Background:Prolonged TNF-induced H3K27 acetylation (H3K27ac) and increased mRNA stability in rheumatoid arthritis (RA) synovial fibroblasts (SF) are leading to a sustained inflammatory response. Underlying enzymes coordinately regulating these pathways have not been identified so far. The histone acetyltransferases cAMP-response element binding protein binding protein (CBP) and p300 are writers of activating H3K27ac marks and close homologues with widely accepted redundant functions.Objectives:To analyze individual functions of CBP and p300 in regulating the inflammatory response of RA SF.Methods:SF were isolated from patients with RA undergoing joint replacement surgery. The expression of CBP and p300 was silenced by transfection of antisense LNA gapmeRs (12.5 nM). SF were stimulated with TNF (10 ng/ml) for 24h. Actinomycin D (10 µg/ml) was added 4h after TNF-treatment for 2h and 4h (n=3) to test mRNA stability. Transcriptomes were determined by RNA-seq (Illumina NovaSeq 6000, n=6). We mapped raw reads from RNA-seq reference genome using STAR. Counts for genes were obtained using Feature counts. We searched for differential expression genes (DEG) across experimental conditions using general linear models (glm) implemented in ‘edgeR’ package of R. Significantly affected genes (± fold change > 1.5, FDR < 0.05, top 3000 genes included) entered pathway enrichment analysis for Gene Ontology (GO) biological process, and KEGG pathways in DAVID. Changes in the mRNA (n=12-14) and protein expression (n=6-12) were confirmed by quantitative Real-time PCR and ELISA. The levels of activating histone marks H3K27ac and nuclear localization of p50 and p65 were analyzed by Western blotting.Results:DEG revealed that silencing of p300 affected the expression of 6026 and 5138 genes in unstimulated and stimulated SF, respectively. In contrast, only 285 and 1911 genes were affected by CBP silencing in unstimulated and stimulated SF, respectively. In TNF-stimulated SF, pathway enrichment analysis of DEG revealed a key role of CBP in regulating the “type I interferon signaling pathway” (p=2.12x10-6). Both, silencing of CBP and p300 regulated genes enriched in the “TNF signaling pathway” (CBP: p=0.005; p300: p=0.031). In contrast to CBP silencing that had anti-inflammatory effects, silencing of p300 had pro-and anti-inflammatory effects. ELISA experiments suggested that silencing of CBP reduced the secretion of IL6 (p<0.01), CCL2, CXC3L1 (p<0.05), and CXCL12 (p<0.001). Silencing of p300 reduced the secretion of CCL2 (p<0.001) and CXC3L1 (p<0.05) but increased the expression of IL8 (p<0.001) and CXCL2 (p<0.05). Western blotting revealed that neither CBP, nor p300 silencing affected the nuclear expression of the NF-ĸB subunits p65 and p50. Silencing of p300 reduced the levels of H3K27ac by 30% in unstimulated SF, and by 61.4% (p<0.05) in presence of TNF. In addition to regulating H3K27ac, silencing of p300 regulated the expression of TNF-induced cytokines by increasing the mRNA stability of IL8, IL6 and CCL2 mRNA but not of CXCL2. Silencing of CBP reduced H3K27ac by 43.5% only in presence of TNF and did not affect TNF-induced mRNA stability of cytokines. This is in line with the enrichment of the GO biological process “regulation of mRNA stability” (p=2.61x10-8) being enriched only after silencing of p300.Conclusion:Our results suggested that p300 is the major writer for H3K27ac marks in SF. Additionally, p300 regulated cytokine expression by affecting mRNA stability in a target-specific manner. We identified overlapping and distinct functions for CBP and p300 in regulating the inflammatory response of SF.Disclosure of Interests:Monika Krosel: None declared, Marcel Gabathuler: None declared, Kellie Walker: None declared, Matija Tomsic: None declared, Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, Caroline Ospelt Consultant of: Consultancy fees from Gilead Sciences., Kerstin Klein: None declared
Pathway enrichment analysis of -omics data