Vontell, A. M. | bioRxiv Science

bioRxiv2026

Comparative modes of chromatin engagement by PAX::FOXO1 fusions in rhabdomyosarcoma

Fusion positive rhabdomyosarcoma (FP-RMS) is an aggressive soft-tissue sarcoma that most frequently affects children and adolescents. Treatment options and outcomes for children with this cancer remain poor, non-specific, and broadly toxic despite decades of research. The defining molecular drivers of the more aggressive fusion-positive subtype of the disease arise from chromosomal translocations that fuse PAX3 or PAX7 to FOXO1 to form PAX3::FOXO1 or PAX7::FOXO1, encoding fusion oncoprotein transcription factors. Despite their high degree of similarity, PAX3::FOXO1 correlates with worse patient overall survival than PAX7::FOXO1. Previous work from our groups and others has revealed evidence focused in chromatin accessibility contexts that PAX3::FOXO1 has key characteristics of a pioneer transcription factor, a specialized subclass of transcription factors that can bind nucleosomal DNA prior to generation of local accessibility. However, evidence at the genome scale for PAX3/7::FOXO1 direct nucleosome targeting, prior to the accessibility step in pioneering, has remained elusive and challenging to capture methodologically for RMS fusion oncoproteins. In this work, we compare the cellular functions of these PAX::FOXO1 fusions, including new approaches for identifying nucleosome targeting at the genome scale. We find that in zebrafish RMS initiation models, the fusions initially activate similar neural transcriptional programs but to different extents, and we further evaluate their mechanisms in RMS cells at the genome scale with modified MNase XChIP to detect nucleosome and subnucleosome fusion/chromatin binding. In establishing our cross-species comparative oncology approach, we report, to our knowledge, the first high resolution nucleosome positioning data in rhabdomyosarcoma. We find that both PAX::FOXO1 fusions bind nucleosomal DNA, but with varied motif preferences and histone mark co-localization patterns. Altogether, we establish the nucleosome targeting functions of PAX7::FOXO1 and PAX3::FOXO1 pioneering and uncover key mechanistic distinctions for chromatin engagement of the two most common RMS fusion oncoproteins. HIGHLIGHTSO_LIPartially overlapping gene signatures are activated by PAX3/7::FOXO1 in vivo C_LIO_LIModified MNase ChIP reveals PAX3/7::FOXO1 bind nucleosomal and subnucleosomal DNA C_LIO_LIPAX7::FOXO1 binds degenerate paired/homeobox motifs within nucleosome targets C_LIO_LIEach fusion engages distinct nucleosomal gene targets C_LI

cancer biology↗

bioRxiv2024

Genetic Analysis and Functional Assessment of a TGFBR2 Variant in Micrognathia and Cleft Palate

Cleft lip and cleft palate are among the most common congenital anomalies and are the result of incomplete fusion of embryonic craniofacial processes or palatal shelves, respectively. We know that genetics play a large role in these anomalies but the list of known causal genes is far from complete. As part of a larger sequencing effort of patients with micrognathia and cleft palate we identified a candidate variant in transforming growth factor beta receptor 2 (TGFBR2) which is rare, changing a highly conserved amino acid, and predicted to be pathogenic by a number of metrics. The family history and population genetics would suggest this specific variant would be incompletely penetrant, but this gene has been convincingly implicated in craniofacial development. In order to test the hypothesis this might be a causal variant, we used genome editing to create the orthologous variant in a new mouse model. Surprisingly, Tgfbr2V387M mice did not exhibit craniofacial anomalies or have reduced survival suggesting this is, in fact, not a causal variant for cleft palate/ micrognathia. The discrepancy between in silico predictions and mouse phenotypes highlights the complexity of translating human genetic findings to mouse models. We expect these findings will aid in interpretation of future variants seen in TGFBR2 from ongoing sequencing of patients with congenital craniofacial anomalies.

developmental biology↗