Objective IRF1 both mediates responses to type I interferons and the

Objective IRF1 both mediates responses to type I interferons and the induction of interferons. preparation using a validated antibody to IRF1. The effect of IRF1 on target gene AGK2 expression was confirmed using an overexpression system in cell lines and co-immunoprecipitation was used to define protein interactions. Results IRF1 binding around transcribed regions was increased in SLE patient monocytes but histone modifications at potential IRF1 binding sites without detectable IRF1 binding were also increased. IRF1 overexpression was sufficient to drive transcription of target genes. IRF1 overexpression was also able to alter histone modifications at a focus set AGK2 of target genes and the use of an IRF1 inhibitor decreased both expression and histone modifications at target genes. IRF1 was found to interact with a select set of histone modifying enzymes and other transcription factors. Conclusions IRF1 is an important signaling protein in the interferon pathway. IRF1 not only activates gene expression as a transcription factor but may perpetuate disease by leading to a dysregulated epigenome. Introduction Systemic lupus erythematosus (SLE) is the quintessential systemic autoimmune disease. The current model for the development of SLE entails an imbalance between the production of apoptotic cells and their clearance with the residual nucleic acid-protein complexes driving a type I interferon response maturation of dendritic cells and B cells with an associated loss of tolerance (1 2 A central role for type I interferons was acknowledged in early studies from your 1970s identifying elevated interferon levels in the serum of patients with lupus (3). The interferon regulatory factors (IRFs) are a family of transcription factors that regulate host defense. In monocytes IRF1 enhances TLR-dependent gene expression (4). Low levels of type I interferons activate JAK and STAT pathways and maintain monocytes and macrophages in a primed state to respond rapidly to infectious difficulties. This priming alters the epigenetic scenery and has been demonstrated to enhance the expression of interferon-β IL-6 and TNF (5 6 This altered epigenetic landscape translates into increased promoter occupancy by activating transcription factors (7-9). The combination of type I interferons and TNF leads to sustained activation of IRF1 and STAT1 driving a self-reinforcing circuit that could alter the pattern of gene regulation through chromatin alterations (10). In a previous study we found that 63% of the genes with H4 hyperacetylation in AGK2 SLE experienced potential binding sites for IRF1 within the upstream region (11). IRF1 is known to associate with p300/CBP and PCAF potentially providing a Rabbit polyclonal to ZNF19. mechanism for the hyperacetylation (12). IRF1 is usually notable from your perspective of the female preponderance of SLE because it is one of the major downstream mediators of prolactin signaling. Prolactin is usually immune stimulatory (13) and can break B cell tolerance (14). Hyperprolactinemia has been reported in 15-33 % of patients with SLE (15) and bromocriptine which inhibits secretion of prolactin has been shown to reduce SLE clinical activity (16 17 AGK2 Thus IRF1 provides a potential nexus of female hormones inflammation and type I interferon signals. Studies from murine lupus models also support a role for IRF1. The IRF1KO bred onto the MRL/lpr background ameliorated the classic MRL/lpr skin disease (18). The mice also experienced decreased autoantibodies less glomerular immune complex deposition diminished glomerulonephritis less proteinuria and improved survival (18). In a separate model IRF1 KO mice experienced markedly ameliorated autoantibody production and renal disease (19). Therefore IRF1 central to interferon-mediated induction of gene expression appears to be pivotal in the lupus disease process. In this study we evaluated the role of IRF1 in modulating the epigenome and characterized its binding pattern in SLE. We found that both defined and potential gene targets of IRF1 experienced a significantly altered chromatin pattern and we found that we could replicate much of the effect by overexpressing IRF1 in cell lines. Methods Patients and cell purification Main human monocytes were purified from seven healthy controls and nine SLE patients by elutriation and adherence (6). Monocytes were more than 90% positive for CD14 staining. All controls and patients were female and an average of.