The splicing factor XAB2 interacts with ERCC1-XPF and XPG for R-loop processing

RNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csb(m/m) developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders

Μελέτη του μηχανισμού ομόλογης αλληλεπίδρασης των Tnfα αλληλίων σε μακροφάγα ποντικού

Several trans-allelic phenomena have been described in the past although their mode of action on gene regulation lacks mechanistic insight. Such a rare phenomenon in mammals is somatic homologous pairing. We have investigated the homologous pairing of the Tnfα locus in LPS stimulated mouse macrophages and found that it occurs early upon activation prior to the Tnfα gene expression switch from monoallelic to biallelic. The proposed regulatory mechanism involves two complementary long non-coding RNAs transcribed from the LT/TNF locus, a protein kinase with transactivation potential and a protein considered to be the mammalian homolog of the Drosophila GAGA-factor involved in transvection. We believe that homologous pairing as a part of genome organization in mammals will be of general interest and propose a mechanism for the regulation of mono- to bi-allelic switch of gene expression.Αρκετά δια-αλληλικά φαινόμενα έχουν περιγραφεί στο παρελθόν, παρότι ο ρόλος τους στη ρύθμιση γονιδίων δεν έχει ακόμα αποσαφηνιστεί. Ένα τέτοιο σπάνιο φαινόμενο στα θηλαστικά είναι η σωματική ομόλογη αλληλεπίδραση. Μελετήσαμε την ομόλογη αλληλεπίδραση του γενετικού τόπου του Tnfα σε μακροφάγα ποντικού ενεργοποιημεένα με λιποπολυσακχαρίτη (LPS) και βρήκαμε ότι συμβαίνει νωρίς κατά την ενεργοποίηση και πριν την αλλαγή της έκφρασης του Tnfα γονιδίου από μόνο- σε δι-αλληλική. Ο προτεινόμενος ρυθμιστικός μηχανισμός περιλαμβάνει δύο συμπληρωματικά μεγάλα μη-κωδικά RNAs που μεταγράφονται από τον LT/TNF γενετικό τόπο, μια πρωτεϊνική κινάση με δυνατότητα ενεργοποίησης από απόσταση και μια πρωτεΐνη που θεωρείται να είναι το ομόλογο του παράγοντα GAGA της Δροσόφιλα, στα θηλαστικά. Πιστεύουμε ότι η ομόλογη αλληλεπίδραση, ως μέρος της γονιδιωματικής οργάνωσης των θηλαστικών θα έχει γενικότερο ενδιαφέρον και προτείνουμε ένα μηχανισμό για τη ρύθμιση της γονιδιακής έκφρασης από μόνο- σε δι-αλληλική

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Ομόλογη αλληλεπίδραση του γενετικού τόπου του TNFa στο ποντίκι

Lipopolysaccharide (LPS) stimulation of murine macrophages triggers the activation of signalling pathways that lead to the nuclear translocation of Nf-θB and the subsequent expression of TNFα., among other pro-inflammatory cytokines. In the present project we have studied the events that take place in the cell nucleus before and during the expression of TNFα and, specifically, we aimed in identifying the protein factors that regulate the inter-chromosomal interactions between the two murine TNF alleles. We found that the homologous association of the two TNFα alleles takes place 30min upon the stimulation of macrophages with LPS. The highest level of TNF expression followed this homologous interaction, at 1h upon LPS stimulation and accordingly the simultaneous transition from mono- to by-allelic expression was detected at the same time point. In correspondence to the model of transvection and the GAGA factor that mediates homologous pairing in Drosophila, we used a series of molecular and genetic approaches to identify the protein complexes that regulate these long-range interactions in mammals

<i>Tnfα</i> mRNA expression in wild type and <i>miR-155</i>^<i>-/-</i> murine macrophages.

<p>(A) <i>Hprt1-</i>normalized relative mRNA expression levels of <i>Tnfα</i> analyzed by qRT-PCR in murine TEPMs isolated from either wild type or <i>miR-155</i>^<i>-/-</i> mice. (B) RNA FISH single-stack confocal images portraying the reduced nuclear and cytoplasmic <i>Tnfα</i> RNA in <i>miR-155</i>^<i>-/-</i> BMDMs, compared to wild type cells (scale bar 4 μm). (C) RNA-DNA FISH experiments for the <i>Tnfα</i> allelic expression profile in wild type and <i>miR-155</i>^<i>-/-</i> TEPMs (sample size, WT: 183 nuclei, <i>miR-155</i>^<i>-/-</i> 191 nuclei). (D) The <i>Tnfα</i> alleles distance was normalized for the volume of each cell (ND) and the frequencies of cells with a normalized distance from 0 to 1 were plotted (scale bar, 2 μm). For the far right graph, the frequency of cells with an allele ND < 0.1 was plotted (sample size, WT: 183 nuclei, <i>miR-155</i>^<i>-/-</i> 191 nuclei). (n.d.: not detected)</p

Transcription stress at telomeres leads to cytosolic DNA release and paracrine senescence.

Transcription stress has been linked to DNA damage -driven aging, yet the underlying mechanism remains unclear. Here, we demonstrate that Tcea1-/- cells, which harbor a TFIIS defect in transcription elongation, exhibit RNAPII stalling at oxidative DNA damage sites, impaired transcription, accumulation of R-loops, telomere uncapping, chromatin bridges, and genome instability, ultimately resulting in cellular senescence. We found that R-loops at telomeres causally contribute to the release of telomeric DNA fragments in the cytoplasm of Tcea1-/- cells and primary cells derived from naturally aged animals triggering a viral-like immune response. TFIIS-defective cells release extracellular vesicles laden with telomeric DNA fragments that target neighboring cells, which consequently undergo cellular senescence. Thus, transcription stress elicits paracrine signals leading to cellular senescence, promoting aging

<i>Tnfα</i> allelic expression profile in tolerant wild type and <i>SeT</i>^-/- TEPMs.

<p>(A) Survival curves of wild type and <i>SeT</i>^<i>-/-</i> mice upon sublethal LPS dose intraperitoneal injection and re-challenging with a lethal LPS dose. The survival rate of each germline is defined by the percentage of surviving individuals over the total number of mice per genotype. (B) Single-stack confocal images portraying the mono-/bi-allelic expression profile of <i>Tnfα</i> in tolerant and one hour LPS-restimulated TEPMs (scale bar 2 μm). The graph represents the frequency of alleles expressed in either a mono- or a bi-allelic manner (sample size, WT: 646 nuclei, <i>SeT</i>^<i>-/-</i>: 890 nuclei).</p

Designing intelligent games adapting to children’s playing maturity

Play is a voluntary activity in which individuals involve for pleasure. It is very important for children because through playing they learn to explore, develop and master physical and social skills. Play development is part of the child’s growth and maturation process since birth. As such, it is widely used in the context of Occupational Therapy (OT). Occupational therapists use activity analysis to shape play activities for therapeutic use and promote an environment where the child can approach various activities while playing. This paper builds on knowledge stemming from the processes and theories used in OT and activity analysis to present the design, implementation and deployment of a new version of the popular farm game as deployed within an Ambient Intelligence (AmI) simulation space. Within this space, an augmented interactive table and a three-dimensional avatar are employed to extend the purpose and objectives of the game, thus also expanding its applicability to the age group of preschool children from 3 to 6 years old. More importantly, through the environment, the game monitors and follows the progress of each young player, adapts accordingly and provides important information regarding the abilities and skills of the child and their development over time. The developed game was evaluated through a small scale study with children of the aforementioned age groups, their parents, and child care professionals. The outcomes of the evaluation were positive for all target groups and provided significant evidence regarding its potential to offer novel play experience to children, but also act as a valuable tool to child care professionals

<i>Tnfα</i> expression in wild type and <i>SeT</i> deficient mice.

<p>(A) Relative <i>Tnfα</i> mRNA expression levels, calculated by the 2^-ΔΔC(t) method, showing overexpression of <i>Tnfα</i> in <i>SeT</i> deficient compared to wild type murine macrophages (BMDMs) upon LPS stimulation in a time frame of six hours. (B) Confocal-derived Z-sections portraying the homologous pairing of <i>Tnfα</i> alleles in wild type and CX3CR1-Cre <i>SeT</i>^<i>fl/fl</i> BMDMs, one hour upon LPS stimulation (scale bar 2 μm). Bar graph depicting the deregulated pattern of paired alleles between wild type and CX3CR1-Cre <i>SeT</i>^<i>fl/fl</i> BMDMs upon LPS stimulation for three hours. Only partially or completely overlapping DNA FISH signals were calculated as paired alleles (sample size, WT: 552 nuclei, CX3CR1-Cre <i>SeT</i>^<i>fl/fl</i>: 495 nuclei). (C) Single-stack confocal images showing the frequency of mono- versus bi-allelic expression of <i>Tnfα</i> in wild type and CX3CR1-Cre <i>SeT</i>^<i>fl/fl</i> BMDMs (scale bar 2 μm) (sample size, WT: 552 nuclei, CX3CR1-Cre <i>SeT</i>^<i>fl/fl</i>: 495 nuclei). (D) Survival rates of wild type and <i>SeT</i>^<i>-/-</i> mice expressed in percentage of mice out of the total number of individuals per genotype (12 mice per genotype) responding to the intraperitoneal injection of lethal LPS dose.</p