Abstract
Recurrent C11orf95-RELA fusions (RELAFUS) are the hallmark of supratentorial ependymomas. The presence of RELA as the fusion partner indicates a close association of aberrant NF-κB activity with tumorigenesis. However, the oncogenic role of the C11orf95 has not been determined. Here, we performed ChIP-seq analyses to explore genomic regions bound by RELAFUS and H3K27ac proteins in human 293 T and mouse ependymoma cells. We then utilized published RNA-Seq data from human and mouse RELAFUS tumors and identified target genes that were directly regulated by RELAFUS in these tumors. Subsequent transcription factor motif analyses of RELAFUS target genes detected a unique GC-rich motif recognized by the C11orf95 moiety, that is present in approximately half of RELAFUS target genes. Luciferase assays confirmed that a promoter carrying this motif is sufficient to drive RELAFUS-dependent gene expression. Further, the RELAFUS target genes were found to be overlapped with Rela target genes primarily via non-canonical NF-κB binding sites. Using a series of truncation and substitution mutants of RELAFUS, we also show that the activation domain in the RELAFUS moiety is necessary for the regulation of gene expression of these RELAFUS target genes. Lastly, we performed an anti-cancer drug screening with mouse ependymoma cells and identified potential anti-ependymoma drugs that are related to the oncogenic mechanism of RELAFUS. These findings suggested that RELAFUS might induce ependymoma formation through oncogenic pathways orchestrated by both C11orf95 and RELA target genes. Thus, our study unveils a complex gene function of RELAFUS as an oncogenic transcription factor in RELAFUS positive ependymomas.
Highlights
Ependymomas are primary glial tumors that can occur in all ages and locations of the central nervous system [39]
HA tag ChIP‐seq analyses identified unique genomic binding sites of RELAFUS1 Accumulating evidence suggests that—in addition to known NF-κB targets— non-NF-κB target genes contribute to the tumorigenesis of RELA fusion (RELAFUS) [31, 35, 41, 45]
We focused our subsequent analysis on genes bound by RELAFUS1 at ± 10 kb of the transcription start sites (TSSs), since these genes were significantly up-regulated compared to genes bound at ±30, 40, or 50 kb of the TSSs (Fig. 1f )
Summary
Ependymomas are primary glial tumors that can occur in all ages and locations of the central nervous system [39]. Recurrent C11orf95-RELA fusion (RELAFUS) genes were identified in a large fraction of supratentorial ependymomas [45]. RELAFUS1 (Type 1) and RELAFUS2 (Type 2), the two most frequent fusion variants are potent driver oncogenes capable of inducing human ependymoma-like tumors in mice, and likely represent the tumorinitiating events in human patients [41, 45, 60]. Given that the fusion protein preferentially localizes in the nucleus, persistent activation of the NF-κB pathway is thought as the primary mechanism for the RELAFUS -driven ependymoma formation as bolstered by high NF-κB activity in human and mouse RELAFUS tumors [41, 45]. Expression of wild type RELA or activating RELA mutants failed to induce brain tumor formation in mice, strongly suggesting an important role of non-NF-κB pathways in the RELAFUS-driven ependymoma formation [41, 45]
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