UTX demethylase activity is required for satellite cell-mediated muscle regeneration.

The X chromosome-encoded histone demethylase UTX (also known as KDM6A) mediates removal of repressive trimethylation of histone H3 lysine 27 (H3K27me3) to establish transcriptionally permissive chromatin. Loss of UTX in female mice is embryonic lethal. Unexpectedly, male UTX-null mice escape embryonic lethality due to expression of UTY, a paralog that lacks H3K27 demethylase activity, suggesting an enzyme-independent role for UTX in development and thereby challenging the need for active H3K27 demethylation in vivo. However, the requirement for active H3K27 demethylation in stem cell-mediated tissue regeneration remains untested. Here, we employed an inducible mouse KO that specifically ablates Utx in satellite cells (SCs) and demonstrated that active H3K27 demethylation is necessary for muscle regeneration. Loss of UTX in SCs blocked myofiber regeneration in both male and female mice. Furthermore, we demonstrated that UTX mediates muscle regeneration through its H3K27 demethylase activity, as loss of demethylase activity either by chemical inhibition or knock-in of demethylase-dead UTX resulted in defective muscle repair. Mechanistically, dissection of the muscle regenerative process revealed that the demethylase activity of UTX is required for expression of the transcription factor myogenin, which in turn drives differentiation of muscle progenitors. Thus, we have identified a critical role for the enzymatic activity of UTX in activating muscle-specific gene expression during myofiber regeneration and have revealed a physiological role for active H3K27 demethylation in vivo.

A KAP1 phosphorylation switch controls MyoD function during skeletal muscle differentiation.

The transcriptional activator MyoD serves as a master controller of myogenesis. Often in partnership with Mef2 (myocyte enhancer factor 2), MyoD binds to the promoters of hundreds of muscle genes in proliferating myoblasts yet activates these targets only upon receiving cues that launch differentiation. What regulates this off/on switch of MyoD function has been incompletely understood, although it is known to reflect the action of chromatin modifiers. Here, we identify KAP1 (KRAB [Krüppel-like associated box]-associated protein 1)/TRIM28 (tripartite motif protein 28) as a key regulator of MyoD function. In myoblasts, KAP1 is present with MyoD and Mef2 at many muscle genes, where it acts as a scaffold to recruit not only coactivators such as p300 and LSD1 but also corepressors such as G9a and HDAC1 (histone deacetylase 1), with promoter silencing as the net outcome. Upon differentiation, MSK1-mediated phosphorylation of KAP1 releases the corepressors from the scaffold, unleashing transcriptional activation by MyoD/Mef2 and their positive cofactors. Thus, our results reveal KAP1 as a previously unappreciated interpreter of cell signaling, which modulates the ability of MyoD to drive myogenesis.

Trichostatin A enhances vascular repair by injected human endothelial progenitors through increasing the expression of TAL1-dependent genes.

A major goal of cell therapy for vascular diseases is to promote revascularization through the injection of endothelial stem/progenitor cells. The gene regulatory mechanisms that underlie endothelial progenitor-mediated vascular repair, however, remain elusive. Here, we identify the transcription factor TAL1/SCL as a key mediator of the vascular repair function of primary human endothelial colony-forming cells (ECFCs). Genome-wide analyses in ECFCs demonstrate that TAL1 activates a transcriptional program that promotes cell adhesion and migration. At the mechanistic level, we show that TAL1 upregulates the expression of migratory and adhesion genes through recruitment of the histone acetyltransferase p300. Based on these findings, we establish a strategy that enhances the revascularization efficiency of ECFCs after ischemia through ex vivo priming with the histone deacetylase inhibitor TSA. Thus, small molecule epigenetics drugs are effective tools for modifying the epigenome of stem/progenitor cells prior to transplantation as a means to enhance their therapeutic potential.

Dystrophic muscle environment induces changes in cell plasticity.

Fibro-adipogenic progenitors (FAPs) reside in the muscle, where they facilitate myofiber regeneration. Under normal conditions, FAPs lack myogenic potential and thus do not directly contribute to regenerated myofibers. Surprisingly, Saccone and colleagues (pp. 841-857) demonstrated that the dystrophic muscle environment causes FAPs to adopt a chromatin state that imparts these cells with myogenic potential. In this context, treatment of muscle with deacetylase inhibitors activates a BAF60c-myomiR transcriptional network in FAPs, blocking adipogenesis and driving muscle differentiation.

Tissue-specific splicing of a ubiquitously expressed transcription factor is essential for muscle differentiation.

Alternate splicing contributes extensively to cellular complexity by generating protein isoforms with divergent functions. However, the role of alternate isoforms in development remains poorly understood. Mef2 transcription factors are essential transducers of cell signaling that modulate differentiation of many cell types. Among Mef2 family members, Mef2D is unique, as it undergoes tissue-specific splicing to generate a muscle-specific isoform. Since the ubiquitously expressed (Mef2Dα1) and muscle-specific (Mef2Dα2) isoforms of Mef2D are both expressed in muscle, we examined the relative contribution of each Mef2D isoform to differentiation. Using both in vitro and in vivo models, we demonstrate that Mef2D isoforms act antagonistically to modulate differentiation. While chromatin immunoprecipitation (ChIP) sequencing analysis shows that the Mef2D isoforms bind an overlapping set of genes, only Mef2Dα2 activates late muscle transcription. Mechanistically, the differential ability of Mef2D isoforms to activate transcription depends on their susceptibility to phosphorylation by protein kinase A (PKA). Phosphorylation of Mef2Dα1 by PKA provokes its association with corepressors. Conversely, exon switching allows Mef2Dα2 to escape this inhibitory phosphorylation, permitting recruitment of Ash2L for transactivation of muscle genes. Thus, our results reveal a novel mechanism in which a tissue-specific alternate splicing event has evolved that permits a ubiquitously expressed transcription factor to escape inhibitory signaling for temporal regulation of gene expression.

Comparative expression profiling identifies differential roles for Myogenin and p38α MAPK signaling in myogenesis.

Skeletal muscle differentiation is mediated by a complex gene expression program requiring both the muscle-specific transcription factor Myogenin (Myog) and p38α MAPK (p38α) signaling. However, the relative contribution of Myog and p38α to the formation of mature myotubes remains unknown. Here, we have uncoupled the activity of Myog from that of p38α to gain insight into the individual roles of these proteins in myogenesis. Comparative expression profiling confirmed that Myog activates the expression of genes involved in muscle function. Furthermore, we found that in the absence of p38α signaling, Myog expression leads to the down-regulation of genes involved in cell cycle progression. Consistent with this, the expression of Myog is sufficient to induce cell cycle exit. Interestingly, p38α-defective, Myog-expressing myoblasts fail to form multinucleated myotubes, suggesting an important role for p38α in cell fusion. Through the analysis of p38α up-regulated genes, the tetraspanin CD53 was identified as a candidate fusion protein, a role confirmed both ex vivo in primary myoblasts, and in vivo during myofiber regeneration in mice. Thus, our study has revealed an unexpected role for Myog in mediating cell cycle exit and has identified an essential role for p38α in cell fusion through the up-regulation of CD53.

Turning on myogenin in muscle: a paradigm for understanding mechanisms of tissue-specific gene expression.

Expression of the myogenin (Myog) gene is restricted to skeletal muscle cells where the transcriptional activator turns on a gene expression program that permits the transition from proliferating myoblasts to differentiating myotubes. The strict temporal and spatial regulation on Myog expression in the embryo makes it an ideal gene to study the developmental regulation of tissue-specific expression. Over the last 20 years, our knowledge of the regulation of Myog expression has evolved from the identification of the minimal promoter elements necessary for the gene to be transcribed in muscle, to a mechanistic understanding of how the proteins that bind these DNA elements work together to establish transcriptional competence. Here we present our current understanding of the developmental regulation of gene expression gained from studies of the Myog gene.

Teashirt-3, a novel regulator of muscle differentiation, associates with BRG1-associated factor 57 (BAF57) to inhibit myogenin gene expression.

In adult muscles and under normal physiological conditions, satellite cells are found in a quiescent state but can be induced to enter the cell cycle by signals resulting from exercise, injury-induced muscle regeneration, or specific disease states. Once activated, satellite cells proliferate, self-renew, and differentiate to form myofibers. In the present study, we found that the zinc finger-containing factor Teashirt-3 (TSHZ3) was expressed in quiescent satellite cells of adult mouse skeletal muscles. We showed that following treatment with cardiotoxin TSHZ3 was strongly expressed in satellite cells of regenerating muscles. Moreover, immunohistochemical analysis indicated that TSHZ3 was expressed in both quiescent and activated satellite cells on intact myofibers in culture. TSHZ3 expression was maintained in myoblasts but disappeared with myotube formation. In C2C12 myoblasts, we showed that overexpression of Tshz3 impaired myogenic differentiation and promoted the down-regulation of myogenin (Myog) and up-regulation of paired-box factor 7 (Pax7). Moreover, knockdown experiments revealed a selective effect of Tshz3 on Myog regulation, and transcriptional reporter experiments indicated that TSHZ3 repressed Myog promoter. We identified the BRG1-associated factor 57 (BAF57), a subunit of the SWI/SNF complex, as a partner of TSHZ3. We showed that TSHZ3 cooperated with BAF57 to repress MYOD-dependent Myog expression. These results suggest a novel mechanism for transcriptional repression by TSHZ3 in which TSHZ3 and BAF57 cooperate to modulate MyoD activity on the Myog promoter to regulate skeletal muscle differentiation.

Teashirt 3 regulates development of neurons involved in both respiratory rhythm and airflow control.

Neonatal breathing in mammals involves multiple neuronal circuits, but its genetic basis remains unclear. Mice deficient for the zinc finger protein Teashirt 3 (TSHZ3) fail to breathe and die at birth. Tshz3 is expressed in multiple areas of the brainstem involved in respiration, including the pre-Bötzinger complex (preBötC), the embryonic parafacial respiratory group (e-pF), and cranial motoneurons that control the upper airways. Tshz3 inactivation led to pronounced cell death of motoneurons in the nucleus ambiguus and induced strong alterations of rhythmogenesis in the e-pF oscillator. In contrast, the preBötC oscillator appeared to be unaffected. These deficits result in impaired upper airway function, abnormal central respiratory rhythm generation, and altered responses to pH changes. Thus, a single gene, Tshz3, controls the development of diverse components of the circuitry required for breathing.