SMCHD1 activates the expression of genes required for the expansion of human myoblasts.

SMCHD1 is an epigenetic regulatory protein known to modulate the targeted repression of large chromatin domains. Diminished SMCHD1 function in muscle fibers causes Facioscapulohumeral Muscular Dystrophy (FSHD2) through derepression of the D4Z4 chromatin domain, an event which permits the aberrant expression of the disease-causing gene DUX4. Given that SMCHD1 plays a broader role in establishing the cellular epigenome, we examined whether loss of SMCHD1 function might affect muscle homeostasis through additional mechanisms. Here we show that acute depletion of SMCHD1 results in a DUX4-independent defect in myoblast proliferation. Genomic and transcriptomic experiments determined that SMCHD1 associates with enhancers of genes controlling cell cycle to activate their expression. Amongst these cell cycle regulatory genes, we identified LAP2 as a key target of SMCHD1 required for the expansion of myoblasts, where the ectopic expression of LAP2 rescues the proliferation defect of SMCHD1-depleted cells. Thus, the epigenetic regulator SMCHD1 can play the role of a transcriptional co-activator for maintaining the expression of genes required for muscle progenitor expansion. This DUX4-independent role for SMCHD1 in myoblasts suggests that the pathology of FSHD2 may be a consequence of defective muscle regeneration in addition to the muscle wasting caused by spurious DUX4 expression.

UTX inhibition as selective epigenetic therapy against TAL1-driven T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous group of hematological tumors composed of distinct subtypes that vary in their genetic abnormalities, gene expression signatures, and prognoses. However, it remains unclear whether T-ALL subtypes differ at the functional level, and, as such, T-ALL treatments are uniformly applied across subtypes, leading to variable responses between patients. Here we reveal the existence of a subtype-specific epigenetic vulnerability in T-ALL by which a particular subgroup of T-ALL characterized by expression of the oncogenic transcription factor TAL1 is uniquely sensitive to variations in the dosage and activity of the histone 3 Lys27 (H3K27) demethylase UTX/KDM6A. Specifically, we identify UTX as a coactivator of TAL1 and show that it acts as a major regulator of the TAL1 leukemic gene expression program. Furthermore, we demonstrate that UTX, previously described as a tumor suppressor in T-ALL, is in fact a pro-oncogenic cofactor essential for leukemia maintenance in TAL1-positive (but not TAL1-negative) T-ALL. Exploiting this subtype-specific epigenetic vulnerability, we propose a novel therapeutic approach based on UTX inhibition through in vivo administration of an H3K27 demethylase inhibitor that efficiently kills TAL1-positive primary human leukemia. These findings provide the first opportunity to develop personalized epigenetic therapy for T-ALL patients.

The Bromodomains of the mammalian SWI/SNF (mSWI/SNF) ATPases Brahma (BRM) and Brahma Related Gene 1 (BRG1) promote chromatin interaction and are critical for skeletal muscle differentiation.

Skeletal muscle regeneration is mediated by myoblasts that undergo epigenomic changes to establish the gene expression program of differentiated myofibers. mSWI/SNF chromatin remodeling enzymes coordinate with lineage-determining transcription factors to establish the epigenome of differentiated myofibers. Bromodomains bind to acetylated lysines on histone N-terminal tails and other proteins. The mutually exclusive ATPases of mSWI/SNF complexes, BRG1 and BRM, contain bromodomains with undefined functional importance in skeletal muscle differentiation. Pharmacological inhibition of mSWI/SNF bromodomain function using the small molecule PFI-3 reduced differentiation in cell culture and in vivo through decreased myogenic gene expression, while increasing cell cycle-related gene expression and the number of cells remaining in the cell cycle. Comparative gene expression analysis with data from myoblasts depleted of BRG1 or BRM showed that bromodomain function was required for a subset of BRG1- and BRM-dependent gene expression. Reduced binding of BRG1 and BRM after PFI-3 treatment showed that the bromodomain is required for stable chromatin binding at target gene promoters to alter gene expression. Our findings demonstrate that mSWI/SNF ATPase bromodomains permit stable binding of the mSWI/SNF ATPases to promoters required for cell cycle exit and establishment of muscle-specific gene expression.

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.

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.

Polycomb repressive complex 1 provides a molecular explanation for repeat copy number dependency in FSHD muscular dystrophy.

Repression of repetitive elements is crucial to preserve genome integrity and has been traditionally ascribed to constitutive heterochromatin pathways. FacioScapuloHumeral Muscular Dystrophy (FSHD), one of the most common myopathies, is characterized by a complex interplay of genetic and epigenetic events. The main FSHD form is linked to a reduced copy number of the D4Z4 macrosatellite repeat on 4q35, causing loss of silencing and aberrant expression of the D4Z4-embedded DUX4 gene leading to disease. By an unknown mechanism, D4Z4 copy-number correlates with FSHD phenotype. Here we show that the DUX4 proximal promoter (DUX4p) is sufficient to nucleate the enrichment of both constitutive and facultative heterochromatin components and to mediate a copy-number dependent gene silencing. We found that both the CpG/GC dense DNA content and the repetitive nature of DUX4p arrays are important for their repressive ability. We showed that DUX4p mediates a copy number-dependent Polycomb Repressive Complex 1 (PRC1) recruitment, which is responsible for the copy-number dependent gene repression. Overall, we directly link genetic and epigenetic defects in FSHD by proposing a novel molecular explanation for the copy number-dependency in FSHD pathogenesis, and offer insight into the molecular functions of repeats in chromatin regulation.

Tendon contains more stem cells than bone at the rotator cuff repair site.

BACKGROUND: The rotator cuff (RC) repair failure rate is high. Tendon and bone represent sources of mesenchymal stem cells (MSCs), but the number of MSCs from each has not been compared. Bone channeling may increase bone-derived MSC numbers participating in enthesis re-formation at the "footprint" repair site. The effect of preoperative channeling on increasing bone MSC numbers has never been reported. We asked (1) whether bone contains more MSCs than tendon at the time of arthroscopic repair and (2) whether bone preoperative channeling at the RC repair site increases the number of bone-derived MSCs at the time of surgery. METHODS: In 23 participants undergoing arthroscopic RC repair, bone was sampled from the footprint and tendon was sampled from the distal supraspinatus. We randomized participants to the channeling or no-channeling group 5 to 7 days before surgery. We enumerated MSCs from both tissues using the colony-forming unit-fibroblast (CFU-F) assay (10 per group). We identified MSC identity using flow cytometry and MSC tri-differentiation capacity (n = 3). RESULTS: Tendon CFU-F per gram exceeded bone CFU-F per gram for both groups (479 ± 173 CFU-F/g vs. 162 ± 54 CFU-F/g for channeling [P = .036] and 1334 ± 393 CFU-F/g vs. 284 ± 88 CFU-F/g for no channeling [P = .009]). Ninety-nine percent of cultured cells satisfied the MSC definition criteria. CONCLUSIONS: The distal supraspinatus tendon contained more MSCs per gram than the humeral footprint. Tendon may represent an important and overlooked MSC source for postoperative enthesis re-formation. Further studies are needed to evaluate the repair role of tendon MSCs and to recommend bone channeling in RC repair.

Nuclear matrix-associated protein SMAR1 regulates alternative splicing via HDAC6-mediated deacetylation of Sam68.

Pre-mRNA splicing is a complex regulatory nexus modulated by various trans-factors and their posttranslational modifications to create a dynamic transcriptome through alternative splicing. Signal-induced phosphorylation and dephosphorylation of trans-factors are known to regulate alternative splicing. However, the role of other posttranslational modifications, such as deacetylation/acetylation, methylation, and ubiquitination, that could modulate alternative splicing in either a signal-dependent or -independent manner remain enigmatic. Here, we demonstrate that Scaffold/matrix-associated region-binding protein 1 (SMAR1) negatively regulates alternative splicing through histone deacetylase 6 (HDAC6)-mediated deacetylation of RNA-binding protein Sam68 (Src-associated substrate during mitosis of 68 kDa). SMAR1 is enriched in nuclear splicing speckles and associates with the snRNAs that are involved in splice site recognition. ERK-MAPK pathway that regulates alternative splicing facilitates ERK-1/2-mediated phosphorylation of SMAR1 at threonines 345 and 360 and localizes SMAR1 to the cytoplasm, preventing its interaction with Sam68. We showed that endogenously, SMAR1 through HDAC6 maintains Sam68 in a deacetylated state. However, knockdown or ERK-mediated phosphorylation of SMAR1 releases the inhibitory SMAR1-HDAC6-Sam68 complex, facilitating Sam68 acetylation and alternative splicing. Furthermore, loss of heterozygosity at the Chr.16q24.3 locus in breast cancer cells, wherein the human homolog of SMAR1 (BANP) has been mapped, enhances Sam68 acetylation and CD44 variant exon inclusion. In addition, tail-vein injections in mice with human breast cancer MCF-7 cells depleted for SMAR1 showed increased CD44 variant exon inclusion and concomitant metastatic propensity, confirming the functional role of SMAR1 in regulation of alternative splicing. Thus, our results reveal the complex molecular mechanism underlying SMAR1-mediated signal-dependent and -independent regulation of alternative splicing via Sam68 deacetylation.

Rbfox proteins regulate tissue-specific alternative splicing of Mef2D required for muscle differentiation.

Among the Mef2 family of transcription factors, Mef2D is unique in that it undergoes tissue-specific splicing to generate an isoform that is essential for muscle differentiation. However, the mechanisms mediating this muscle-specific processing of Mef2D remain unknown. Using bioinformatics, we identified Rbfox proteins as putative modulators of Mef2D muscle-specific splicing. Accordingly, we found direct and specific Rbfox1 and Rbfox2 binding to Mef2D pre-mRNA in vivo. Gain- and loss-of-function experiments demonstrated that Rbfox1 and Rbfox2 cooperate in promoting Mef2D splicing and subsequent myogenesis. Thus, our findings reveal a new role for Rbfox proteins in regulating myogenesis through activation of essential muscle-specific splicing events.

Signal-dependent incorporation of MyoD-BAF60c into Brg1-based SWI/SNF chromatin-remodelling complex.

Tissue-specific transcriptional activators initiate differentiation towards specialized cell types by inducing chromatin modifications permissive for transcription at target loci, through the recruitment of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin-remodelling complex. However, the molecular mechanism that regulates SWI/SNF nuclear distribution in response to differentiation signals is unknown. We show that the muscle determination factor MyoD and the SWI/SNF subunit BAF60c interact on the regulatory elements of MyoD-target genes in myoblasts, prior to activation of transcription. BAF60c facilitates MyoD binding to target genes and marks the chromatin for signal-dependent recruitment of the SWI/SNF core to muscle genes. BAF60c phosphorylation on a conserved threonine by differentiation-activated p38α kinase is the signal that promotes incorporation of MyoD-BAF60c into a Brg1-based SWI/SNF complex, which remodels the chromatin and activates transcription of MyoD-target genes. Our data support an unprecedented two-step model by which pre-assembled BAF60c-MyoD complex directs recruitment of SWI/SNF to muscle loci in response to differentiation cues.

Constitutive heterochromatin reorganization during somatic cell reprogramming.

Induced pluripotent stem (iPS) cell reprogramming is a gradual epigenetic process that reactivates the pluripotent transcriptional network by erasing and establishing repressive epigenetic marks. In contrast to loci-specific epigenetic changes, heterochromatin domains undergo epigenetic resetting during the reprogramming process, but the effect on the heterochromatin ultrastructure is not known. Here, we characterize the physical structure of heterochromatin domains in full and partial mouse iPS cells by correlative electron spectroscopic imaging. In somatic and partial iPS cells, constitutive heterochromatin marked by H3K9me3 is highly compartmentalized into chromocentre structures of densely packed chromatin fibres. In contrast, chromocentre boundaries are poorly defined in pluripotent embryonic stem and full iPS cells, and are characterized by unusually dispersed 10 nm heterochromatin fibres in high Nanog-expressing cells, including pluripotent cells of the mouse blastocyst before differentiation. This heterochromatin reorganization accompanies retroviral silencing during conversion of partial iPS cells by MEK/GSK3 2i inhibitor treatment. Thus, constitutive heterochromatin is compacted in partial iPS cells but reorganizes into dispersed 10 nm chromatin fibres as the fully reprogrammed iPS cell state is acquired.

Differential genomic targeting of the transcription factor TAL1 in alternate haematopoietic lineages.

TAL1/SCL is a master regulator of haematopoiesis whose expression promotes opposite outcomes depending on the cell type: differentiation in the erythroid lineage or oncogenesis in the T-cell lineage. Here, we used a combination of ChIP sequencing and gene expression profiling to compare the function of TAL1 in normal erythroid and leukaemic T cells. Analysis of the genome-wide binding properties of TAL1 in these two haematopoietic lineages revealed new insight into the mechanism by which transcription factors select their binding sites in alternate lineages. Our study shows limited overlap in the TAL1-binding profile between the two cell types with an unexpected preference for ETS and RUNX motifs adjacent to E-boxes in the T-cell lineage. Furthermore, we show that TAL1 interacts with RUNX1 and ETS1, and that these transcription factors are critically required for TAL1 binding to genes that modulate T-cell differentiation. Thus, our findings highlight a critical role of the cellular environment in modulating transcription factor binding, and provide insight into the mechanism by which TAL1 inhibits differentiation leading to oncogenesis in the T-cell lineage.

Maintenance of gene silencing by the coordinate action of the H3K9 methyltransferase G9a/KMT1C and the H3K4 demethylase Jarid1a/KDM5A.

Chromatin remodeling is essential for controlling the expression of genes during development. The histone-modifying enzyme G9a/KMT1C can act both as a coactivator and a corepressor of transcription. Here, we show that the dual function of G9a as a coactivator vs. a corepressor entails its association within two distinct protein complexes, one containing the coactivator Mediator and one containing the corepressor Jarid1a/KDM5A. Functionally, G9a is important in stabilizing the Mediator complex for gene activation, whereas its repressive function entails a coordinate action with the histone H3 lysine 4 (H3K4) demethylase Jarid1a for the maintenance of gene repression. The essential nature of cross-talk between the histone methyltransferase G9a and the demethylase Jarid1a is demonstrated on the embryonic E(y)-globin gene, where the concurrent introduction of repressive histone marks (dimethylated H3K9 and dimethylated H3K27) and removal of activating histone mark (trimethylated H3K4) is required for maintenance of gene silencing. Taken together with our previous demonstration of cross-talk between UTX and MLL2 to mediate activation of the adult ß(maj)-globin gene, these data suggest a model where "active" and "repressive" cross-talk between histone-modifying enzymes coexist on the same multigene locus and play a crucial role in the precise control of developmentally regulated gene expression.

Epigenetic integration of signaling from the regenerative environment.

Skeletal muscle has an extraordinary capacity to regenerate itself after injury due to the presence of tissue-resident muscle stem cells. While these muscle stem cells are the primary contributor to the regenerated myofibers, the process occurs in a regenerative microenvironment where multiple different cell types act in a coordinated manner to clear the damaged myofibers and restore tissue homeostasis. In this regenerative environment, immune cells play a well-characterized role in initiating repair by establishing an inflammatory state that permits the removal of dead cells and necrotic muscle tissue at the injury site. More recently, it has come to be appreciated that the immune cells also play a crucial role in communicating with the stem cells within the regenerative environment to help coordinate the timing of repair events through the secretion of cytokines, chemokines, and growth factors. Evidence also suggests that stem cells can help modulate the extent of the inflammatory response by signaling to the immune cells, demonstrating a cross-talk between the different cells in the regenerative environment. Here, we review the current knowledge on the innate immune response to sterile muscle injury and provide insight into the epigenetic mechanisms used by the cells in the regenerative niche to integrate the cellular cross-talk required for efficient muscle repair.

Progeria-based vascular model identifies networks associated with cardiovascular aging and disease.

Hutchinson-Gilford Progeria syndrome (HGPS) is a lethal premature aging disorder caused by a de novo heterozygous mutation that leads to the accumulation of a splicing isoform of Lamin A termed progerin. Progerin expression deregulates the organization of the nuclear lamina and the epigenetic landscape. Progerin has also been observed to accumulate at low levels during normal aging in cardiovascular cells of adults that do not carry genetic mutations linked with HGPS. Therefore, the molecular mechanisms that lead to vascular dysfunction in HGPS may also play a role in vascular aging-associated diseases, such as myocardial infarction and stroke. Here, we show that HGPS patient-derived vascular smooth muscle cells (VSMCs) recapitulate HGPS molecular hallmarks. Transcriptional profiling revealed cardiovascular disease remodeling and reactive oxidative stress response activation in HGPS VSMCs. Proteomic analyses identified abnormal acetylation programs in HGPS VSMC replication fork complexes, resulting in reduced H4K16 acetylation. Analysis of acetylation kinetics revealed both upregulation of K16 deacetylation and downregulation of K16 acetylation. This correlates with abnormal accumulation of error-prone nonhomologous end joining (NHEJ) repair proteins on newly replicated chromatin. The knockdown of the histone acetyltransferase MOF recapitulates preferential engagement of NHEJ repair activity in control VSMCs. Additionally, we find that primary donor-derived coronary artery vascular smooth muscle cells from aged individuals show similar defects to HGPS VSMCs, including loss of H4K16 acetylation. Altogether, we provide insight into the molecular mechanisms underlying vascular complications associated with HGPS patients and normative aging.

GSK3 temporally regulates neurogenin 2 proneural activity in the neocortex.

The neocortex is comprised of six neuronal layers that are generated in a defined temporal sequence. While extrinsic and intrinsic cues are known to regulate the sequential production of neocortical neurons, how these factors interact and function in a coordinated manner is poorly understood. The proneural gene Neurog2 is expressed in progenitors throughout corticogenesis, but is only required to specify early-born, deep-layer neuronal identities. Here, we examined how neuronal differentiation in general and Neurog2 function in particular are temporally controlled during murine neocortical development. We found that Neurog2 proneural activity declines in late corticogenesis, correlating with its phosphorylation by GSK3 kinase. Accordingly, GSK3 activity, which is negatively regulated by canonical Wnt signaling, increases over developmental time, while Wnt signaling correspondingly decreases. When ectopically activated, GSK3 inhibits Neurog2-mediated transcription in cultured cells and Neurog2 proneural activities in vivo. Conversely, a reduction in GSK3 activity promotes the precocious differentiation of later stage cortical progenitors without influencing laminar fate specification. Mechanistically, we show that GSK3 suppresses Neurog2 activity by influencing its choice of dimerization partner, promoting heterodimeric interactions with E47 (Tcfe2a), as opposed to Neurog2-Neurog2 homodimer formation, which occurs when GSK3 activity levels are low. At the functional level, Neurog2-E47 heterodimers have a reduced ability to transactivate neuronal differentiation genes compared with Neurog2-Neurog2 homodimers, both in vitro and in vivo. We thus conclude that the temporal regulation of Neurog2-E47 heterodimerization by GSK3 is a central component of the neuronal differentiation "clock" that coordinates the timing and tempo of neocortical neurogenesis in mouse.

Caspase 3/caspase-activated DNase promote cell differentiation by inducing DNA strand breaks.

Caspase 3 is required for the differentiation of a wide variety of cell types, yet it remains unclear how this apoptotic protein could promote such a cell-fate decision. Caspase signals often result in the activation of the specific nuclease caspase-activated DNase (CAD), suggesting that cell differentiation may be dependent on a CAD-mediated modification in chromatin structure. In this study, we have investigated if caspase 3/CAD plays a role in initiating the DNA strand breaks that are known to occur during the terminal differentiation of skeletal muscle cells. Here, we show that inhibition of caspase 3 or reduction of CAD expression leads to a dramatic loss of strand-break formation and a block in the myogenic program. Caspase-dependent induction of differentiation results in CAD targeting of the p21 promoter, and loss of caspase 3 or CAD leads to a significant down-regulation in p21 expression. These results show that caspase 3/CAD promotes cell differentiation by directly modifying the DNA/nuclear microenvironment, which enhances the expression of critical regulatory genes.

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.