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Margaret Buckingham - One of the best experts on this subject based on the ideXlab platform.
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PAX3 and PAX7 as upstream regulators of myogenesis.
Seminars in cell & developmental biology, 2015Co-Authors: Margaret Buckingham, Frederic RelaixAbstract:Like other subclasses within the PAX transcription factor family, PAX3 and PAX7 play important roles in the emergence of a number of different tissues during development. PAX3 regulates neural crest and, together with its orthologue PAX7, is also expressed in parts of the central nervous system. In this chapter we will focus on their role in skeletal muscle. Both factors are key regulators of myogenesis where PAX3 plays a major role during early skeletal muscle formation in the embryo while Pax7 predominates during post-natal growth and muscle regeneration in the adult. We review the expression and functions of these factors in the myogenic context. We also discuss mechanistic aspects of PAX3/7 function and modulation of their activity by interaction with other proteins, as well as the post-transcriptional and transcriptional regulation of their expression.
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Transcriptome analyses based on genetic screens for PAX3 myogenic targets in the mouse embryo
BMC Genomics, 2010Co-Authors: Mounia Lagha, Frederic Relaix, Ana Cumano, Takahiko Sato, Béatrice Regnault, Aimée Zuniga, Jonathan Licht, Margaret BuckinghamAbstract:Background PAX3 is a key upstream regulator of the onset of myogenesis, controlling progenitor cell survival and behaviour as well as entry into the myogenic programme. It functions in the dermomyotome of the somite from which skeletal muscle derives and in progenitor cell populations that migrate from the somite such as those of the limbs. Few PAX3 target genes have been identified. Identifying genes that lie genetically downstream of PAX3 is therefore an important endeavour in elucidating the myogenic gene regulatory network. Results We have undertaken a screen in the mouse embryo which employs a PAX3 ^ GFP allele that permits isolation of PAX3 expressing cells by flow cytometry and a PAX3 ^ PAX3-FKHR allele that encodes PAX3-FKHR in which the DNA binding domain of PAX3 is fused to the strong transcriptional activation domain of FKHR. This constitutes a gain of function allele that rescues the PAX3 mutant phenotype. Microarray comparisons were carried out between PAX3 ^ GFP/+ and PAX3 ^ GFP/PAX3-FKHR preparations from the hypaxial dermomyotome of somites at E9.5 and forelimb buds at E10.5. A further transcriptome comparison between PAX3-GFP positive and negative cells identified sequences specific to myogenic progenitors in the forelimb buds. Potential PAX3 targets, based on changes in transcript levels on the gain of function genetic background, were validated by analysis on loss or partial loss of function PAX3 mutant backgrounds. Sequences that are up- or down-regulated in the presence of PAX3-FKHR are classified as somite only, somite and limb or limb only. The latter should not contain sequences from PAX3 positive neural crest cells which do not invade the limbs. Verification by whole mount in situ hybridisation distinguishes myogenic markers. Presentation of potential PAX3 target genes focuses on signalling pathways and on transcriptional regulation. Conclusions PAX3 orchestrates many of the signalling pathways implicated in the activation or repression of myogenesis by regulating effectors and also, notably, inhibitors of these pathways. Important transcriptional regulators of myogenesis are candidate PAX3 targets. Myogenic determination genes, such as Myf5 are controlled positively, whereas the effect of PAX3 on genes encoding inhibitors of myogenesis provides a potential brake on differentiation. In the progenitor cell population, Pax7 and also Hdac5 which is a potential repressor of Foxc2 , are subject to positive control by PAX3 .
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muscle stem cell behavior is modified by microrna 27 regulation of PAX3 expression
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Colin G Crist, Didier Rocancourt, Ana Cumano, Didier Montarras, Giorgia Pallafacchina, Simon J Conway, Margaret BuckinghamAbstract:Skeletal muscle stem cells are regulated by PAX3/7. During development, PAX3 is required for the maintenance of these cells in the somite and their migration to sites of myogenesis; high levels of PAX3 interfere with muscle cell differentiation, both in the embryo and in the adult. Quantitative fine-tuning of PAX3 is critical, and microRNAs provide a potential mechanism. We identify microRNA-27b (miR-27b), which directly targets the 3′-UTR of PAX3 mRNA, as such a regulator. miR-27b is expressed in the differentiating skeletal muscle of the embryonic myotome and in activated satellite cells of adult muscle. In vivo overexpression of a miR-27b transgene in PAX3-positive cells in the embryo leads to down-regulation of PAX3, resulting in interference with progenitor cell migration and in premature differentiation. In a complementary experiment, miR-27b inhibitors were transfected into cultures of adult muscle satellite cells that normally express miR-27b at the onset of differentiation, when PAX3 protein levels undergo rapid down-regulation. Interference with miR-27b function results in continuing PAX3 expression leading to more proliferation and a delay in the onset of differentiation. Pax7 levels are not affected. Introduction of miR-27b antagomirs at a site of muscle injury in vivo also affects PAX3 expression and regeneration in vivo. We therefore conclude that miR-27b regulates PAX3 protein levels and this down-regulation ensures rapid and robust entry into the myogenic differentiation program.
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Skeletal Muscle Differentiation of Embryonic Mesoangioblasts Requires PAX3 Activity
Stem cells (Dayton Ohio), 2009Co-Authors: Graziella Messina, Frederic Relaix, Dario Sirabella, Stefania Monteverde, Beatriz G. Gálvez, Rossana Tonlorenzi, Esther Schnapp, Luciana De Angelis, Silvia Brunelli, Margaret BuckinghamAbstract:Mesoangioblasts have been characterized as a population of vessel-associated stem cells able to differentiate into several mesodermal cell types, including skeletal muscle. Here, we report that the paired box transcription factor PAX3 plays a crucial role in directing mouse mesoangioblasts toward skeletal myogenesis in vitro and in vivo. Mesoangioblasts isolated from the aorta of PAX3 null embryos are severely impaired in skeletal muscle differentiation, whereas most other differentiation programs are not affected by the absence of PAX3. Moreover, PAX3(-/-) null mesoangioblasts failed to rescue the myopathic phenotype of the alpha-sarcoglycan mutant mouse. In contrast, mesoangioblasts from PAX3 gain of function, PAX3(PAX3-FKHR/+), mice display enhanced myogenesis in vitro and are more efficient in regenerating new muscle fibers in this model of muscular dystrophy. These data demonstrate that PAX3 is required for the differentiation of mesoangioblast stem cells into skeletal muscle, in keeping with its role in orchestrating entry into the myogenic program.
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Regulation of skeletal muscle stem cell behavior by PAX3 and Pax7.
Cold Spring Harbor symposia on quantitative biology, 2008Co-Authors: Mounia Lagha, Frederic Relaix, Didier Montarras, Takahiko Sato, L. Bajard, Philippe Daubas, M. Esner, Margaret BuckinghamAbstract:Pax genes have important roles in the regulation of stem cell behavior, leading to tissue differentiation. In the case of skeletal muscle, PAX3 and Pax7 perform this function both during development and on regeneration in the adult. The myogenic determination gene Myf5 is directly activated by PAX3, leading to the formation of skeletal muscle. Fgfr4 is also a direct PAX3 target and Sprouty1, which encodes an intracellular inhibitor of fibroblast growth factor (FGF) signaling, is under PAX3 control. Orchestration of FGF signaling, through Fgfr4/Sprouty1, modulates the entry of cells into the myogenic program, thus controling the balance between stem cell self-renewal and tissue differentiation. This and other aspects of PAX3/7 function in regulating the behavior of skeletal muscle stem cells are discussed.
Frederic Relaix - One of the best experts on this subject based on the ideXlab platform.
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PAX3 and PAX7 as upstream regulators of myogenesis.
Seminars in cell & developmental biology, 2015Co-Authors: Margaret Buckingham, Frederic RelaixAbstract:Like other subclasses within the PAX transcription factor family, PAX3 and PAX7 play important roles in the emergence of a number of different tissues during development. PAX3 regulates neural crest and, together with its orthologue PAX7, is also expressed in parts of the central nervous system. In this chapter we will focus on their role in skeletal muscle. Both factors are key regulators of myogenesis where PAX3 plays a major role during early skeletal muscle formation in the embryo while Pax7 predominates during post-natal growth and muscle regeneration in the adult. We review the expression and functions of these factors in the myogenic context. We also discuss mechanistic aspects of PAX3/7 function and modulation of their activity by interaction with other proteins, as well as the post-transcriptional and transcriptional regulation of their expression.
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alveolar rhabdomyosarcoma associated proteins PAX3 foxo1a and pax7 foxo1a suppress the transcriptional activity of myod target genes in muscle stem cells
Oncogene, 2013Co-Authors: F Calhabeu, Frederic Relaix, Shinichiro Hayashi, Jennifer E Morgan, Peter S ZammitAbstract:Rhabdomyosarcoma (RMS) is the commonest soft-tissue sarcoma in childhood and is characterized by expression of myogenic proteins, including the transcription factors MyoD and myogenin. There are two main subgroups, embryonal RMS and alveolar RMS (ARMS). Most ARMS are associated with chromosomal translocations that have breakpoints in introns of either PAX3 or PAX7, and FOXO1A. These translocations create chimeric transcription factors termed PAX3/FOXO1A and PAX7/FOXO1A respectively. Upon ectopic PAX3/FOXO1A expression, together with other genetic manipulation in mice, both differentiating myoblasts and satellite cells (the resident stem cells of postnatal muscle) can give rise to tumours with ARMS characteristics. As PAX3 and PAX7 are part of transcriptional networks that regulate muscle stem cell function in utero and during early postnatal life, PAX3/FOXO1A and PAX7/FOXO1A may subvert normal PAX3 and PAX7 functions. Here we examined how PAX3/FOXO1A and PAX7/FOXO1A affect myogenesis in satellite cells. PAX3/FOXO1A or PAX7/FOXO1A inhibited myogenin expression and prevented terminal differentiation in murine satellite cells: the same effect as dominant-negative (DN) PAX3 or Pax7 constructs. The transcription of MyoD-target genes myogenin and muscle creatine kinase were suppressed by PAX3/FOXO1A or PAX7/FOXO1A in C2C12 myogenic cells again as seen with PAX3/7DN. PAX3/FOXO1A or PAX7/FOXO1A did not inhibit the transcriptional activity of MyoD by perturbing MyoD expression, localization, phosphorylation or interaction with E-proteins. Chromatin immunoprecipitation on the myogenin promoter showed that PAX3/FOXO1A or PAX7/FOXO1A did not prevent MyoD from binding. However, PAX3/FOXO1A or PAX7/FOXO1A reduced occupation of the myogenin promoter by RNA polymerase II and decreased acetylation of histone H4, but did not directly bind to the myogenin promoter. Together, these observations reveal that PAX3/FOXO1A and PAX7/FOXO1A act to prevent myogenic differentiation via suppression of the transcriptional activation of MyoD-target genes.
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Transcriptome analyses based on genetic screens for PAX3 myogenic targets in the mouse embryo
BMC Genomics, 2010Co-Authors: Mounia Lagha, Frederic Relaix, Ana Cumano, Takahiko Sato, Béatrice Regnault, Aimée Zuniga, Jonathan Licht, Margaret BuckinghamAbstract:Background PAX3 is a key upstream regulator of the onset of myogenesis, controlling progenitor cell survival and behaviour as well as entry into the myogenic programme. It functions in the dermomyotome of the somite from which skeletal muscle derives and in progenitor cell populations that migrate from the somite such as those of the limbs. Few PAX3 target genes have been identified. Identifying genes that lie genetically downstream of PAX3 is therefore an important endeavour in elucidating the myogenic gene regulatory network. Results We have undertaken a screen in the mouse embryo which employs a PAX3 ^ GFP allele that permits isolation of PAX3 expressing cells by flow cytometry and a PAX3 ^ PAX3-FKHR allele that encodes PAX3-FKHR in which the DNA binding domain of PAX3 is fused to the strong transcriptional activation domain of FKHR. This constitutes a gain of function allele that rescues the PAX3 mutant phenotype. Microarray comparisons were carried out between PAX3 ^ GFP/+ and PAX3 ^ GFP/PAX3-FKHR preparations from the hypaxial dermomyotome of somites at E9.5 and forelimb buds at E10.5. A further transcriptome comparison between PAX3-GFP positive and negative cells identified sequences specific to myogenic progenitors in the forelimb buds. Potential PAX3 targets, based on changes in transcript levels on the gain of function genetic background, were validated by analysis on loss or partial loss of function PAX3 mutant backgrounds. Sequences that are up- or down-regulated in the presence of PAX3-FKHR are classified as somite only, somite and limb or limb only. The latter should not contain sequences from PAX3 positive neural crest cells which do not invade the limbs. Verification by whole mount in situ hybridisation distinguishes myogenic markers. Presentation of potential PAX3 target genes focuses on signalling pathways and on transcriptional regulation. Conclusions PAX3 orchestrates many of the signalling pathways implicated in the activation or repression of myogenesis by regulating effectors and also, notably, inhibitors of these pathways. Important transcriptional regulators of myogenesis are candidate PAX3 targets. Myogenic determination genes, such as Myf5 are controlled positively, whereas the effect of PAX3 on genes encoding inhibitors of myogenesis provides a potential brake on differentiation. In the progenitor cell population, Pax7 and also Hdac5 which is a potential repressor of Foxc2 , are subject to positive control by PAX3 .
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Skeletal Muscle Differentiation of Embryonic Mesoangioblasts Requires PAX3 Activity
Stem cells (Dayton Ohio), 2009Co-Authors: Graziella Messina, Frederic Relaix, Dario Sirabella, Stefania Monteverde, Beatriz G. Gálvez, Rossana Tonlorenzi, Esther Schnapp, Luciana De Angelis, Silvia Brunelli, Margaret BuckinghamAbstract:Mesoangioblasts have been characterized as a population of vessel-associated stem cells able to differentiate into several mesodermal cell types, including skeletal muscle. Here, we report that the paired box transcription factor PAX3 plays a crucial role in directing mouse mesoangioblasts toward skeletal myogenesis in vitro and in vivo. Mesoangioblasts isolated from the aorta of PAX3 null embryos are severely impaired in skeletal muscle differentiation, whereas most other differentiation programs are not affected by the absence of PAX3. Moreover, PAX3(-/-) null mesoangioblasts failed to rescue the myopathic phenotype of the alpha-sarcoglycan mutant mouse. In contrast, mesoangioblasts from PAX3 gain of function, PAX3(PAX3-FKHR/+), mice display enhanced myogenesis in vitro and are more efficient in regenerating new muscle fibers in this model of muscular dystrophy. These data demonstrate that PAX3 is required for the differentiation of mesoangioblast stem cells into skeletal muscle, in keeping with its role in orchestrating entry into the myogenic program.
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Activation of PAX3 target genes is necessary but not sufficient for neurogenesis in the ophthalmic trigeminal placode.
Developmental biology, 2008Co-Authors: Carolynn M. Dude, Frederic Relaix, C.-y. Kelly Kuan, James R. Bradshaw, Nicholas D. E. Greene, Michael R. Stark, Clare V.h. BakerAbstract:Abstract Vertebrate cranial neurogenic placodes are relatively simple model systems for investigating the control of sensory neurogenesis. The ophthalmic trigeminal (opV) placode, for which the earliest specific marker is the paired domain homeodomain transcription factor PAX3, forms cutaneous sensory neurons in the ophthalmic lobe of the trigeminal ganglion. We previously showed that PAX3 expression in avian opV placode cells correlates with specification and commitment to a PAX3+, cutaneous sensory neuron fate. PAX3 can act as a transcriptional activator or repressor, depending on the cellular context. We show using mouse Splotch2H mutants that PAX3 is necessary for the normal neuronal differentiation of opV placode cells. Using an electroporation construct encoding a PAX3–Engrailed fusion protein, which represses PAX3 target genes, we show that activation of PAX3 target genes is required cell-autonomously within chick opV placode cells for expression of the opV placode markers FGFR4 and Ngn2, maintenance of the preplacodal marker Eya2, expression of PAX3 itself (suggesting that PAX3 autoregulates), neuronal differentiation and delamination. Mis-expression of PAX3 in head ectoderm is sufficient to induce FGFR4 and Ngn2 expression, but neurons do not differentiate, suggesting that additional signals are necessary to enable PAX3+ cells to differentiate as neurons. Mis-expression of PAX3 in the Pax2+ otic and epibranchial placodes also downregulates Pax2 and disrupts otic vesicle closure, suggesting that PAX3 is sufficient to alter the identity of these cells. Overall, our results suggest that activation of PAX3 target genes is necessary but not sufficient for neurogenesis in the opV placode.
Frederic G. Barr - One of the best experts on this subject based on the ideXlab platform.
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The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3.
Modern pathology : an official journal of the United States and Canadian Academy of Pathology Inc, 2020Co-Authors: William J. Fredericks, Frederic G. Barr, Jeannette L Bennicelli, Naomi Galili, Sunil Mukhopadhyay, Giovanni Rovera, Frank J. RauscherAbstract:Alveolar rhabdomyosarcomas are pediatric solid tumors with a hallmark cytogenetic abnormality: translocation of chromosomes 2 and 13 [t(2;13) (q35;q14)]. The genes on each chromosome involved in this translocation have been identified as the transcription factor-encoding genes PAX3 and FKHR. The NH2-terminal paired box and homeodomain DNA-binding domains of PAX3 are fused in frame to COOH-terminal regions of the chromosome 13-derived FKHR gene, a novel member of the forkhead DNA-binding domain family. To determine the role of the fusion protein in transcriptional regulation and oncogenesis, we identified the PAX3-FKHR fusion protein and characterized its function(s) as a transcription factor relative to wild-type PAX3. Antisera specific to PAX3 and FKHR were developed and used to examine PAX3 and PAX3-FKHR expression in tumor cell lines. Sequential immunoprecipitations with anti-PAX3 and anti-FKHR sera demonstrated expression of a 97-kDa PAX3-FKHR fusion protein in the t(2;13)-positive rhabdomyosarcoma Rh30 cell line and verified that a single polypeptide contains epitopes derived from each protein. The PAX3-FKHR protein was localized to the nucleus in Rh30 cells, as was wild-type PAX3, in t(2;13)-negative A673 cells. In gel shift assays using a canonical PAX binding site (e5 sequence), we found that DNA binding of PAX3-FKHR was significantly impaired relative to that of PAX3 despite the two proteins having identical PAX DNA-binding domains. However, the PAX3-FKHR fusion protein was a much more potent transcriptional activator than PAX3 as determined by transient cotransfection assays using e5-CAT reporter plasmids. The PAX3-FKHR protein may function as an oncogenic transcription factor by enhanced activation of normal PAX3 target genes.
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PAX3 foxo1 is essential for tumour initiation and maintenance but not recurrence in a human myoblast model of rhabdomyosarcoma
The Journal of Pathology, 2017Co-Authors: Puspa R Pandey, Bishwanath Chatterjee, Mary E Olanich, Javed Khan, Markku Miettinen, Stephen M Hewitt, Frederic G. BarrAbstract:: The PAX3-FOXO1 fusion gene is generated by a 2;13 chromosomal translocation and is a characteristic feature of an aggressive subset of rhabdomyosarcoma (RMS). To dissect the mechanism of oncogene action during RMS tumourigenesis and progression, doxycycline-inducible PAX3-FOXO1 and constitutive MYCN expression constructs were introduced into immortalized human myoblasts. Although myoblasts expressing PAX3-FOXO1 or MYCN alone were not transformed in focus formation assays, combined PAX3-FOXO1 and MYCN expression resulted in transformation. Following intramuscular injection into immunodeficient mice, myoblasts expressing PAX3-FOXO1 and MYCN formed rapidly growing RMS tumours, whereas myoblasts expressing only PAX3-FOXO1 formed tumours after a longer latency period. Doxycycline withdrawal in myoblasts expressing inducible PAX3-FOXO1 and constitutive MYCN following tumour formation in vivo or focus formation in vitro resulted in tumour regression or smaller foci associated with myogenic differentiation and cell death. Following regression, most tumours recurred in the absence of doxycycline. Analysis of recurrent tumours revealed a subset without PAX3-FOXO1 expression, and cell lines derived from these recurrent tumours showed transformation in the absence of doxycycline. The doxycycline-independent oncogenicity in these recurrent tumour-derived lines persisted even after PAX3-FOXO1 was inactivated with a CRISPR/Cas9 editing strategy. Whereas cell lines derived from primary tumours were dependent on PAX3-FOXO1 and differentiated following doxycycline withdrawal, recurrent tumour-derived cells without PAX3-FOXO1 expression did not differentiate under these conditions. These findings indicate that PAX3-FOXO1 collaborates with MYCN during early RMS tumourigenesis to dysregulate proliferation and inhibit myogenic differentiation and cell death. Although most cells in the primary tumours are dependent on PAX3-FOXO1, recurrent tumours can develop by a PAX3-FOXO1-independent mechanism, in which rare cells are postulated to acquire secondary transforming events that were activated or selected by initial PAX3-FOXO1 expression. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.
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High Expression of the PAX3-FKHR Oncoprotein Is Required to Promote Tumorigenesis of Human Myoblasts
The American journal of pathology, 2009Co-Authors: Shujuan J. Xia, Dara D. Holder, Bruce R. Pawel, Chune Zhang, Frederic G. BarrAbstract:PAX3-FKHR is a fusion oncoprotein generated by the 2;13 chromosomal translocation in alveolar rhabdomyosarcoma (ARMS), a cancer associated with the skeletal muscle lineage. Previous studies determined that high-level PAX3-FKHR expression is a consistent feature in ARMS tumors. To investigate the relationship between expression and phenotype in human myogenic cells, PAX3-FKHR was introduced into immortalized human myoblasts to produce a low overall PAX3-FKHR expression level. Although PAX3-FKHR alone failed to exert transforming activity, a combination of PAX3-FKHR and MYCN induced transforming activity in cell culture assays. Furthermore, myoblasts expressing PAX3-FKHR with or without MYCN formed tumors in SCID mice. These tumors demonstrated invasive features and expressed myogenic markers, consistent with rhabdomyosarcoma. Comparisons of tumor and parental cells revealed that only a subset of parental cells developed into tumors and that tumor cells expressed high PAX3-FKHR levels compared with transduced parental cells. Subcloning of parental PAX3-FKHR/MYCN-transduced myoblasts identified rare high PAX3-FKHR-expressing subclones with high transforming and tumorigenic activity; however, most subclones expressed low PAX3-FKHR and showed neither transforming nor tumorigenic activity. Finally, RNA interference experiments in myoblast-derived tumor and ARMS cells revealed that high PAX3-FKHR expression plays a crucial role in regulating proliferation, transformation, and differentiation. These findings support the premise that high PAX3-FKHR-expressing cells are selected during tumorigenesis.
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identification of PAX3 fkhr regulated genes differentially expressed between alveolar and embryonal rhabdomyosarcoma focus on mycn as a biologically relevant target
Genes Chromosomes and Cancer, 2008Co-Authors: Gabriela E Mercado, Chune Zhang, Donna M Gustafson, Marc Ladanyi, Frederic G. BarrAbstract:Rhabdomyosarcoma is a family of myogenic soft tissue tumors subdivided into two main subtypes: alveolar (ARMS) and embryonal (ERMS). ARMS is characterized by a frequent 2;13 chromosomal translocation that creates a PAX3-FKHR fusion transcription factor. To identify downstream targets of PAX3-FKHR, we introduced an inducible form of PAX3-FKHR into human RD ERMS cells. Microarray analysis identified 39 genes (29 upregulated and 10 downregulated) that are modulated by PAX3-FKHR in RD cells and differentially expressed between ERMS and PAX3-FKHR-positive ARMS tumors. Functional annotation demonstrated that genes involved in regulation of transcription and development, particularly neurogenesis, are represented in this group. MYCN was one notable neural-related transcription factor-encoding gene identified in this set, and its regulation by PAX3-FKHR was further confirmed at the RNA and protein levels. The findings of cycloheximide inhibition and time-course studies are consistent with the hypothesis that the PAX3-FKHR protein acts directly on the MYCN gene at the transcriptional level. Functional studies established that MYCN cooperates with PAX3-FKHR to enhance oncogenic activity. In conclusion, we identified a selected set of biologically relevant genes modulated by PAX3-FKHR, and demonstrated that PAX3-FKHR contributes to the expression of MYCN and in turn MYCN collaborates with PAX3-FKHR in tumorigenesis. © 2008 Wiley-Liss, Inc.
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Co-expression of alternatively spliced forms of PAX3, PAX7, PAX3-FKHR and PAX7-FKHR with distinct DNA binding and transactivation properties in rhabdomyosarcoma
International journal of cancer, 2005Co-Authors: Eric J. Lawrence, Donna Strzelecki, Prerna Rajput, Shujuan J. Xia, Dina M. Gottesman, Frederic G. BarrAbstract:PAX3 and PAX7 encode transcription factors implicated in the pathogenesis of rhabdomyosarcoma (RMS), including alveolar RMS in which chromosomal translocations generate PAX3-FKHR and PAX7-FKHR fusions. Previous studies of wild-type PAX3 and PAX7 identified alternative splicing events that modify the paired box and generate 2 isoforms of PAX3 (Q+ and Q−) and 4 isoforms of PAX7 (Q+GL+, Q+GL−, Q−GL+, Q−GL−). In our study, we investigated alternative splicing of the wild-type and fusion forms of PAX3 and PAX7 in alveolar and embryonal RMS and assessed the functional implications. For PAX3 and PAX3-FKHR, the Q+ and Q− isoforms were consistently co-expressed in RMS tumors with slightly higher levels of the Q+ isoform. For PAX7 and PAX7-FKHR, there was a consistent pattern of co-expression of the 4 isoforms in RMS tumors: Q+GL− > Q+GL+ ≥ Q−GL− > Q−GL+. DNA binding analysis demonstrated that PAX3 and PAX3-FKHR Q− isoforms exhibit higher affinity than corresponding Q+ isoforms for class I sites and no difference for class II sites. For PAX7 and PAX7-FKHR, the relative affinity was Q−GL− > Q+GL− > Q−GL+ ≥ Q+GL+ for class I sites and Q−GL−, Q+GL− > Q−GL+, Q+GL+ for class II sites. Finally, the transcriptional activities of the PAX3-FKHR and PAX7-FKHR isoforms on reporter plasmids varied over a 5-fold and 50-fold range, respectively, in accord with the differences in DNA binding activity. In conclusion, these studies reveal that PAX3, PAX7 and their fusions with FKHR are each expressed in RMS tumors as a consistent mixture of functionally distinct isoforms. © 2005 Wiley-Liss, Inc.
Didier Rocancourt - One of the best experts on this subject based on the ideXlab platform.
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muscle stem cell behavior is modified by microrna 27 regulation of PAX3 expression
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Colin G Crist, Didier Rocancourt, Ana Cumano, Didier Montarras, Giorgia Pallafacchina, Simon J Conway, Margaret BuckinghamAbstract:Skeletal muscle stem cells are regulated by PAX3/7. During development, PAX3 is required for the maintenance of these cells in the somite and their migration to sites of myogenesis; high levels of PAX3 interfere with muscle cell differentiation, both in the embryo and in the adult. Quantitative fine-tuning of PAX3 is critical, and microRNAs provide a potential mechanism. We identify microRNA-27b (miR-27b), which directly targets the 3′-UTR of PAX3 mRNA, as such a regulator. miR-27b is expressed in the differentiating skeletal muscle of the embryonic myotome and in activated satellite cells of adult muscle. In vivo overexpression of a miR-27b transgene in PAX3-positive cells in the embryo leads to down-regulation of PAX3, resulting in interference with progenitor cell migration and in premature differentiation. In a complementary experiment, miR-27b inhibitors were transfected into cultures of adult muscle satellite cells that normally express miR-27b at the onset of differentiation, when PAX3 protein levels undergo rapid down-regulation. Interference with miR-27b function results in continuing PAX3 expression leading to more proliferation and a delay in the onset of differentiation. Pax7 levels are not affected. Introduction of miR-27b antagomirs at a site of muscle injury in vivo also affects PAX3 expression and regeneration in vivo. We therefore conclude that miR-27b regulates PAX3 protein levels and this down-regulation ensures rapid and robust entry into the myogenic differentiation program.
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a novel genetic hierarchy functions during hypaxial myogenesis PAX3 directly activates myf5 in muscle progenitor cells in the limb
Genes & Development, 2006Co-Authors: Lola Bajard, Frederic Relaix, Didier Rocancourt, Mounia Lagha, Philippe Daubas, Margaret BuckinghamAbstract:We address the molecular control of myogenesis in progenitor cells derived from the hypaxial somite. Null mutations in PAX3, a key regulator of skeletal muscle formation, lead to cell death in this domain. We have developed a novel allele of PAX3 encoding a PAX3–engrailed fusion protein that acts as a transcriptional repressor. Heterozygote mouse embryos have an attenuated mutant phenotype, with partial conservation of the hypaxial somite and its myogenic derivatives, including some hindlimb muscles. At these sites, expression of Myf5 is compromised, showing that PAX3 acts genetically upstream of this myogenic determination gene. We have characterized a 145-base-pair (bp) regulatory element, at −57.5 kb from Myf5, that directs transgene expression to the mature somite, notably to myogenic cells of the hypaxial domain that form ventral trunk and limb muscles. A PAX3 consensus site in this sequence binds PAX3 in vitro and in vivo. Multimers of the 145-bp sequence direct transgene expression to sites of PAX3 function, and an assay of its activity in the chick embryo shows PAX3 dependence. Mutation of the PAX3 site abolishes all expression controlled by the 145-bp sequence in transgenic mouse embryos. We conclude that PAX3 directly regulates Myf5 in the hypaxial somite and its derivatives.
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a PAX3 pax7 dependent population of skeletal muscle progenitor cells
Nature, 2005Co-Authors: Frederic Relaix, Didier Rocancourt, Ahmed Mansouri, Margaret BuckinghamAbstract:During vertebrate development, successive phases of embryonic and fetal myogenesis lead to the formation and growth of skeletal muscles 1 . Although the origin and molecular regulation of the earliest embryonic muscle cells is well understood 2 , less is known about later stages of myogenesis. We have identified a new cell population that expresses the transcription factors PAX3 and Pax7 (paired box proteins 3 and 7) but no skeletal-musclespecific markers. These cells are maintained as a proliferating population in embryonic and fetal muscles of the trunk and limbs throughout development. Using a stable green fluorescent protein (GFP) reporter targeted to PAX3, we demonstrate that they constitute resident muscle progenitor cells that subsequently become myogenic and form skeletal muscle. Late in fetal development, these cells adopt a satellite cell position characteristic of progenitor cells in postnatal muscle. In the absence of both PAX3 and Pax7, further muscle development is arrested and only the early embryonic muscle of the myotome forms. Cells failing to express PAX3 or Pax7 die or assume a nonmyogenic fate. We conclude that this resident PAX3/Pax7dependent progenitor cell population constitutes a source of myogenic cells of prime importance for skeletal muscle formation, a finding also of potential value in the context of cell therapy for muscle disease. During the onset of skeletal myogenesis in the embryo, PAX3 is required for the survival of the ventro-lateral dermomyotome, the part of the somite that gives rise to hypaxial body 3,4 and limb 5 musculature. PAX3 is also implicated in the determination of myo
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A PAX3/Pax7-dependent population of skeletal muscle progenitor cells.
Nature, 2005Co-Authors: Frederic Relaix, Didier Rocancourt, Ahmed Mansouri, Margaret BuckinghamAbstract:During vertebrate development, successive phases of embryonic and fetal myogenesis lead to the formation and growth of skeletal muscles. Although the origin and molecular regulation of the earliest embryonic muscle cells is well understood, less is known about later stages of myogenesis. We have identified a new cell population that expresses the transcription factors PAX3 and Pax7 (paired box proteins 3 and 7) but no skeletal-muscle-specific markers. These cells are maintained as a proliferating population in embryonic and fetal muscles of the trunk and limbs throughout development. Using a stable green fluorescent protein (GFP) reporter targeted to PAX3, we demonstrate that they constitute resident muscle progenitor cells that subsequently become myogenic and form skeletal muscle. Late in fetal development, these cells adopt a satellite cell position characteristic of progenitor cells in postnatal muscle. In the absence of both PAX3 and Pax7, further muscle development is arrested and only the early embryonic muscle of the myotome forms. Cells failing to express PAX3 or Pax7 die or assume a non-myogenic fate. We conclude that this resident PAX3/Pax7-dependent progenitor cell population constitutes a source of myogenic cells of prime importance for skeletal muscle formation, a finding also of potential value in the context of cell therapy for muscle disease.
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divergent functions of murine PAX3 and pax7 in limb muscle development
Genes & Development, 2004Co-Authors: Frederic Relaix, Didier Rocancourt, Ahmed Mansouri, Margaret BuckinghamAbstract:Pax genes encode evolutionarily conserved transcription factors that play critical roles in development. PAX3 and Pax7 constitute one of the four Pax subfamilies. Despite partially overlapping expression domains, mouse mutations for PAX3 and Pax7 have very different consequences. To investigate the mechanism of these contrasting phenotypes, we replaced PAX3 by Pax7 by using gene targeting in the mouse. Pax7 can substitute for PAX3 function in dorsal neural tube, neural crest cell, and somite development, but not in the formation of muscles involving long-range migration of muscle progenitor cells. In limbs in which PAX3 is replaced by Pax7, the severity of the muscle phenotype increases as the number of Pax7 replacement alleles is reduced, with the forelimb more affected than the hindlimb. We show that this hypomorphic activity of Pax7 is due to defects in delamination, migration, and proliferation of muscle precursor cells with inefficient activation of c-met in the hypaxial domain of the somite. Despite this, overall muscle patterning is retained. We conclude that functions already prefigured by the single PAX3/7 gene present before vertebrate radiation are fulfilled by Pax7 as well as PAX3, whereas the role of PAX3 in appendicular muscle formation has diverged, reflecting the more recent origin of this mode of myogenesis.
Jonathan A Epstein - One of the best experts on this subject based on the ideXlab platform.
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PAX3 and hippo signaling coordinate melanocyte gene expression in neural crest
Cell Reports, 2014Co-Authors: Lauren J Manderfield, Kurt A Engleka, Haig Aghajanian, Mudit Gupta, Steven Yang, L Li, Julie E Baggs, John B Hogenesch, Eric N Olson, Jonathan A EpsteinAbstract:Summary Loss of PAX3, a developmentally regulated transcription factor expressed in premigratory neural crest, results in severe developmental defects and embryonic lethality. Although PAX3 mutations produce profound phenotypes, the intrinsic transcriptional activation exhibited by PAX3 is surprisingly modest. We postulated the existence of transcriptional coactivators that function with PAX3 to mediate developmental functions. A high-throughput screen identified the Hippo effector proteins Taz and Yap65 as PAX3 coactivators. Synergistic coactivation of target genes by PAX3-Taz/Yap65 requires DNA binding by PAX3, is Tead independent, and is regulated by Hippo kinases Mst1 and Lats2. In vivo, PAX3 and Yap65 colocalize in the nucleus of neural crest progenitors in the dorsal neural tube. Neural crest deletion of Taz and Yap65 results in embryo-lethal neural crest defects and decreased expression of the PAX3 target gene, Mitf . These results suggest that PAX3 activity is regulated by the Hippo pathway and that Pax factors are Hippo effectors.
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Identification of minimal enhancer elements sufficient for PAX3 expression in neural crest and implication of Tead2 as a regulator of PAX3.
Development (Cambridge England), 2004Co-Authors: Rita C. Milewski, Christopher B. Brown, Neil C. Chi, Jonathan A EpsteinAbstract:PAX3 is a transcription factor that is required by pre-migratory neural crest cells, which give rise to the peripheral nervous system, melanocytes, some vascular smooth muscle, and numerous other derivatives [corrected]. Both mice and humans with PAX3 deficiency exhibit neural crest-related developmental defects [corrected]. PAX3 is also expressed in the dorsal neural tube, and by myogenic progenitors in the presomitic mesoderm and the hypaxial somites. Molecular pathways that regulate PAX3 expression in the roof plate probably represent early upstream signals in neural crest induction. We have identified an enhancer region in the PAX3 genomic locus that is sufficient to recapitulate expression in neural crest precursors in transgenic mice. We show that Tead2, a member of the Tead box family of transcription factors, binds to a neural crest enhancer and activates PAX3 expression. Tead2, and its co-activator YAP65, are co-expressed with PAX3 in the dorsal neural tube, and mutation of the Tead2 binding site in the context of PAX3 transgenic constructs abolishes neural expression. In addition, a Tead2-Engrailed fusion protein is able to repress retinoic acid-induced PAX3 expression in P19 cells and in vivo. These results suggest that Tead2 is an endogenous activator of PAX3 in neural crest.
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Migration of cardiac neural crest cells in Splotch embryos
Development, 2000Co-Authors: Jonathan A Epstein, D Lang, Minmin Lu, Christopher B. Brown, Jun Li, F Chen, M. Thomas, Andy WesselsAbstract:PAX3 encodes a transcription factor expressed during mid-gestation in the region of the dorsal neural tube that gives rise to migrating neural crest populations. In the absence of PAX3, both humans and mice develop with neural crest defects. Homozygous Splotch embryos that lack PAX3 die by embryonic day 13.5 with cardiac defects that resemble those induced by neural crest ablation in chick models. This has led to the hypothesis that PAX3 is required for cardiac neural crest migration. However, cardiac derivatives of PAX3-expressing precursor cells have not been previously defined, and PAX3-expressing cells within the heart have not been well demonstrated. Hence, the precise role of PAX3 during cardiac development remains unclear. Here, we use a Cre-lox method to fate map PAX3-expressing neural crest precursors to the cardiac outflow tract. We show that although PAX3 itself is extinguished prior to neural crest populating the heart, derivatives of these precursors contribute to the aorticopulmonary septum. We further show that neural crest cells are found in the outflow tract of Splotch embryos, albeit in reduced numbers. This indicates that contrary to prior reports, PAX3 is not required for cardiac neural crest migration. Using a neural tube explant culture assay, we demonstrate that neural crest cells from Splotch embryos show normal rates of proliferation but altered migratory characteristics. These studies suggest that PAX3 is required for fine tuning the migratory behavior of the cardiac neural crest cells while it is not essential for neural crest migration.
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PAX3 functions in cell survival and in pax7 regulation.
Development (Cambridge England), 1999Co-Authors: Anne-gaëlle Borycki, Fuzi Jin, Charles P. Emerson, Jonathan A EpsteinAbstract:In developing vertebrate embryos, PAX3 is expressed in the neural tube and in the paraxial mesoderm that gives rise to skeletal muscles. PAX3 mutants develop muscular and neural tube defects; furthermore, PAX3 is essential for the proper activation of the myogenic determination factor gene, MyoD, during early muscle development and PAX3 chromosomal translocations result in muscle tumors, providing evidence that PAX3 has diverse functions in myogenesis. To investigate the specific functions of PAX3 in development, we have examined cell survival and gene expression in presomitic mesoderm, somites and neural tube of developing wild-type and PAX3 mutant (Splotch) mouse embryos. Disruption of PAX3 expression by antisense oligonucleotides significantly impairs MyoD activation by signals from neural tube/notochord and surface ectoderm in cultured presomitic mesoderm (PSM), and is accompanied by a marked increase in programmed cell death. In PAX3 mutant (Splotch) embryos, MyoD is activated normally in the hypaxial somite, but MyoD-expressing cells are disorganized and apoptosis is prevalent in newly formed somites, but not in the neural tube or mature somites. In neural tube and somite regions where cell survival is maintained, the closely related Pax7 gene is upregulated, and its expression becomes expanded into the dorsal neural tube and somites, where PAX3 would normally be expressed. These results establish that PAX3 has complementary functions in MyoD activation and inhibition of apoptosis in the somitic mesoderm and in repression of Pax7 during neural tube and somite development.
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Tumor-Specific PAX3-FKHR Transcription Factor, but Not PAX3, Activates the Platelet-Derived Growth Factor Alpha Receptor
Molecular and cellular biology, 1998Co-Authors: Jonathan A Epstein, Baoliang Song, Maha M. Lakkis, Chiayeng WangAbstract:The t(2;13) chromosomal translocation occurs at a high frequency in alveolar rhabdomyosarcoma, a common pediatric tumor of muscle. This translocation results in the production of a chimeric fusion protein derived from two developmentally regulated transcription factors, PAX3 and FKHR. The two DNA binding modules, the paired domain and the homeodomain, of PAX3 are fused in frame to the transactivation domain of FKHR. Previously, tumor-specific PAX3-FKHR has been shown to bind to DNA sequences normally recognized by wild-type PAX3 and to exhibit relatively enhanced transcriptional activity. The DNA binding sites used to demonstrate that PAX3-FKHR is a more potent transcriptional activator than PAX3 have included recognition sequences for the paired domain of PAX3. In this report, we demonstrate the ability of PAX3-FKHR to activate the product of a growth control gene, platelet-derived growth factor alpha receptor (PDGFαR), by recognizing a paired-type homeodomain binding site located in the PDGFαR promoter. PAX3 alone cannot mediate transcriptional activation of this promoter under the conditions tested. This provides the first evidence that chromosomal translocation results in altered target gene specificity of PAX3-FKHR and suggests a transcriptional target that may play a significant role in oncogenic activity and rhabdomyosarcoma development.
