Rôle de l'histone déméthylase JMJD2A dans la régulation transcriptionnelle au cours de la différentiation musculaire

La chromatine est une structure dynamique, qui peut être modulée par divers mécanismes, dont la méthylation des histones. L'état plus ou moins compacté de la chromatine est une composante importante de la régulation de la transcription des gènes. L'objectif de ma thèse a été de déterminer le rôle de l'histone déméthylase JMJD2A dans le contrôle de l'expression des gènes dont l'activation est associée à une déméthylation de la lysine 9 de l'histone H3 (H3K9). La différenciation musculaire est caractérisée par une sortie du cycle cellulaire (associée à la répression des gènes cibles de E2F) suivie de l'induction de gènes spécifiques du muscle. Dans ce cadre, j'ai mis en évidence l'existence d'une nouvelle isoforme de JMJD2A induite lors de la différenciation musculaire et essentielle à ce processus. Ce nouveau variant, appelé DN-JMJD2A, est une forme tronquée en N-terminal de la protéine pleine taille produite à partir d'un ARNm interne au gène JMJD2A. Une approche par perte de fonction (siRNA) et puces d'expression a montré que cette petite isoforme est nécessaire à l'expression des gènes musculaires ainsi qu'au bon déroulement du processus global de différenciation musculaire, alors que la forme pleine taille n'est pas requise. DN-JMJD2A est recrutée sur le promoteur de la myogénine (facteur de transcription essentiel à la différenciation) et participe à la déméthylation locale de H3K9 lors de l'induction transcriptionnelle de ce gène. Des analyses à grande échelle (ChIP on chips) menées dans des cellules prolifératives ou différenciées ont également permis d'identifier les gènes cibles de JMJD2A dans ce modèle. D'autre part, DN-JMJD2A est aussi impliquée dans le contrôle du cycle cellulaire. En effet, la déplétion des deux isoformes de JMJD2A (par siRNA) induit une inhibition de la prolifération caractérisée par un arrêt à la transition G1/S, tandis que la déplétion de la protéine pleine taille seule n'a pas d'effet. De plus, seule la surexpression de DN-JMJD2A (et pas de la forme pleine taille) confère aux cellules un avantage prolifératif. Des expériences d'immunoprécipitation de chromatine (ChIP) montrent que JMJD2A est recrutée au niveau des promoteurs des gènes cibles de E2F en corrélation l'induction de leur expression, suggérant que le rôle de DN-JMJD2A dans le contrôle du cycle cellulaire passe par la régulation des gènes cibles de E2F, impliqué dans l'entrée et l'avancée des cellules en phase S. Dans leur ensemble, les résultats obtenus au cours de ma thèse mettent en évidence le rôle crucial de DN-JMJD2A à la fois dans le contrôle de la différenciation musculaire et de la prolifération cellulaire, soulignant l'importance des variants d'enzymes de modification sans activité catalytique dans la régulation de l'expression des gènes.Chromatin modifications, such as histone methylation, are key components in the transcriptional control of gene expression. During my PhD, I investigated the function of the JMJD2A histone demethylase in the regulation of gene expression. During skeletal muscle differentiation, determined cells exit from the cell cycle and the expression of tissue-specific genes is induced. In this context, I described for the first time the existence of a second variant of the histone demethylase JMJD2A and its essential role in the regulation of muscle specific genes transcription. Indeed, I showed that a short isoform of JMJD2A truncated for the N-terminal demethylase domain is induced during differentiation. This isoform, called DN-JMJD2A, is necessary for the differentiation process through regulation of muscle specific genes expression, at a step earlier than myogenin induction. Although JMJD2A short isoform does not contain a demethylase domain, its role on muscle gene expression is direct, being recruited on myogenin promoter at the onset of differentiation to demethylate H3K9, suggesting that it recruits another demethylase specific of H3K9. Genome wide targets of JMJD2A were also identified in proliferative and differentiated cells by ChIP on chip. In addition, other results show that DN-JMJD2A regulates cell proliferation. Indeed, the depletion of both isoforms of JMJD2A leads to proliferation defects, characterized by an accumulation of the cells in G1 phase, whereas the depletion of JMJD2A full length only does not. Moreover, cells that overexpress DN-JMJD2A grow faster than control cells or cells expressing JMJD2A full lenght. ChIP experiments showed that JMJD2A is recruited on some E2F-target genes in correlation with the induction of their expression, suggesting that it can participate to their regulation during the cell cycle. Globally, this work uncovers the existence of a new isoform of JMJD2A, DN-JMJD2A, and highlights its role in the control of muscle differentiation and cell proliferation. It also underlines the importance of catalytic variants of histone modifying enzymes in transcriptional regulation

Neuromesodermal progenitors and the making of the spinal cord

Neuromesodermal progenitors (NMps) contribute to both the elongating spinal cord and the adjacent paraxial mesoderm. It has been assumed that these cells arise as a result of patterning of the anterior neural plate. However, as the molecular mechanisms that specify NMps in vivo are uncovered, and as protocols for generating these bipotent cells from mouse and human pluripotent stem cells in vitro are established, the emerging data suggest that this view needs to be revised. Here, we review the characteristics, regulation, in vitro derivation and in vivo induction of NMps. We propose that these cells arise within primitive streak-associated epiblast via a mechanism that is separable from that which establishes neural fate in the anterior epiblast. We thus argue for the existence of two distinct routes for making central nervous system progenitors

PIXSIC: A Wireless Intracerebral Radiosensitive Probe in Freely Moving Rats

International audienceThe aim of this study was to demonstrate the potential of a wireless pixelated β+-sensitive intracerebral probe (PIXSIC) for in vivo positron emission tomographic (PET) radiopharmacology in awake and freely moving rodents. The binding of [ 11 C]raclopride to D 2 dopamine receptors was measured in anesthetized and awake rats following injection of the radiotracer. Competitive binding was assessed with a cold raclopride injection 20 minutes later. The device can accurately monitor binding of PET ligands in freely moving rodents with a high spatiotemporal resolution. Reproducible time-activity curves were obtained for pixels throughout the striatum and cerebellum. A significantly lower [ 11 C]raclopride tracer–specific binding was observed in awake animals. These first results pave the way for PET tracer pharmacokinetics measurements in freely moving rodents

Global regulation of heterochromatin spreading by Leo1

Heterochromatin plays important roles in eukaryotic genome regulation. However, the repressive nature of heterochromatin combined with its propensity to self-propagate necessitates robust mechanisms to contain heterochromatin within defined boundaries and thus prevent silencing of expressed genes. Here we show that loss of the PAF complex (PAFc) component Leo1 compromises chromatin boundaries, resulting in invasion of heterochromatin into flanking euchromatin domains. Similar effects are seen upon deletion of other PAFc components, but not other factors with related functions in transcription-associated chromatin modification, indicating a specific role for PAFc in heterochromatin regulation. Loss of Leo1 results in reduced levels of H4K16 acetylation at boundary regions, while tethering of the H4K16 acetyltransferase Mst1 to boundary chromatin suppresses heterochromatin spreading in leo1 Delta cells, suggesting that Leo1 antagonises heterochromatin spreading by promoting H4K16 acetylation. Our findings reveal a previously undescribed role for PAFc in regulating global heterochromatin distribution

A New Isoform of the Histone Demethylase JMJD2A/KDM4A Is Required for Skeletal Muscle Differentiation

In proliferating myoblasts, muscle specific genes are silenced by epigenetic modifications at their promoters, including histone H3K9 methylation. Derepression of the promoter of the gene encoding the myogenic factor myogenin (Myog) is key for initiation of muscle differentiation. The mechanism of H3K9 demethylation at the Myog promoter is unclear, however. Here, we identify an isoform of the histone demethylase JMJD2A/KDM4A that lacks the N-terminal demethylase domain (ΔN-JMJD2A). The amount of ΔN-JMJD2A increases during differentiation of C2C12 myoblasts into myotubes. Genome-wide expression profiling and exon-specific siRNA knockdown indicate that, in contrast to the full-length protein, ΔN-JMJD2A is necessary for myotube formation and muscle-specific gene expression. Moreover, ΔN-JMJD2A promotes MyoD-induced conversion of NIH3T3 cells into muscle cells. ChIP-on-chip analysis indicates that ΔN-JMJD2A binds to genes mainly involved in transcriptional control and that this binding is linked to gene activation. ΔN-JMJD2A is recruited to the Myog promoter at the onset of differentiation. This binding is essential to promote the demethylation of H3K9me2 and H3K9me3. We conclude that induction of the ΔN-JMJD2A isoform is crucial for muscle differentiation: by directing the removal of repressive chromatin marks at the Myog promoter, it promotes transcriptional activation of the Myog gene and thus contributes to initiation of muscle-specific gene expression

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Recent advances in direct C–H arylation: Methodology, selectivity and mechanism in oxazole series

Catalytic direct (hetero)arylation of (hetero)arenes is an attractive alternative to traditional Kumada, Stille, Negishi and Suzuki–Miyaura cross-coupling reactions, notably as it avoids the prior preparation and isolation of (hetero)arylmetals. Developments of this methodology in the oxazole series are reviewed in this article. Methodologies, selectivity, mechanism and future aspects are presented

Mechanism selection for regiocontrol in base-assisted, palladium-catalysed direct C-H coupling with halides: first approach for oxazole- and thiazole-4-carboxylates.

International audienceBoth base-assisted non-concerted metallation-deprotonation (nCMD) and concerted metallation-deprotonation (CMD) have been identified as two potent operating mechanisms in palladium-catalysed direct C-H coupling of oxazole and thiazole-4-carboxylate esters with halides through base- and solvent-effect experiments. Novel C2- and C5-selective CMD direct arylation procedures in oxazole- and thiazole-4-carboxylate series were then designed by controlling the balance between electronic and steric factors. Notably, charge interactions between the palladium catalyst and substrate were identified as a parameter for controlling selectivity and reducing the impact of steric factors in the CMD reaction

PIXSIC, a Pixelated β(+)-Sensitive Probe for Radiopharmacological Investigations in Rat Brain: Binding Studies with [(18)F]MPPF.

International audienc