Specific enhancer selection by IRF3, IRF5 and IRF9 is determined by ISRE half-sites, 5' and 3' flanking bases, collaborating transcription factors and the chromatin environment in a combinatorial fashion.

IRF3, IRF5 and IRF9 are transcription factors, which play distinct roles in the regulation of antiviral and inflammatory responses. The determinants that mediate IRF-specific enhancer selection are not fully understood. To uncover regions occupied predominantly by IRF3, IRF5 or IRF9, we performed ChIP-seq experiments in activated murine dendritic cells. The identified regions were analysed with respect to the enrichment of DNA motifs, the interferon-stimulated response element (ISRE) and ISRE half-site variants, and chromatin accessibility. Using a machine learning method, we investigated the predictability of IRF-dominance. We found that IRF5-dominant regions differed fundamentally from the IRF3- and IRF9-dominant regions: ISREs were rare, while the NFKB motif and special ISRE half-sites, such as 5'-GAGA-3' and 5'-GACA-3', were enriched. IRF3- and IRF9-dominant regions were characterized by the enriched ISRE motif and lower frequency of accessible chromatin. Enrichment analysis and the machine learning method uncovered the features that favour IRF3 or IRF9 dominancy (e.g. a tripartite form of ISRE and motifs for NF-κB for IRF3, and the GAS motif and certain ISRE variants for IRF9). This study contributes to our understanding of how IRF members, which bind overlapping sets of DNA sequences, can initiate signal-dependent responses without activating superfluous or harmful programmes.

Signal Integration of IFN-I and IFN-II With TLR4 Involves Sequential Recruitment of STAT1-Complexes and NFκB to Enhance Pro-inflammatory Transcription.

Atherosclerosis is a chronic inflammatory disease of the blood vessels, characterized by atherosclerotic lesion formation. Vascular Smooth Muscle Cells (VSMC), macrophages (MΦ), and dendritic cells (DC) play a crucial role in vascular inflammation and atherosclerosis. Interferon (IFN)α, IFNγ, and Toll-like receptor (TLR)4 activate pro-inflammatory gene expression and are pro-atherogenic. Gene expression regulation of many pro-inflammatory genes has shown to rely on Signal Integration (SI) between IFNs and TLR4 through combinatorial actions of the Signal Transducer and Activator of Transcription (STAT)1 complexes ISGF3 and γ-activated factor (GAF), and Nuclear Factor-κB (NFκB). Thus, IFN pre-treatment ("priming") followed by LPS stimulation leads to enhanced transcriptional responses as compared to the individual stimuli. To characterize the mechanism of priming-induced IFNα + LPS- and IFNγ + LPS-dependent SI in vascular cells as compared to immune cells, we performed a comprehensive genome-wide analysis of mouse VSMC, MΦ, and DC in response to IFNα, IFNγ, and/or LPS. Thus, we identified IFNα + LPS or IFNγ + LPS induced genes commonly expressed in these cell types that bound STAT1 and p65 at comparable γ-activated sequence (GAS), Interferon-stimulated response element (ISRE), or NFκB sites in promoter proximal and distal regions. Comparison of the relatively high number of overlapping ISRE sites in these genes unraveled a novel role of ISGF3 and possibly STAT1/IRF9 in IFNγ responses. In addition, similar STAT1-p65 co-binding modes were detected for IFNα + LPS and IFNγ + LPS up-regulated genes, which involved recruitment of STAT1 complexes preceding p65 to closely located GAS/NFκB or ISRE/NFκB composite sites already upon IFNα or IFNγ treatment. This STAT1-p65 co-binding significantly increased after subsequent LPS exposure and correlated with histone acetylation, PolII recruitment, and amplified target gene transcription in a STAT1-p65 co-bound dependent manner. Thus, co-binding of STAT1-containing transcription factor complexes and NFκB, activated by IFN-I or IFN-II together with LPS, provides a platform for robust transcriptional activation of pro-inflammatory genes. Moreover, our data offer an explanation for the comparable effects of IFNα or IFNγ priming on TLR4-induced activation in vascular and immune cells, with important implications in atherosclerosis.