Given that virus-induced IFN-I expression mainly depends on TBK1-IRF3 activation in the cytoplasm, Cao and colleagues sought to identify new nuclear viral DNA sensors that could recognize viral DNA in the nucleus and then translocate to the cytoplasm to activate the TBK1-IRF3 pathway. To identify potential nuclear DNA sensors, Wang et al. searched the proteins that were precipitated by the biotinylated genomic DNA of HSV-1 (F strain) and translocated from the nucleus to the cytoplasm 2 h after HSV-1 infection, using 2D SDS-PAGE and mass spectrometry. This screen allowed them to identify 23 potential nuclear viral DNA sensors, among which hnRNPA2B1 was validated to bind both selfand pathogen-derived DNA, but not native nucleosomes. hnRNPA2B1, an RNA-binding protein, has been reported to function as an mA reader and is involved in RNA transport, processing or splicing. Wang et al. found that Hnrnpa2b1 knockdown in vitro or knockout in mice in vivo impaired DNA virusbut not RNA virus-induced IFN-I production, thus promoting viral replication without affecting the expression of pro-inflammatory cytokines such as TNF-α and IL-6. These results suggested that hnRNPA2B1 was a bona fide IFN-I-inducing nuclear DNA sensor. How does hnRNPA2B1 initiate the DNA virus-induced IFN-I response? Wang et al. found that upon binding viral DNA in the cell nucleus, hnRNPA2B1 could translocate to the cytoplasm and activate TBK1 through the tyrosine kinase Src and endoplasmic reticulum adaptor STING. Hnrnpa2b1 deficiency impaired the phosphorylation of TBK1 and IRF3, thus decreasing the kinase activity of TBK1 after HSV-1 infection. Next, they investigated the mechanisms of hnRNPA2B1 nucleocytoplasmic translocation and found that HSV-1 infection induced the homodimerization of hnRNPA2B1. Mutation of the dimer interface in the RRM (RNA recognition motif) domain abrogated hnRNPA2B1 dimerization and nucleocytoplasmic translocation upon HSV-1 infection. In addition, through mutational screening of arginine, serine, and threonine of hnRNPA2B1, they found that a mutation of Arg226 (R226A) within the arginine-glycine-glycine (RGG) domain significantly enhanced Ifnb1 expression compared to wild-type hnRNPA2B1. It was previously reported that hnRNPA2B1 could be methylated at arginine residues within the RGG domain. Indeed, arginine methylation assay revealed that R226 was the key site for arginine mono-methylation of hnRNPA2B1, which was decreased after HSV-1 infection. Wang et al. further demonstrated that the demethylation of hnRNPA2B1 at Arg226 was mediated by the arginine demethylase JMJD6. hnRNPA2B1 with dimer interface mutation was unable to associate with JMJD6 after HSV-1 infection and showed increased arginine methylation levels compared to full-length hnRNPA2B1. Thus, dimerization of hnRNPA2B1 upon recognizing viral DNA is required for its demethylation, activation, nucleocytoplasmic translocation and the subsequent initiation of IFN-β expression (Fig. 1). Are there cross-talks between this new nuclear DNA hnRNPA2B1 sensor pathway and other well-known cytoplasmic DNA sensor pathways? Wang et al. found that hnRNPA2B1 overexpression increased HSV-1-induced TBK1 activation and Ifnb1 expression in cgas L929 cells, suggesting that hnRNPA2B1 was able to induce IFN activation in a cGAS-independent manner. Hnrnpa2b1 deficiency attenuated IFN-I production induced by HSV-1 and Vaccinia virus (VACV), another DNA virus, replicating in the cytoplasm. They also found that hnRNPA2B1 bound cgas, Ifi16 and Sting mRNAs as an RNA-binding protein to transport those mRNAs from nucleus to cytoplasm. Interestingly, hnRNPA2B1 was constitutively associated with RNA mA demethylases FTO at steady state to keep the mA levels of those mRNAs at low levels. This association was abrogated after HSV-1 infection, thus increasing the mA levels and promoting nucleocytoplasmic trafficking to enhance the translation of these mRNAs without affecting their transcription and stability. They demonstrated that Hnrnpa2b1 deficiency significantly decreased the mA levels of cgas, Ifi16 and Sting mRNAs, and led to the nuclear retention of cgas, Ifi16 and Sting mRNAs. Thus, hnRNPA2B1 can also enhance the efficient induction of antiviral IFN-I production mediated by cGAS, IFI16, and STING through mA machinery (Fig. 1).

Bidirectional mitochondrial introgression between Korean cobitid fish mediated by hybridogenetic hybrids.

Abstract: Genomic introgression through interspecific hybridization has been observed in some species of the freshwater fish family Cobitidae. Within this family, a Cobitis hankugensis-Iksookimia longicorpa diploid-triploid hybrid species complex on the Korean peninsula is unique in displaying hybridogenesis, a unisexual reproduction mode that allows hybrids to mediate the transfer of mitochondrial DNA (but not nuclear DNA) between the two parent species. However, populations of the parental species in the wild have never been examined for the potential effect of introgression on their genomes. To address the genetic consequences of unisexual hybridization on the parental species, we examined genetic structure of the two parental species, C. hankugensis and I. longicorpa, in three independent natural habitats where they coexist with their hybrid complex using DNA sequence data of one mitochondrial gene and three nuclear genes. We found that mitochondrial introgression between the two species was extensive in all the examined localities, while there was no evidence of nuclear introgression across the species boundary. This result indicates that the hybridogenetic individuals mediate mitochondrial introgression from one species to the other, producing mito-nuclear mosaic genomes such as C. hankugensis nuclear genomes associated with I. longicorpa mitochondrial DNA and the reverse. The direction and degree of introgression varied among the three localities, but the underlying mechanisms for this observation proved elusive. Introgression might depend on which species serves as the predominant sperm or ovum donor or the environmental conditions of the localities. The present study suggests that introgressive hybridization between pure C. hankugensis and I. longicorpa species is highly likely where the two species co-occur with hybridogenetic individuals, but the consequence of introgression could be variable due to the history and environmental characteristics of particular populations across the parental species' ranges.

Archaic mitochondrial DNA inserts in modern day nuclear genomes.

Abstract: Traces of interbreeding of Neanderthals and Denisovans with modern humans in the form of archaic DNA have been detected in the genomes of present-day human populations outside sub-Saharan Africa. Up to now, only nuclear archaic DNA has been detected in modern humans; we therefore attempted to identify archaic mitochondrial DNA (mtDNA) residing in modern human nuclear genomes as nuclear inserts of mitochondrial DNA (NUMTs). We analysed 221 high-coverage genomes from Oceania and Indonesia using an approach which identifies reads that map both to the nuclear and mitochondrial DNA. We then classified reads according to the source of the mtDNA, and found one NUMT of Denisovan mtDNA origin, present in 15 analysed genomes; analysis of the flanking region suggests that this insertion is more likely to have happened in a Denisovan individual and introgressed into modern humans with the Denisovan nuclear DNA, rather than in a descendant of a Denisovan female and a modern human male. Here we present our pipeline for detecting introgressed NUMTs in next generation sequencing data that can be used on genomes sequenced in the future. Further discovery of such archaic NUMTs in modern humans can be used to detect interbreeding between archaic and modern humans and can reveal new insights into the nature of such interbreeding events.