Aster H. Juan

The NAD+-Dependent SIRT1 Deacetylase Translates a Metabolic Switch into Regulatory Epigenetics in Skeletal Muscle Stem Cells

Stem cells undergo a shift in metabolic substrate utilization during specification and/or differentiation, a process that has been termed metabolic reprogramming. Here, we report that during the transition from quiescence to proliferation, skeletal muscle stem cells experience a metabolic switch from fatty acid oxidation to glycolysis. This reprogramming of cellular metabolism decreases intracellular NAD(+) levels and the activity of the histone deacetylase SIRT1, leading to elevated H4K16 acetylation and activation of muscle gene transcription. Selective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program in SCs. Moreover, mice with muscle-specific inactivation of the SIRT1 deacetylase domain display reduced myofiber size, impaired muscle regeneration, and derepression of muscle developmental genes. Overall, these findings reveal how metabolic cues can be mechanistically translated into epigenetic modifications that regulate skeletal muscle stem cell biology.

Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells

Satellite cells (SCs) sustain muscle growth and empower adult skeletal muscle with vigorous regenerative abilities. Here, we report that EZH2, the enzymatic subunit of the Polycomb-repressive complex 2 (PRC2), is expressed in both Pax7+/Myf5⁻ stem cells and Pax7+/Myf5+ committed myogenic precursors and is required for homeostasis of the adult SC pool. Mice with conditional ablation of Ezh2 in SCs have fewer muscle postnatal Pax7+ cells and reduced muscle mass and fail to appropriately regenerate. These defects are associated with impaired SC proliferation and derepression of genes expressed in nonmuscle cell lineages. Thus, EZH2 controls self-renewal and proliferation, and maintains an appropriate transcriptional program in SCs.

Enhancer timing of Hox gene expression: deletion of the endogenous Hoxc8 early enhancer

The proper expression of Hox genes is necessary for the accurate patterning of the body plan. The elucidation of the developmental genetic basis of transcriptional regulation of Hox genes by the study of their cis-regulatory elements provides crucial information regarding the establishment of axial specification. In this report, we investigate the role of the early enhancer (EE) of the murine Hoxc8 gene to better understand its role in pattern formation. Previous reports show that knockouts of the endogenous Hoxc8 coding region result in a combination of neural, behavioral and skeletal phenotypes. In this report, we limit ourselves to a consideration of the skeletal abnormalities. Early reports from our laboratory based on exogenous transgenic reporter constructs implicate a 200 bp non-coding element 3 kb upstream of the Hoxc8 promoter as a crucial enhancer that regulates the transcription of Hoxc8. In the present work, we have deleted this regulatory region from the endogenous genome using embryonic stem cell technology. Our results show that the deletion of the EE results in a significant delay in the temporal expression of Hoxc8. We also show that the deletion of the EE does not eliminate the expression of the Hoxc8 protein, but delays the attainment of control levels of expression and anterior and posterior boundaries of expression on the AP axis. The temporal delay in Hoxc8 expression is sufficient to produce phenocopies of many of the axial skeletal defects associated with the complete absence of Hoxc8 gene product as previously reported for the Hoxc8-null mutation. Our results are consistent with emerging evidence that the precise temporal expression of Hox genes is crucial for the establishment of regional identities. The fact that the EE deletion does not eliminate Hoxc8 expression indicates the existence of a Hoxc8 transcriptional regulatory apparatus independent to some degree of the Hoxc8 EE. In a comparison of our results with those reported previously by others investigating temporal control of Hox gene expression, we have discovered a structural similarity between the Hoxc8 EE reported here and a transcriptional control element located in the Hoxd11 region. We speculate that a distributed system of expression timing control may exist that is similar the one we propose for Hoxc8. Last, our data is consistent with the position that disparate regulatory pathways are responsible for the expression of Hoxc8 in the organogenesis of somites, neural tube and limb bud.

MicroRNA let-7 establishes expression of β2-adrenergic receptors and dynamically down-regulates agonist-promoted down-regulation

Although β2-adrenergic receptors (β2AR) are expressed on most cell types, mechanisms that establish expression levels and regulate expression by chronic agonist remain unclear. The 3′ UTR of ADRB2 has a conserved 8-nucleotide seed region that we hypothesized is targeted by the let-7 family of miRNAs leading to translational repression. In luciferase assays with transfected cells, luc-β2WT3′UTR had decreased expression when cotransfected with let-7f, but a mutated luc-β23′UTR lacking the seed was unaffected by let-7f; a mutated let-7f also had no effect on luc-β2WT3′UTR expression. ADRB2 mRNA was in greater abundance in immunoprecipitates of Ago2, a core component of the miRNA-induced silencing complex, when cells were transfected with let-7f, but not with a mutated let-7f, indicating a direct interaction with the silencing mechanism. H292 cells transfected with let-7f caused ∼60% decrease in native β2AR expression, but transfection with let-7f–specific locked nucleic acid anti-miRNA increased β2AR expression by ∼twofold. We considered that an increase in let-7f leading to greater repression of translation contributes to agonist-promoted down-regulation. Paradoxically, in cells and in lungs from mice treated in vivo, an ∼50% decrease in let-7f occurs during long-term agonist exposure, indicating a counterregulatory event. Consistent with this notion, let-7f locked nucleic acid transfection caused depressed agonist-promoted down-regulation. Thus, let-7f miRNA regulates baseline β2AR expression and decreases in let-7f evoked by agonist attenuate down-regulation. This positive feedback loop has not previously been described for a G protein-coupled receptor and its miRNA. Methods to decrease let-7f expression in targeted cells may increase therapeutic responses to β-agonist by increasing β2AR expression or minimizing tachyphylaxis.

MicroRNA-214 and polycomb group proteins: A regulatory circuit controlling differentiation and cell fate decisions

Comment on: Juan AH, et al. Mol Cell. 2009; 36:61-74.

Polycomb Ezh2 controls the fate of GABAergic neurons in the embryonic cerebellum.

Although the genetic interactions between signaling pathways and transcription factors have been largely decoded, much remains to be learned about the epigenetic regulation of cerebellar development. Here, we report that cerebellar deletion of Ezh2, the methyltransferase subunit of the PRC2 complex, results in reduced H3K27me3 and profound transcriptional dysregulation, including that of a set of transcription factors directly involved in cerebellar neuronal cell-type specification and differentiation. Such transcriptional changes lead to increased GABAergic interneurons and decreased Purkinje cells. Transcriptional changes also inhibit the proliferation of granule precursor cells derived from the rhombic lip. The loss of both cell types ultimately results in cerebellar hypoplasia. These findings indicate Ezh2/PRC2 plays crucial roles in regulating neurogenesis from both cerebellar germinal zones. Summary: Cerebellar deletion of Ezh2 leads to increased GABAergic interneurons, decreased Purkinje cells and blocked proliferation of granule precursor cells derived from the rhombic lip.

A Muscle-Specific Enhancer RNA Mediates Cohesin Recruitment and Regulates Transcription In trans

The enhancer regions of the myogenic master regulator MyoD give rise to at least two enhancer RNAs. Core enhancer eRNA (CEeRNA) regulates transcription of the adjacent MyoD gene, whereas DRReRNA affects expression of Myogenin in trans. We found that DRReRNA is recruited at the Myogenin locus, where it colocalizes with Myogenin nascent transcripts. DRReRNA associates with the cohesin complex, and this association correlates with its transactivating properties. Despite being expressed in undifferentiated cells, cohesin is not loaded on Myogenin until the cells start expressing DRReRNA, which is then required for cohesin chromatin recruitment and maintenance. Functionally, depletion of either cohesin or DRReRNA reduces chromatin accessibility, prevents Myogenin activation, and hinders muscle cell differentiation. Thus, DRReRNA ensures spatially appropriate cohesin loading in trans to regulate gene expression.

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

Epigenetic Regulation Shapes the Stem Cells State

Pluripotent stem cells are endowed with the dual capacity to self-renew and to differentiate towards all lineages. Genetic and genome-wide studies in different model organisms have provided compelling evidence for the importance of epigenetic factors both in the maintenance of pluripotency and in the establishment of cell lineage commitment, during embryonic differentiation and in regenerative events occurring in postnatal life. In this special issue, we have collected reviews and reports highlighting the plasticity of the epigenome in embryonic, induced pluripotent and adult stem cells, providing readers with an overview of different molecular mechanisms, spanning from DNA methylation, histone modifications and variants, and regulatory RNAs. In response to signals from the external niche and/or to intracellular signaling pathways, embryonic and adult stem cells engage epigenetic factors in the transition process towards differentiation. L. Fagnocchi et al. have summarized the current understanding of the cross-talk between extrinsic/intrinsic signaling pathways and epigenetic factors and how they cooperatively regulate the fate of different stem cell lineages. Together with signaling molecules from the niche, metabolites and cofactors derived from the environment modulate intracellular pathways and the epigenetic response. A. J. Harvey et al. review several examples of metabolites and cofactors, which interface metabolic pathways and epigenetic targets, affecting histone marks and transcription. DNA methylation, once believed to be an irreversible signature restricted to germ cells and embryo development, is now recognized as a dynamic modification, occurring in all cell types. R. C. Laker and J. G. Ryall present recent advances in our knowledge of the role of DNA methylation and hydroxymethylation in skeletal muscle stem cells, with an emphasis on recent whole genome sequencing results that show genomic enrichment for these modifications outside promoter regions and underscore their plastic role in sensing environmental cues. Recently, the novel function of long noncoding RNAs (lncRNAs) in maintaining pluripotency of ESCs has been explored. A. Rosa and M. Ballarino present an overview of the underlying molecular mechanisms of lncRNAs in regulating ESC pluripotency and differentiation. Another class of noncoding RNAs are presented in the review by A. D. Haase, in which PIWI-interacting RNAs (piRNAs) are described. piRNAs developed transcription and posttranscription strategies to limit the spread of transposon elements, which are mobile genetic elements threatening genomic integrity. The author describes piRNAs as an RNA-based immune system guarding the genome integrity through non-self-memory and adaptive protection against transposons. Adult stem cells hold great promise for their clinical relevance in regenerative medicine. In the article by S. Consalvi et al., the authors describe many of the epigenetic regulators involved in the differentiation of skeletal muscle stem cells. The authors focus predominantly on the processes of histone acetylation and deacetylation but also describe a potentially novel role for noncoding RNAs in the epigenetic regulation of differentiation and the potential for epigenetic modulation of skeletal muscle stem cells for the treatment of Duchenne muscular dystrophy (DMD). In the review by F. A. Choudry and M. Frontini, the authors give an overview on the changes of the epigenetic landscape within the haematopoietic stem cell (HSC) compartment occurring in the elderly, which may be linked to increased occurrence of myeloproliferative disorders, myeloid malignancy, and thrombosis observed in the elderly. Epigenetic changes in the HSC compartment affect HSC activity, survival, and function and they might lead to the selection and expansion of particular HSC clones generating myeloid and platelet skewing of the haematopoietic system distinctive of the elderly population. The review by L. Rouhana and J. Tasaki focuses on the process of tissue regeneration in lower order organisms. The authors discuss the careful integration of DNA methylation, histone modifications, and noncoding RNAs in the regulation of regeneration, as well as the important role of programmed cell death. In contrast to changes to the DNA sequence, epigenetic modifications are reversible and are therefore considered promising therapeutic targets for the use of stem cells in the treatment of human diseases. In their review, R. Fernandez-Santiago and M. Ezquerra describe how induced pluripotent stem cells are becoming a valuable model for neurodegenerative disorders, recapitulating key disease-associated molecular events. Furthermore, these authors highlight the potential of epigenetic regulation of patient-specific iPSC-derived neural models to develop novel therapeutic approaches for human disorders. During the cellular reprogramming of somatic cells, distinctive chromatin status coupled with gene expression changes is an important determinant for the reprogramming efficiency towards pluripotency. In the research paper contributed by F. Dong et al., the authors showed that redistribution of histone variants H2A.Z during the reprogramming process alters nucleosome stability to increase expression of genes that promote reprogramming. Together with kinase inhibitors, cocktails of epigenetic modulators can be used to promote reprogramming and to probe stem cells functions. In their report, Y.-C. Han et al. describe a novel method to induce neuronal stem cells from mouse embryonic fibroblasts, with the use of small molecules, and suggest that the reprogramming is enhanced by histone demethylation and histone acetylation and decreased DNA methylation. Transdifferentiation is an alternative approach to somatic reprogramming of induced pluripotent stem cells, which allows the direct conversion of one cell type into another, bypassing safety concerns related to the pluripotent cell state. G. Palazzolo and colleagues present an original research paper documenting a transdifferentiation process used to convert fibroblasts from golden retriever dogs with muscular dystrophy (GRMD) directly to cardiac-like myocytes. While the induced cells do not exhibit spontaneous contraction in vitro, when transplanted into the hearts of neonatal mice, the induced cells were found to participate in cardiac myogenesis. Overall, this special issue highlights recent advances in our understanding of epigenetic regulation of stem cells and describes several new approaches to investigate stem cell biology to model human disorders and develop novel therapies for disease states. Giuseppina Caretti Libera Berghella Aster Juan Lucia Latella James Ryall