Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes

Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1’s intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42’s unresponsiveness. Rather, Zfp42’s promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.We thank the Montpellier Ressources Imagerie facility (BioCampus Montpellier, Centre National de la Recherche Scientifique [CNRS], INSERM, University of Montpellier) and for computer resources from CINECA (ISCRA grant thanks to computer resources from INFN and CINECA [ISCRA Grant HP10C8JWU7]). G.C., Q.S., and F.B. were supported by a grant from the European Research Council (Advanced Grant 3DEpi, 788972) and by the CNRS. This work was funded by EMBO and the Wellcome Trust (ALTF1554-2016 and 206475/Z/17/Z; to M.I.R.) as well as the Deutsche Forschungsgemeinschaft (KR3985/7-3 and MU 880/16-1 to S.M.)

Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial

Background Non-alcoholic steatohepatitis (NASH) is a common type of chronic liver disease that can lead to cirrhosis. Obeticholic acid, a farnesoid X receptor agonist, has been shown to improve the histological features of NASH. Here we report results from a planned interim analysis of an ongoing, phase 3 study of obeticholic acid for NASH. Methods In this multicentre, randomised, double-blind, placebo-controlled study, adult patients with definite NASH,non-alcoholic fatty liver disease (NAFLD) activity score of at least 4, and fibrosis stages F2–F3, or F1 with at least oneaccompanying comorbidity, were randomly assigned using an interactive web response system in a 1:1:1 ratio to receive oral placebo, obeticholic acid 10 mg, or obeticholic acid 25 mg daily. Patients were excluded if cirrhosis, other chronic liver disease, elevated alcohol consumption, or confounding conditions were present. The primary endpointsfor the month-18 interim analysis were fibrosis improvement (≥1 stage) with no worsening of NASH, or NASH resolution with no worsening of fibrosis, with the study considered successful if either primary endpoint was met. Primary analyses were done by intention to treat, in patients with fibrosis stage F2–F3 who received at least one dose of treatment and reached, or would have reached, the month 18 visit by the prespecified interim analysis cutoff date. The study also evaluated other histological and biochemical markers of NASH and fibrosis, and safety. This study is ongoing, and registered with ClinicalTrials.gov, NCT02548351, and EudraCT, 20150-025601-6. Findings Between Dec 9, 2015, and Oct 26, 2018, 1968 patients with stage F1–F3 fibrosis were enrolled and received at least one dose of study treatment; 931 patients with stage F2–F3 fibrosis were included in the primary analysis (311 in the placebo group, 312 in the obeticholic acid 10 mg group, and 308 in the obeticholic acid 25 mg group). The fibrosis improvement endpoint was achieved by 37 (12%) patients in the placebo group, 55 (18%) in the obeticholic acid 10 mg group (p=0·045), and 71 (23%) in the obeticholic acid 25 mg group (p=0·0002). The NASH resolution endpoint was not met (25 [8%] patients in the placebo group, 35 [11%] in the obeticholic acid 10 mg group [p=0·18], and 36 [12%] in the obeticholic acid 25 mg group [p=0·13]). In the safety population (1968 patients with fibrosis stages F1–F3), the most common adverse event was pruritus (123 [19%] in the placebo group, 183 [28%] in the obeticholic acid 10 mg group, and 336 [51%] in the obeticholic acid 25 mg group); incidence was generally mild to moderate in severity. The overall safety profile was similar to that in previous studies, and incidence of serious adverse events was similar across treatment groups (75 [11%] patients in the placebo group, 72 [11%] in the obeticholic acid 10 mg group, and 93 [14%] in the obeticholic acid 25 mg group). Interpretation Obeticholic acid 25 mg significantly improved fibrosis and key components of NASH disease activity among patients with NASH. The results from this planned interim analysis show clinically significant histological improvement that is reasonably likely to predict clinical benefit. This study is ongoing to assess clinical outcomes

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Lessons from non-canonical splicing

Recent improvements in experimental and computational techniques that are used to study the transcriptome have enabled an unprecedented view of RNA processing, revealing many previously unknown non-canonical splicing events. This includes cryptic events located far from the currently annotated exons and unconventional splicing mechanisms that have important roles in regulating gene expression. These non-canonical splicing events are a major source of newly emerging transcripts during evolution, especially when they involve sequences derived from transposable elements. They are therefore under precise regulation and quality control, which minimizes their potential to disrupt gene expression. We explain how non-canonical splicing can lead to aberrant transcripts that cause many diseases, and also how it can be exploited for new therapeutic strategies

Variation in general supportive and preventive intensive care management of traumatic brain injury: a survey in 66 neurotrauma centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study

Abstract Background General supportive and preventive measures in the intensive care management of traumatic brain injury (TBI) aim to prevent or limit secondary brain injury and optimize recovery. The aim of this survey was to assess and quantify variation in perceptions on intensive care unit (ICU) management of patients with TBI in European neurotrauma centers. Methods We performed a survey as part of the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. We analyzed 23 questions focused on: 1) circulatory and respiratory management; 2) fever control; 3) use of corticosteroids; 4) nutrition and glucose management; and 5) seizure prophylaxis and treatment. Results The survey was completed predominantly by intensivists (n = 33, 50%) and neurosurgeons (n = 23, 35%) from 66 centers (97% response rate). The most common cerebral perfusion pressure (CPP) target was > 60 mmHg (n = 39, 60%) and/or an individualized target (n = 25, 38%). To support CPP, crystalloid fluid loading (n = 60, 91%) was generally preferred over albumin (n = 15, 23%), and vasopressors (n = 63, 96%) over inotropes (n = 29, 44%). The most commonly reported target of partial pressure of carbon dioxide in arterial blood (PaCO2) was 36–40 mmHg (4.8–5.3 kPa) in case of controlled intracranial pressure (ICP) < 20 mmHg (n = 45, 69%) and PaCO2 target of 30–35 mmHg (4–4.7 kPa) in case of raised ICP (n = 40, 62%). Almost all respondents indicated to generally treat fever (n = 65, 98%) with paracetamol (n = 61, 92%) and/or external cooling (n = 49, 74%). Conventional glucose management (n = 43, 66%) was preferred over tight glycemic control (n = 18, 28%). More than half of the respondents indicated to aim for full caloric replacement within 7 days (n = 43, 66%) using enteral nutrition (n = 60, 92%). Indications for and duration of seizure prophylaxis varied, and levetiracetam was mostly reported as the agent of choice for both seizure prophylaxis (n = 32, 49%) and treatment (n = 40, 61%). Conclusions Practice preferences vary substantially regarding general supportive and preventive measures in TBI patients at ICUs of European neurotrauma centers. These results provide an opportunity for future comparative effectiveness research, since a more evidence-based uniformity in good practices in general ICU management could have a major impact on TBI outcome

Étude du repliement tridimensionnel de la chromatine en domaines topologiques

My thesis project consisted in studying the mechanisms of the three-dimensional genome folding in eukaryotic cells. The organization of chromosomes is closely related to the regulation of many biological processes, such as gene expression control, DNA replication or genomic stability. The Hi-C "chromosome conformation capture" method, which allows the mapping of interactions between DNA regions, has revealed that the genome of many species is organized into domains enriched in chromatin interactions, the "Topologically Associating Domains" (TADs). TADs have emerged as major players of genome regulation by their ability to spatially define functional domains. However, chromosome conformation capture methods generate averaged interaction profiles that generally come from an ensemble of cells. Determining the nature and the folding of TADs in individual cells is therefore crucial to better understand the structure-function relationship of these domains. During my thesis, I used a combination of fluorescent DNA labeling and super-resolution microscopy to characterize the organization of chromosomes in single cells. In Drosophila, TADs coincide with the partitioning of the chromatin into distinct epigenetic domains. In this species, we could characterize the folding of the chromosomes into a series of discrete units that correspond to TADs, reflecting the mutual exclusion of transcriptionally active and inactive regions. These results indicate that Drosophila TADs form physical domains that characterize a higher-order layer of chromosome folding in individual cells. In mammals, the majority of TADs emerge through the action of the cohesin complex and the CCCTC-binding factor (CTCF) bound at their borders. The application of super-resolution imaging in mouse embryonic stem cells and neuronal progenitor cells revealed the high degree of cell-to-cell heterogeneity of TAD folding, ranging from condensed and globular objects to dispersed and stretched conformations. We were able to observe their organization into discrete subdomains which seem to represent a general property of the folding of the chromatin fiber at the nanoscale. Furthermore, our data indicate that the physical intermingling of the chromatin is highly favored within TADs in a large majority of cells. Depletion of CTCF abolishes the TAD-dependent spatial organization of the chromatin fiber, highlighting the role of this protein in generating physical barriers between adjacent TADs. Altogether, our results demonstrate that the dynamic folding of TAD is compatible with the establishment of chromosomal environments in which contacts are privileged, and thus reconcile the probabilistic nature of chromatin folding with the proposed role of TADs in the spatial definition of functional genomic units.Mon projet de thèse a consisté à étudier les mécanismes du repliement tridimensionnel du génome dans les cellules eucaryotes. L’organisation des chromosomes est étroitement liée à la régulation de nombreuses fonctions biologiques, telles que le contrôle de l’expression génique, la réplication de l’ADN ou encore la stabilité génomique. La méthode de « chromosome conformation capture » Hi-C, qui permet la cartographie des interactions entre régions d’ADN, a révélé que chez de nombreuses espèces, le génome est organisé en domaines enrichis en interactions chromatiniennes, les « Topologically Associating Domains » (TADs). Les TADs sont apparus être des acteurs majeurs de la régulation du génome par leur capacité à définir spatialement des domaines fonctionnels. Cependant, les méthodes de chromosome conformation capture générèrent des profils d’interactions généralement moyennés à partir d’ensemble de cellules. Déterminer la nature du repliement des TADs en cellules individuelles est donc crucial pour comprendre la relation structure-fonction de ces domaines génomiques. Au cours de ma thèse, j’ai utilisé des techniques de marquage fluorescent d'ADN combinés à de la microscopie en super-résolution afin d’étudier l’organisation des chromosomes en cellules uniques. Chez la drosophile, les TADs coïncident avec le partitionnement de la chromatine en domaines épigénétiques distincts. Nous avons pu caractériser chez cette espèce que les chromosomes sont organisés en une série d’unités discrètes qui correspondent aux TADs, reflétant l’exclusion mutuelle de régions transcriptionnellement actives et inactives. Ces résultats indiquent que les TADs de drosophile forment des domaines physiques qui caractérisent un niveau d’organisation structurale des chromosomes en cellules uniques. Chez les mammifères, la majorité des TADs est formée grâce à l’action du complexe cohésine et à la présence de la protéine CCCTC-binding factor (CTCF) à leurs frontières. L'application de l'imagerie à super-résolution dans des cellules souches embryonnaires et des cellules progénitrices neuronales de souris nous a permis de caractériser l’hétérogénéité du repliement des TAD d’une cellule à l’autre. Nous avons notamment pu observer leur organisation en sous-domaines globulaires qui semblent représenter une propriété générale du repliement de la chromatine à l’échelle de la centaine de nanomètres. De plus, nos résultats indiquent que les interactions chromatiniennes sont fortement favorisées à l’intérieur des TADs dans la majorité des cellules. La déplétion de CTCF abolie l’organisation spatiale de la fibre de chromatine associée aux TAD, soulignant le rôle de cette protéine dans la génération de barrières physiques entre TAD adjacents. Ces données démontrent que le repliement dynamique des TAD est compatible avec l'établissement d'environnements chromosomiques dans lesquels les contacts sont privilégiés, et réconcilient ainsi la nature probabiliste du repliement de la chromatine avec le rôle proposé des TAD dans la définition spatiale d’unités génomiques fonctionnelles

Three-dimensional chromatin folding into topologically associating domains

Mon projet de thèse a consisté à étudier les mécanismes du repliement tridimensionnel du génome dans les cellules eucaryotes. L’organisation des chromosomes est étroitement liée à la régulation de nombreuses fonctions biologiques, telles que le contrôle de l’expression génique, la réplication de l’ADN ou encore la stabilité génomique. La méthode de « chromosome conformation capture » Hi-C, qui permet la cartographie des interactions entre régions d’ADN, a révélé que chez de nombreuses espèces, le génome est organisé en domaines enrichis en interactions chromatiniennes, les « Topologically Associating Domains » (TADs). Les TADs sont apparus être des acteurs majeurs de la régulation du génome par leur capacité à définir spatialement des domaines fonctionnels. Cependant, les méthodes de chromosome conformation capture générèrent des profils d’interactions généralement moyennés à partir d’ensemble de cellules. Déterminer la nature du repliement des TADs en cellules individuelles est donc crucial pour comprendre la relation structure-fonction de ces domaines génomiques. Au cours de ma thèse, j’ai utilisé des techniques de marquage fluorescent d'ADN combinés à de la microscopie en super-résolution afin d’étudier l’organisation des chromosomes en cellules uniques. Chez la drosophile, les TADs coïncident avec le partitionnement de la chromatine en domaines épigénétiques distincts. Nous avons pu caractériser chez cette espèce que les chromosomes sont organisés en une série d’unités discrètes qui correspondent aux TADs, reflétant l’exclusion mutuelle de régions transcriptionnellement actives et inactives. Ces résultats indiquent que les TADs de drosophile forment des domaines physiques qui caractérisent un niveau d’organisation structurale des chromosomes en cellules uniques. Chez les mammifères, la majorité des TADs est formée grâce à l’action du complexe cohésine et à la présence de la protéine CCCTC-binding factor (CTCF) à leurs frontières. L'application de l'imagerie à super-résolution dans des cellules souches embryonnaires et des cellules progénitrices neuronales de souris nous a permis de caractériser l’hétérogénéité du repliement des TAD d’une cellule à l’autre. Nous avons notamment pu observer leur organisation en sous-domaines globulaires qui semblent représenter une propriété générale du repliement de la chromatine à l’échelle de la centaine de nanomètres. De plus, nos résultats indiquent que les interactions chromatiniennes sont fortement favorisées à l’intérieur des TADs dans la majorité des cellules. La déplétion de CTCF abolie l’organisation spatiale de la fibre de chromatine associée aux TAD, soulignant le rôle de cette protéine dans la génération de barrières physiques entre TAD adjacents. Ces données démontrent que le repliement dynamique des TAD est compatible avec l'établissement d'environnements chromosomiques dans lesquels les contacts sont privilégiés, et réconcilient ainsi la nature probabiliste du repliement de la chromatine avec le rôle proposé des TAD dans la définition spatiale d’unités génomiques fonctionnelles.My thesis project consisted in studying the mechanisms of the three-dimensional genome folding in eukaryotic cells. The organization of chromosomes is closely related to the regulation of many biological processes, such as gene expression control, DNA replication or genomic stability. The Hi-C "chromosome conformation capture" method, which allows the mapping of interactions between DNA regions, has revealed that the genome of many species is organized into domains enriched in chromatin interactions, the "Topologically Associating Domains" (TADs). TADs have emerged as major players of genome regulation by their ability to spatially define functional domains. However, chromosome conformation capture methods generate averaged interaction profiles that generally come from an ensemble of cells. Determining the nature and the folding of TADs in individual cells is therefore crucial to better understand the structure-function relationship of these domains. During my thesis, I used a combination of fluorescent DNA labeling and super-resolution microscopy to characterize the organization of chromosomes in single cells. In Drosophila, TADs coincide with the partitioning of the chromatin into distinct epigenetic domains. In this species, we could characterize the folding of the chromosomes into a series of discrete units that correspond to TADs, reflecting the mutual exclusion of transcriptionally active and inactive regions. These results indicate that Drosophila TADs form physical domains that characterize a higher-order layer of chromosome folding in individual cells. In mammals, the majority of TADs emerge through the action of the cohesin complex and the CCCTC-binding factor (CTCF) bound at their borders. The application of super-resolution imaging in mouse embryonic stem cells and neuronal progenitor cells revealed the high degree of cell-to-cell heterogeneity of TAD folding, ranging from condensed and globular objects to dispersed and stretched conformations. We were able to observe their organization into discrete subdomains which seem to represent a general property of the folding of the chromatin fiber at the nanoscale. Furthermore, our data indicate that the physical intermingling of the chromatin is highly favored within TADs in a large majority of cells. Depletion of CTCF abolishes the TAD-dependent spatial organization of the chromatin fiber, highlighting the role of this protein in generating physical barriers between adjacent TADs. Altogether, our results demonstrate that the dynamic folding of TAD is compatible with the establishment of chromosomal environments in which contacts are privileged, and thus reconcile the probabilistic nature of chromatin folding with the proposed role of TADs in the spatial definition of functional genomic units

Principles of genome folding into topologically associating domains

International audienceUnderstanding the mechanisms that underlie chromosome folding within cell nuclei is essential to determine the relationship between genome structure and function. The recent application of "chromosome conformation capture" techniques has revealed that the genome of many species is organized into domains of preferential internal chro-matin interactions called "topo logically associating domains" (TADs). This chromosome compartmentalization has emerged as a key feature of higher-order genome organization and function through evolution. Although TADs have now been described in a wide range of organisms, they appear to have specific characteristics in terms of size, structure, and proteins involved in their formation. Here, we depict the main features of these domains across species and discuss the relation between chromatin structure, genome activity, and epigenome, highlighting mecha-nistic principles of TAD formation. We also consider the potential influence of TADs in genome evolution

Higher-Order Chromosomal Structures Mediate Genome Function

International audienceHow chromosomes are organized within the tridimensional space of the nucleus and how can this organization affect genome function have been long-standing questions on the path to understanding genome activity and its link to disease. In the last decade, high-throughput chromosome conformation capture techniques, such as Hi-C, have facilitated the discovery of new principles of genome folding. Chromosomes are folded in multiple high-order structures, with local contacts between enhancers and promoters, intermediate-level contacts forming Topologically Associating Domains (TADs) and higher-order chromatin structures sequestering chromatin into active and repressive compartments. However, despite the increasing evidence that genome organization can influence its function, we are still far from understanding the underlying mechanisms. Deciphering these mechanisms represents a major challenge for the future, which large, international initiatives, such as 4DN, HCA and LifeTime, aim to collaboratively tackle by using a conjunction of state-of-the-art population-based and single-cell approaches