SWI/SNF-like chromatin remodeling factor Fun30 supports point centromere function in S. cerevisiae

Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture

Pw1/Peg3 règle la croissance du tissu musculaire et l'auto-renouvellement des cellules satellites

Pw1/Peg3 est un gène d’empreinte parental exprimé par l’allèle paternel. Il est exprimé dans l’ensemble des populations de cellules souches, y compris les cellules satellites du tissu musculaire. Nous avons découvert que la perte constitutive de Pw1/Peg3 entraîne une perte de la masse musculaire, résultat d’une diminution du nombre de fibres musculaires. Le nombre de fibres réduit est présent dès la naissance. De plus, les souris double KO ont un nombre de fibres encore inférieur, suggérant que l’allèle maternel est fonctionnel pendant le développement pré-natal, et des analyses de souris hybrides C57BL6J/CAST/Ei révèlent une expression bi-allélique de Pw1/Peg3 d’environ 10%. Pw1/Peg3 est également fortement exprimé après blessure du muscle squelettique. Chez les souris Pw1/Peg3 KO, nous avons observé que les cellules satellites montrent une réduction de leur capacité d’auto-renouvèlement à la suite d’une blessure. Pw1/Peg3 est également exprimé dans une sous-population de cellules souches interstitielles, les PICS. Afin de déterminer le rôle spécifique de Pw1/Peg3 dans les cellules satellites nous avons croisé notre allèle conditionnel Pw1/Peg3 avec la lignée Pax7-Cre-ER. Ces souris ont un phénotype présentant un défaut de régénération prononcé, montrant ainsi un rôle clair et direct de Pw1/Peg3 dans la fonction régénératrice des cellules satellites. En résumé, l’ensemble de ces données montre un rôle de Pw1/Peg3 dans le développement fœtal et la détermination du nombre de fibres musculaires par son action dans l’auto-renouvellement des cellules satellites du tissu musculaire.Pw1/Peg3 is a parentally imprinted gene expressed from the paternal allele. It is expressed in all adult progenitor/stem cell populations examined to date including muscle satellite cells. We examined the impact of loss-of-function of Pw1/Peg3 in skeletal muscle, a tissue that greatly contributes to body mass. We found that constitutive loss of Pw1/Peg3 results in reduced muscle mass resulting from a decrease in muscle fiber number. The reduced fiber number is present at birth. Mice lacking both the paternal and maternal alleles display a lower fiber number as compared to mice carrying the paternal deletion, suggesting that the maternal allele is functional during prenatal development. Hybrid analyses (C57BL6J and Cast/Ei) of muscle tissue reveal a bi-allelic expression of Pw1/Peg3 around 10%. Pw1/Peg3 is strongly up-regulated in response to muscle injury. Using the constitutive Pw1/Peg3 knock out mouse, we observed that satellite cells display a reduced self-renewal capacity following muscle injury. Pw1/Peg3 is expressed in satellite cells as well as a subset of muscle interstitial cells (PICs). To determine the specific role of Pw1/Peg3 in satellite cells, we crossed our conditional Pw1/Peg3 allele with the Pax7-CreER line. Interestingly, these mice displayed a more pronounced phenotype of impaired regeneration revealing a clear and direct role for Pw1/Peg3 in satellite cells. Taken together, our data show that Pw1/Peg3 plays a role during fetal development in the determination of muscle fiber number that is gene-dosage dependent and plays a specific role in muscle satellite cell self-renewal

The imprinted gene Pw1/Peg3 regulates skeletal muscle growth, satellite cell metabolic state, and self-renewal.

Pw1/Peg3 is an imprinted gene expressed from the paternally inherited allele. Several imprinted genes, including Pw1/Peg3, have been shown to regulate overall body size and play a role in adult stem cells. Pw1/Peg3 is expressed in muscle stem cells (satellite cells) as well as a progenitor subset of muscle interstitial cells (PICs) in adult skeletal muscle. We therefore examined the impact of loss-of-function of Pw1/Peg3 during skeletal muscle growth and in muscle stem cell behavior. We found that constitutive loss of Pw1/Peg3 function leads to a reduced muscle mass and myofiber number. In newborn mice, the reduction in fiber number is increased in homozygous mutants as compared to the deletion of only the paternal Pw1/Peg3 allele, indicating that the maternal allele is developmentally functional. Constitutive and a satellite cell-specific deletion of Pw1/Peg3, revealed impaired muscle regeneration and a reduced capacity of satellite cells for self-renewal. RNA sequencing analyses revealed a deregulation of genes that control mitochondrial function. Consistent with these observations, Pw1/Peg3 mutant satellite cells displayed increased mitochondrial activity coupled with accelerated proliferation and differentiation. Our data show that Pw1/Peg3 regulates muscle fiber number determination during fetal development in a gene-dosage manner and regulates satellite cell metabolism in the adult

A Novel Mutant Allele of Pw1/Peg3 Does Not Affect Maternal Behavior or Nursing Behavior

International audienceParental imprinting is a mammalian-specific form of epigenetic regulation in which one allele of a gene is silenced depending on its parental origin. Parentally imprinted genes have been shown to play a role in growth, metabolism, cancer, and behavior. Although the molecular mechanisms underlying parental imprinting have been largely elucidated, the selective advantage of silencing one allele remains unclear. The mutant phenotype of the imprinted gene, Pw1/Peg3, provides a key example to illustrate the hypothesis on a coadaptation between mother and offspring, in which Pw1/Peg3 is required for a set of essential maternal behaviors, such as nursing, nest building, and postnatal care. We have generated a novel Pw1/Peg3 mutant allele that targets the last exon for the PW1 protein that contains >90% of the coding sequence resulting in a loss of Pw1/Peg3 expression. In contrast to previous reports that have targeted upstream exons, we observe that maternal behavior and lactation are not disrupted upon loss of Pw1/Peg3. Both paternal and homozygous Pw1/Peg3 mutant females nurse and feed their pups properly and no differences are detected in either oxytocin neuron number or oxytocin plasma levels. In addition, suckling capacities are normal in mutant pups. Consistent with previous reports, we observe a reduction of postnatal growth. These results support a general role for Pw1/Peg3 in the regulation of body growth but not maternal care and lactation

The imprinted gene Pw1/Peg3 regulates skeletal muscle growth, satellite cell metabolic state, and self-renewal

Abstract Pw1/Peg3 is an imprinted gene expressed from the paternally inherited allele. Several imprinted genes, including Pw1/Peg3, have been shown to regulate overall body size and play a role in adult stem cells. Pw1/Peg3 is expressed in muscle stem cells (satellite cells) as well as a progenitor subset of muscle interstitial cells (PICs) in adult skeletal muscle. We therefore examined the impact of loss-of-function of Pw1/Peg3 during skeletal muscle growth and in muscle stem cell behavior. We found that constitutive loss of Pw1/Peg3 function leads to a reduced muscle mass and myofiber number. In newborn mice, the reduction in fiber number is increased in homozygous mutants as compared to the deletion of only the paternal Pw1/Peg3 allele, indicating that the maternal allele is developmentally functional. Constitutive and a satellite cell-specific deletion of Pw1/Peg3, revealed impaired muscle regeneration and a reduced capacity of satellite cells for self-renewal. RNA sequencing analyses revealed a deregulation of genes that control mitochondrial function. Consistent with these observations, Pw1/Peg3 mutant satellite cells displayed increased mitochondrial activity coupled with accelerated proliferation and differentiation. Our data show that Pw1/Peg3 regulates muscle fiber number determination during fetal development in a gene-dosage manner and regulates satellite cell metabolism in the adult

<i>Pw1</i> deletion does not result in significant decrease in oxytocin production and release.

<p><b>A.</b> Left panel: schematic sagittal section of the adult mouse brain showing sectioning direction (arrow) on interaural coordinates. Right panel: schematic coronal section of the adult mouse brain showing the paraventricular nuclei (PVN) and the supraoptic nuclei (SON) in pink. <b>B-C.</b> Immunohistochemistry for oxytocin-expressing neurons in the PVN (B) and SON (C) of postpartum female brains (<i>Pw1</i>^<i>+/+</i>, n = 7; <i>Pw1</i>^<i>m+/p-</i>, n = 7; <i>Pw1</i>^<i>m-/p+</i>, n = 5; <i>Pw1-/-</i>, n = 6). Scale bar: 50μm. <b>D.</b> Total number of oxytocin (OT) positive neurons per nuclei as stained as in Fig 3B and 3C (<i>Pw1</i>^<i>+/+</i>, n = 7; <i>Pw1</i>^<i>m+/p-</i>, n = 7; <i>Pw1</i>^<i>m-/p+</i>, n = 5; <i>Pw1-/-</i>, n = 6). Bottom panel: total number of oxytocin (OT) positive neurons per medial preoptic area (MPOA) (<i>Pw1</i>^<i>+/+</i>, n = 6; <i>Pw1-/-</i>, n = 6). No significant differences were found between all four genotypes. <b>E.</b> Number of oxytocin-positive neurons per section as stained as in Fig 3B and 3C for <i>Pw1</i>^<i>+/+</i> and <i>Pw1</i>^<i>-/-</i> postpartum female brains. <b>F.</b> Oxytocin plasma level in virgin (V) and postpartum (PP) females (V: <i>n</i> = 11, 9, 7, and 8; PP: n = 8, 8, 6, and 8; for <i>Pw1</i>^<i>+/+</i>, <i>Pw1</i>^<i>m+/p-</i>, <i>Pw1</i>^<i>m-/p+</i>, <i>Pw1</i>^<i>-/-</i> females, respectively). <i>Pw1</i>^<i>-/-</i> postpartum females tend to have a lower oxytocin plasma level but this observation is not statistically significant. In all graphs, values represent mean ± s.e.m. Statistical analysis was performed using nonparametric one-way ANOVA (Kruskal-Wallis test) (Fig 3D), multiple t-tests (Fig 3E) or two-way ANOVA test (Fig 3F). *P<0.05, **P<0.01, and ***P<0.001. NS: non-significant.</p

Reproduction parameters of <i>Pw1</i> mutant mice are comparable to <i>Pw1</i>^<i>+/+</i> mice.

<p>Reproduction parameters of <i>Pw1</i> mutant mice are comparable to <i>Pw1</i>^<i>+/+</i> mice.</p

Lactation is not compromised in <i>Pw1</i> mutant mice.

<p><b>A.</b> Birth weight of <i>Pw1</i>^<i>+/+</i> pups born to <i>Pw1</i>^<i>+/+</i>, <i>Pw1</i>^<i>m+/p-</i>, or <i>Pw1</i>^<i>m-/p+</i> mothers is unchanged (n = 7, 13, and 8 pups, respectively). <b>B.</b> Early postnatal growth of wild-type progeny of <i>Pw1</i>^<i>m+/p-</i> mothers is comparable to wild-type progeny of <i>Pw1</i>^<i>+/+</i> mothers. Weights were measured at postnatal days 2, 7, 10, 14, and 21, prior to weaning (n = 15, and n = 14 pups from at least 7 breedings <i>Pw1</i>^<i>+/+</i> x <i>Pw1</i>^<i>+/+</i>, and 7 breedings <i>Pw1</i>^<i>m+/p-</i> x <i>Pw1</i>^<i>+/+</i>, respectively). No significant differences were found. <b>C.</b> Early postnatal growth of <i>Pw1</i>^<i>m-/p+</i> progeny to <i>Pw1</i>^<i>m+/p-</i> and <i>Pw1</i>^<i>-/-</i> mothers crossed with a <i>Pw1</i>^<i>+/+</i> male are comparable. Weights have been measured at postnatal days 2, 7, 10, 14, and 21, prior to weaning (n = 9, n = 11, for breedings <i>Pw1</i>^<i>m+/p-</i> x <i>Pw1</i>^<i>+/+</i> and <i>Pw1</i>^<i>-/-</i> x <i>Pw1</i>^<i>+/+</i>, respectively). No significant differences were found. <b>D.</b> Milk intake was assessed by measuring the gain of pup weight after a 2 hour starvation period at postnatal day 7. Milk intake of <i>Pw1</i>^<i>m+/p-</i> pups was similar to <i>Pw1</i>^<i>+/+</i> (<i>Pw1</i>^<i>+/+</i>: n = 20 pups; <i>Pw1</i>^<i>m+/p-</i>: n = 19 pups obtained from 5 independent breedings). The two-sided arrow indicates the 2 hour time-window when the pups were starved. <b>E.</b> Milk spot in day 0 pups (arrow). <b>F and G.</b> Percentage of postnatal day 2 pups showing a significant milk spot size from the following breedings: a female <i>Pw1</i>^<i>m+/p-</i> crossed with a male <i>Pw1</i>^<i>+/+</i> (F) and a female <i>Pw1</i>^<i>m+/p-</i> crossed with a male <i>Pw1</i>^<i>m+/p-</i> (G). The number of pups used is indicated on bars, with the number of independent breedings indicated in brackets. In all graphs, values represent mean ± s.e.m. Statistical analysis was performed using two-way ANOVA test.</p

<i>Pw1</i> knockout strategy and characterization.

<p><b>A.</b><i>Pw1</i> knockout construct. Pink, blue, and green arrows-arrowheads correspond to location of <i>Pw1</i> primers. <b>B.</b> Expression levels of <i>Pw1</i> wildtype and <i>Pw1</i> truncated knockout alleles from semi-quantitative RT-PCR analysis in postnatal day 0 (P0) and 2 months old (2 mo) <i>Pw1</i>^<i>+/+</i> (+/+), <i>Pw1</i>^<i>m+/p-</i> (+/-), <i>Pw1</i>^<i>m-/p+</i> (-/+), and <i>Pw1</i>^<i>-/-</i> (-/-) brains (n = 3). <b>C.</b> Expression level of <i>Pw1</i> wild-type allele from real time PCR normalized to <i>Hprt1</i> gene (n = 3). <b>D.</b> PW1 immunofluorescence (green) on 3–4 months old postpartum female hypothalamus (retrochiasmatic area) (n≥4). Nuclei were counterstained by DAPI. Scale bar: 50μm. <b>E.</b> Western blot analysis showing levels of PW1 at P0 in <i>Pw1</i>^<i>+/+</i> (+/+), <i>Pw1</i>^<i>m+/p-</i> (+/-), <i>Pw1</i>^<i>m-/p+</i> (-/+), and <i>Pw1</i>^<i>-/-</i> (-/-) brains (n = 3). <b>F. Left panel:</b> Postnatal growth of <i>Pw1</i>^<i>+/+</i> (n = 26), <i>Pw1</i>^<i>m+/p-</i> (n = 12), <i>Pw1</i>^<i>m-/p+</i> (n = 5), and <i>Pw1</i>^<i>-/-</i> (n = 5) female mice. <b>Right panel:</b> Postnatal growth of <i>Pw1</i>^<i>+/+</i> (n = 18), <i>Pw1</i>^<i>m+/p-</i> (n = 10), <i>Pw1</i>^<i>m-/p+</i> (n = 9), and <i>Pw1</i>^<i>-/-</i> (n = 4) male mice. Paternal loss of <i>Pw1</i> leads to a reduced postnatal growth. <b>G.</b> Data shown in F presented additionally as percentage of <i>Pw1</i>^<i>+/+</i> littermates weight. In all graphs except panel G, values represent mean ± s.e.m. Statistical analysis was performed using two-way ANOVA test. *P<0.05, **P<0.01, and ***P<0.001. NS: non-significant.</p

Inhibition of the Activin Receptor Type-2B Pathway Restores Regenerative Capacity in Satellite Cell-Depleted Skeletal Muscle

Degenerative myopathies typically display a decline in satellite cells coupled with a replacement of muscle fibers by fat and fibrosis. During this pathological remodeling, satellite cells are present at lower numbers and do not display a proper regenerative function. Whether a decline in satellite cells directly contributes to disease progression or is a secondary result is unknown. In order to dissect these processes, we used a genetic model to reduce the satellite cell population by ~70–80% which leads to a nearly complete loss of regenerative potential. We observe that while no overt tissue damage is observed following satellite cell depletion, muscle fibers atrophy accompanied by changes in the stem cell niche cellular composition. Treatment of these mice with an Activin receptor type-2B (AcvR2B) pathway blocker reverses muscle fiber atrophy as expected, but also restores regenerative potential of the remaining satellite cells. These findings demonstrate that in addition to controlling fiber size, the AcvR2B pathway acts to regulate the muscle stem cell niche providing a more favorable environment for muscle regeneration