GSE120806
Genome-wide collaboration of canonical and non-canonical STAT1 complexes with NF-?B to control signal integration between Interferons and TLR4 in vascular and immune cells [ChIP-seq]
Atherosclerosis is a disease of large and medium-sized muscular arteries and is characterized by vascular inflammation and lipid-laden plaque formation within the intima of the vessel wall. Atherosclerosis is initiated by recruitment of blood leukocytes to the injured vascular endothelium and leads to altered contractility of Vascular Smooth Muscle Cells (VSMCs), acute and chronic luminal obstruction, abnormalities of blood flow and diminished oxygen supply to target organs. The pro-inflammatory pathways activated by Toll-like Receptors (TLRs), and Interferons (IFNs) have been identified as key components of atherogenesis. These pathways culminate in the activation of Signal Transducers and Activator of Transcription (STAT)1-containing complexes ISGF3 and GAF, and Nuclear factor-?B (NF?B) that coordinate expression of multiple chemokines, adhesion molecules, antiviral and antimicrobial proteins in vascular and immune cells. IFN-I and IFN-II participate in signaling cross-talk with TLR4 through combinatorial actions of ISGF3 and GAF with NF-kB leading to enhanced gene expression. Currently it is not clear how this signal integration is regulated at the genome-wide level and if similar mechanisms are activated by the different IFNs in vascular cells as compared to macrophages and dendritic cells. Therefore, we compared genome-wide transcriptional responses of mouse VSMCs, macrophages (BMDMs) and dendritic cells (DCs) in response to IFNa, IFN? or LPS alone, or after combined treatment using RNA-seq. Consequently, commonly up-regulated genes in VSMCs, BMDMs and DCs could be identified after combined treatment with IFNa+LPS, as well as after combined treatment with IFN?+LPS. Generally, in all three cell-types combined treatment with IFNa+LPS or IFN?+LPS resulted in a synergistic increase in gene expression as compared to single treatments. Also, significant functional overlap could be observed between commonly upregulated genes in response to IFNa+LPS and IFN?+LPS. Using ChIP-seq on chromatin from VSMCs, STAT1 and p65 bound IFNa+LPS and IFN?+LPS up-regulated genes were identified, containing GAF/GAS, ISGF3/ISRE or NF-kB binding motifs located in promoter regions, but also to up- and downstream genomic regions. Comparing IFNa+LPS and IFN?+LPS commonly upregulated genes identified a substantial overlap of ISRE-containing genes. Interestingly, STAT1 as part of ISGF3 was shown to bind the promoter of these ISRE-containing genes in response to IFNa as well as IFN?. Moreover, different binding modes of STAT1-p65 co-binding were detected, including GAS-NF?B, ISRE-NF?B or GAS-ISRE-NF?B, shared by IFNa+LPS and IFN?+LPS up-regulated genes. This cooperative involvement involved a STAT1-dependent role in the nearby recruitment of p65 already upon single IFNa or IFN? treatment, via closely located GAS-NF?B or ISRE-NF?B binding sites in promoter regions. More important, this STAT1-p65 co-binding was significantly increased upon subsequent LPS exposure and resulted in amplified transcriptional activity of IFNa+LPS and IFN?+LPS upregulated genes. Overall design: ChIP-seq samples were derived from IFN alpha- (8 hours), IFN gamma- (8 hours), LPS- (4hours), IFN alpha+LPS- and IFN gamma+LPS-treated Vascular Smooth Muscle Cells (VSMC) in duplicates.