Fig. 5
RELAFUS1 transcriptionally regulates the target gene expression through DNA binding on the RELAFUS1-MEME-2 sequence. a Venn diagram showing the number of the RELAFUS1 target genes overlapped between 293T and mEPN cells. 41 common targets were shown in the right table. SE-located genes were highlighted in yellow. b, c, i, j RELAFUS1-HA, RELAFUS1−S486E-HA and H3K27ac binding profiles surrounding the human C11orf95 and LMX1B in 293T/tv-a, and mouse 2700081O15Rik and Lmx1b loci in mEPN (H1203 and H41) cells. Rela binding profile in MEFs was also shown in mouse 2700081O15Rik locus. The position of the MEME-2 and κB sites are shown as a blue vertical bar on positive (+) and negative (−) DNA strands. d Boxplots of C11orf95 and LMX1B mRNA expression in human RELAFUS positive (n = 14) and negative (n = 54) ependymomas. e, f Boxplots of 2700081O15Rik and Lmx1b mRNA expression in mouse normal brain (NB) and RCAS-RELAFUS1-driven ependymoma or PDGFA-driven glioma tissues in the N/tv-a (e) or N/tv-a;Ink4a-Arf−/−;Ptenfl/fl (KO) (f) mice (n = 4 in each group). All box plots showing mRNA expression extend from the 25th to 75th percentiles. Whiskers of the box plots extend to the most extreme data point (d–f). Gene expression analysis was done using unpaired two-tailed t-test (d, e) or Ordinary one-way ANOVA (f). **p < 0.01; ****p < 0.0001. g Relative C11orf95 and LMX1B mRNA expression in 293T cells. Data are displayed as the relative ratio to GFP cells. qPCR data (mean ± SD) for C11orf95 and LMX1B expression are displayed as the relative ratio to GFP cells (n = 3, in technical quadruplicate). Analysis was done using paired two-tailed t-test. *p < 0.05; **p < 0.01 (See Methods). h Relative Nanoluc reporter activity to the upstream sequence of C11orf95 gene normalized to the Firefly luciferase activity and GFP cells in the 293T cells (n = 6, in technical triplicate). Analysis was done using paired two-tailed t-test. ****p < 0.0001 k Relative Lmx1b mRNA expression in H1203 cells introduced with the indicated dCas-sgRNA. Data are displayed as the relative ratio to sgGFP vector-infected cells. Data from two independent experiments with two technical replicates are shown in the figures. Each qPCR was performed in technical quadruplicate. Analysis was done using paired two-tailed t-test. **p < 0.01; ***p < 0.001