Search

ObjectivesRecent insights supporting the safety of live-attenuated vaccines and novel studies on the immunogenicity of vaccinations in the era of biological disease-modifying antirheumatic drugs in paediatric patients with autoimmune/inflammatory rheumatic diseases (pedAIIRD) necessitated updating the EULAR recommendations.MethodsRecommendations were developed using the EULAR standard operating procedures. Two international expert committees were formed to update the vaccination recommendations for both paediatric and adult patients with AIIRD. After a systematic literature review, separate recommendations were formulated for paediatric and adult patients. For pedAIIRD, six overarching principles and seven recommendations were formulated and provided with the level of evidence, strength of recommendation and Task Force level of agreement.ResultsIn general, the National Immunisation Programmes (NIP) should be followed and assessed yearly by the treating specialist. If possible, vaccinations should be administered prior to immunosuppressive drugs, but necessary treatment should never be postponed. Non-live vaccines can be safely given to immunosuppressed pedAIIRD patients. Mainly, seroprotection is preserved in patients receiving vaccinations on immunosuppression, except for high-dose glucocorticoids and B-cell depleting therapies. Live-attenuated vaccines should be avoided in immunosuppressed patients. However, it is safe to administer the measles–mumps–rubella booster and varicella zoster virus vaccine to immunosuppressed patients under specific conditions. In addition to the NIP, the non-live seasonal influenza vaccination should be strongly considered for immunosuppressed pedAIIRD patients.ConclusionsThese recommendations are intended for paediatricians, paediatric rheumatologists, national immunisation agencies, general practitioners, patients and national rheumatology societies to attain safe and effective vaccination and optimal infection prevention in immunocompromised pedAIIRD patients.

The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.

DNA folding and dynamics along with major nuclear functions are determined by chromosome structural properties, which remain, thus far, elusive in vivo. Here, we combine polymer modeling and single particle tracking experiments to determine the physico-chemical parameters of chromatin in vitro and in living yeast. We find that the motion of reconstituted chromatin fibers can be recapitulated by the Rouse model using mechanical parameters of nucleosome arrays deduced from structural simulations. Conversely, we report that the Rouse model shows some inconsistencies to analyze the motion and structural properties inferred from yeast chromosomes determined with chromosome conformation capture techniques (specifically, Hi-C). We hence introduce the Rouse model with Transient Internal Contacts (RouseTIC), in which random association and dissociation occurs along the chromosome contour. The parametrization of this model by fitting motion and Hi-C data allows us to measure the kinetic parameters of the contact formation reaction. Chromosome contacts appear to be transient; associated to a lifetime of seconds and characterized by an attractive energy of –0.3 to –0.5 kBT. We suggest attributing this energy to the occurrence of histone tail-DNA contacts and notice that its amplitude sets chromosomes in ‘theta’ conditions, in which they are poised for compartmentalization and phase separation.

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that wild-type RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I., This work was supported by the Agence Nationale de la Recherche (ANDY) and the IDEX of Toulouse University (Clemgene and Nudgene). We thank Margaux Cescato, Lilian Guillot and Jorge Perez-Fernandez for strain and plasmid constructions. YCp50-26 carrying RPA49 was kindly provided by P. Thuriaux. We also thank Melanie Panarotto for the Miller spread analysis. This work benefited from exchanges with all unit members, including Anthony Henras, Frederic Beckouët, Lise Dauban, and Marta Kwapisz. We thank Stephanie Balor and Vanessa Soldan from the METI platform for the TEM acquisitions. CFT was supported by grants BFU2013-48374-P and BFU2017-87397-P of the Spanish Ministry of Economy and Competitiveness, and by the Ramón Areces Foundation.

International audience; Branched chain amino acids (BCAA) are essential elements of the human diet, which display increased plasma levels in obesity and regained particular interest as potential biomarkers for development of diabetes. To define determinants of insulin resistance (IR) we investigated 73 genes involved in BCAA metabolism or regulation by fine-scale haplotype mapping in two European populations with metabolic syndrome. French and Romanians (n = 465) were genotyped for SNPs (Affymetrix) and enriched by imputation (BEAGLE 4.1) at 1000 genome project density. Initial association hits detected by sliding window were refined (HAPLOVIEW 3.1 and PHASE 2.1) and correlated to homeostasis model assessment (HOMAIR) index, in vivo insulin sensitivity (SI) and BCAA plasma levels (ANOVA). Four genomic regions were associated with IR located downstream of MUT, AACS, SLC6A15 and PRKCA genes (P between 9.3 and 3.7 x 10-5). Inferred haplotypes up to 13 SNPs length were associated with IR (e.g. MUT gene with P < 4.9 x 10-5; Bonferroni 1.3 x 10-3) and synergistic to HOMAIR. SNPs in the same regions were also associated with one order of magnitude lower P values (e.g. rs20167284 in the MUT gene with P < 1.27 x 10-4) and replicated in Mediterranean samples (n = 832). In French, influential haplotypes (OR > 2.0) were correlated with in vivo insulin sensitivity (1/SI) except for SLC6A15 gene. Association of these genes with BCAA levels was variable, but influential haplotypes confirmed implication of MUT from BCAA metabolism as well as a role of regulatory genes (AACS and PRKCA) and suggested potential changes in transcriptional activity. These data drive attention towards new regulatory regions involved in IR in relation with BCAA and show the ability of haplotypes in phased DNA to detect signals complimentary to SNPs, which may be useful in designing genetic markers for clinical applications in ethnic populations.

How spatial organization of the genome depends on nuclear shape is unknown, mostly because accurate nuclear size and shape measurement is technically challenging. In large cell populations of the yeast Saccharomyces cerevisiae, we assessed the geometry (size and shape) of nuclei in three dimensions with a resolution of 30 nm. We improved an automated fluorescence localization method by implementing a post-acquisition correction of the spherical microscopic aberration along the z-axis, to detect the three dimensional (3D) positions of nuclear pore complexes (NPCs) in the nuclear envelope. Here, we used a method called NucQuant to accurately estimate the geometry of nuclei in 3D throughout the cell cycle. To increase the robustness of the statistics, we aggregated thousands of detected NPCs from a cell population in a single representation using the nucleolus or the spindle pole body (SPB) as references to align nuclei along the same axis. We could detect asymmetric changes of the nucleus associated with modification of nucleolar size. Stereotypical modification of the nucleus toward the nucleolus further confirmed the asymmetric properties of the nuclear envelope., Summary: This novel method to explore 3D geometry of the nuclear envelope with enhanced resolution and post-acquisition correction of z-axis aberration revealed increased NPC density near the SPB and the nucleolus.

In budding yeast, RNA polymerase III–transcribed genes preferentially associate with the nucleolar and nuclear periphery when permitted by the Rabl-like orientation of interphase chromosomes., The association of RNA polymerase III (Pol III)–transcribed genes with nucleoli seems to be an evolutionarily conserved property of the spatial organization of eukaryotic genomes. However, recent studies of global chromosome architecture in budding yeast have challenged this view. We used live-cell imaging to determine the intranuclear positions of 13 Pol III–transcribed genes. The frequency of association with nucleolus and nuclear periphery depends on linear genomic distance from the tethering elements—centromeres or telomeres. Releasing the hold of the tethering elements by inactivating centromere attachment to the spindle pole body or changing the position of ribosomal DNA arrays resulted in the association of Pol III–transcribed genes with nucleoli. Conversely, ectopic insertion of a Pol III–transcribed gene in the vicinity of a centromere prevented its association with nucleolus. Pol III–dependent transcription was independent of the intranuclear position of the gene, but the nucleolar recruitment of Pol III–transcribed genes required active transcription. We conclude that the association of Pol III–transcribed genes with the nucleolus, when permitted by global chromosome architecture, provides nucleolar and/or nuclear peripheral anchoring points contributing locally to intranuclear chromosome organization.

Introduction: Insulin gene VNTR was associated with polycystic ovary syndrome (PCOS) in some studies but not in others. This couldb be due to the heterogeneity of the definition of PCOS and/or the use of inappropriate gene mapping strategies. Material and methods: In this investigation, the association of VNTR with PCOS was explored in a population of women from Central Europe (377 cases and 105 controls) in whom PCOS was diagnosed according to Rotterdam criteria. Seven SNPs: rs3842756 (G/A), rs3842755 (G/T), rs3842754 (C/T), rs3842753 (A/C), rs3842752 (C/T), rs3842748 (G/C), and rs689 (T/A) were genotyped in a portion of the population (160 cases and 95 controls) by sequencing or by SSO-PCR. Analysis of linkage disequilibrium (LD) pattern allowed selecting three tagSNPs (rs3842754, rs3842748, and rs689), which were genotyped in the rest of the population by KASPar. Results: Six haplotypes were reconstructed, among which three (h1, h2 and h6) were more frequent. Statistical analysis allowed observation of the association of the SNP rs3842748, through its GC genotype, with obesity in PCOS (P = 0.049; OR CI95% 1,59 [1.00–2.51]) and in classical PCOS (YPCOS) (P = 0.010), as well as the correlation of the SNP rs689 and the pair of haplotypes h1/h1 with higher levels of testosteronaemia in the PCOS group, although this was at the limit of significance (P = 0.054) Conclusion: These results are in accordance with some studies in literature and highlight the role of insulin gene VNTR in complex metabolic disorders. (Endokrynol Pol 2015; 66 (3): 198–206)

The FTO (fat mass and obesity associated) gene was associated with different metabolic disorders in populations from different origins but with great difference between African and non-African populations. North-African populations combine many genetic backgrounds, among which African, Berber and Caucasian components, which makes North-Africans a good model for studying the genetic association of FTO. In the present investigation we explored the association of FTO gene with polycystic ovary syndrome (PCOS) in a population from Tunisia (n=278). Single nucleotide polymorphisms (SNPs) used in this study were previously associated in non-African populations: rs8050136 (A/C), rs9939609 (A/T), rs9930506 (G/A), or in both African and non-African populations: rs8057044 (A/G). Genotyping was performed by allelic discrimination method on StepOne real-time PCR system or KASPar technology. Linkage disequilibrium (LD) pattern was assessed by HAPLOVIEW and reconstruction of haplotypes was performed by PHASE, while statistical analyses were performed using StatView and GoldenHelix programs. Among the 13 haplotypes in the population, three (h1, h7 and h13) were strongly associated with PCOS notably h13 (P

Chromosome mechanical properties determine DNA folding and dynamics, and underlie all major nuclear functions. Here we combine modeling and real-time motion tracking experiments to infer the physical parameters describing chromatin fibers. In vitro, motion of nucleosome arrays can be accurately modeled by assuming a Kuhn length of 35-55 nm. In vivo, the amplitude of chromosome fluctuations is drastically reduced, and depends on transcription. Transcription activation increases chromatin dynamics only if it involves gene relocalization, while global transcriptional inhibition augments the fluctuations, yet without relocalization. Chromatin fiber motion is accounted for by a model of equilibrium fluctuations of a polymer chain, in which random contacts along the chromosome contour induce an excess of internal friction. Simulations that reproduce chromosome conformation capture and imaging data corroborate this hypothesis. This model unravels the transient nature of chromosome contacts, characterized by a life time of ∼2 seconds and a free energy of formation of ∼1 kBT.

Transcription by RNA polymerase I (Pol-I) is the main driving force behind ribosome biogenesis, a fundamental cellular process that requires the coordinated transcription of all three nuclear polymerases. Increased Pol-I transcription and the concurrent increase in ribosome biogenesis has been linked to the high rates of proliferation in cancers. The ellipticine family contains a number of potent anticancer therapeutic agents, some having progressed to stage I and II clinical trials; however, the mechanism by which many of the compounds work remains unclear. It has long been thought that inhibition of Top2 is the main reason behind the drugs antiproliferative effects. Here we report that a number of the ellipticines, including 9-hydroxyellipticine, are potent and specific inhibitors of Pol-I transcription, with IC50 in vitro and in cells in the nanomolar range. Essentially, the drugs did not affect Pol-II and Pol-III transcription, demonstrating a high selectivity. We have shown that Pol-I inhibition occurs by a p53-, ATM/ATR-, and Top2-independent mechanism. We discovered that the drug influences the assembly and stability of preinitiation complexes by targeting the interaction between promoter recognition factor SL1 and the rRNA promoter. Our findings will have an impact on the design and development of novel therapeutic agents specifically targeting ribosome biogenesis. Background: rRNA synthesis by Pol-I is a key and rate-limiting stage of ribosome biogenesis. Results: Ellipticines selectively inhibit Pol-I transcription both in vitro and in cells. Conclusion: Interactions of essential transcription factor SL1 with the promoter is the primary target of the drugs. Significance: This study reveals a novel class of Pol-I inhibitors and analyses their mechanism of action.

Insulin gene VNTR was associated with polycystic ovary syndrome (PCOS) in some studies but not in others. This couldb be due to the heterogeneity of the definition of PCOS and/or the use of inappropriate gene mapping strategies.In this investigation, the association of VNTR with PCOS was explored in a population of women from Central Europe (377 cases and 105 controls) in whom PCOS was diagnosed according to Rotterdam criteria. Seven SNPs: rs3842756 (G/A), rs3842755 (G/T), rs3842754 (C/T), rs3842753 (A/C), rs3842752 (C/T), rs3842748 (G/C), and rs689 (T/A) were genotyped in a portion of the population (160 cases and 95 controls) by sequencing or by SSO-PCR. Analysis of linkage disequilibrium (LD) pattern allowed selecting three tagSNPs (rs3842754, rs3842748, and rs689), which were genotyped in the rest of the population by KASPar.Six haplotypes were reconstructed, among which three (h1, h2 and h6) were more frequent. Statistical analysis allowed observation of the association of the SNP rs3842748, through its GC genotype, with obesity in PCOS (P = 0.049; OR CI95% 1,59 [1.00-2.51]) and in classical PCOS (YPCOS) (P = 0.010), as well as the correlation of the SNP rs689 and the pair of haplotypes h1/h1 with higher levels of testosteronaemia in the PCOS group, although this was at the limit of significance (P = 0.054) CONCLUSION: These results are in accordance with some studies in literature and highlight the role of insulin gene VNTR in complex metabolic disorders.

IS911 transposition involves a free circular transposon intermediate where the terminal inverted repeat sequences are connected. Transposase synthesis is usually driven by a weak promoter, p(IRL), in the left end (IRL). Circle junction formation creates a strong promoter, p(junc), with a -35 sequence located in the right end and the -10 sequence in the left. p(junc) assembly would permit an increase in synthesis of transposase from the transposon circle, which would be expected to stimulate integration. Insertion results in p(junc) disassembly and a return to the low p(IRL)- driven transposase levels. We demonstrate that p(junc) plays an important role in regulating IS911 transposition. Inactivation of p(junc) strongly decreased IS911 transposition when transposase was produced in its natural configuration. This novel feedback mechanism permits transient and controlled activation of integration only in the presence of the correct (circular) intermediate. We have also investigated other members of the IS3 and other IS families. Several, but not all, IS3 family members possess p(junc) equivalents, underlining that the regulatory mechanisms adopted to fine-tune transposition may be different.

Ribosome biogenesis is a major metabolic effort for growing cells. In Saccharomyces cerevisiae, Hmo1, an abundant high-mobility group box protein (HMGB) binds to the coding region of the RNA polymerase I transcribed ribosomal RNAs genes and the promoters of similar to 70% of ribosomal protein genes. In this study, we have demonstrated the functional conservation of eukaryotic HMGB proteins involved in ribosomal DNA (rDNA) transcription. We have shown that when expressed in budding yeast, human UBF1 and a newly identified Sp-Hmo1 (Schizosaccharomyces pombe) localize to the nucleolus and suppress growth defect of the RNA polymerase I mutant rpa49-Delta. Owing to the multiple functions of both proteins, Hmo1 and UBF1 are not fully interchangeable. By deletion and domains swapping in Hmo1, we identified essential domains that stimulate rDNA transcription but are not fully required for stimulation of ribosomal protein genes expression. Hmo1 is organized in four functional domains: a dimerization module, a canonical HMGB motif followed by a conserved domain and a C-terminal nucleolar localization signal. We propose that Hmo1 has acquired species-specific functions and shares with UBF1 and Sp-Hmo1 an ancestral function to stimulate rDNA transcription.

Comprehensive analysis of the intranuclear territories and motion of budding yeast chromosome XII loci suggests that long-range chromosome architecture is mainly determined by the physical principles of polymers., Chromosomes architecture is viewed as a key component of gene regulation, but principles of chromosomal folding remain elusive. Here we used high-throughput live cell microscopy to characterize the conformation and dynamics of the longest chromosome of Saccharomyces cerevisiae (XII). Chromosome XII carries the ribosomal DNA (rDNA) that defines the nucleolus, a major hallmark of nuclear organization. We determined intranuclear positions of 15 loci distributed every ∼100 kb along the chromosome, and investigated their motion over broad time scales (0.2–400 s). Loci positions and motions, except for the rDNA, were consistent with a computational model of chromosomes based on tethered polymers and with the Rouse model from polymer physics, respectively. Furthermore, rapamycin-dependent transcriptional reprogramming of the genome only marginally affected the chromosome XII internal large-scale organization. Our comprehensive investigation of chromosome XII is thus in agreement with recent studies and models in which long-range architecture is largely determined by the physical principles of tethered polymers and volume exclusion.

The Pol I subunits Rpa34 and Rpa49, required for elongation and 35S rRNA production, promote clustering of neighboring Pol I complexes to enhance the rRNA gene transcription cycle., RNA polymerase I (Pol I) produces large ribosomal RNAs (rRNAs). In this study, we show that the Rpa49 and Rpa34 Pol I subunits, which do not have counterparts in Pol II and Pol III complexes, are functionally conserved using heterospecific complementation of the human and Schizosaccharomyces pombe orthologues in Saccharomyces cerevisiae. Deletion of RPA49 leads to the disappearance of nucleolar structure, but nucleolar assembly can be restored by decreasing ribosomal gene copy number from 190 to 25. Statistical analysis of Miller spreads in the absence of Rpa49 demonstrates a fourfold decrease in Pol I loading rate per gene and decreased contact between adjacent Pol I complexes. Therefore, the Rpa34 and Rpa49 Pol I–specific subunits are essential for nucleolar assembly and for the high polymerase loading rate associated with frequent contact between adjacent enzymes. Together our data suggest that localized rRNA production results in spatially constrained rRNA production, which is instrumental for nucleolar assembly.

In DNA site-specific recombination catalysed by tyrosine recombinases, two pairs of DNA strands are sequentially exchanged between separate duplexes and the mechanisms that confer directionality to this theoretically reversible reaction remain unclear. The tyrosine recombinase TnpI acts at the internal resolution site (IRS) of the transposon Tn4430 to resolve intermolecular transposition products. Recombination is catalysed at the IRS core sites (IR1-IR2) and is regulated by adjacent TnpI-binding motifs (DR1 and DR2). These are dispensable accessory sequences that confer resolution selectivity to the reaction by stimulating synapsis between directly repeated IRSs. Here, we show that formation of the DR1-DR2-containing synapse imposes a specific order of activation of the TnpI catalytic subunits in the complex so that the IR1-bound subunits catalyse the first strand exchange and the IR2-bound subunits the second strand exchange. This ordered pathway was demonstrated for a complete recombination reaction using a TnpI catalytic mutant (TnpI-H234L) partially defective in DNA rejoining. The presence of the DR1- and DR2-bound TnpI subunits was also found to stabilize transient recombination intermediates, further displacing the reaction equilibrium towards product formation. Implication of TnpI/IRS accessory elements in the initial architecture of the synapse and subsequent conformational changes taking place during strand exchange is discussed.

During ribosome biogenesis, the RNA precursor to mature rRNAs undergoes numerous post-transcriptional chemical modifications of bases, including conversions of uridines to pseudouridines. In archaea and eukaryotes, these conversions are performed by box H/ACA small ribonucleoprotein particles (box H/ACA RNPs), which contain a small guide RNA responsible for the selection of substrate uridines and four proteins, including the pseudouridine synthase, Cbf5p. So far, no in vitro reconstitution of eukaryotic box H/ACA RNPs from purified components has been achieved, principally due to difficulties in purifying recombinant eukaryotic Cbf5p. In this study, we present the purification of a truncated derivative of yeast Cbf5p (Cbf5Δp) that retains the highly conserved TRUB and PUA domains. We have used band retardation assays to show that Cbf5Δp on its own binds to box H/ACA small nucleolar (sno)RNAs. We demonstrate that the conserved H and ACA boxes enhance the affinity of the protein for the snoRNA. Furthermore, like its archaeal homologs, Cbf5Δp can bind to a single stem–loop-box ACA RNA. Finally, we report the first enzymatic footprinting analysis of a Cbf5–RNA complex. Our results are compatible with the view that two molecules of Cbf5p interact with a binding platform constituted by the 5′ end of the RNA, the single-stranded hinge domain containing the conserved H box, and the 3′ end of the molecule, including the conserved ACA box.