Fig. 6
Characterization of the novel enhancer and promoter in the 3′ end of the IL1RAPL1 gene. a Schematic map of the enhancer/promoter region (chromosome X: 29,675,519-29,684,072 bp, hg19). Repetitive elements are shaded. Regions used for the promoter/enhancer (long and core) luciferase assays are indicated. Downward-facing black arrows show the location of gRNAs used for deleting the interval. Inward-facing black arrows correspond to oligonucleotide primers used for PCR screening of mutant clones. Below are ChIP-Seq profiles for RPE1 and S40 to H3K9me3, H3K4me2 and H3K27Ac (peak heights of 10, 150 and 150, respectively). The y-axis on the genome browser image is normalized read density. Colored translucent bands corresponding to the first half of the ChIP-Seq normalized read density have been added to assist in visualizing change regions. The location of a DNaseI hypersensitive cluster is indicated. b Graph of the results of a dual luciferase assay showing the ratio of Firefly to Renilla activity (y-axis) for the indicated enhancer constructs (x-axis) displayed as oneway ANOVA. P values calculated using Tukey–Kramer honest significant difference. The green diamonds show the mean of the triplicates (central horizontal line) and 95% confidence interval between the apexes of the diamond. Circles on the far right show all pairs Tukey–Kramer 0.05 p value spread with significance presented by the angle of intersection between circles. Data shown is representative of data obtained from at least two replicate experiments performed in triplicate. c IL1RAPL1 locus RNA-Seq data for RPE1 and SETDB1 KO clones showing forward- and reverse-strand profiles, and d profiles centered on the ERVL-MaLR element (black labeled box) (chromosome X: 29,659,850-29,660,300, hg38). e Sashimi plots showing quantitative visualization of splice junctions for the novel forward (top, chromosome X: 29,678,310-29,973,937 bp) and reverse (bottom, chromosome X: 29,560,159-29,678,231) transcripts originating from the ERVL-MaLR element for RPE1 and two SETDB KO clones. Boxed numbers in the top left corners indicate exon-coverage data range. f Graph of the data for the ERVL-MaLR promoter dual luciferase assay as in part-b above