PncCCND1_B Engages an Inhibitory Protein Network to Downregulate CCND1 Expression upon DNA Damage.

Abstract

Simple SummaryEwing sarcoma is a pediatric tumor characterized by chromosomal translocations, giving rise to the oncogene EWS-FLI1, which triggers the transcription of genes involved in neoplastic transformation including CCND1. In this work, we found that exposure to etoposide, a topoisomerase II inhibitor usually administered in combination with other drugs in the standard regimen for Ewing sarcoma treatment, induced cell death and reduced CCND1 levels. Etoposide acts, at least in part, by enhancing the expression of the pncCCND1_B, a promoter-associated noncoding RNA transcribed from the promoter region of the CCND1 gene. PncCCND1_B regulates in cis CCND1 expression by forming a molecular complex with the RNA binding protein Sam68 and the DNA/RNA helicase DHX9. Upon exposure to etoposide, the increase in pncCCND1_B coupled with the decrease in DHX9 expression promote epigenetic changes and formation of DNA:RNA hybrids at the promoter region of CCND1 gene, which downregulate its expression.Promoter-associated noncoding RNAs (pancRNAs) represent a class of noncoding transcripts driven from the promoter region of protein-coding or non-coding genes that operate as cis-acting elements to regulate the expression of the host gene. PancRNAs act by altering the chromatin structure and recruiting transcription regulators. PncCCND1_B is driven by the promoter region of CCND1 and regulates CCND1 expression in Ewing sarcoma through recruitment of a multi-molecular complex composed of the RNA binding protein Sam68 and the DNA/RNA helicase DHX9. In this study, we investigated the regulation of CCND1 expression in Ewing sarcoma cells upon exposure to chemotherapeutic drugs. Pan-inhibitor screening indicated that etoposide, a drug used for Ewing sarcoma treatment, promotes transcription of pncCCND1_B and repression of CCND1 expression. RNA immunoprecipitation experiments showed increased binding of Sam68 to the pncCCND1_B after treatment, despite the significant reduction in DHX9 protein. This effect was associated with the formation of DNA:RNA duplexes at the CCND1 promoter. Furthermore, Sam68 interacted with HDAC1 in etoposide treated cells, thus contributing to chromatin remodeling and epigenetic changes. Interestingly, inhibition of the ATM signaling pathway by KU 55,933 treatment was sufficient to inhibit etoposide-induced Sam68-HDAC1 interaction without rescuing DHX9 expression. In these conditions, the DNA:RNA hybrids persist, thus contributing to the local chromatin inactivation at the CCND1 promoter region. Altogether, our results show an active role of Sam68 in DNA damage signaling and chromatin remodeling on the CCND1 gene by fine-tuning transitions of epigenetic complexes on the CCND1 promoter.