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Additional file 4 of C11orf95-RELA fusion drives aberrant gene expression through the unique epigenetic regulation for ependymoma formation

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posted on 2021-03-09, 04:40 authored by Tatsuya Ozawa, Syuzo Kaneko, Frank Szulzewsky, Zhiwei Qiao, Mutsumi Takadera, Yoshitaka Narita, Tadashi Kondo, Eric C. Holland, Ryuji Hamamoto, Koichi Ichimura
Additional file 4: Supplementary Fig. 3. RELAFUS1 binds on specific DNA regions through the unique DNA-binding motif. (A) Top three transcription factor binding motifs enriched within the RELAFUS1-S486E-HA peaks identified by the Multiple Em for Motif Elicitation (MEME) tool in 293T/tv-a cells. E-value, enrichment p-value. (B) Rela binding motif most enriched within the Rela peaks identified by the MEME tool in TNF-treated MEFs. E-value, enrichment p-value. (C–F) Schematic of a luciferase reporter construct to measure a Nanoluc reporter activity through the RELAFUS1 motif (C). Top three or five RELAFUS1 binding motif sequences for MEME-1 (D), 2 (E) and 3 (F) motifs were inserted in the upstream of a minimal promoter (blue box) in tandem, respectively. The DNA sequences for each motif used in the reporter assay are shown below the sequence logos, respectively. (G) RCAS vector expression in 293T cells. To measure Nanoluc reporter activity through the RELAFUS1-MEME-2 motif sequence, RCAS-GFP or RELAFUS1-HA vector with the relevant luciferase reporter vectors was transiently transfected in 293T cells. After 24 hours of the transfection, the cell lysates were subjected to luciferase reporter assay and immunoblot analysis with the indicated antibodies. (H) RELAFUS1-HA and RELAFUS1-S486E-HA binding profiles surrounding the human NFKBIA locus in 293T/tv-a cells. The position of the κB site was shown as a blue vertical bar on positive (+) and negative (−) DNA strands. MEME-2 site was not identified in this locus. (JPEG 472 KB)

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KAKENHI Children’s Cancer Association of Japan The Mother and Child Health Foundation JST CREST JSPS Grant-in-Aid for Scientific Research The Takeda Science Foundation

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