DNA-binding landscape of IRF3, IRF5 and IRF7 dimers: implications for dimer-specific gene regulation

Figures

• 

  Figure 1.. PBM-based analysis of IRF3/5/7 DNA binding. (A) Schematic of IRF dimers and PBM probe design. (B) Phosphomimetic IRF variants used in our PBM experiments. Positions of Ser/Thr to Asp mutations are indicated. (C) PBM z-score distributions for IRF dimer binding to 10 044 synthetic SNV probes. Highlighted are z-scores for literature described IRF binding sites and a high-affinity consensus site bound by all three IRF dimers. (D) PBM-derived DNA-binding logos for IRF3/5/7.

• 

  Figure 2.. IRF3/5/7 DNA-binding preferences. (A) Pairwise comparison of z-scores for IRF3, IRF5 and IRF7 binding to 10 044 synthetic IRF sites (black dots) and 644 random background sequences (blue dots). (B) Pairwise comparison of PBM z-scores (same sequences as in (A)) for IRF7 performed at two single concentrations (i.e. pseudo-replicate experiments). (C) Shown are the same pairwise comparison data as in (A) with all binding sequences matching the pattern 5′-GATANNGATA-3′ (N is any base) highlighted in red.

• 

  Figure 3.. IRF3/5/7 binding to ISREs with alternate half-site spacing. (A) Binding of IRF3/5/7 to binding sites found in the PRDI and PRDIII elements of the IFNB promoter (27). Two sites (P1, P2) have a 2-bp spacer between GAAA-type half-sites (2-bp sites); one site (P3) has a 3-bp spacer. Error bars show the standard deviation of PBM-based z-scores to five replicate DNA probe sequences (z-score for each individual sequence was determined by fitting to experiments at different concentrations as described in Materials and Methods). (B) Differential binding (i.e. delta z-scores) of IRF3/5/7 to identical sets of 120 sequence-matched 2- and 3-bp sites that differ by a single base (as shown in schematic). A schematic is shown illustrating how the ‘delta z-score’ for each sequence-matched pairs is calculated. The delta z-score distribution for all 120 sequence pairs is shown for IRF3/5/7. (C) EMSAs are shown for IRF3 binding to three DNA site variants: (monomer)—a single ISRE half-site (i.e. 5′-AANNGAAA-3′); (proximal dimer)—site with a 2-bp spacer between half-sites (2-bp site); (extended dimer)—site with an 8-bp spacer between half-site (8-bp site). (D) Schematic illustrating the proposed…

• 

  Figure 4.. Wild-type and mutant IRF dimer binding to IFNA VREs. (A) Binding profiles of wild-type and mutant IRFs to human IFNA VREs. Binding sites are illustrated as 10-bp blocks in register with the canonical 5-GAAANNGAAA-3′ elements of an IRF binding site. Low-affinity binding sites (z-score < 4.0) are not shown (see Materials and Methods). Z-scores for each panel range from 0 to 13.0. IFNA gene names are shown. Coordinates for VREs elements are provided (Supplementary File 2). (B) Binding profiles to mouse IFNA VREs are shown, all details as in (A). (C) Binding profiles of wild-type IRF3/5/7 to known IRF-binding regulatory elements are shown. Profile details as in (A,B). Coordinates for regulatory element DNA sequences are provided (Supplementary File 2). (D) DNA-binding logos for wild-type and mutant IRF5/7 are shown. Wild-type logos are identical to those in Figure 1 and are shown for contrast. Regions in which the DNA base specificity is altered in the mutant IRFs are highlighted with red shading. DNA logos generated as described in Materials and Methods (i.e. SNV approach). (E) Multiple protein-sequence alignment for human and mouse IRF3/5/7 is shown. Alignment is limited to the…

• 

  Figure 5.. Comparison of DNA binding and gene activation by ISRE variants. (A) Schematic for promoter variants based on IFNA14 VRE. Promoter variants contain simultaneous alterations of C-site and D-site with the binding site shown in the box. Wild-type C-site (WT-C) is shown for context. (B, D) PBM-determined z-scores are shown for IRF3/5/7 to binding site sequence variants listed in (A). Error bars are calculated as in Figure 3. (C, E) Reporter gene activation levels for promoter variants in HEK293T cells over-expressing the phosphomimetic, constitutively active IRF dimers: IRF3(6D), IRF5(4D) and IRF7(6D) (Methods and Materials). Normalized fold-induction (relative to I7–2 promoter levels) is shown for each promoter. Mean and standard error of the mean (SEM) values were calculated over at least 15 replicate measurements (Supplementary File 6).