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Direct interaction of MYCN and p53 regulate transcriptional responses in neuroblastoma [RNA-Seq]

ABSTRACT: The net transcriptome of a cancer cell is defined by relative levels of transcription factors, activators, suppressors and co-factors. These in turn are controlled by epigenetic, genetic and metabolic restraints. Here we used RNA-Seq, ChIP-qPCR, and ChIP-Seq to determine the impact of MYCN protein levels on p53 and MYCN mediated transcription in neuroblastoma, a p53 wild-type neural crest derived pediatric malignancy. Of note, about 25% of neuroblastoma is MYCN amplified and expresses 10-100 fold higher levels of MYCN protein than non-amplified tumors. We hypothesized that p53 functions would be globally altered by extreme MYCN levels and this could help to explain aggressive biology and poor long term survival which distinguishes MYCN-amplified neuroblastoma. In fact, we found that in the context of high MYCN levels, activation of p53 results in marked changes in transcription of specific p53 and MYCN target loci regulating apoptosis, DNA repair and cell cycle progression. Co-immunoprecipitation of endogenous and GST-fused proteins, as well as ChIP-qPCR confirms formation of p53/MYCN complexes and enrichment of both transcription factors at active loci. This p53/MYC interaction is independent of MYC/MAX complexes. Together, these data demonstrate MYCN to be a direct co-factor modulating p53 transcriptional output in MYCN amplified cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE83327 | GEO | 2018/05/17

REPOSITORIES: GEO

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Project description:In neuroblastoma, amplification of the oncogenic basic helix-loop-helix (bHLH) transcription factor (TF) MYCN is the defining prognosticator of high-risk disease, occurs in one-third of neuroblastoma, and drastically reduces overall survival rates. As a proto-oncogene, targeted MYCN overexpression in peripheral neural crest is sufficient to initiate disease in mouse models. In MYCN amplified neuroblastoma, elevated expression of the factor is crucial to maintain tumor stemness and is associated with increased proliferation and aberrant cell cycle progression, as these tumors lack the ability to arrest in G1 in response to irradiation. MYCN down-regulation broadly reverses these oncogenic phenotypes in a variety of neuroblastoma models and recent thereapeutic strategies to indirectly target MYCN production or protein stability have reduced tumor growth in vivo. These observations motivate an investigation of MYCN binding in MYCN amplified tumors as it remains fundamentally unclear how elevated levels of the factor occupy the genome and alter transcriptional programs in neuroblastoma. Here we present the first dynamic chromatin and transcriptional landscape of direct MYCN perturbation in neuroblastoma. We find that at oncogenic levels, MYCN associates with E-box (CANNTG) binding motifs in an affinity dependent manner across most active cis-regulatory promoters and enhancers. MYCN shutdown globally reduces histone acetylation and transcription, consistent with prior descriptions of MYC proteins as non-linear amplifiers of gene expression. We establish that MYCN load at the promoter and proximal enhancers predicts transcriptional responsiveness to MYCN shutdown and that MYCN enhancer binding occurs prominently at the most strongly occupied and down-regulated genes, suggesting a role for these tissue specific elements in predicating MYCN responsive âtargetâ genes. At these invaded enhancers, we identify the lineage specific bHLH TWIST1 as a key collaborator and dependency of oncogenic MYCN. These data suggest that MYCN enhancer invasion helps shape transcriptional amplification of the neuroblastoma gene expression program to promote tumorigenesis. ChIP-Seq in SHEP21, BE2C, KELLY, and NGP neuroblastoma cell lines for H3K27ac, H3K4me3, RNA PolII, MYCN, BRD4, or TWIST1

Project description:In neuroblastoma, amplification of the oncogenic basic helix-loop-helix (bHLH) transcription factor (TF) MYCN is the defining prognosticator of high-risk disease, occurs in one-third of neuroblastoma, and drastically reduces overall survival rates1,2. As a proto-oncogene, targeted MYCN overexpression in peripheral neural crest is sufficient to initiate disease in mouse models3. In MYCN amplified neuroblastoma, elevated expression of the factor is crucial to maintain tumor stemness4,5 and is associated with increased proliferation and aberrant cell cycle progression, as these tumors lack the ability to arrest in G1 in response to irradiation6-9. MYCN down-regulation broadly reverses these oncogenic phenotypes in a variety of neuroblastoma models10-12 and recent thereapeutic strategies to indirectly target MYCN production or protein stability have reduced tumor growth in vivo13-15. These observations motivate an investigation of MYCN binding in MYCN amplified tumors as it remains fundamentally unclear how elevated levels of the factor occupy the genome and alter transcriptional programs in neuroblastoma. Here we present the first dynamic chromatin and transcriptional landscape of direct MYCN perturbation in neuroblastoma. We find that at oncogenic levels, MYCN associates with E-box (CANNTG) binding motifs in an affinity dependent manner across most active cis-regulatory promoters and enhancers. MYCN shutdown globally reduces histone acetylation and transcription, consistent with prior descriptions of MYC proteins as non-linear amplifiers of gene expression. We establish that MYCN load at the promoter and proximal enhancers predicts transcriptional responsiveness to MYCN shutdown and that MYCN enhancer binding occurs prominently at the most strongly occupied and down-regulated genes, suggesting a role for these tissue specific elements in predicating MYCN responsive â??targetâ?? genes. At these invaded enhancers, we identify the lineage specific bHLH TWIST1 as a key collaborator and dependency of oncogenic MYCN. These data suggest that MYCN enhancer invasion helps shape transcriptional amplification of the neuroblastoma gene expression program to promote tumorigenesis. ATAC-Seq in SHEP21, BE2C, KELLY, NGP, and MM1S cell lines

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Direct interaction of MYCN and p53 regulate transcriptional responses in neuroblastoma [RNA-Seq]

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