Pax3 is a developmental transcription factor that is required for neural tube and neural crest development. We previously showed that inactivating the p53 tumor suppressor protein prevents neural tube and cardiac neural crest defects in Pax3-mutant mouse embryos. This demonstrates that Pax3 regulates these processes by blocking p53 function. Here we investigated the mechanism by which Pax3 blocks p53 function.

The transcription factor, Pax3, alters transcription by binding directly to promoter regions harbouring sequences recognized by either its paired domain or its homeodomain. We demonstrated previously that the promoter regions of many of the genes whose expression was altered during a Pax3-induced mesenchymal-to-epithelial transition harboured sequences recognized by lymphoid enhancer factor-1 (Lef1). Given the apparent lack of DNA-binding consensus sequences for Pax3 in these promoters, it was hypothesized that Pax3 might alter transcriptional activity of promoters harbouring Lef1-binding sites independent of Pax3 binding to DNA. We describe here a novel mode of Pax3-dependent regulation of transcription that is mediated through DNA-independent binding to Lef1. Specifically, we demonstrate that Pax3 binds to Lef1, determined in binding assays and co-immunoprecipitation of endogenous Pax3 and Lef1. Binding assays employing deletion mutants of Pax3 and Lef1 determined that association was mediated through the homeodomain of Pax3 and the first half of the Lef1 DNA-binding domain. The significance of this association was demonstrated in transcriptional assays using a luciferase reporter gene downstream of a model promoter harbouring Lef1 DNA-binding consensus sites. Pax3 augmented Lef1-dependent transactivation from this promoter. This increase in transcriptional activity occurred in the absence and presence of added beta-catenin. Chromatin immunoprecipitation assays demonstrated further that Pax3-association to complexes bound to DNA harbouring Lef1 consensus sequences was dependent on Lef1. These data reveal a novel mode of transcriptional regulation by Pax3. This mode of transcriptional regulation suggests further that Pax3 activity may directly effect the expression of factors regulated by signal transduction pathways dependent on Lef1.

Pax3 is expressed early during embryonic development in spatially restricted domains including limb muscle, neural crest, and neural tube. Pax3 functions at the nodal point in melanocyte stem cell differentiation, cardiogenesis and neurogenesis. Additionally Pax3 has been implicated in migration and differentiation of precursor cell populations. Currently there are questions about how Pax3 regulates these diverse functions. In this study we found that in the absence of functional Pax3, as in Splotch embryos, the neural crest cells undergo premature neurogenesis, as evidenced by increased Brn3a positive staining in neural tube explants, in comparison with wild-type. Premature neurogenesis in the absence of functional Pax3 may be due to a change in the regulation of basic helix-loop-helix transcription factors implicated in proliferation and differentiation. Using promoter-luciferase activity measurements in transient co-transfection experiments and electro-mobility shift assays, we show that Pax3 regulates Hairy and enhancer of split homolog-1 (Hes1) and Neurogenin2 (Ngn2) by directly binding to their promoters. Chromatin immunoprecipitation assays confirmed that Pax3 bound to cis-regulatory elements within Hes1 and Ngn2 promoters. These observations suggest that Pax3 regulates Hes1 and Ngn2 and imply that it may couple migration with neural stem cell maintenance and neurogenesis.

PAX3 (paired box 3) is known to have an important role in melanocyte development through modulation of microphthalmia-associated transcription factor transcription. Here we found that PAX3 transcriptional activity could be regulated through FGF2 (basic fibroblast growth factor)-STAT3 (signal transducer and activator of transcription 3) signaling in the pigment cells. To study its function in vivo, we have generated a transgenic mouse model expressing PAX3 driven by tyrosinase promoter in a tissue-specific fashion. These animals exhibit hyperpigmentation in the epidermis, evident in the skin color of their ears and tails. We showed that the darker skin color results from both increased melanocyte numbers and melanin synthesis. Together, our study delineated a novel pathway in the melanocyte lineage, linking FGF2-STAT3 signaling to increased PAX3 transcription. Moreover, our results suggest that this pathway might contribute to the regulation of melanocyte numbers and melanin levels, and thereby provide an alternative strategy to induce pigmentation.

Paired box 3 (PAX3) is expressed early during embryonic development in spatially restricted domains in the nervous system and in some mesodermally-derived structure. In recent years, it is found to be overexpressed in different types of cancer tissues and cell lines including glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing sarcomas and gastric cancers, suggesting that it may function as an oncogene in these cancers. However, its role in thyroid cancer remains totally unclear. The aim of this study was to explore the functions and related molecular mechanism of PAX3 in thyroid tumorigenesis. Using quantitative RT-PCR (qRT-PCR) and Methylation-specific PCR (MSP) assays, we demonstrated that PAX3 was frequently down-regulated by promoter methylation in both primary thyroid cancer tissues and thyroid cancer cell lines. In addition, our data showed that ectopic expression of PAX3 dramatically inhibited thyroid cancer cell proliferation, colony formation, migration and invasion, induced cell cycle arrest and apoptosis and retarded tumorigenic potential in nude mice. Mechanically, PAX3 exerted its tumor suppressor function by inhibiting the activity of major signaling pathways including the phosphatidylinositol-3-kinase (PI3K)/Akt and MAPK/Erk pathways, and enhancing expression and activity of transcription factor FOXO3a. Altogether, our findings provided insight into the role of PAX3 as a novel functional tumor suppressor in thyroid cancer through modulating the activities of PI3K/Akt and MAPK signaling pathways and transcription factor FOXO3a, and demonstrated that epigenetic alterations such as promoter methylation should be a major mechanism of PAX3 inactivation in this cancer.

Paired box 3 (PAX3) is a transcription factor and critical regulator of pigment cell development during embryonic development. However, while there have been several studies on PAX3, its expression patterns and precise role remain to be clarified. The present study is an in-depth computational study of tumor-associated gene information, with specific emphasis on the expression of PAX3 in melanoma, using Oncomine along with an investigation of corresponding expression profiles in an array of cancer cell lines through Cancer Cell Line Encyclopedia analysis. Based on Kaplan-Meier analysis, the prognostic value of high PAX3 expression in tissues from patients with melanoma compared with normal tissues was assessed. PAX3 was more highly expressed in male patients with melanoma compared with female patients with melanoma. Using Oncomine and Coexpedia analysis, it was demonstrated that PAX3 expression was clearly associated with SRY-box 10 expression. The survival analysis results revealed that high PAX3 mRNA expression was associated with worse survival rates in patients with melanoma. These results suggested that PAX3 may be a biomarker and essential prognostic factor for melanoma, and provided an important theoretical basis for the development of melanoma treatments.

Pax3 is a transcription factor expressed in the dorsal neural tube and somite of the developing embryo. It plays critical roles in pre-migratory neural crest cells and in myogenic precursors of skeletal muscle. Pax3-deficient Splotch embryos display neural tube and neural crest defects and lack hypaxial muscles. We have created a new allele of Splotch by replacing the first coding exon with a gene encoding Cre recombinase. This functions as a null allele and no Pax3 protein is detected in homozygous embryos. Heterozygous Pax3(Cre/+) mice display a white belly spot, as do Splotch heterozygotes. Homozygous Pax3(Cre/Cre) embryos are embryonic lethal. We have used Pax3(Cre/+) mice to fate-map Pax3 derivatives in the developing mouse. As expected, neural crest and some somitic derivatives are identified. However, we also detect previously unappreciated derivatives of Pax3-expressing precursors in the colonic epithelium of the hindgut and within the urogenital system.

Melanoma is responsible for an estimated 62,000 new American cancer diagnoses and is projected to cause nearly 8000 deaths in 2008 alone. Although the histogenesis of the tumor is not well understood, it is thought to originate from a rare melanocyte stem cell that resides in the skin. The transcription factor PAX3 has a well-established role in the development of melanocytes during embryogenesis, and has recently been characterized as a molecular switch in the mature melanocyte. Based on this function, PAX3 promotes a melanocytic phenotype but blocks terminal differentiation. This mechanism may also contribute to the uncontrolled cell growth and loss of terminal differentiation in melanomas. Here, we find PAX3 expression in 8/8 melanoma cell lines. We also find that PAX3 is commonly expressed in primary melanoma samples (21/58) but significantly less frequently in benign pigmented lesions (9/75). Further analysis of our melanoma set revealed that PAX3 expression is strongly correlated with younger patients with low or no evidence of sun damage. Our data suggest that PAX3-expressing melanomas may be less environmentally dependent and more genetically linked.

Alveolar rhabdomyosarcoma (ARMS) has a high propensity to metastasize, leading to its aggressiveness and a poor survival rate among those with the disease. More than 80% of aggressive ARMSs harbor a PAX3-FKHR fusion transcription factor, which regulates cell migration and promotes metastasis, most likely by regulating the fusion protein's transcriptional targets. Therefore, identifying druggable transcription targets of PAX3-FKHR that are also downstream effectors of PAX3-FKHR-mediated cell migration and metastasis may lead to novel therapeutic approaches for treating ARMS.

The protein encoded by paired-box homeotic gene 3 (PAX3) is a key regulator of the microphthalmia-associated transcription factor (Mitf) in the melanocyte lineage. Here, we show that PAX3 expression in skin is directly inhibited by TGF-beta/Smads. UV irradiation represses TGF-beta in keratinocytes, and the repression of TGF-beta/Smads upregulates PAX3 in melanocytes, which is associated with a UV-induced melanogenic response and consequent pigmentation. Furthermore, the TGF-beta-PAX3 signaling pathway interacts with the p53-POMC/MSH-MC1R signaling pathway, and both are crucial in melanogenesis. The activation of p53-POMC/MSH-MC1R signaling is required for the UV-induced melanogenic response because PAX3 functions in synergy with SOX10 in a cAMP-response element (CRE)-dependent manner to regulate the transcription of Mitf. This study will provide a rich foundation for further research on skin cancer prevention by enabling us to identify targeted small molecules in the signaling pathways of the UV-induced melanogenic response that are highly likely to induce naturally protective pigmentation.

Pax3 and Pax7 are closely related transcription factors that are widely expressed in the developing nervous system and somites. During the normal development in the central nervous system (CNS), Pax3 and Pax7 are mainly expressed in the dorsal part of the neural tube. Further analysis revealed that Pax3 and Pax7 shared redundant functions in the spinal cord development. However, it is still unknown whether Pax3 and Pax7 play a role in neuronal differentiation. In this study, Pax3 and Pax7 genes were overexpressed in Neuro-2a, the mouse neuroblastoma cell line. CCK-8 and EdU assay results showed that overexpression of Pax3 inhibited cell viability and proliferation of Neuro-2a cells, whereas the overexpression of Pax7 had no significant difference on their cell viability and proliferation. Overexpression of Pax3 not only increased the percentage of cells in the S phase and G0/G1 phase, but also decreased that in the G2 phase. Moreover, the total neurite lengths of Neuro-2a cells were significantly shorter in Pax3 overexpressed group than those in negative control group and showed no significant difference between Pax7 overexpressed group and negative control group. These results suggested that Pax3 not only inhibited the cell viability and proliferation but also affected the cell cycle and the neurite outgrowth of Neuro-2a cells. RNA sequencing analysis showed up-regulated genes in Pax3 overexpressed group were involved in cell cycle machinery, which may reveal the potential mechanism of Neuro-2a cells proliferation.

Recent characterizations of pioneer transcription factors provide insights into their structures and patterns of chromatin recognition associated with their roles in cell fate commitment and transformation. Intersecting with these basic science concepts, identification of pioneer factors (PFs) fused together as driver translocations in childhood cancers raises questions of whether these fusions retain the fundamental ability to invade repressed chromatin, consistent with their monomeric PF constituents. This study defines the cellular and chromatin localization of PAX3-FOXO1, an oncogenic driver of childhood rhabdomyosarcoma (RMS), derived from a fusion of PFs. To quantitatively define its chromatin-targeting functions and capacity to drive epigenetic reprogramming, we developed a ChIP-seq workflow with per-cell normalization (pc-ChIP-seq). Our quantitative localization studies address structural variation in RMS genomes and reveal insights into inactive chromatin localization of PAX3-FOXO1. Taken together, our studies are consistent with pioneer function for a driver oncoprotein in RMS, with repressed chromatin binding and nucleosome-motif targeting.

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.

Transcription factor PAX3/Pax3 contributes to diverse cell lineages during embryonic development and is important in tumourigenesis. We found that PAX3 is re-expressed in neuroblastoma and malignant neuroblastic (N-type) neuroblastoma cells had significantly higher PAX3 protein expression than their benign substrate-adherent (S-type) counterparts. Knock-down of PAX3 expression by siRNA transfection resulted in persistent cell growth inhibition in both types of neuroblastoma cell, owing to G1 cell cycle arrest and progressive apoptosis. Inhibition of PAX3 expression significantly decreased the attachment of S-type SH-EP1 cells to extra-cellular matrix proteins, fibronectin, laminin and collagen IV. Migration and invasion of both neuroblastoma cell types were markedly reduced after PAX3 down-regulation. PAX3 knock-down significantly augmented the cytotoxic effect of chemotherapeutic agents, etoposide, vincristine and cisplatin, commonly used to treat neuroblastoma. Microarray analyses revealed that particularly signalling pathways involving cell cycle, apoptosis, cell adhesion, cytoskeletal remodelling and development were altered by PAX3 down-regulation. Changes in PAX3 downstream genes identified by microarray analyses were validated in 47 genes by quantitative PCR. These novel findings lead us to propose that PAX3 might contribute to oncogenic characteristics of neuroblastoma cells by regulating a variety of crucial signalling pathways.

The PAX3 transcription factor is the key regulator of melanocyte development during embryogenesis and is also frequently found in melanoma cells. While PAX3 is known to regulate melanocyte differentiation, survival, proliferation and migration during development, it is not clear if its function is maintained in adult melanocytes and melanoma cells. To clarify this we have assessed which genes are targeted by PAX3 in these cells. We show here that similar to its roles in development, PAX3 regulates complex differentiation networks in both melanoma cells and melanocytes, in order to maintain cells as "stem" cell-like (via NES and SOX9). We show also that mediators of migration (MCAM and CSPG4) are common to both cell types but more so in melanoma cells. By contrast, PAX3-mediated regulation of melanoma cell proliferation (through TPD52) and survival (via BCL2L1 and PTEN) differs from that in melanocytes. These results suggest that by controlling cell proliferation, survival and migration as well as maintaining a less differentiated "stem" cell like phenotype, PAX3 may contribute to melanoma development and progression.

Melanoma is a highly aggressive disease that is difficult to treat owing to rapid tumor growth, apoptotic resistance and high metastatic potential. The MET proto-oncogene (MET) tyrosine kinase receptor promotes many of these cellular processes, but while MET is often overexpressed in melanoma, the mechanism driving this overexpression is unknown. As the MET gene is rarely mutated or amplified in melanoma, MET overexpression may be driven to increased activation through promoter elements. In this report, we find that transcription factors PAX3 and ETS1 directly interact to synergistically activate MET expression. Inhibition of PAX3 and ETS1 expression in melanoma cells leads to a significant reduction of MET receptor levels. The 300-bp 5' proximal MET promoter contains a PAX3 response element and two ETS1 consensus motifs. Although ETS1 can moderately activate both of these sites without cofactors, robust MET promoter activation of the first site is PAX dependent and requires the presence of PAX3, whereas the second site is PAX independent. The induction of MET by ETS1 via this second site is enhanced by hepatocyte growth factor-dependent ETS1 activation, thereby MET indirectly promotes its own expression. We further find that expression of a dominant-negative ETS1 reduces the ability of melanoma cells to grow both in culture and in vivo. Thus, we discover a pathway where ETS1 advances melanoma through the expression of MET via PAX-dependent and -independent mechanisms.

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.

Melanoma is a very aggressive neoplasm with a propensity to undergo progression and invasion early in its evolution. The molecular pathways underpinning invasion in melanoma are now just beginning to be elucidated, but a clear understanding of the transition from non-invasive to invasive melanoma cells remains elusive. Microphthalmia-associated transcription factor (MITF), is thought to be a central player in melanoma biology, and it controls many aspects of the phenotypic expression of the melanocytic lineage. However, recently the paired box transcription factor PAX3 was shown to transcriptionally activate POU3F2/BRN2, leading to direct repression of MITF expression. Here we present a theory to explain melanoma phenotype switching and discuss the predictions that this theory makes. One prediction is that independent and opposing roles for MITF and PAX3 in melanoma would be expected, and we present empirical evidence supporting this: in melanoma tissues PAX3 expression occurs independently of MITF, and PAX3 does not play a key role in melanoma cell proliferation. Furthermore, we show that knockdown of PAX3 inhibits cell migration in a group of "lower MITF" melanoma cell lines, while knockdown of MITF promotes cell migration in a complementary "higher MITF" group of melanoma cell lines. Moreover, the morphological effects of knocking down PAX3 versus MITF in melanoma cells were found to differ. While these data support the notion of independent roles for MITF and PAX3, additional experiments are required to provide robust examination of the proposed genetic switch theory. Only upon clear delineation of the mechanisms associated with progression and invasion of melanoma cells will successful treatments for invasive melanoma be developed.

The PAX3-FOXO1 fusion protein is the key oncogenic driver in fusion positive rhabdomyosarcoma (FP-RMS), an aggressive soft tissue malignancy with a particularly poor prognosis. Identifying key downstream targets of PAX3-FOXO1 will provide new therapeutic opportunities for treatment of FP-RMS. Herein, we demonstrate that Forkhead Box F1 (FOXF1) transcription factor is uniquely expressed in FP-RMS and is required for FP-RMS tumorigenesis. The PAX3-FOXO1 directly binds to FOXF1 enhancers and induces FOXF1 gene expression. CRISPR/Cas9 mediated inactivation of either FOXF1 coding sequence or FOXF1 enhancers suppresses FP-RMS tumorigenesis even in the presence of PAX3-FOXO1 oncogene. Knockdown or genetic knockout of FOXF1 induces myogenic differentiation in PAX3-FOXO1-positive FP-RMS. Over-expression of FOXF1 decreases myogenic differentiation in primary human myoblasts. In FP-RMS tumor cells, FOXF1 protein binds chromatin near enhancers associated with FP-RMS gene signature. FOXF1 cooperates with PAX3-FOXO1 and E-box transcription factors MYOD1 and MYOG to regulate FP-RMS-specific gene expression. Altogether, FOXF1 functions downstream of PAX3-FOXO1 to promote FP-RMS tumorigenesis.

OTX2 is a potent oncogene that promotes tumor growth in Group 3 medulloblastoma. However, the mechanisms by which OTX2 represses neural differentiation are not well characterized. Here, we perform extensive multiomic analyses to identify an OTX2 regulatory network that controls Group 3 medulloblastoma cell fate. OTX2 silencing modulates the repressive chromatin landscape, decreases levels of PRC2 complex genes and increases the expression of neurodevelopmental transcription factors including PAX3 and PAX6. Expression of PAX3 and PAX6 is significantly lower in Group 3 medulloblastoma patients and is correlated with reduced survival, yet only PAX3 inhibits self-renewal in vitro and increases survival in vivo. Single cell RNA sequencing of Group 3 medulloblastoma tumorspheres demonstrates expression of an undifferentiated progenitor program observed in primary tumors and characterized by translation/elongation factor genes. Identification of mTORC1 signaling as a downstream effector of OTX2-PAX3 reveals roles for protein synthesis pathways in regulating Group 3 medulloblastoma pathogenesis.