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Tissue regeneration declines with ageing but little is known about whether this arises from changes in stem-cell heterogeneity. Here, in homeostatic skeletal muscle, we identify two quiescent stem-cell states distinguished by relative CD34 expression: CD34High, with stemness properties (genuine state), and CD34Low, committed to myogenic differentiation (primed state). The genuine-quiescent state is unexpectedly preserved into later life, succumbing only in extreme old age due to the acquisition of primed-state traits. Niche-derived IGF1-dependent Akt activation debilitates the genuine stem-cell state by imposing primed-state features via FoxO inhibition. Interventions to neutralize Akt and promote FoxO activity drive a primed-to-genuine state conversion, whereas FoxO inactivation deteriorates the genuine state at a young age, causing regenerative failure of muscle, as occurs in geriatric mice. These findings reveal transcriptional determinants of stem-cell heterogeneity that resist ageing more than previously anticipated and are only lost in extreme old age, with implications for the repair of geriatric muscle., The authors acknowledge funding from MINECO-Spain (grant no. RTI2018-096068), ERC2016-AdG-741966, LaCaixa-HEALTH-HR17-00040, MDA, UPGRADE-H2020-825825, AFM and DPP-Spain to P.M.-C; María-de-Maeztu-Program for Units of Excellence to UPF (grant no. MDM-2014-0370) and the Severo-Ochoa-Program for Centers of Excellence to CNIC (grant no. SEV-2015-0505). This work was also supported by NIAMS IRP through NIH grants nos AR041126 and AR041164 to V.S. and utilized computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov); ASI, Ricerca Finalizzata, Ateneo Sapienza to A.M.; AIRC (grant no. 23257); ASI (grant no. MARS-PRE, DC-VUM-2017-006); H2020-MSCA-RISE-2014 (645648) to M.S. and a FNR core grant (grant no. C15/BM/10397420) to A.d.S. L.G.P. was partially supported by an FPI fellowship and an EMBO fellowship (grant no. ALTF 420-2017); and S.C., X.H. and V.M. by FI, Severo-Ochoa and PFI Fellowships (Spain), respectively.

Summary It remains challenging to generate reproducible, high-quality cDNA libraries from RNA derived from rare cell populations. Here, we describe a protocol for high-throughput RNA-seq library preparation, including isolation of 200 skeletal muscle stem cells from mouse tibialis anterior muscle by fluorescence-activated cell sorting and cDNA preparation. We also describe RNA extraction and cDNA preparation from differentiating mouse embryonic stem cells. For complete details on the use and execution of this protocol, please refer to Juan et al. (2016) and Garcia-Prat et al. (2016)., Graphical abstract, Highlights • FACS isolation of 200 muscle stem cells (MuSCs) from one mouse tibialis anterior muscle • cDNA library construction for deep sequencing by direct lysis of 200 MuSCs • cDNA library construction for deep sequencing from 5,000–10,000 embryonic stem cells, It remains challenging to generate reproducible, high-quality cDNA libraries from RNA derived from rare cell populations. Here, we describe a protocol for high-throughput RNA-seq library preparation, including isolation of 200 skeletal muscle stem cells from mouse tibialis anterior muscle by fluorescence-activated cell sorting and cDNA preparation. We also describe RNA extraction and cDNA preparation from differentiating mouse embryonic stem cells.

Dedicated stem cells ensure postnatal growth, repair and homeostasis of skeletal muscle. Following injury, muscle stem cells (MuSCs) exit from quiescence and divide to reconstitute the stem cell pool and give rise to muscle progenitors. The transcriptomes of pooled MuSCs have provided a rich source of information for describing the genetic programs of distinct static cell states; however, bulk microarray and RNA sequencing provide only averaged gene expression profiles, blurring the heterogeneity and developmental dynamics of asynchronous MuSC populations. Instead, the granularity required to identify distinct cell types, states, and their dynamics can be afforded by single cell analysis. We were able to compare the transcriptomes of thousands of MuSCs and primary myoblasts isolated from homeostatic or regenerating muscles by single cell RNA sequencing. Using computational approaches, we could reconstruct dynamic trajectories and place, in a pseudotemporal manner, the transcriptomes of individual MuSC within these trajectories. This approach allowed for the identification of distinct clusters of MuSCs and primary myoblasts with partially overlapping but distinct transcriptional signatures, as well as the description of metabolic pathways associated with defined MuSC states.

Dedicated stem cells ensure post-natal growth, repair, and homeostasis of skeletal muscle. Following injury, muscle stem cells (MuSCs) exit from quiescence and divide to reconstitute the stem cell pool and give rise to muscle progenitors. The transcriptomes of pooled MuSCs have provided a rich source of information for describing the genetic programs of distinct static cell states; however, bulk microarray and RNA-seq provide only averaged gene expression profiles, blurring the heterogeneity and developmental dynamics of asynchronous MuSC populations. Instead, the granularity required to identify distinct cell types, states, and their dynamics can be afforded by single-cell analysis. We were able to compare the transcriptomes of thousands of MuSCs and primary myoblasts isolated from homeostatic or regenerating muscles by single-cell RNA- sequencing. Using computational approaches, we could reconstruct dynamic trajectories and place, in a pseudotemporal manner, the transcriptomes of individual MuSC within these trajectories. This approach allowed for the identification of distinct clusters of MuSCs and primary myoblasts with partially overlapping but distinct transcriptional signatures, as well as the description of metabolic pathways associated with defined MuSC states.

In mammals, gene silencing by the RNA-induced silencing complex (RISC) is a well-understood cytoplasmic posttranscriptional gene regulatory mechanism. Here, we show that embryonic stem cells (ESCs) contain high levels of nuclear AGO proteins and that in ESCs nuclear AGO protein activity allows for the onset of differentiation. In the nucleus, AGO proteins interact with core RISC components, including the TNRC6 proteins and the CCR4-NOT deadenylase complex. In contrast to cytoplasmic miRNA-mediated gene silencing that mainly operates on cis-acting elements in mRNA 3' untranslated (UTR) sequences, in the nucleus AGO binding in the coding sequence and potentially introns also contributed to post-transcriptional gene silencing. Thus, nuclear localization of AGO proteins in specific cell types leads to a previously unappreciated expansion of the miRNA-regulated transcriptome.

Summary Spt6 coordinates nucleosome dis- and re-assembly, transcriptional elongation, and mRNA processing. Here, we report that depleting Spt6 in embryonic stem cells (ESCs) reduced expression of pluripotency factors, increased expression of cell-lineage-affiliated developmental regulators, and induced cell morphological and biochemical changes indicative of ESC differentiation. Selective downregulation of pluripotency factors upon Spt6 depletion may be mechanistically explained by its enrichment at ESC super-enhancers, where Spt6 controls histone H3K27 acetylation and methylation and super-enhancer RNA transcription. In ESCs, Spt6 interacted with the PRC2 core subunit Suz12 and prevented H3K27me3 accumulation at ESC super-enhancers and associated promoters. Biochemical as well as functional experiments revealed that Spt6 could compete for binding of the PRC2 methyltransferase Ezh2 to Suz12 and reduce PRC2 chromatin engagement. Thus, in addition to serving as a histone chaperone and transcription elongation factor, Spt6 counteracts repression by opposing H3K27me3 deposition at critical genomic regulatory regions.

While 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 led to increased GABAergic interneurons and decreased.Purkinje cells. Transcriptional changes also inhibited the proliferation of granule precursor cells derived from the rhombic lip. The loss of both cell types ultimately resulted in cerebellar hypoplasia. These findings indicate Ezh2/PRC2 plays critical roles in regulating neurogenesis from both cerebellar germinal zones.

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

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.

Mouse naked cuticle 2 (mNkd2), the mammalian homolog of the Drosophila segment polarity gene naked cuticle (nkd), encodes an EF hand protein that regulates early Wg activity by acting as an inducible antagonist. The transcription factor, Hoxc8, a member of the homeobox gene family, is vital for growth and differentiation. Chromatin immunoprecipitation (ChIP) assay, an electrophoretic mobility shift assay (EMSA), and a reporter assay demonstrated that endogenous Hoxc8 protein binds directly to the enhancer region of the mNkd2 gene, implying a Hoxc8-dependent transcriptional activity. Introduction of exogenous Hoxc8 into NIH3T3 cell lines lacking wild-type Hoxc8 dramatically reduced expression of mNkd2 mRNA. If, as the results suggest, mNkd2 is a direct target of Hoxc8, it represents a novel mechanism by which Hoxc8 might cross-talk with the Wnt signaling pathway by regulating mNkd2.

Hox genes encode transcription factors that control spatial patterning during embryogenesis. To date, downstream targets of Hox genes have proven difficult to identify. Here, we describe studies designed to identify target genes under the control of the murine transcription factor Hoxc8 . We used a mouse 16,463 gene oligonucleotide microarray to identify mRNAs whose expression was altered by the overexpression of Hoxc8 in C57BL/6J mouse embryo fibroblasts (MEF) in cell culture ( in vitro ). We identified a total of 34 genes whose expression was changed by 2-fold or greater: 16 genes were up-regulated, and 18 genes were down-regulated. The majority of genes encoded proteins involved in critical biological processes, such as cell adhesion, migration, metabolism, apoptosis, and tumorigenesis. Two genes showed high levels of regulation: ( i ) secreted phosphoprotein 1 ( Spp1 ), also known as osteopontin ( OPN ), was down-regulated 4.8-fold, and ( ii ) frizzled homolog 2 ( Drosophila ) ( Fzd2 ) was up-regulated 4.4-fold. Chromatin immunoprecipitation (ChIP) analysis confirmed the direct interaction between the OPN promoter and Hoxc8 protein in vivo , supporting the view that OPN is a direct transcriptional target of Hoxc8 .

Signal-directed chromatin recruitment of mammalian Polycomb complexes is a fundamental component of epigenetic regulation. In this issue, Yi et al. (2016) reveal how mTORC1 activation deploys the ribosomal serine/threonine kinase S6K1 and Polycomb proteins at genomic regulatory regions to repress expression of anti-adipogenic developmental regulators.

MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression by inhibiting translation or promoting degradation of specific target messenger RNAs (mRNAs). Alteration of the levels of a number of miRNAs is common in solid and hematological tumors. We have shown previously that miR-214 regulates Ezh2 in skeletal muscle and embryonic stem cells. The current study was aimed at examining the role of miR-214 in breast cancer where miR-214 levels are reduced but whether this phenomenon bears a functional relevance is unknown. MiR-214 expression was inversely correlated with Ezh2 mRNA and protein levels in breast cancer cell lines and at least one copy of the miR-214 alleles was found to be deleted in 24% (6/25) of primary breast tumors. Experimental increase of miR-214 in breast cancer cell lines correlated with reduction of Ezh2 protein levels, a known marker of invasion and aggressive breast cancer behavior. Supporting a direct targeting mechanism, miR-214 decreased luciferase activity from a construct containing the Ezh2 3′ untranslated region. Expression of miR-214 specifically reduced cell proliferation of breast cancer cells and inhibited the invasive potential of a highly metastatic breast cancer cell line. These findings indicate that reduced miR-214 levels may contribute to breast tumorigenesis by allowing abnormally elevated Ezh2 accumulation and subsequent unchecked cell proliferation and invasion.

Satellite cells (SCs) are the main source of adult skeletal muscle stem cells responsible for muscle growth and regeneration. By interpreting extracellular cues, developmental regulators control quiescence, proliferation, and differentiation of SCs by influencing coordinate gene expression. The scope of this review is limited to the description and discussion of protein complexes that introduce and decode heritable histone and chromatin modifications and how these modifications are relevant for SC biology.

Adult skeletal muscle regenerates in response to traumatic injuries or degenerative conditions. This property is afforded mainly by satellite cells (SCs), a heterogeneous population of resident committed myogenic progenitors and noncommitted stem cells (Sherwood et al. 2004; Collins et al. 2005; Montarras et al. 2005; Kuang et al. 2007). In the mouse, postnatal SCs are mitotically active for the initial 2 wk after birth. After this period, they enter quiescence and their number declines. However, following muscle injury or degeneration, adult SCs undergo intense proliferation and efficiently differentiate. To replenish the reservoir, a subset of dividing SCs returns to the niche following a process of asymmetric and symmetric cell division (Shinin et al. 2006; Conboy et al. 2007; Kuang et al. 2007; Shea et al. 2010). Approximately 10% of noncommitted Pax7+/Myf5− SCs can asymmetrically generate a self-renewing, noncommitted Pax7+/Myf5− cell and a committed Pax7+/Myf5+ daughter cell in vivo. The noncommitted Pax7+/Myf5− cell returns to the niche to maintain the SC reservoir, while the committed Pax7+/Myf5+ SC undergoes several rounds of cell division and the ensuing cells eventually differentiate into pre-existing or newly formed myofibers (Kuang et al. 2008). Polycomb group (PcG) proteins regulate differentiation of totipotent embryonic stem (ES) cells and maintenance of multipotent and progenitor stem cell populations (Sauvageau and Sauvageau 2010). The Polycomb-repressive complex 2 (PRC2) subunit EZH2 methylates histone H3 Lys 27 (H3K27me3), establishing an epigenetic mark that identifies repressed chromatin regions. Ablation of PRC2 members in ES cells impairs their differentiation (Pasini et al. 2007; Chamberlain et al. 2008; Shen et al. 2008) and results in unscheduled expression of mixed cell lineage genes (Boyer et al. 2006; Lee et al. 2006). While PcG establishes and maintains positional patterning of the body axis through regulation of Hox genes in both Drosophila and mammals, its role in conferring cell identity by repressing inappropriate cell lineage-specific transcription in animal development has not been demonstrated. Indeed, derepression of mixed cell lineage genes does not occur in epidermal, neuronal, or pancreatic cells of Ezh2 conditional null mice (Chen et al. 2009; Ezhkova et al. 2009; Hirabayashi et al. 2009). We generated mice in which Ezh2 was conditionally ablated in SCs (Ezh2 muscle knockout, Ezh2mKO). While EZH2 was dispensable for fetal muscle development, it was required for postnatal muscle growth and adult muscle regeneration, ensuring appropriate homeostasis of the SC pool. Unlike other progenitor cells, reduced H3K27me3 in Ezh2mKO SCs was accompanied by RNA polymerase II (PolII) recruitment and transcriptional activation of genes normally repressed in SCs and expressed in other cell lineages, including cardiac progenitors, retinal cones, neurons, and chondrocytes. Thus, our findings indicate that EZH2, which regulates proliferation and maintains transcriptional identity of adult muscle stem cells, is an important molecular component of adult skeletal myogenesis.

Although β 2 -adrenergic receptors (β 2 AR) 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-β 2 WT3′UTR had decreased expression when cotransfected with let-7f , but a mutated luc-β 2 3′UTR lacking the seed was unaffected by let-7f ; a mutated let-7f also had no effect on luc-β 2 WT3′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 β 2 AR expression, but transfection with let-7f –specific locked nucleic acid anti-miRNA increased β 2 AR 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 β 2 AR 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 β 2 AR expression or minimizing tachyphylaxis.

Satellite cells (SCs) are the main source of adult skeletal muscle stem cells responsible for muscle growth and regeneration. By interpreting extracellular cues, developmental regulators control quiescence, proliferation, and differentiation of SCs by influencing coordinate gene expression. The scope of this review is limited to the description and discussion of protein complexes that introduce and decode heritable histone and chromatin modifications and how these modifications are relevant for SC biology.

During vertebrate development, gene expression is tightly controlled by dynamic regulatory circuits which determine and maintain cell lineages. These regulatory mechanisms, including transcription factor-DNA interactions and epigenetic programming, insure proper spatial and temporal gene expression. In addition to the aforementioned mechanisms, microRNAs (miRNAs) have been shown to play a critical role in controlling a wide range of cellular processes. miRNAs are 18–25 nucleotide long non-coding RNAs that repress mRNA translation or modulate mRNA degradation by binding to the 3’-untranslated region of target mRNAs. The primary miRNA transcripts are transcribed by RNA polymerase II and further processed to mature miRNAs by the Drosha/DGCR8 and Dicer complexes. Individual miRNAs can target hundreds of mRNAs and their expression is often associated with specific cell types or developmental stages.1,2 This suggests that even a single miRNA can have a significant impact on numerous biological processes. Several studies have shown that microRNA-214 (miR-214) is instrumental in determining cell fate in several cell types.3,4,6 Yet the modalities by which miR-214 modulates cell lineage specification can be quite diverse. In zebrafish, miR-214 is required for muscle cell fate decision during somitogenesis. Inhibition of miR-214 expression decreases slow-muscle cell types in the developing somites. One miR-214 target in zebrafish is suppressor of fused (su(fu)), a negative regulator of Hedgehog signaling. By repressing su(fu) expression in different somite compartments, miR-214 mediates muscle cell fate transition through the Hedgehog pathway.3 In mouse skeletal muscle, miR-214 shows robust expression in differentiating myoblasts. Overexpression of miR-214 in muscle cells results in premature expression of muscle genes and acceleration of muscle differentiation while blockage of its expression promotes myoblast proliferation and dampens myogenesis.4 Genetic ablation of a region containing the murine mir-214 locus leads to several developmental defects, including reduced skeletal muscle mass.5 Unlike zebrafish, in mouse skeletal muscle, miR-214 targets the Polycomb group protein (PcG) Enhancer of zest homologue 2 (Ezh2) to regulate muscle cell differentiation.4 Ezh2 trimethylates lysine 27 of histone H3 (H3K27me3) and represses gene transcription. It has been shown that Ezh2 is developmentally regulated during myogenesis and blocks muscle differentiation by imposing H3K27me3 on muscle specific genes. It is critical to remove Ezh2 binding and its cognate methylation for appropriate muscle gene activation.7 Therefore, repression of Ezh2 by miR-214 is essential for initiating muscle differentiation.4 Interestingly, this miR-214-dependent Ezh2 regulation is also observed in embryonic stem (ES) cells. In ES cells induced to differentiate by retinoic acid, upregulation of miR-214 expression coincides with reduction of Ezh2 protein. Ectopic expression of miR-214 in pluripotent ES cells reduces Ezh2 protein level thus derepressing transcription of developmental regulators leading to loss of ES cell pluripotency.4 In Xenopus, miR-214 is highly expressed in multipotent retinal progenitors at early embryonic stages. By repressing Xotx2 and Xvsx, two key regulators of late retinal neurons, miR-214 controls the developmental timing of these progenitors and determines their fate.6 The importance of miR-214 in cell fate commitment suggests that miR-214 expression also needs to be precisely controlled. The relevance of miRNA dynamic regulation is well illustrated in skeletal muscle where miR-214 transcription is regulated by a double-negative feedback loop in which miR-214 is repressed by Ezh2 and activated by myoD/myogenin.4 In addition of being regulated at the transcriptional level, the processing of primary-214 transcripts to mature miRNAs is controlled by p72 Dead-box RNA helicase subunits in the mouse Drosha complex.8 Of note, p72 itself associates with MyoD to promote muscle differentiation.9 Thus, p72 helicase could promote differentiation by both co-activating MyoD-dependent transcription and favoring miR-214 processing. Figure 1 summarizes a working model of the regulatory network involving miR-214 during muscle differentiation. Muscle cell fate is determined by a series of events including transcription and biogenesis of miR-214 and the feedback loop between miR-214 and its target. The mechanisms discussed here may serve as a potential model for miRNA mediated regulatory networks in other biological systems. Figure 1 In proliferating myoblasts, where it is highly expressed, Ezh2 represses primary-miR-214 transcription as well as other muscle specific genes to maintain the myoblasts in undifferented state. Upon differentiation, Ezh2 expression is reduced and miR-214 ...

Arthur Manuscript date: 2010 October 9, Polycomb group (PcG) proteins exert essential functions in the most disparate biological processes. The contribution of PcG proteins to cell commitment and differentiation relates to their ability to repress transcription of developmental regulators in embryonic stem (ES) cells and in committed cell lineages, including skeletal muscle cells (SMC). PcG proteins are preferentially removed from transcribed regions, but the underlying mechanisms remain unclear. Here, PcG proteins are found to occupy and repress transcription from an intronic region containing the microRNA miR-214 in undifferentiated SMC. Differentiation coincides with PcG disengagement, recruitment of the developmental regulators MyoD and myogenin, and activation of miR-214 transcription. Once transcribed, miR-214 negatively feeds back on PcG by targeting the Ezh2 3′UTR, the catalytic subunit of the PRC2 complex. miR-214-mediated Ezh2 protein reduction accelerates SMC differentiation and promotes unscheduled transcription of developmental regulators in ES cells. Thus, miR-214 and Ezh2 establish a regulatory loop controlling PcG-dependent gene expression during differentiation., National Institute of Arthritis and Musculoskeletal and Skin Diseases (U.S.) (Intramural Research Program)

We are interested in investigating the function of Hoxc8 in skeletogenesis during mouse development. Previous studies have shown that deregulation of Hoxc8 expression in the mouse leads to several skeletal defects, such as homeotic transformation in the thoracic vertebrae, abnormal development of the rib cage, and overproliferation of chondrocytes in the hypertrophic area. By deleting a crucial enhancer of Hoxc8 in vivo, we found that precise temporal expression of Hoxc8 is important for determining the correct identity of the vertebral column in early embryos. We also identified downstream targets of Hoxc8 relevant to osteoblast differentiation at later developmental stages.

The transcription factor,Hoxc8, is a member of the homeobox gene family that is vital for growth and differentiation. Previously, we identified 34 genes whose expression levels were changed at least 2-fold by forced expression ofHoxc8in C57BL/6J mouse embryo fibroblast cells using a mouse 16,463-gene oligonucleotide microarray. In the present study, we used the combined power of microarray profiling, global Hoxc8 DNA-binding site analysis, and high-throughput chromatin immunoprecipitation assays to identify direct and biologically relevant targets ofHoxc8 in vivo. Here we show that 19 of the 34 responsive genes contain Hoxc8 consensus DNA-binding sequence(s) in their regulatory regions. Chromatin immunoprecipitation analysis indicated that Hoxc8-DNA interaction was detected in five of the 19 candidate genes. All of these five target genes have been implicated in oncogenesis, cell adhesion, proliferation, and apoptosis. Overall, the genes described here should aid in the understanding of global regulatory networks ofHoxgenes and to provide valuable insight into the molecular basis ofHoxc8in development and carcinogenesis.

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 Hoxc8expression 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.

The polycomb repressive complex 2 (PRC2) methylates lysine 27 of histone H3 (H3K27) through its catalytic subunit Ezh2. PRC2-mediated di- and tri-methylation (H3K27me2/H3K27me3) have been interchangeably associated with gene repression. However, it remains unclear whether these two degrees of H3K27 methylation have different functions. In this study, we have generated isogenic mouse embryonic stem cells (ESCs) with a modified H3K27me2/H3K27me3 ratio. Our findings document dynamic developmental control in the genomic distribution of H3K27me2 and H3K27me3 at regulatory regions in ESCs. They also reveal that modifying the ratio of H3K27me2 and H3K27me3 is sufficient for the acquisition and repression of defined cell lineage transcriptional programs and phenotypes and influences induction of the ESC ground state., National Institute of Arthritis and Musculoskeletal and Skin Diseases (U.S.). Intramural Research Program