Helicase CHD4 is an epigenetic coregulator of PAX3-FOXO1 in alveolar rhabdomyosarcoma
Maria Böhm, … , Raffaella Santoro, Beat W. Schäfer
Maria Böhm, … , Raffaella Santoro, Beat W. Schäfer
Published October 17, 2016
Citation Information: J Clin Invest. 2016;126(11):4237-4249. https://doi.org/10.1172/JCI85057.
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Research Article Oncology

Helicase CHD4 is an epigenetic coregulator of PAX3-FOXO1 in alveolar rhabdomyosarcoma

Abstract

A vast number of cancer genes are transcription factors that drive tumorigenesis as oncogenic fusion proteins. Although the direct targeting of transcription factors remains challenging, therapies aimed at oncogenic fusion proteins are attractive as potential treatments for cancer. There is particular interest in targeting the oncogenic PAX3-FOXO1 fusion transcription factor, which induces alveolar rhabdomyosarcoma (aRMS), an aggressive cancer of skeletal muscle cells for which patient outcomes remain dismal. In this work, we have defined the interactome of PAX3-FOXO1 and screened 60 candidate interactors using siRNA-mediated depletion to identify candidates that affect fusion protein activity in aRMS cells. We report that chromodomain helicase DNA binding protein 4 (CHD4), an ATP-dependent chromatin remodeler, acts as crucial coregulator of PAX3-FOXO1 activity. CHD4 interacts with PAX3-FOXO1 via short DNA fragments. Together, they bind to regulatory regions of PAX3-FOXO1 target genes. Gene expression analysis suggested that CHD4 coregulatory activity is essential for a subset of PAX3-FOXO1 target genes. Depletion of CHD4 reduced cell viability of fusion-positive but not of fusion-negative RMS in vitro, which resembled loss of PAX3-FOXO1. It also caused specific regression of fusion-positive xenograft tumors in vivo. Therefore, this work identifies CHD4 as an epigenetic coregulator of PAX3-FOXO1 activity, providing rational evidence for CHD4 as a potential therapeutic target in aRMS.

Authors

Maria Böhm, Marco Wachtel, Joana G. Marques, Natalie Streiff, Dominik Laubscher, Paolo Nanni, Kamel Mamchaoui, Raffaella Santoro, Beat W. Schäfer

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Figure 6

CHD4 colocalizes to PAX3-FOXO1 binding sites.

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CHD4 colocalizes to PAX3-FOXO1 binding sites. (A) Expression levels of i...
(A) Expression levels of indicated PAX3-FOXO1 target genes were quantified by quantitative real-time PCR after CHD4 knockdown in 2 FP-RMS cell lines. Bar charts are geometric means from 4 independent experiments with 95% CI (P < 0.05; Dunnett’s multiple comparison test). Fold change of mRNA expression was normalized to uninduced cells with PAX3-FOXO1 knockdown serving as positive control. (B) ChIP was performed in RH4 cells using PAX3/7 and CHD4 antibodies on known PAX3-FOXO1 DNA binding sites in target genes, and only beads without antibody served as negative control (Beads G). Bar charts indicate the mean ± SEM of at least 3 independent biological replicates. Abundance of precipitated DNA fragments was measured by quantitative PCR, and results are presented as the percentage of input. The GAPDH promoter region served as negative control. (C) Effect on PAX3-FOXO1 binding upon CHD4 silencing (48 hours of incubation with doxycycline) was determined by ChIP as described in B and is shown as fold enrichment over PAX3-FOXO1 ChIP signal in the presence of CHD4 (no doxycycline control).