Fig 2: Features of enhancer activity measured from human genomic DNA.a, Venn diagram showing the concordance of biological and in silico replicate IDR peaks from genomic STARR-seq in HepG2 cells, demonstrating that the IDR method yields similar peak-calls (~90% specificity if biological replicate analysis is considered ground truth) when it is used as an internal control approach (see Methods). b, Genome browser snapshot of enhancer activity and TF binding at the BBC3 gene locus in GP5d and HepG2 cells, demonstrating the excellent signal-to-noise ratio of the STARR-seq data. Both plus and minus-strand STARR-seq signal is shown, as well as ATAC-seq coverage and ChIP-seq for H3K27ac, TP53, and HNF4A. Cell type–specific STARR-seq signals agree with tissue-specific TF binding with a common TP53-bound enhancer and a HepG2-sepcific HNF4A-bound enhancer highlighted with pink boxes. Genomic sequences at these loci with TP53 and HNF4A motifs are shown. c, Overlap between genomic STARR-seq peaks and genomic features (regulatory element features, ChIP-seq peaks for individual DNA-binding proteins and their motifs) in GP5d cells (see Methods). The total number of peaks for each experiment is the sum of the values inside its circle. The significance of the overlaps between STARR-seq and the other measurements (two-tailed Fisher’s exact test, see Methods for details): ATAC-seq: P < 2.2251×10−308; H3K27ac ChIP-seq: P < 2.2251×10−308; superenhancers: P < 2.2251×10−308; TT-seq enhancers: P < 2.2251×10−308; TP53 ChIP-seq: P < 2.2251×10−308; CTCF ChIP-seq: P = 4.1888×10−111; FOXA1 ChIP-seq: P < 2.2251×10−308. d, Top, overlap of genomic STARR−seq peaks with TP53 ChIP-seq peaks and the p53 motif in HepG2 cells. The motif-match overlap was calculated using 24,176 highest affinity matches in the genome (all with score > 9). Bottom, overlap between STARR+, ATAC- peaks and ChIP-seq data for H3K4me1 and H3K9me3 histone modifications in HepG2 cells. Collectively, the results shown in panels c and d demonstrate that although p53 and IRF motifs are the strongest activators in the cells (see Fig. 1c, Fig. 2f), they contribute to a relatively small proportion of the overall enhancer activity, based on the overlap analysis of genomic STARR-seq peaks with TP53 ChIP-seq peaks and with p53 and IRF3 motifs (see also Supplementary Note).

Fig 3: IFIT1 Facilitates Sin3A Corepressor Removal and IRF3 Recruitment to Multiple ISG Loci(A–C) Control and IFIT1 shRNA-expressing THP1 cells were stimulated with 100 ng/mL LPS for the indicated times, and Sin3A binding was measured by ChIP-qPCR at the (A) IFNB1, (B) IRF7, and (C) IRF8 promoters.(D–F) Control and IFIT1 shRNA-expressing THP1 cells were stimulated with 100 ng/mL LPS for the indicated times, and IRF3 binding was measured by ChIP-qPCR at the (D) IFNB1, (E) IRF7, and (F) IRF8 promoters.Data are representative of three independent experiments and are expressed as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (two-way ANOVA followed by Sidak’s multiple comparison test). See also Figure S5.

Fig 4: Signaling Responses in IFIT1-Depleted Cells(A) Control and IFIT1 shRNA-expressing THP1 cells were stimulated with 100 ng/mL LPS for the indicated times, and cytosolic and nuclear fractions were subjected to western blot analysis for phos-pho-p65/NF-?B (Ser536), phospho p38 and MAPK (Thr180/Tyr182), p105/NF-?B, and p50/NF-?B. GAPDH and heterogenous nuclear ribonucleoprotein L (hnRNPL) were detected as positive controls for cytosolic and nuclear fractions.(B) Control and IFIT1 shRNA-expressing THP1 cells were stimulated with 100 ng/mL LPS for the indicated times, and whole-cell lysates were subjected to western blot analysis for phospho-IRF3 (S386), phospho-STAT1 (Tyr701), IFIT1, and actin (loading control).(C) WT and Ifit1-/- BMDM cells were stimulated with 100 ng/mL LPS for the indicated times, and whole-cell lysates were subjected to western blot analysis for phospho-STAT1 (Tyr701), phospho-p38 and MAPK (Thr180/Tyr182), phospho-JNK (Thr183/Tyr185), phospho-ERK (Thr202/Tyr204), and GAPDH (loading control).(D) WT and Ifit1-/- mouse BMDM transfected with non-targeting control (NTC) or IFIT3 siRNA were stimulated with 100 ng/mL LPS and Ifnb1 mRNA was measured by qPCR.Data are representative of three independent experiments. Data in (D) are expressed as mean ± SD; **p < 0.01, ***p < 0.001 (two-way ANOVA followed by Sidak’s multiple comparison test). See also Figure S3.