Epigenetic reprogramming of human embryonic stem cells into skeletal muscle cells and generation of contractile myospheres

Direct generation of a homogeneous population of skeletal myoblasts from human embryonic stem cells (hESCs) and formation of three-dimensional contractile structures for disease modeling in vitro are current challenges in regenerative medicine. Previous studies reported on the generation of myoblasts from ESC-derived embryoid bodies (EB), but not from undifferentiated ESCs, indicating the requirement for mesodermal transition to promote skeletal myogenesis. Here, we show that selective absence of the SWI/SNF component BAF60C (encoded by SMARCD3) confers on hESCs resistance to MyoD-mediated activation of skeletal myogenesis. Forced expression of BAF60C enables MyoD to directly activate skeletal myogenesis in hESCs by instructing MyoD positioning and allowing chromatin remodeling at target genes. BAF60C/MyoD-expressing hESCs are epigenetically committed myogenic progenitors, which bypass the mesodermal requirement and, when cultured as floating clusters, give rise to contractile three-dimensional myospheres composed of skeletal myotubes. These results identify BAF60C as a key epigenetic determinant of hESC commitment to the myogenic lineage and establish the molecular basis for the generation of hESC-derived myospheres exploitable for 'disease in a dish' models of muscular physiology and dysfunction

Dr1 (NC2) is present at tRNA genes and represses their transcription in human cells

Dr1 (also known as NC2{beta}) was identified as a repressor of RNA polymerase (pol) II transcription. It was subsequently shown to inhibit pol III transcription when expressed at high levels in vitro or in yeast cells. However, endogenous Dr1 was not detected at pol III-transcribed genes in growing yeast. In contrast, we demonstrate that endogenous Dr1 is present at pol III templates in human cells, as is its dimerization partner DRAP1 (also called NC2{alpha}). Expression of tRNA by pol III is selectively enhanced by RNAi-mediated depletion of endogenous human Dr1, but we found no evidence that DRAP1 influences pol III output in vivo. A stable association was detected between endogenous Dr1 and the pol III-specific transcription factor Brf1. This interaction may recruit Dr1 to pol III templates in vivo, as crosslinking to these sites increases following Brf1 induction. On the basis of these data, we conclude that the physiological functions of human Dr1 include regulation of pol III transcription

Dynamics of cellular states of fibro-adipogenic progenitors during myogenesis and muscular dystrophy

Fibro-adipogenic progenitors (FAPs) are currently defined by their anatomical position, expression of non-specific membrane-associated proteins, and ability to adopt multiple lineages in vitro. Gene expression analysis at single-cell level reveals that FAPs undergo dynamic transitions through a spectrum of cell states that can be identified by differential expression levels of Tie2 and Vcam1. Different patterns of Vcam1-negative Tie2highor Tie2lowand Tie2low/Vcam1-expressing FAPs are detected during neonatal myogenesis, response to acute injury and Duchenne Muscular Dystrophy (DMD). RNA\ua0sequencing analysis identified cell state-specific transcriptional profiles that predict functional interactions with satellite and inflammatory cells. In particular, Vcam1-expressing FAPs, which exhibit a pro-fibrotic expression profile, are transiently activated by acute injury in concomitance with the inflammatory response. Aberrant persistence of Vcam1-expressing FAPs is detected in DMD muscles or upon macrophage depletion, and is associated with muscle fibrosis, thereby revealing how disruption of inflammation-regulated FAPs dynamics leads to a pathogenic outcome

Brahma is required for cell cycle arrest and late muscle gene expression during skeletal myogenesis

Although the two catalytic subunits of the SWI/SNF chromatin-remodeling complex—Brahma (Brm) and Brg1—are almost invariably co-expressed, their mutually exclusive incorporation into distinct SWI/SNF complexes predicts that Brg1- and Brm-based SWI/SNF complexes execute specific functions. Here, we show that Brg1 and Brm have distinct functions at discrete stages of muscle differentiation. While Brg1 is required for the activation of muscle gene transcription at early stages of differentiation, Brm is required for Ccnd1 repression and cell cycle arrest prior to the activation of muscle genes. Ccnd1 knockdown rescues the ability to exit the cell cycle in Brm-deficient myoblasts, but does not recover terminal differentiation, revealing a previously unrecognized role of Brm in the activation of late muscle gene expression independent from the control of cell cycle. Consistently, Brm null mice displayed impaired muscle regeneration after injury, with aberrant proliferation of satellite cells and delayed formation of new myofibers. These data reveal stage-specific roles of Brm during skeletal myogenesis, via formation of repressive and activatory SWI/SNF complexes

TFIIB recognition elements control the TFIIA-NC2 axis in transcriptional regulation

TFIIB recognizes DNA sequence-specific motifs that can flank the TATA elements of the promoters of protein-encoding genes. The TFIIB recognition elements (BREu and BREd) can have positive or negative effects on transcription in a promoter context-dependent manner. Here we show that the BREs direct the selective recruitment of TFIIA and NC2 to the promoter. We find that TFIIA preferentially associates with BRE-containing promoters while NC2 is recruited to promoters that lack consensus BREs. The functional relevance of the BRE-dependent recruitment of TFIIA and NC2 was determined by small interfering RNA-mediated knockdown of TFIIA and NC2, both of which elicited BRE-dependent effects on transcription. Our results confirm the established functional reciprocity of TFIIA and NC2. However, our findings show that TFIIA assembly at BRE-containing promoters results in reduced transcriptional activity, while NC2 acts as a positive factor at promoters that lack functional BREs. Taken together, our results provide a basis for the selective recruitment of TFIIA and NC2 to the promoter and give new insights into the functional relationship between core promoter elements and general transcription factor activity

Déterminants de la consommation des antibiotiques et de la résistance de Staphylococcus Aureus à la méticiline dans les établissements de santé

En France, les établissements de santé ont été incités à surveiller leurs consommations des antibiotiques et leurs résistances bactériennes. Nos objectifs étaient d'étudier les déterminants de la consommation des antibiotiques et de la résistance de Staphylococcus aureus à la méticilline (SARM) dans 99 établissements de santé de l'interrégion Sud-Ouest afin de préciser les modalités d'interprétation des données et d'étudier les liens existants avec les politiques mises en place. Au delà de la typologie habituellement utilisée d'autres critères d'ajustement doivent être pris en compte pour une comparaison inter établissements. Les politiques de bon usage des antibiotiques et de lutte contre les infections nosocomiales étaient associées à une plus forte consommation d'antibiotiques et à une incidence plus élevée de SARM. L'incidence de SARM était corrélée à la consommation des fluoroquinolones mais la méthode ne permettait pas de préjuger d'une relation de causalité. L'analyse des données agrégées peut aider à l'interprétation des variations observées pour une comparaison des indicateurs dans les établissements de santé.Due to the high resistance rate and excessive use of antibiotics, French government has recommended that hospitals should monitor antibiotics consumption and incidence of methicillin-resistant Staphylococcus aureus (MRSA). Our aim was to determine factors that correlate with antibiotics use and Staphylococcus aureus resistance among 99 hospitals in south western France and to study relationship between these two indicators and policies developed by hospitals. Hospital type and hospitals areas stratification seems to be not enough homogenous to make valid comparisons. Number of beds could be used to explain difference when indicator of case mix is not available. Antibiotics policies and infection control program were associated with high consumption and high resistance rates. Fluoroquinolone use correlated with MRSA incidence but the relationships between antibiotic use and MRSA are complex and aggregated data do not prove causality link. However, such aggregated data may be helpful for comparison purpose.BORDEAUX2-BU Santé (330632101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF