Régulation épigénétique de la différenciation du muscle squelettique

LSD1 and PHF2 are lysine de-methylases that can de-methylate both histone proteins, influencing gene expression and non-histone proteins, affecting their activity or stability. Functional approaches using Lsd1 or Phf2 inactivation in mouse have demonstrated the involvement of these enzymes in the engagement of progenitor cells into differentiation. One of the best-characterized examples of how progenitor cells multiply and differentiate to form functional organ is myogenesis. It is initiated by the specific timing expression of the specific regulatory genes; among these factors, MYOD is a key regulator of the engagement into differentiation of muscle progenitor cells. Although the action of MYOD during muscle differentiation has been extensively studied, still little is known about the chromatin remodeling events associated with the activation of MyoD expression. Among the regulatory regions of MyoD expression, the Core Enhancer region (CE), which transcribes for a non-coding enhancer RNA (CEeRNA), has been demonstrated to control the initiation of MyoD expression during myoblast commitment. We identified LSD1 and PHF2 as key activators of the MyoD CE. In vitro and in vivo ablation of LSD1 or inhibition of LSD1 enzymatic activity impaired the recruitment of RNA PolII on the CE, resulting in a failed expression of the CEeRNA. According to our results, forced expression of the CEeRNA efficiently rescue MyoD expression and myoblast fusion in the absence of LSD1. Moreover PHF2 interacts with LSD1 regulating its protein stability. Indeed in vitro ablation of PHF2 results in a massive LSD1 degradation and thus absence of CEeRNA expression. However, all the histone modifications occurring on the CE region upon activation cannot be directly attributed to LSD1 or PHF2 enzymatic activity. These results raise the question of the identity of LSD1 and PHF2 partners, which co-participate to CEeRNA expression and thus to the engagement of myoblast cells into differentiation.LSD1 et PHF2 sont des déméthylases de lysines capables de déméthyler à la fois les protéines histones qui influencent l’expression génique et les protéines non histones en affectant leurs activités ou stabilités. Des approches fonctionnelles d’inactivation de Lsd1 ou Phf2 chez la souris ont démontré l’implication de ces enzymes dans l'engagement des cellules progénitrices au cours de la différenciation. La myogenèse est l'un des exemples les mieux caractérisés sur la façon dont les cellules progénitrices se multiplient et se différencient pour former un organe fonctionnel. Elle est initiée par une expression temporelle spécifique des gènes régulateurs cibles. Parmi ces facteurs, MYOD est un régulateur clé de l'engagement dans la différenciation des cellules progénitrices musculaires. Bien que l’action de MYOD au cours de la différenciation cellulaire ait été largement étudiée, peu de chose sont connus sur les événements de remodelage de la chromatine associés à l'activation de l'expression de MyoD. Parmi les régions régulatrices de l'expression de MyoD, la région Core Enhancer (CE) qui est transcrite en ARN activateur non codant (CEeRNA) a été démontrée pour contrôler l'initiation de l'expression de MyoD au cours de l'engagement de myoblastes dans la différenciation.Nous avons identifié LSD1 et PHF2 comme des activateurs clés du CE de MyoD. L'invalidation in vitro et in vivo de LSD1 ou l'inhibition de l'activité enzymatique de LSD1 empêche le recrutement de l'ARN PolII sur le CE, empêchant l’expression du CEeRNA. D’après nos résultats, l'expression forcée du CEeRNA restaure efficacement l'expression de MyoD et la fusion myoblastique en l'absence de LSD1. De plus, PHF2 interagit avec LSD1 en régulant sa stabilité protéique.En effet, l'ablation in vitro de PHF2 entraîne une dégradation massive de LSD1 et donc une absence d'expression du CEeRNA. Cependant, toutes les modifications d'histones qui ont lieu dans la région du CE lors de l'activation de la différenciation ne peuvent pas être directement attribuées à l'activité enzymatique de LSD1 ou PHF2. Ces résultats soulèvent la question de l'identité des partenaires de LSD1 et PHF2, qui co-participeraient à l'expression du CEeRNA et donc à l'engagement des myoblastes dans la différenciation cellulaire

Le rôle inattendu des gouttelettes lipidiques dans la régulation du destin des cellules souches musculaires

Les cellules souches musculaires (CSM) sont des cellules souches résidentes du muscle squelettique responsables de la régénération de ce dernier. Il est de plus en plus évident que la capacité des CSM à s’auto-renouveler ou à se différencier est influencée par le métabolisme cellulaire. Une nouvelle étude a récemment établi que les gouttelettes lipidiques (GL) sont de nouveaux régulateurs du devenir des CSM. En effet, les GL se répartissent différemment selon l’état des CSM au cours du processus de régénération, les CSM avec peu de GL étant plus enclines à s’auto-renouveler tandis que les CSM contenant beaucoup de GL s’engagent dans la différenciation. Ces résultats soulignent que le renouvellement correct des GL est nécessaire pour décider du destin des CSM. Ceci pose la question du mécanisme moléculaire sous-jacent de la régulation du métabolisme des lipides dans la détermination du destin des CSM

Response [NEUROMUSCULAR DISORDERS]

This is a letter to the Editor in response to comments from Spatafora et al. Neuromuscul DisThe letter by Spadafora and coworkers questions the pathogenic role of heterozygous CAV3 T78M variant in a patient we recently described (Ricci et al, 2012) because the same heterozygous variant has been found in 3, out of 195, healthy unrelated controls. As an alternative hypothesis to explain our patient carrying both heterozygous D4Z4 reduction and CAV3 T78M variant the authors suggests that the dramatic reduction of caveolin-3 we observed in the muscle biopsy might be due to mutations in other genes. This interesting hypothesis is consistent with several reports suggesting that other genetic modifiers may be implied in Cav-3 deficiency, so that the same CAV3 mutation can lead to heterogeneous clinical phenotypes and muscle histopathological changes (REF). In particular the study by Traverso et al. (2008) suggested that the T78M mutation might results pathogenic only in homozygous feature through a loss-of-function mechanism. In our view the finding that CAV3 T78M mutation is a common variant with a frequency of 1.5% might disclose a different scenario. In fact by analyzing DNA elements at 4q35 in more than 800 Italian and Brazilian samples of normal individuals unrelated to any FSHD patients, we have recently discovered that 1.3 % of healthy unrelated subjects carry alleles of 21-35 kb in size on chromosome 4q associated with the 4APAS161 permissive haplotype (Scionti et al 2012). This discovery points at the possibility that in the heterozygous state D4Z4 reduction might produce a subclinical sensitized condition that requires other epigenetic mechanisms or a contributing factor to cause overt myopathy. In some rare cases, it could be by becoming homozygous (Scionti et al 2012b) while in others, it might be by the simultaneous heterozygosity for a different and recessive myopathy, as suggested by many reports in which the FSHD contractions are found in association with a second molecular defect (REF). The clinical case, we have recently reported, seems to fall in this complex picture. The observation of Spatafora et al supports the idea that D4Z4 reduced allele as well CAV3 T78M variant, are not per se sufficient to cause a disease phenotype. In our patient, whose geographical origin is Calabria, the simultaneous mutations (heterozygous CAV3 T78M and 35 kb 4A161PAS D4Z4 allele) could play a synergistic effect in reaching disease threshold and determining overlapping phenotype.In light of this finding particular attention should be paid to the geographical origin of similar cases. As suggested by Spatafora et al, the frequency of the CAV3 T78M variant on the whole Italian territory should be established. In conclusion these population studies could have important relevance for prenatal genetic counseling of healthy carriers who are at risk of transmitting the mutation to next generation

H2A.Z is dispensable for both basal and activated transcription in post-mitotic mouse muscles

International audienceWhile the histone variant H2A.Z is known to be required for mitosis, it is also enriched in nucleosomes surrounding the transcription start site of active promoters, implicating H2A.Z in transcription. However, evidence obtained so far mainly rely on correlational data generated in actively dividing cells. We have exploited a paradigm in which transcription is uncoupled from the cell cycle by developing an in vivo system to inactivate H2A.Z in terminally differentiated post-mitotic muscle cells. ChIP-seq, RNA-seq and ATAC-seq experiments performed on H2A.Z KO post-mitotic muscle cells show that this histone variant is neither required to maintain nor to activate transcription. Altogether, this study provides in vivo evidence that in the absence of mitosis H2A.Z is dispensable for transcription and that the enrichment of H2A.Z on active promoters is a marker but not an active driver of transcription

Facioscapulohumeral muscular dystrophy: New insights from compound heterozygotes and implication for prenatal genetic counselling

Background: Facioscapulohumeral muscular dystrophy (FSHD) is considered an autosomal dominant disease with a prevalence of 1 in 20 000. Almost all patients with FSHD carry deletions of integral copies of tandem 3.3 kb repeats (D4Z4) located on chromosome 4q35. However, FSHD families have been reported in which individuals carrying a D4Z4-reduced allele remain asymptomatic. Recently, it has been proposed that the D4Z4-reduced allele is pathogenic only in association with the permissive haplotype, 4APAS. Methods and results: Through the Italian National Registry for FSHD (INRF), genotype-phenotype correlations were extensively studied in 11 non-consanguineous families in which two D4Z4-reduced alleles segregate. Overall, 68 subjects carrying D4Z4-reduced alleles were examined, including 15 compound heterozygotes. It was found that in four families the only FSHD-affected subject was the compound heterozygote for the D4Z4-reduced allele, and 52.6% of subjects carrying a single D4Z4-reduced 4A161PAS haplotype were non-penetrant carriers; moreover, the population frequency of the 4A161PAS haplotype associated with a D4Z4-reduced allele was found to be as high as 1.2%. Conclusions: This study reveals a high frequency of compound heterozygotes in the Italian population and the presence of D4Z4-reduced alleles with the 4A161PAS pathogenic haplotype in the majority of non-penetrant subjects in FSHD families with compound heterozygosity. These data suggest that carriers of FSHD-sized alleles with 4A161PAS haplotype are more common in the general population than expected on the basis of FSHD prevalence. These findings challenge the notion that FSHD is a fully penetrant autosomal dominant disorder uniquely associated with the 4A161PAS haplotype, with relevant repercussions for genetic counselling and prenatal diagnosis

Risk factors for renal calcifications and determinants of hypercalciuria in patients with chronic, post-surgical hypoparathyroidism

Conventional therapy with oral calcium supplements and activated vitamin D is the most diffuse and available therapy for chronic hypoparathyroidism (HypoPT). This treatment does not replace the lack of PTH and is associated with renal complications. We report the results of a case control study with a prospective design which included 178 adult patients with differentiated thyroid cancer treated with total thyroidectomy with a follow-up longer that 3 years after surgery: 89 with PoHypoPT treated with conventional therapy and 89 without PoHypoPT, matched for age and sex. Both groups were balanced for gender, age, time since thyroidectomy, supplementation with cholecalciferol, dose of levothyroxine and dietary calcium intake. Half of the patients were stable on treatment with calcitriol alone, 45% with calcitriol and calcium carbonate, and 4 with calcium carbonate alone. All patients underwent biochemical tests and renal ultrasound. Twenty-fou- hour urinary calcium, creatinine, sodium, potassium, chloride, sulfate, uric acid, phosphate, oxalate, citrate, volume and Ph were measured. The biochemical control of patients with PoHypoPT was satisfactory, but only one-third of patients was at target according to ESE guidelines. Patients with PoHypoPT, compared with those without PoHypoPT, had significantly lower alb-Ca and PTH and increased serum phosphate, calcium-phosphate product, and 24-h urinary calcium, but there was no difference in estimated GFR. Renal calcifications were detected in 26 (29.2%) patients with PoHypoPT and in 11(12.4%) without. We found a positive association between renal calcification and age (P=0.03) and plasma PTH (P=0.01), but no association with hypercalciuria or other urinary parameters. The median 24-h urinary calcium was significantly higher in patients with PoHypoPT than in those without (248 vs 162 mg, P< 0.01) Urinary calcium in patients with PoHypoPT was positively associated with serum calcium (P <0.001), urinary magnesium (P <0.001), and urinary volume (P= 0.003), and negatively associated with serum albumin (P = 0.025), urinary oxalate (P <0.001) and creatinine (P= 0.008). Our study confirms that conventional therapy in patients with chronic PoHypoPT is suboptimal. 24-h urinary calcium and the rate of renal calcification are higher in patients with chronic PoHypoPT compared with controls. We found no significant difference in renal function (eGFR) between patients with chronic PoHypoPT compared with controls. We found no association between renal calcification and hypercalciuria and/or other urinary stone risk factors. Further prospective studies including a large number of patients would be necessary to better define the risk factor for renal calcifications in patients with PoHypoP

LSD1 Controls Timely MyoD Expression via MyoD Core Enhancer Transcription

International audienceMyoD is a master regulator of myogenesis. Chromatin modifications required to trigger MyoD expression are still poorly described. Here, we demonstrate that the histone demethylase LSD1/KDM1a is recruited on the MyoD core enhancer upon muscle differentiation. Depletion of Lsd1 in myoblasts precludes the removal of H3K9 methylation and the recruitment of RNA polymerase II on the core enhancer, thereby preventing transcription of the non-coding enhancer RNA required for MyoD expression (CEeRNA). Consistently, Lsd1 conditional inactivation in muscle progenitor cells during embryogenesis prevented transcription of the CEeRNA and delayed MyoD expression. Our results demonstrate that LSD1 is required for the timely expression of MyoD in limb buds and identify a new biological function for LSD1 by showing that it can activate RNA polymerase II-dependent transcription of enhancers