JARID2 and the PRC2 complex regulate the cell cycle in skeletal muscle.

JARID2 is a noncatalytic member of the polycomb repressive complex 2 (PRC2) which methylates of histone 3 lysine 27 (H3K27). In this work, we show that JARID2 and the PRC2 complex regulate the cell cycle in skeletal muscle cells to control proliferation and mitotic exit. We found that the stable depletion of JARID2 leads to increased proliferation and cell accumulation in S phase. The regulation of the cell cycle by JARID2 is mediated by direct repression of both cyclin D1 and cyclin E1, both of which are targets of PRC2-mediated H3K27 methylation. Intriguingly, we also find that the retinoblastoma protein (RB1) is a direct target of JARID2 and the PRC2 complex. The depletion of JARID2 is not sufficient to activate RB1. However, the ectopic expression of RB1 can suppress cyclin D1 expression in JARID2-depleted cells. Transient depletion of JARID2 in skeletal muscle cells leads to a transient up-regulation of cyclin D1 that is quickly suppressed with no resulting effect on proliferation, Taken together, we show that JARID2 and the PRC2 complex regulate skeletal muscle proliferation in a precise manner that involves the repression of cyclin D1, thus restraining proliferation and repressing RB1, which is required for mitotic exit and terminal differentiation.

Alternative splicing of MEF2C pre-mRNA controls its activity in normal myogenesis and promotes tumorigenicity in rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Many cellular disruptions contribute to the progression of this pediatric cancer, including aberrant alternative splicing. The MEF2 family of transcription factors regulates many developmental programs, including myogenesis. MEF2 gene transcripts are subject to alternate splicing to generate protein isoforms with divergent functions. We found that MEF2Cα1 was the ubiquitously expressed isoform that exhibited no myogenic activity and that MEF2Cα2, the muscle-specific MEF2C isoform, was required for efficient differentiation. We showed that exon α in MEF2C was aberrantly alternatively spliced in RMS cells, with the ratio of α2/α1 highly down-regulated in RMS cells compared with normal myoblasts. Compared with MEF2Cα2, MEF2Cα1 interacted more strongly with and recruited HDAC5 to myogenic gene promoters to repress muscle-specific genes. Overexpression of the MEF2Cα2 isoform in RMS cells increased myogenic activity and promoted differentiation in RMS cells. We also identified a serine protein kinase, SRPK3, that was down-regulated in RMS cells and found that expression of SRPK3 promoted the splicing of the MEF2Cα2 isoform and induced differentiation. Restoration of either MEF2Cα2 or SPRK3 inhibited both proliferation and anchorage-independent growth of RMS cells. Together, our findings indicate that the alternative splicing of MEF2C plays an important role in normal myogenesis and RMS development. An improved understanding of alternative splicing events in RMS cells will potentially reveal novel therapeutic targets for RMS treatment.

Myogenin recruits the histone chaperone facilitates chromatin transcription (FACT) to promote nucleosome disassembly at muscle-specific genes.

Facilitates chromatin transcription (FACT) functions to reorganize nucleosomes by acting as a histone chaperone that destabilizes and restores nucleosomal structure. The FACT complex is composed of two subunits: SSRP1 and SPT16. We have discovered that myogenin interacts with the FACT complex. Transfection of FACT subunits with myogenin is highly stimulatory for endogenous muscle gene expression in 10T1/2 cells. We have also found that FACT subunits do not associate with differentiation-specific genes while C2C12 cells are proliferating but are recruited to muscle-specific genes as differentiation initiates and then dissociate as differentiation proceeds. The recruitment is dependent on myogenin, as knockdowns of myogenin show no recruitment of the FACT complex. These data suggest that FACT is involved in the early steps of gene activation through its histone chaperone activities that serve to open the chromatin structure and facilitate transcription. Consistent with this hypothesis, we find that nucleosomes are depleted at muscle-specific promoters upon differentiation and that this activity is dependent on the presence of FACT. Our results show that the FACT complex promotes myogenin-dependent transcription and suggest that FACT plays an important role in the establishment of the appropriate transcription profile in a differentiated muscle cell.

TBX2 blocks myogenesis and promotes proliferation in rhabdomyosarcoma cells.

Rhabdomyosarcomas (RMSs) are the most frequent soft tissue sarcomas in children that share many features of developing skeletal muscle. We have discovered that a T-box family member, TBX2, is highly upregulated in tumor cells of both major RMS subtypes. TBX2 is a repressor that is often overexpressed in cancer cells and is thought to function in bypassing cell growth control, including repression of p14 and p21. The cell cycle regulator p21 is required for the terminal differentiation of skeletal muscle cells and is silenced in RMS cells. We have found that TBX2 interacts with the myogenic regulatory factors MyoD and myogenin and inhibits the activity of these factors. TBX2 is expressed in primary myoblasts and C2C12 cells, but is strongly downregulated upon differentiation. TBX2 recruits the histone deacetylase HDAC1 and is a potent inhibitor of the expression of muscle-specific genes and the cell cycle regulators, p21 and p14. TBX2 promotes the proliferation of RMS cells and either depletions of TBX2 or dominant negative TBX2 upregulate p21- and muscle-specific genes. Significantly, depletion or interference with TBX2 completely inhibits tumor growth in a xenograft assay, highlighting the oncogenic role of TBX2 in RMS cells. Thus, the data demonstrate that elevated expression of TBX2 contributes to the pathology of RMS cells by promoting proliferation and repressing differentiation-specific gene expression. These results show that deregulated TBX2 serves as an oncogene in RMS, suggesting that TBX2 may serve as a new diagnostic marker or therapeutic target for RMS tumors.

Loss of MEF2D expression inhibits differentiation and contributes to oncogenesis in rhabdomyosarcoma cells.

BACKGROUND: Rhabdomyosarcoma (RMS) is a highly malignant pediatric cancer that is the most common form of soft tissue tumors in children. RMS cells have many features of skeletal muscle cells, yet do not differentiate. Thus, our studies have focused on the defects present in these cells that block myogenesis. METHODS: Protein and RNA analysis identified the loss of MEF2D in RMS cells. MEF2D was expressed in RD and RH30 cells by transient transfection and selection of stable cell lines, respectively, to demonstrate the rescue of muscle differentiation observed. A combination of techniques such as proliferation assays, scratch assays and soft agar assays were used with RH30 cells expressing MEF2D to demonstrate the loss of oncogenic growth in vitro and xenograft assays were used to confirm the loss of tumor growth in vivo. RESULTS: Here, we show that one member of the MEF2 family of proteins required for normal myogenesis, MEF2D, is largely absent in RMS cell lines representing both major subtypes of RMS as well as primary cells derived from an embryonal RMS model. We show that the down regulation of MEF2D is a major cause for the failure of RMS cells to differentiate. We find that MyoD and myogenin are bound with their dimerization partner, the E proteins, to the promoters of muscle specific genes in RMS cells. However, we cannot detect MEF2D binding at any promoter tested. We find that exogenous MEF2D expression can activate muscle specific luciferase constructs, up regulate p21 expression and increase muscle specific gene expression including the expression of myosin heavy chain, a marker for skeletal muscle differentiation. Restoring expression of MEF2D also inhibits proliferation, cell motility and anchorage independent growth in vitro. We have confirmed the inhibition of tumorigenicity by MEF2D in a tumor xenograft model, with a complete regression of tumor growth. CONCLUSIONS: Our data indicate that the oncogenic properties of RMS cells can be partially attributed to the loss of MEF2D expression and that restoration of MEF2D may represent a useful therapeutic strategy to decrease tumorigenicity.

CIITA is silenced by epigenetic mechanisms that prevent the recruitment of transactivating factors in rhabdomyosarcoma cells.

Rhabdomyosarcomas (RMS) are highly malignant pediatric sarcomas. We have discovered that the gene encoding the major histocompatibilty complex class II transactivator, CIITA, is silenced in cells representing both major subtypes of RMS. Silencing of CIITA prevents the IFN-γ inducible expression of MHC class II genes in these cells. Overexpression of CIITA in these cells can restore MHC expression. We have found that IFN-γ signaling is intact in these cells, but pSTAT1 and IRF1 do not bind to the CIITA PIV promoter. The CIITA promoter is not hypermethylated in RD (ERMS) cells but does show a modestly enhanced methylation status in SJRH30 (ARMS) cells. We have found that histone acetylation, which normally increases on the CIITA PIV promoter following IFN-γ treatment, is blocked in both types of RMS cells. In RD cells, treatment with a histone deacetylase inhibitor (TSA) reverses the silencing of CIITA. In SJRH30 cells, treatment with DNA methyltransferase inhibitors and TSA cooperatively restores CIITA expression. Surprisingly, we have also shown that the expression of two components of the immunoproteasome, which are embedded in the class II locus, is stimulated by IFN-γ in certain RMS cells in the absence of stimulation by CIITA. CIITA overexpression can also activate the expression of these genes, indicating that the immunoproteasome genes LMP2 and LMP7 can be activated by both CIITA dependent and CIITA independent pathways.

Transcriptional analysis of the titin cap gene.

Mutations in titin cap (Tcap), also known as telethonin, cause limb-girdle muscular dystrophy type 2G (LGMD2G). Tcap is one of the titin interacting Z-disc proteins involved in the regulation and development of normal sarcomeric structure. Given the essential role of Tcap in establishing and maintaining normal skeletal muscle architecture, we were interested in determining the regulatory elements required for expression of this gene in myoblasts. We have defined a highly conserved 421 bp promoter proximal promoter fragment that contains two E boxes and multiple putative Mef2 binding sequences. This promoter can be activated by MyoD and myogenin in NIH3T3 fibroblast cells, and maintains the differentiated cell-specific expression pattern of the endogenous Tcap in C2C12 cells. We find that while both E boxes are required for full activation by MyoD or myogenin in NIH3T3 cells, the promoter proximal E box has a greater contribution to activation of this promoter in C2C12 cells and to activation by MyoD in NIH3T3 cells. Together, the data suggest an important role for MyoD in activating Tcap expression through the promoter proximal E box. We also show that myogenin is required for normal expression in vivo and physically binds to the Tcap promoter during embryogenesis.

Myogenin is required for assembly of the transcription machinery on muscle genes during skeletal muscle differentiation.

Skeletal muscle gene expression is governed by the myogenic regulatory family (MRF) which includes MyoD (MYOD1) and myogenin (MYOG). MYOD1 and MYOG are known to regulate an overlapping set of muscle genes, but MYOD1 cannot compensate for the absence of MYOG in vivo. In vitro, late muscle genes have been shown to be bound by both factors, but require MYOG for activation. The molecular basis for this requirement was unclear. We show here that MYOG is required for the recruitment of TBP and RNAPII to muscle gene promoters, indicating that MYOG is essential in assembling the transcription machinery. Genes regulated by MYOD1 and MYOG include genes required for muscle fusion, myomaker and myomerger, and we show that myomaker is fully dependent on activation by MYOG. We also sought to determine the role of MYOD1 in MYOG dependent gene activation and unexpectedly found that MYOG is required to maintain Myod1 expression. However, we also found that exogenous MYOD1 was unable to compensate for the loss of Myog and activate muscle gene expression. Thus, our results show that MYOD1 and MYOG act in a feed forward loop to maintain each other's expression and also show that it is MYOG, and not MYOD1, that is required to load TBP and activate gene expression on late muscle gene promoters bound by both factors.

Proteasomal Regulation of Mammalian SPT16 in Controlling Transcription.

FACT (facilitates chromatin transcription), an essential and evolutionarily conserved heterodimer from yeast to humans, controls transcription and is found to be upregulated in various cancers. However, the basis for such upregulation is not clearly understood. Our recent results deciphering a new ubiquitin-proteasome system regulation of the FACT subunit SPT16 in orchestrating transcription in yeast hint at the involvement of the proteasome in controlling FACT in humans, with a link to cancer. To test this, we carried out experiments in human embryonic kidney (HEK293) cells, which revealed that human SPT16 undergoes ubiquitylation and that its abundance is increased following inhibition of the proteolytic activity of the proteasome, thus implying proteasomal regulation of human SPT16. Furthermore, we find that the increased abundance/expression of SPT16 in HEK293 cells alters the transcription of genes, including ones associated with cancer, and that the proteasomal degradation of SPT16 is impaired in kidney cancer (Caki-2) cells to upregulate SPT16. Like human SPT16, murine SPT16 in C2C12 cells also undergoes ubiquitylation and proteasomal degradation to regulate transcription. Collectively, our results reveal a proteasomal regulation of mammalian SPT16, with physiological relevance in controlling transcription, and implicate such proteasomal control in the upregulation of SPT16 in cancer.

The ERG1A K+ Channel Is More Abundant in Rectus abdominis Muscle from Cancer Patients Than that from Healthy Humans.

BACKGROUND: The potassium channel encoded by the ether-a-gogo-related gene 1A (erg1a) has been detected in the atrophying skeletal muscle of mice experiencing either muscle disuse or cancer cachexia and further evidenced to contribute to muscle deterioration by enhancing ubiquitin proteolysis; however, to our knowledge, ERG1A has not been reported in human skeletal muscle. METHODS AND RESULTS: Here, using immunohistochemistry, we detect ERG1A immunofluorescence in human Rectus abdominis skeletal muscle sarcolemma. Further, using single point brightness data, we report the detection of ERG1A immunofluorescence at low levels in the Rectus abdominis muscle sarcolemma of young adult humans and show that it trends toward greater levels (10.6%) in healthy aged adults. Interestingly, we detect ERG1A immunofluorescence at a statistically greater level (53.6%; p < 0.05) in the skeletal muscle of older cancer patients than in age-matched healthy adults. Importantly, using immunoblot, we reveal that lower mass ERG1A protein is 61.5% (p < 0.05) more abundant in the skeletal muscle of cachectic older adults than in healthy age-matched controls. Additionally, we report that the ERG1A protein is detected in a cultured human rhabdomyosarcoma line that may be a good in vitro model for the study of ERG1A in muscle. CONCLUSIONS: The data demonstrate that ERG1A is detected more abundantly in the atrophied skeletal muscle of cancer patients, suggesting it may be related to muscle loss in humans as it has been shown to be in mice experiencing muscle atrophy as a result of malignant tumors.

The PRC2 complex directly regulates the cell cycle and controls proliferation in skeletal muscle.

The polycomb repressive complex 2 (PRC2) is an important developmental regulator responsible for the methylation of histone 3 lysine 27 (H3K27). Here, we show that the PRC2 complex regulates the cell cycle in skeletal muscle cells to control proliferation and mitotic exit. Depletions of the catalytic subunit of the PRC2 complex, EZH2, have shown that EZH2 is required for cell viability, suggesting that EZH2 promotes proliferation. We found that EZH2 directly represses both positive and negative cell cycle genes, thus enabling the PRC2 complex to tightly control the cell cycle. We show that modest inhibition or depletion of EZH2 leads to enhanced proliferation and an accumulation of cells in S phase. This effect is mediated by direct repression of cyclin D1 (Ccnd1) and cyclin E1 (Ccne1) by the PRC2 complex. Our results show that PRC2 has pleiotropic effects on proliferation as it serves to restrain cell growth, yet clearly has a function required for cell viability as well. Intriguingly, we also find that the retinoblastoma protein gene (Rb1) is a direct target of the PRC2 complex. However, modest depletion of EZH2 is not sufficient to maintain Rb1 expression, indicating that the PRC2 dependent upregulation of cyclin D1 is sufficient to inhibit Rb1 expression. Taken together, our results show that the PRC2 complex regulates skeletal muscle proliferation in a complex manner that involves the repression of Ccnd1 and Ccne1, thus restraining proliferation, and the repression of Rb1, which is required for mitotic exit and terminal differentiation.

The transcription elongation factor TCEA3 induces apoptosis in rhabdomyosarcoma.

TCEA3 is one of three genes representing the transcription elongation factor TFIIS family in vertebrates. TCEA3 is upregulated during skeletal muscle differentiation and acts to promote muscle specific gene expression during myogenesis. Rhabdomyosarcoma (RMS) is a pediatric cancer derived from the muscle lineage, but the expression or function of TCEA3 in RMS was uncharacterized. We found that TCEA3 expression was strongly inhibited in RMS cell lines representing both ERMS and ARMS subtypes of RMS. TCEA3 expression correlates with DNA methylation and we show that TBX2 is also involved in the repression of TCEA3 in RMS cell lines. Ectopic expression of TCEA3 inhibited proliferation of RMS cell lines and initiated apoptosis through both the intrinsic and extrinsic pathways. We found that only pan-caspase inhibitors could block apoptosis in the presence of TCEA3. While expression of TCEA3 is highest in skeletal muscle, expression has been detected in other tissues as well, including breast, ovarian and prostate. We found that ectopic expression of TCEA3 also promotes apoptosis in HeLa, MCF7, MDA-231, and PC3 cell lines, representing cervical, breast, and prostate cancer, respectively. Restoration of TCEA3 expression in RMS cell lines enhanced sensitivity to chemotherapeutic drugs, including TRAIL. Thus, TCEA3 presents a novel target for therapeutic strategies to promote apoptosis and enhance sensitivity to current chemotherapeutic drugs.

IFN-γ and CIITA modulate IL-6 expression in skeletal muscle.

Interleukin 6 (IL-6) is a secreted cytokine that is an important mediator of the immune response in numerous tissues, including skeletal muscle. IL-6 is considered a myokine as it can be secreted by muscle. IL-6 is secreted following exercise, where it exerts both pro-myogenic effects as well as anti-myogenic effects such as promoting atrophy and muscle wasting. The regulation of IL-6 in skeletal muscle is not well understood. The purpose of this study was to determine if IFN-γ and TNF-ɑ stimulate IL-6 in skeletal muscle. We found that both IFN-γ and TNF-α stimulate IL-6 in skeletal muscle, but the stimulation is not cooperative as seen in monocytes. We have previously shown that the IFN-γ stimulated class II major histocompatibility complex transactivator (CIITA) mediates many of the effects of IFN-γ in skeletal muscle and we show here that CIITA directly stimulates IL-6. The regulation of IL-6 by CIITA is clearly complex, as we found that CIITA both stimulates and restrains IL-6 expression. To show that these effects could be observed in a physiological setting, mice were treated with IFN-γ and we found that both CIITA and IL-6 were upregulated in skeletal muscle.

The ERG1a potassium channel increases basal intracellular calcium concentration and calpain activity in skeletal muscle cells.

BACKGROUND: Skeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation. It occurs in response to several stimuli including disease, injury, starvation, and normal aging. Currently, there is no truly effective pharmacological therapy for atrophy; therefore, exploration of the mechanisms contributing to atrophy is essential because it will eventually lead to discovery of an effective therapeutic target. The ether-a-go-go related gene (ERG1A) K+ channel has been shown to contribute to atrophy by upregulating ubiquitin proteasome proteolysis in cachectic and unweighted mice and has also been implicated in calcium modulation in cancer cells. METHODS: We transduced C2C12 myotubes with either a human ERG1A encoded adenovirus or an appropriate control virus. We used fura-2 calcium indicator to measure intracellular calcium concentration and Calpain-Glo assay kits (ProMega) to measure calpain activity. Quantitative PCR was used to monitor gene expression and immunoblot evaluated protein abundances in cell lysates. Data were analyzed using either a Student's t test or two-way ANOVAs and SAS software as indicated. RESULTS: Expression of human ERG1A in C2C12 myotubes increased basal intracellular calcium concentration 51.7% (p < 0.0001; n = 177). Further, it increased the combined activity of the calcium-activated cysteine proteases, calpain 1 and 2, by 31.9% (p < 0.08; n = 24); these are known to contribute to degradation of myofilaments. The increased calcium levels are likely a contributor to the increased calpain activity; however, the change in calpain activity may also be attributable to increased calpain protein abundance and/or a decrease in levels of the native calpain inhibitor, calpastatin. To explore the enhanced calpain activity further, we evaluated expression of calpain and calpastatin genes and observed no significant differences. There was no change in calpain 1 protein abundance; however, calpain 2 protein abundance decreased 40.7% (p < 0.05; n = 6). These changes do not contribute to an increase in calpain activity; however, we detected a 31.7% decrease (p < 0.05; n = 6) in calpastatin which could contribute to enhanced calpain activity. CONCLUSIONS: Human ERG1A expression increases both intracellular calcium concentration and combined calpain 1 and 2 activity. The increased calpain activity is likely a result of the increased calcium levels and decreased calpastatin abundance.

The transcription elongation factor TCEA3 promotes the activity of the myogenic regulatory factors.

The transcription elongation factor TFIIS is encoded by a three member gene family in vertebrates. Here we show that one member of this family, TCEA3, is upregulated during skeletal muscle differentiation and acts to promote gene activation by the myogenic regulatory family of transcription factors, which includes MyoD and myogenin. We show that myogenin is a direct regulator of Tcea3. Myogenin binds to the Tcea3 promoter and is required to recruit RNA polymerase II. TCEA3 can bind to both myogenin and MyoD and is co-recruited with the MRFs to promoters dependent on the MRFs. Depletion of myogenin inhibits the recruitment of TCEA3, suggesting that the interaction of TCEA3 with the MRFs serves to aid in recruitment to target promoters. Like TFIIS, we show that TCEA3 interacts with RNA polymerase II. TCEA3 travels with the elongating RNA polymerase II in the coding region of genes and depletions of TCEA3 inhibit the recruitment of RNA polymerase II to promoters. In proliferating cells, TCEA3 expressed at low levels and is present in both the nucleus and cytoplasm. However, upon differentiation, TCEA3 is upregulated and transported exclusively to the nucleus. Thus, our data show that TCEA3 is a required co-factor for MRF driven gene expression during myogenesis.

TBX3 represses TBX2 under the control of the PRC2 complex in skeletal muscle and rhabdomyosarcoma.

TBX2 and TBX3 function as repressors and are frequently implicated in oncogenesis. We have shown that TBX2 represses p21, p14/19, and PTEN in rhabdomyosarcoma (RMS) and skeletal muscle but the function and regulation of TBX3 were unclear. We show that TBX3 directly represses TBX2 in RMS and skeletal muscle. TBX3 overexpression impairs cell growth and migration and we show that TBX3 is directly repressed by the polycomb repressive complex 2 (PRC2), which methylates histone H3 lysine 27 (H3K27me). We found that TBX3 promotes differentiation only in the presence of early growth response factor 1 (EGR1), which is differentially expressed in RMS and is also a target of the PRC2 complex. The potent regulation axis revealed in this work provides novel insight into the effects of the PRC2 complex in normal cells and RMS and further supports the therapeutic value of targeting of PRC2 in RMS.

Target gene selectivity of the myogenic basic helix-loop-helix transcription factor myogenin in embryonic muscle.

The myogenic regulatory factors MyoD and myogenin are crucial for skeletal muscle development. Despite their importance, the mechanisms by which these factors selectively regulate different target genes are unclear. The purpose of the present investigation was to compare embryonic skeletal muscle from myogenin(+/+) and myogenin(-/-) mice to identify genes whose expression was dependent on the presence of myogenin but not MyoD and to determine whether myogenin-binding sites could be found within regulatory regions of myogenin-dependent genes independent of MyoD. We identified a set of 140 muscle-expressed genes whose expression in embryonic tongue muscle of myogenin(-/-) mice was downregulated in the absence of myogenin, but in the presence of MyoD. Myogenin bound within conserved regulatory regions of several of the downregulated genes, but MyoD bound only to a subset of these same regions, suggesting that many downregulated genes were selective targets of myogenin. The regulatory regions activated gene expression in cultured myoblasts and fibroblasts overexpressing myogenin or MyoD, indicating that expression from exogenously introduced DNA could not recapitulate the selectivity for myogenin observed in vivo. The results identify new target genes for myogenin and show that myogenin's target gene selectivity is not based solely on binding site sequences.

JARID2 and the PRC2 complex regulate skeletal muscle differentiation through regulation of canonical Wnt signaling.

BACKGROUND: JARID2 is a non-catalytic member of the polycomb repressive complex 2 (PRC2), which is known to regulate developmental target genes in embryonic stem cells. Here, we provide mechanistic insight into the modulation of Wnt signaling by JARID2 during murine skeletal muscle differentiation. RESULTS: We show that JARID2 is expressed in proliferating myoblasts, but downregulated upon muscle differentiation. Unexpectedly, depletion of JARID2 or the catalytic subunit of the PRC2 complex, EZH2, inhibited differentiation, suggesting that JARID2 and the PRC2 complex are required to initiate this process. Expression of the myogenic regulatory factors required to promote differentiation, MYOD and MYOG, was downregulated in the absence of JARID2, even though decreases in the methylation of histone H3 lysine 27 (H3K27me3) were observed on both promoters. We found that activation of the Wnt signaling pathway upregulated MYOD and restored differentiation. Activation of the Wnt pathway in JARID2 depleted cells caused ß-catenin to translocate to the nucleus, where it bound to and activated the Myod1 promoter. We show that the Wnt antagonist SFRP1 is highly upregulated in the absence of JARID2 and is a direct target of JARID2 and the PRC2 complex. Ectopic expression of SFRP1 blocked MYOD and late muscle gene expression and inhibited the translocation of ß-catenin to the nucleus. Finally, we show that JARID2 and SFRP1 are inversely correlated in melanoma, confirming that the JARID2-mediated repression of SFRP1 extends beyond skeletal muscle and has important implications in many cellular systems, including cancer. CONCLUSIONS: We show that JARID2 and the PRC2 complex regulate muscle differentiation by modulating Wnt signaling through the direct repression of Wnt antagonists.

EGR1 interacts with TBX2 and functions as a tumor suppressor in rhabdomyosarcoma.

EGR1, one of the immediate-early response genes, can function as a tumor suppressor gene or as an oncogene in cancer. The function of EGR1 has not been fully characterized in rhabdomyosarcoma (RMS), a pediatric cancer derived from the muscle linage. We found that EGR1 is downregulated in the alveolar RMS (ARMS) subtype but expressed at levels comparable to normal skeletal muscle in embryonal RMS (ERMS). We found that overexpression of EGR1 in ARMS significantly decreased cell proliferation, mobility, and anchorage-independent growth while also promoting differentiation. We found that EGR1 interacts with TBX2, which we have shown functions as an oncogene in RMS. The interaction inhibits EGR1 dependent gene expression, which includes the cell cycle regulators p21 and PTEN as well as other important cell growth drivers such as NDRG1 and CST6. We also found that EGR1 induced apoptosis by triggering the intrinsic apoptosis pathway. EGR1 also activated two pro-apoptotic factors, BAX and dephosphorylated BAD, which are both located upstream of the caspase cascades in the intrinsic pathway. EGR1 also sensitized RMS cells to chemotherapeutic agents, suggesting that activating EGR1 may improve therapeutic targeting by inducing apoptosis. Our results establish the important role of EGR1 in understanding RMS pathology.

Transcriptional control: an activating role for arginine methylation.

A rare histone modification, arginine methylation, has been linked to activation of hormone-responsive genes. Interestingly, methylation of a lysine residue in the same histone is present prior to hormone activation, but is excluded from the active loci.