ABSTRACT
Alveolar rhabdomyosarcoma (ARMS) is an aggressive childhood cancer of striated muscle characterized by the presence of the PAX3-FOXO1A or PAX7-FOXO1A chimeric oncogenic transcription factor. Identification of their targets is essential for understanding ARMS pathogenesis. To this aim, we analyzed transcriptomic data from rhabdomyosarcoma samples and found that P-cadherin expression is correlated with PAX3/7-FOXO1A presence. We then show that expression of a PAX3 dominant negative variant inhibits P-cadherin expression in ARMS cells. Using mouse models carrying modified Pax3 alleles, we demonstrate that P-cadherin is expressed in the dermomyotome and lies genetically downstream from the myogenic factor Pax3. Moreover, in vitro gel shift analysis and chromatin immunoprecipitation indicate that the P-cadherin gene is a direct transcriptional target for PAX3/7-FOXO1A. Finally, P-cadherin expression in normal myoblasts inhibits myogenesis and induces myoblast transformation, migration and invasion. Conversely, P-cadherin downregulation by small hairpin RNA decreases the transformation, migration and invasive potential of ARMS cells. P-cadherin also favors cadherin switching, which is a hallmark of metastatic progression, by controlling N- and M-cadherin expression and/or localization. Our findings demonstrate that P-cadherin is a direct PAX3-FOXO1A transcriptional target involved in ARMS aggressiveness. Therefore, P-cadherin emerges as a new and attractive target for therapeutic intervention in ARMS.
Subject(s)
Cadherins/metabolism , Forkhead Transcription Factors/metabolism , Paired Box Transcription Factors/metabolism , Rhabdomyosarcoma, Alveolar/metabolism , Animals , Base Sequence , Cadherins/genetics , Cell Movement/genetics , Cell Transformation, Neoplastic/genetics , Forkhead Box Protein O1 , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, Transgenic , Neoplasm Invasiveness/genetics , PAX3 Transcription Factor , PAX7 Transcription Factor/metabolism , Paired Box Transcription Factors/genetics , RNA Interference , RNA, Small Interfering , Rhabdomyosarcoma, Alveolar/pathology , Sequence Alignment , Transcription, GeneticABSTRACT
The RNA polymerase II transcription factor TFIID comprises the TATA binding protein (TBP) and a set of TBP-associated factors (TAF(II)s). TFIID has been extensively characterized for yeast, Drosophila, and humans, demonstrating a high degree of conservation of both the amino acid sequences of the constituent TAF(II)s and overall molecular organization. In recent years, it has been assumed that all the metazoan TAF(II)s have been identified, yet no metazoan homologues of yeast TAF(II)47 (yTAF(II)47) and yTAF(II)65 are known. Both of these yTAF(II)s contain a histone fold domain (HFD) which selectively heterodimerizes with that of yTAF(II)25. We have cloned a novel mouse protein, TAF(II)140, containing an HFD and a plant homeodomain (PHD) finger, which we demonstrated by immunoprecipitation to be a mammalian TFIID component. TAF(II)140 shows extensive sequence similarity to Drosophila BIP2 (dBIP2) (dTAF(II)155), which we also show to be a component of Drosophila TFIID. These proteins are metazoan homologues of yTAF(II)47 as their HFDs selectively heterodimerize with dTAF(II)24 and human TAF(II)30, metazoan homologues of yTAF(II)25. We further show that yTAF(II)65 shares two domains with the Drosophila Prodos protein, a recently described potential dTAF(II). These conserved domains are critical for yTAF(II)65 function in vivo. Our results therefore identify metazoan homologues of yTAF(II)47 and yTAF(II)65.
Subject(s)
Drosophila Proteins , Histones/chemistry , Saccharomyces cerevisiae Proteins , TATA-Binding Protein Associated Factors , Transcription Factors, TFII/chemistry , Transcription Factors/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , COS Cells , Candida albicans/chemistry , Cloning, Molecular , Conserved Sequence , Dimerization , Drosophila , Evolution, Molecular , Genetic Complementation Test , HeLa Cells , Humans , In Situ Hybridization , Mice , Microscopy, Fluorescence , Molecular Sequence Data , Plasmids/metabolism , Precipitin Tests , Protein Structure, Tertiary , Salivary Glands/metabolism , Sequence Homology, Amino Acid , Temperature , Time Factors , Trans-Activators/chemistry , Transcription Factor TFIID , Two-Hybrid System Techniques , Xenopus , ZebrafishABSTRACT
Transcription factor TFIID is a multiprotein complex composed of the TATA binding protein and its associated factors, and is required for accurate and regulated initiation of transcription by RNA polymerase II. The subunit composition of this factor is highly conserved from yeast to mammals. X-ray crystallography and biochemical experiments have shown that the histone fold motif mediates many of the subunit interactions within this complex. These results, together with electron microscopy and yeast genetics, provide insights into the overall organization of this complex.
