THU0038 INDIVIDUAL FUNCTIONS OF THE HISTONE-ACETYLTRANSFERASES CBP AND P300 IN REGULATING THE INFLAMMATORY RESPONSE OF RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS
Authors: Marcel Gabathuler, Monika Krosel, Christoph Kolling, Matija Tomsic, Oliver Distler, Caroline Ospelt, Kerstin Klein
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> The two coactivators of transcription cAMP-response element binding protein (CREB) binding protein (CBP) and p300 are close homologues. They are widely accepted as redundant proteins and unique functions have not been investigated in depth. Both proteins contain a histone acetyltransferase (HAT) activity for writing of cell type specific activating H3K27 histone acetylation marks, and a bromodomain, which functions as a reader of acetylated lysine residues on histone tails. Inhibitors targeting the bromodomains are in drug development for inflammatory and malignant diseases. <h3>Objectives:</h3> To analyze individual functions of CBP and p300 in regulating the inflammatory response of rheumatoid arthritis (RA) synovial fibroblasts (SF). <h3>Methods:</h3> SF were isolated from knee, shoulder and hand joints of RA patients undergoing joint replacement surgery. The expression of CBP and p300 was silenced by transfection of antisense LNA gapmeRs (12.5 nM). 24h after transfection, cells were stimulated with TNF-α (10 ng/ml, 24h). The levels of H3K27ac in SF were analyzed by Western blotting (n=7). Transcriptomes were determined by RNA-seq (Illumina NovaSeq 6000, n=6). Pathway enrichment analysis of RNAseq data was performed using DAVID bioinformatic resources (fold change &gt; 1.5, FDR &lt; 0.05, top 3000 genes included). Changes in the mRNA expression of potential target genes were confirmed by quantitative Real-time PCR (n=12-14). <h3>Results:</h3> Silencing of p300 reduced the levels of H3K27ac by 30% in unstimulated SF, and by 61.4% (p&lt;0.05) in presence of TNF-α, whereas silencing of CBP reduced H3K27ac by 43.5% only in presence of TNF-α. In line with the changes in the H3K27ac, 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. 184 genes were affected by both, CBP and p300, in unstimulated SF, with 9.2% of genes being regulated in opposite directions. 13.5% of overlapping genes affected by CBP and p300 were regulated in opposite directions in TNF-α-stimulated SF. Principal component (PC) analysis of RNAseq data separated TNF-α-stimulated from unstimulated SF (PC1) and p300 gapmeR-transfected SF (PC2) from controls and CBP gapmeR-transfected SF, which clustered together. The top pathways regulated by CBP were 'cell cycle' and 'focal adhesion' in unstimulated cells and 'DNA replication' and 'cell cycle' after stimulation with TNF-α. Top pathways regulated by p300 in presence and absence of TNF-α were 'proteasome', 'spliceosome' and 'focal adhesion'. The expression of 16 chemokines and cytokines was changed in RNAseq data (fold change &gt;1.5, p&lt;0.05) by either silencing of CBP or p300. Whereas silencing of CBP reduced the expression of all of them, silencing of p300 had pro- and anti-inflammatory effects. We further confirmed expression changes in cytokine and chemokine expression by Real-time PCR. Silencing of CBP reduced the expression of IL6, CCL2 (p&lt;0.01), CXC3L1 (p&lt;0.05), and CXCL10. Silencing of p300 reduced the expression of CCL2 and CXC3L1 (p&lt;0.001) but increased the expression of IL8 (p&lt;0.001) and CXCL2 (p&lt;0.05). <h3>Conclusion:</h3> Our results suggest that p300 is the major writer for H3K27ac marks in SF. We have identified overlapping and distinct functions for CBP and p300 in SF. CBP inhibition has anti-inflammatory effects. In contrast, p300 inhibition has pro- and anti-inflammatory functions.&nbsp; <h3>Disclosure of Interests:</h3> Marcel Gabathuler: None declared, Monika Krosel: None declared, Christoph Kolling: None declared, Matija Tomsic: None declared, Oliver Distler Grant/research support from: Prof. Distler received research funding from Actelion, Bayer, Boehringer Ingelheim and Mitsubishi Tanabe to investigate potential treatments of scleroderma and its complications, Consultant for: Prof. Distler has/had consultancy relationship within the last 3 years with Actelion, AnaMar, Bayer, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, iQvia, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Serodapharm and UCB in the area of potential treatments of scleroderma and its complications. In addition, he had/has consultancy relationship within the last 3 years with A. Menarini, Amgen, Abbvie, GSK, Mepha, MSD, Pfizer and UCB in the field of arthritides and related disorders, Caroline Ospelt: None declared, Kerstin Klein: None declared&nbsp;