Anna Polesskaya

Wnt Signaling Induces the Myogenic Specification of Resident CD45+ Adult Stem Cells during Muscle Regeneration

The observation that CD45(+) stem cells injected into the circulation participate in muscle regeneration raised the question of whether CD45(+) stem cells resident in muscle play a physiological role during regeneration. We found that CD45(+) cells cultured from uninjured muscle were uniformly nonmyogenic. However, CD45(+) cells purified from regenerating muscle readily gave rise to determined myoblasts. The number of CD45(+) cells in muscle rapidly expanded following injury, and a high proportion entered the cell cycle. Investigation of candidate pathways involved in embryonic myogenesis revealed that Wnt signaling was sufficient to induce the myogenic specification of muscle-derived CD45(+) stem cells. Moreover, injection of the Wnt antagonists sFRP2/3 into regenerating muscle markedly reduced CD45(+) stem cell proliferation and myogenic specification. Our data therefore suggest that mobilization of resident CD45(+) stem cells is an important factor in regeneration after injury and highlight the Wnt pathway as a potential therapeutic target for degenerative neuromuscular disease.

The microRNA miR-181 targets the homeobox protein Hox-A11 during mammalian myoblast differentiation

Deciphering the mechanisms underlying skeletal muscle-cell differentiation in mammals is an important challenge. Cell differentiation involves complex pathways regulated at both transcriptional and post-transcriptional levels. Recent observations have revealed the importance of small (20–25 base pair) non-coding RNAs (microRNAs or miRNAs) that are expressed in both lower organisms and in mammals. miRNAs modulate gene expression by affecting mRNA translation or stability. In lower organisms, miRNAs are essential for cell differentiation during development; some miRNAs are involved in maintenance of the differentiated state. Here, we show that miR-181, a microRNA that is strongly upregulated during differentiation, participates in establishing the muscle phenotype. Moreover, our results suggest that miR-181 downregulates the homeobox protein Hox-A11 (a repressor of the differentiation process), thus establishing a functional link between miR-181 and the complex process of mammalian skeletal-muscle differentiation. Therefore, miRNAs can be involved in the establishment of a differentiated phenotype — even when they are not expressed in the corresponding fully differentiated tissue.

A Suv39h-dependent mechanism for silencing S-phase genes in differentiating but not in cycling cells

The Rb/E2F complex represses S‐phase genes both in cycling cells and in cells that have permanently exited from the cell cycle and entered a terminal differentiation pathway. Here we show that S‐phase gene repression, which involves histone‐modifying enzymes, occurs through distinct mechanisms in these two situations. We used chromatin immunoprecipitation to show that methylation of histone H3 lysine 9 (H3K9) occurs at several Rb/E2F target promoters in differentiating cells but not in cycling cells. Furthermore, phenotypic knock‐down experiments using siRNAs showed that the histone methyltransferase Suv39h is required for histone H3K9 methylation and subsequent repression of S‐phase gene promoters in differentiating cells, but not in cycling cells. These results indicate that the E2F target gene permanent silencing mechanism that is triggered upon terminal differentiation is distinct from the transient repression mechanism in cycling cells. Finally, Suv39h‐depleted myoblasts were unable to express early or late muscle differentiation markers. Thus, appropriately timed H3K9 methylation by Suv39h seems to be part of the control switch for exiting the cell cycle and entering differentiation.

CREB-binding Protein/p300 Activates MyoD by Acetylation

The myogenic protein MyoD requires two nuclear histone acetyltransferases, CREB-binding protein (CBP)/p300 and PCAF, to transactivate muscle promoters. MyoD is acetylated by PCAFin vitro, which seems to increase its affinity for DNA. We here show that MyoD is constitutively acetylated in muscle cells.In vitro, MyoD is acetylated both by CBP/p300 and by PCAF on two lysines located at the boundary of the DNA binding domain. MyoD acetylation by CBP/p300 (as well as by PCAF) increases its activity on a muscle-specific promoter, as assessed by microinjection experiments. MyoD mutants that cannot be acetylated in vitro are not activated in the functional assay. Our results provide direct evidence that MyoD acetylation functionally activates the protein and show that both PCAF and CBP/p300 are candidate enzymes for MyoD acetylationin vivo.

CBP/p300 histone acetyl-transferase activity is important for the G1/S transition

Transforming viral proteins such as E1A which force quiescent cells into S phase have two essential cellular target proteins, Rb and CBP/p300. Rb regulates the G1/S transition by controlling the transcription factor E2F. CBP/p300 is a transcriptional co-activator with intrinsic histone acetyl-transferase activity. This activity is regulated in a cell cycle dependent manner and shows a peak at the G1/S transition, suggesting a function for CBP/p300 in this crucial step of the cell cycle. Here, we have artificially modulated CBP/p300 levels in individual cells through microinjection of specific antibodies and expression vectors. We show that CBP/p300 is required for cell proliferation and has an essential function during the G1/S transition. Using the same microinjection system and GFP-reporter vectors, we demonstrate that CBP/p300 is essential for the activity of E2F, a transcription factor that controls the G1/S transition. In addition, our results suggest that CBP HAT activity is required both for the G1/S transition and for E2F activity. Thus CBP/p300 seems to be a versatile protein involved in opposing cellular processes, which raises the question of how its multiple activities are regulated.

CBP/p300 and muscle differentiation: no HAT, no muscle

Terminal differentiation of muscle cells follows a precisely orchestrated program of transcriptional regulatory events at the promoters of both muscle‐specific and ubiquitous genes. Two distinct families of transcriptional co‐activators, GCN5/PCAF and CREB‐binding protein (CBP)/p300, are crucial to this process. While both possess histone acetyl‐transferase (HAT) activity, previous studies have failed to identify a requirement for CBP/p300 HAT function in myogenic differentiation. We have addressed this issue directly using a chemical inhibitor of CBP/p300 in addition to a negative transdominant mutant. Our results clearly demonstrate that CBP/p300 HAT activity is critical for myogenic terminal differentiation. Furthermore, this requirement is restricted to a subset of events in the differentiation program: cell fusion and specific gene expression. These data help to define the requirements for enzymatic function of distinct coactivators at different stages of the muscle cell differentiation program.

Interaction between Acetylated MyoD and the Bromodomain of CBP and/or p300

ABSTRACT Acetylation is emerging as a posttranslational modification of nuclear proteins that is essential to the regulation of transcription and that modifies transcription factor affinity for binding sites on DNA, stability, and/or nuclear localization. Here, we present both in vitro and in vivo evidence that acetylation increases the affinity of myogenic factor MyoD for acetyltransferases CBP and p300. In myogenic cells, the fraction of endogenous MyoD that is acetylated was found associated with CBP or p300. In vitro, the interaction between MyoD and CBP was more resistant to high salt concentrations and was detected with lower doses of MyoD when MyoD was acetylated. Interestingly, an analysis of CBP mutants revealed that the interaction with acetylated MyoD involves the bromodomain of CBP. In live cells, MyoD mutants that cannot be acetylated did not associate with CBP or p300 and were strongly impaired in their ability to cooperate with CBP for transcriptional activation of a muscle creatine kinase-luciferase construct. Taken together, our data suggest a new mechanism for activation of protein function by acetylation and demonstrate for the first time an acetylation-dependent interaction between the bromodomain of CBP and a nonhistone protein.

Simultaneous miRNA and mRNA transcriptome profiling of human myoblasts reveals a novel set of myogenic differentiation-associated miRNAs and their target genes

BackgroundmiRNA profiling performed in myogenic cells and biopsies from skeletal muscles has previously identified miRNAs involved in myogenesis.ResultsHere, we have performed miRNA transcriptome profiling in human affinity-purified CD56+ myoblasts induced to differentiate in vitro. In total, we have identified 60 miRNAs differentially expressed during myogenic differentiation. Many were not known for being differentially expressed during myogenic differentiation. Of these, 14 (miR-23b, miR-28, miR-98, miR-103, miR-107, miR-193a, miR-210, miR-324-5p, miR-324-3p, miR-331, miR-374, miR-432, miR-502, and miR-660) were upregulated and 6 (miR-31, miR-451, miR-452, miR-565, miR-594 and miR-659) were downregulated. mRNA transcriptome profiling performed in parallel resulted in identification of 6,616 genes differentially expressed during myogenic differentiation.ConclusionsThis simultaneous miRNA/mRNA transcriptome profiling allowed us to predict with high accuracy target genes of myogenesis-related microRNAs and to deduce their functions.

Role of the RNA-binding protein IMP-2 in muscle cell motility

ABSTRACT Insulin-like growth factor 2 (IGF-2) mRNA-binding proteins (IMPs) are a family of posttranscriptional regulatory factors with well-understood roles in embryonic development and cancer but with poorly characterized functions in normal adult cells and tissues. We now show that IMP-2, the most ubiquitously expressed member of the family, is abundant in human and mouse adult skeletal myoblasts, where it is indispensable for cell motility and for stabilization of microtubules. To explore the functions of IMP-2, we analyzed the transcripts that were differentially regulated in IMP-2-depleted myoblasts and bound to IMP-2 in normal myoblasts. Among them were the mRNAs of PINCH-2, an important mediator of cell adhesion and motility, and MURF-3, a microtubule-stabilizing protein. By gain- and loss-of-function assays and gel shift experiments, we show that IMP-2 regulates the expression of PINCH-2 and MURF-3 proteins via direct binding to their mRNAs. Upregulation of PINCH-2 in IMP-2-depleted myoblasts is the key event responsible for their decreased motility. Our data reveal how the posttranscriptional regulation of gene expression by IMP-2 contributes to the control of adhesion structures and stable microtubules and demonstrate an important function for IMP-2 in cellular motility.

Adult Stem Cell Specification by Wnt Signaling in Muscle Regeneration

Recently, we describe a biological role for endogenous CD45+ stem cells in maintaining muscle integrity by participating in regeneration. Our experiments further establish that Wnt-signaling is the mechanism by which resident CD45+ adult stem cells are induced to undergo myogenic specification during muscle regeneration. Importantly, our study suggests that targeting the Wnt-pathway represents a promising therapeutic approach for the treatment of neuromuscular degenerative diseases.