Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain ...unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes and are targeted to HR repair via the transcription elongation-associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. DNA double-strand breaks (DSBs) within rDNA induce both rDNA transcriptional repression and nucleolar ...segregation, but the link between the two events remains unclear. Here we found that DSBs induced on rDNA trigger transcriptional repression in a cohesin- and HUSH (human silencing hub) complex-dependent manner throughout the cell cycle. In S/G2 cells, transcriptional repression is further followed by extended resection within the interior of the nucleolus, DSB mobilization at the nucleolar periphery within nucleolar caps, and repair by homologous recombination. We showed that nuclear envelope invaginations frequently connect the nucleolus and that rDNA DSB mobilization, but not transcriptional repression, involves the nuclear envelope-associated LINC complex and the actin pathway. Altogether, our data indicate that rDNA break localization at the nucleolar periphery is not a direct consequence of transcriptional repression but rather is an active process that shares features with the mobilization of persistent DSB in active genes and heterochromatin.
Agriculture is changing to rely on agroecological practices that take into account biodiversity, and the ecological processes occurring in soils. The use of agricultural biostimulants has emerged as ...a valid alternative to chemicals to indirectly sustain plant growth and productivity. Certain BS have been shown to select and stimulate plant beneficial soil microorganisms. However, there is a lack of knowledge on the effects and way of action of the biostimulants operating on soil functioning as well as on the extent and dynamic of these effects. In this study we aimed to decipher the way of action of a seaweed and amino-acids based biostimulant intended to be applied on soil crop residues to increase their microbial mineralization and the further release of nutrients. By setting-up a two-phase experiment (soil plant-growing and soil incubation), our objectives were to (1) determine the effects of the soil biostimulant over time on the active soil bacteria and fungi and the consequences on the organic carbon mineralization in bare soils, and (2) assess the biostimulant effects on soil microorganisms relatively to plant legacy effects in planted soils. We demonstrated that the soil biostimulant had a delayed effect on the active soil microorganisms and activated both plant growth promoting bacteria and saprophytes microorganisms at the medium-term of 49 days. However, the changes in the abundances of active microbial decomposers were not associated to a higher mineralization rate of organic carbon derived from soil and/or litter. The present study assessed the biostimulant beneficial effect on active soil microbial communities as similar as or even higher than the legacy effects of either A. thaliana or T. aestivum plants. We specifically showed that the biostimulant increased the active fungal richness to a higher extent than observed in soils that previously grew the two plants tested.
DNA double-strand breaks (DSBs) elicit the so-called DNA damage response (DDR), largely relying on ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PKcs), two members of the ...PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA system (DSB inducible via AsiSI) combined with high-resolution mapping and advanced microscopy, we uncovered that both ATM and DNA-PKcs spread in cis on a confined region surrounding DSBs, independently of the pathway used for repair. However, once recruited, these kinases exhibit non-overlapping functions on end joining and γH2AX domain establishment. More specifically, we found that ATM is required to ensure the association of multiple DSBs within “repair foci.” Our results suggest that ATM acts not only on chromatin marks but also on higher-order chromatin organization to ensure repair accuracy and survival.
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•Both ATM and DNA-PKcs are recruited at AsiSI-induced DSBs•Once recruited, both kinases exhibit complementary and non-redundant functions•DNA-PKcs activity is required for end joining at all AsiSI-induced DSBs•ATM activity promotes repair accuracy, H2AX phosphorylation, and DSB clustering
By inducing multiple annotated DNA double-strand breaks in the human genome, Caron et al. show that two DNA damage response kinases, ATM and DNA-PKcs, are co-recruited at DSBs but exhibit non-redundant functions in promoting end joining, repair accuracy, H2AX phosphorylation, and DSB clustering.
The posterior signalling centre (PSC), a small group of specialised cells, controls hemocyte (blood cell) homeostasis in the Drosophila larval hematopoietic organ, the lymph gland. This role of the ...PSC is very reminiscent of the "niche," the micro-environment of hematopoietic stem cells in vertebrates. We have recently shown that the PSC acts in a non-cell-autonomous manner to maintain janus tyrosine kinase/signal transducers and activators of transcription (JAK/STAT) signalling in hematopoietic progenitors (prohemocytes), thereby preserving the multipotent character necessary for their differentiation into lamellocytes, a cryptic and dedicated immune cell type required to fight specific immune threats such as wasp parasitism. In this report, on the basis of a knock out generated by homologous recombination, we show that a short type I cytokine-related receptor CG14225/Latran is required for switching off JAK/STAT signalling in prohemocytes. This is a prerequisite to massive differentiation of lamellocytes upon wasp parasitisation. In vivo and cell culture assays indicate that Latran forms heteromers with Domeless, the Drosophila type I cytokine signalling receptor related to mammalian GP130, and antagonises Domeless activity in a dose-dependent manner. Our analysis further shows that a primary immune response to wasp parasitism is a strong decrease in cytokine mRNA levels in the lymph gland, followed by an increase in the latran/domeless ratio. We propose that this sequence of events culminates in the complete inhibition of residual JAK/STAT signalling by Latran. JAK/STAT activity has been associated with several human diseases including leukaemia while knock-out studies in mice point to a central role of this pathway in hematopoiesis and regulation of immune functions. The specific function of Drosophila Latran is, to our knowledge, the first in vivo example of a role for a nonsignalling receptor in controlling a dedicated immune response, and thus raises the question of whether short, nonsignalling receptors also control specific aspects of vertebrate cellular immunity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Specification of muscle identity in Drosophila is a multistep process: early positional information defines competence groups termed promuscular clusters, from which muscle progenitors are selected, ...followed by asymmetric division of progenitors into muscle founder cells (FCs). Each FC seeds the formation of an individual muscle with morphological and functional properties that have been proposed to reflect the combination of transcription factors expressed by its founder. However, it is still unclear how early patterning and muscle-specific differentiation are linked. We addressed this question, using Collier (Col; also known as Knot) expression as both a determinant and read-out of DA3 muscle identity. Characterization of the col upstream region driving DA3 muscle specific expression revealed the existence of three separate phases of cis-regulation, correlating with conserved binding sites for different mesodermal transcription factors. Examination of col transcription in col and nautilus ( nau ) loss-of-function and gain-of-function conditions showed that both factors are required for col activation in the `naïve' myoblasts that fuse with the DA3 FC, thereby ensuring that all DA3 myofibre nuclei express the same identity programme. Together, these results indicate that separate sets of cis-regulatory elements control the expression of identity factors in muscle progenitors and myofibre nuclei and directly support the concept of combinatorial control of muscle identity.
The increasing number of available genomic sequences makes it now possible to study the evolutionary history of specific genes or gene families. Transcription factors (TFs) involved in regulation of ...gene-specific expression are key players in the evolution of metazoan development. The low complexity COE (Collier/Olfactory-1/Early B-Cell Factor) family of transcription factors constitutes a well-suited paradigm for studying evolution of TF structure and function, including the specific question of protein modularity. Here, we compare the structure of coe genes within the metazoan kingdom and report on the mechanism behind a vertebrate-specific exon duplication.
COE proteins display a modular organisation, with three highly conserved domains : a COE-specific DNA-binding domain (DBD), an Immunoglobulin/Plexin/transcription (IPT) domain and an atypical Helix-Loop-Helix (HLH) motif. Comparison of the splice structure of coe genes between cnidariae and bilateriae shows that the ancestral COE DBD was built from 7 separate exons, with no evidence for exon shuffling with other metazoan gene families. It also confirms the presence of an ancestral H1LH2 motif present in all COE proteins which partly overlaps the repeated H2d-H2a motif first identified in rodent EBF. Electrophoretic Mobility Shift Assays show that formation of COE dimers is mediated by this ancestral motif. The H2d-H2a alpha-helical repetition appears to be a vertebrate characteristic that originated from a tandem exon duplication having taken place prior to the splitting between gnathostomes and cyclostomes. We put-forward a two-step model for the inclusion of this exon in the vertebrate transcripts.
Three main features in the history of the coe gene family can be inferred from these analyses: (i) each conserved domain of the ancestral coe gene was built from multiple exons and the same scattered structure has been maintained throughout metazoan evolution. (ii) There exists a single coe gene copy per metazoan genome except in vertebrates. The H2a-H2d duplication that is specific to vertebrate proteins provides an example of a novel vertebrate characteristic, which may have been fixed early in the gnathostome lineage. (iii) This duplication provides an interesting example of counter-selection of alternative splicing.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Plants shape soil microbial communities through their root architecture, their rhizodeposits and return of dead plant material to the soil. These interactions can have a strong influence on the soil ...organic carbon dynamics. However, it is unclear whether the plant species effects on the soil microbial community could influence the organic carbon mineralization through plant legacy effects. Therefore, we examined how and to what extent a short-term plant growing phase affected the total and active soil microorganisms and through a possible plant legacy, also the mineralization of soil organic carbon, a central ecosystem function. Using a controlled pot experiment, we first showed that the two phylogenetically distinct plants, Arabidopsis thaliana and Triticum aestivum, differently shaped the soil microbial communities when recruiting from the same soil community. Although both plants recruited plant-growth promoting bacteria in the vicinity of their roots, A. thaliana had a stronger effect than T. aestivum and also recruited saprophytic fungi, while inhibiting fungal pathogens. Due to plant legacy effects on the soil microbial communities, different microbial successions occurred in the two previously planted soils when subjected to plant litter. By contrast, plant legacy effects on soil basal respiration were not plant-specific, with basal respiration increasing similarly in both cases and moreover did not translate to changes in litter carbon mineralization in the short-term of 49 days. Our results suggest that the soil nutrient dynamics rather than changes in soil microbial community composition drive the organic carbon mineralization of added litter. The present study brings new insights in how the relationships between plants, microorganisms and soil nutrient dynamics affect litter carbon cycling.
•A. thaliana and T. aestivum differently shaped the soil microbial communities.•A. thaliana had a stronger effect, especially for fungal communities.•PGPB were recruited in the vicinity of the roots of both A. thaliana and T. aestivum.•Plant legacy effects traduced in changes in soil organic carbon and nutrient dynamics.•Plant legacy effects linked to nutrient dynamics rather than to microbial community.
Cold tolerance is considered an important factor determining the geographic distribution of insects. We have previously shown that despite its tropical origin, the cockroach
is capable of surviving ...exposures to cold. However, the freezing tolerance of this species had not yet been examined. Low temperature is known to alter membrane integrity in insects, but whether chilling or freezing compromises DNA integrity remains a matter of speculation. In the present study, we subjected the
adults to freezing to determine their supercooling point (SCP) and evaluated whether the cockroaches were capable of surviving partial and complete freezing. Next, we conducted single cell gel electrophoresis (SCGE) assays to determine whether heat, cold and freezing altered hemocyte DNA integrity. The SCP of this species was high and around -4.76°C, which is within the typical range of freezing-tolerant species. Most cockroaches survived to 1 day after partial ice formation (20% mortality), but died progressively in the next few days after cold stress (70% mortality after 4 days). One day after complete freezing, most insects died (70% mortality), and after 4 days, 90% of them had succumbed. The SCGE assays showed substantial levels of DNA damage in hemocytes. When cockroaches were heat-stressed, the level of DNA damage was similar to that observed in the freezing treatment, though all heat-stressed insects survived. The present study shows that
can be considered as moderately freeze-tolerant, and that extreme low temperature stress can affect DNA integrity, suggesting that this cockroach may possess an efficient DNA repair system.
Research to date on abiotic stress responses in plants has been largely focused on the plant itself, but current knowledge indicates that microorganisms can interact with and help plants during ...periods of abiotic stress. In our research, we aim to investigate the interkingdom communication between the plant root and the rhizo-microbiota. Our investigation showed that miRNA plays a pivotal role in this interkingdom communication. Here, we describe a protocol for the analysis of miRNA secreted by the plant root, which includes all of the steps from the isolation of the miRNA to the bioinformatics analysis. Because of their short nucleotide length, Next Generation Sequencing (NGS) library preparation from miRNAs can be challenging due to the presence of dimer adapter contaminants. Therefore, we highlight some strategies we adopt to inhibit the generation of dimer adapters during library preparation. Current screens of miRNA targets mostly focus on the identification of targets present in the same organism expressing the miRNA. Our bioinformatics analysis challenges the barrier of evolutionary divergent organisms to identify candidate sequences of the microbiota targeted by the miRNA of plant roots. This protocol should be of interest to researchers investigating interkingdom RNA-based communication between plants and their associated microorganisms, particularly in the context of holobiont responses to abiotic stresses.
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