Fig. 2: The rs2280381-containing region is a cell-type-dependent enhancer regulating IRF8 expression. | Nature Communications

Fig. 2: The rs2280381-containing region is a cell-type-dependent enhancer regulating IRF8 expression.

From: Lupus enhancer risk variant causes dysregulation of IRF8 through cooperative lncRNA and DNA methylation machinery

Fig. 2

A Flow chart for generating genomic fragment deletion clones using the CRISPR-Cas9 technology. B The genotype of rs2280381 wildtype clones and deletion clones. C RT-qPCR analysis of IRF8 expression in U-937 WT and KO clones (n = 3, biologically independent samples). D WB analysis of IRF8 expression in U-937 WT and KO clones. Blot is representative of n = 3 biologically independent experiments. E Analysis of active enhancer signals (chromatin accessibility and H3K27ac) within the rs2280381-containing region in U-937 cells by ATAC-seq and ChIP-seq. 4C-seq analysis of contact profiles of the IRF8 promoter (F) and the rs2280381 site (G) using a 2 kb window size in the main trend subpanel. Red arrowhead indicates the view point position, and black arrowhead indicates the target position. Gray dots indicate normalized contact intensities. The heat map displays a set of medians of normalized contact intensities calculated at different window sizes. RT-qPCR analysis of IRF8 expression in Raji (H) or Jurkat (I) WT and KO clones (n = 3, biologically independent samples). RT-qPCR analysis of IRF8 expression in CRISPR/Cas9 RNP edited CD19+ B cells (J), CD3+ T cells (K) and CD14+ monocytes (L) (n = 3, biologically independent samples). WT: rs2280381 wildtype, KO: 138 bp fragment containing the rs2280381 deletion. Data are represented as mean ± SEM, and p values are calculated using an unpaired two-tailed Student’s t test (C, H, I) and paired two-tailed Student’s t test (J–L). ns not significant. See also Supplementary Figs. 2–5.

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