Fig. 3: The genomic region harboring rs2431697 is a cell type-specific enhancer regulating miR-146a expression. | Nature Communications

Fig. 3: The genomic region harboring rs2431697 is a cell type-specific enhancer regulating miR-146a expression.

From: SLE non-coding genetic risk variant determines the epigenetic dysfunction of an immune cell specific enhancer that controls disease-critical microRNA expression

Fig. 3: The genomic region harboring rs2431697 is a cell type-specific enhancer regulating miR-146a expression.

a–d RT-qPCR analysis of miR-146a expression in Raji or Jurkat WT and deletion clones (**P = 0.0029, *P = 0.0381, ***P < 0.0001) (n = 3, biological replicates). WT: rs2431697 wild type, D30: 30 bp fragment-harboring rs2431697 deletion. e ChIP-qPCR analysis of active enhancer marks (H3K4me1 and H3K27ac) within the rs2431697-containing region in U-937, Raji, and Jurkat cells (U-937 H3K4me1: ***P < 0.0001, U-937 H3K27ac: ***P = 0.0002, Raji H3K4me1: ***P = 0.0003, Raji H3K27ac: ***P = 0.0004) (n = 3, biological replicates). Fold enrichment was calculated as the ratio of the signal from the target antibody sample to the signal from the IgG control as measured by qPCR. f FAIRE-qPCR analysis of open chromatin and nucleosome occupancy for a rs2431697-centered 5 kb region of the 5q33.3 locus in U-937, Raji, and Jurkat cells (n = 3, biological replicates). Fold enrichment was calculated as the ratio of the signal from the FAIRE sample to the signal from the input control DNA as measured by qPCR. Primer sets and amplicons are indicated by a red arrow. Chr, chromosome. g Experimental scheme of Cas9 RNP delivery to different primary immune cells for genome editing and phenotypic characterization. h–j RT-qPCR analysis of miR-146a expression in CRISPR/Cas9 RNP edited CD14+ monocytes (h) (**P = 0.0059), CD19+ B cells (i) and CD3+ T cells (j) isolated from donor PBMCs (n = 5, biological samples replicates). k Schematic illustration of the construction of humanized mice to study cell type-specific regulation. NSG, NOD-scid IL2Rγnull. l–n In vivo editing of the rs2431697-containing region in humanized mice and RT-qPCR analysis of miR-146a expression in edited CD14+ monocytes (l) (*P = 0.0195), CD19+ B cells (m), and CD3+ T cells (n) (n = 6, biological mouse replicates). Data are represented as mean ± SEM and P-values are calculated using unpaired two-tailed Student’s t-test (a–e, l–n) and paired two-tailed Student’s test (h–j). *P < 0.05; **P < 0.01; ***P < 0.001. See also Supplementary Fig. 9.

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