Fig. 4

IFNAR1.L1M2a inducibly enhances transcription of IFNAR1 during type I IFN signaling. A IFNAR1 and three nearby genes, IFNAR2, IL10RB, and IFNGR2, were all considered to be possible targets of IFNAR1.L1M2a enhancer activity due to their proximity. B RNA sequencing of three wildtype clones (gray) and four IFNAR1.L1M2a.enh knockout clones (purple) for IFNAR1.L1M2a-proximal genes during a 24 h time course of IFNβ treatment. Pairwise differential expression analysis [48] at the untreated timepoint showed slightly but significantly lower baseline expression of IFNAR1 and IFNGR2 in knockout cells, and likelihood ratio tests [48] showed significant differences in response to IFNβ treatment of IFNAR1 and IL10RB. At the 24 h timepoint, IFNAR1 expression was slightly but significantly upregulated compared to the untreated timepoint in wildtype cells but not in knockout cells, and expression of IFNAR1 was significantly lower in knockout cells. C-D Immunofluorescence was used to confirm protein-level differences in IFNAR1 expression between wildtype cells (left, gray) and knockout cells (right, purple) in untreated (top, light) and IFNβ-treated (bottom, dark) conditions. In agreement with RNA-seq data, IFNAR1 expression was induced by IFNβ treatment in wildtype cells, but not in knockout cells. Scale bar indicates 25 microns