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PAX3-FOXO1 directs CBP/P300 via its activation domain to build RNA Pol2 clusters

ABSTRACT: Activation of oncogenic gene expression from long-range enhancers is initiated by the assembly of DNA-binding transcription factors (TF), leading to recruitment of co-activators such as CBP/p300 to modify the local genomic context and facilitate RNA-Polymerase 2 (Pol2) binding. Yet, most TF-to-coactivator recruitment relationships remain unmapped. Here, studying the oncogenic fusion TF PAX3-FOXO1 (P3F) from alveolar rhabdomyosarcoma (aRMS), we show that a single cysteine in the activation domain (AD) of PAX3-FOXO1 forms a small alpha helical hook that recruits CBP/p300 to chromatin. P3F driven transcription requires both this single cysteine, and also CBP/p300. Furthermore, we discover a profound dependence on CBP/p300 for clustering of Pol2 loops that connect P3F to its target genes. In the absence of CBP/p300, Pol2 long range enhancer loops collapse, Pol2 accumulates in CpG islands and fails to exit the gene body. These results reveal a potential novel axis for therapeutic interference with P3F in aRMS and clarify the molecular relationship of P3F and CBP/p300 in sustaining active Pol2 clusters essential for oncogenic transcription.

ORGANISM(S): Homo sapiens

PROVIDER: GSE208146 | GEO | 2023/12/25

REPOSITORIES: GEO

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Project description:Rhabdomyosarcoma (RMS) is a highly malignant soft tissue sarcoma classified into two major histologic subtypes: embryonal (ERMS) and alveolar (ARMS). ARMS subtype is clinically more aggressive, and characterized by an oncogenic fusion protein PAX3-FOXO1 (P3F) that drives oncogenic cellular properties. To understand the role of the fusion oncoprotein in paracrine signaling, we focused on secreted exosomes, which have been demonstrated to contribute to metastasis in multiple tumor types. Mass spectrometry analysis of the protein cargo of exosomes isolated from C2C12 myoblasts transduced with P3F fusion gene revealed 52 deregulated proteins compared to control cells, with 26 enriched and 26 depleted proteins. Using both PANTHER gene classification and IPA software, we found that the main biological processes in which the 52 deregulated proteins are involved include “Catalytic activity”, “Binding”, “Metabolic process” and “Cellular process”. The pathways engaging the 26 enriched proteins include “14-3-3 mediated signaling”, “Cell cycle” and “ERK5, VEGF, IGF1and p70S6K signaling”. Main nodes in which deregulated exosome protein and miRNA intersected revealed pathways conferring protection from stress and promoting plasticity. Physiologically, exosomes derived from P3F-C2C12 cells activated ERK, 4-EBP1 and MMP-2, factors known to increase cell proliferation and invasion. In addition, they increased angiogenesis as evidenced by studies on HUVEC cells, and promoted stemness of MEFs and C2C12 cells. Treating stromal cells with exosomes from P3F-C2C12 cells protected them against effects of hydrogen peroxide (H2O2) in vitro, noting that P3F-C2C12 cells produce less ROS and are more resistant to induced exogenous oxidative stress as compared to control cells. Our findings highlight the role of P3F fusion protein in modulating exosome cargo to confer a protective effect on recipient cells against oxidative stress and promote plasticity and survival, potentially contributing to the known aggressive phenotype of the fusion gene-positive subtype of ARMS.

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PAX3-FOXO1 directs CBP/P300 via its activation domain to build RNA Pol2 clusters

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