Objectives Changes in DNA methylation and histone marks have been associated with diseases such as cancer, rheumatoid arthritis (RA) and systemic lupus erythematosus. Previously, we reported global genomic hypomethylation in late RA synovial fibroblasts (RASF). Now, we searched for specific genes that are differentially methylated in late, early RASF and SF from patients with resolving arthritis (ReSF) in comparison to osteoarthritis synovial fibroblasts (OASF).
Methods Methylation immunoprecipitation (MeDIP) was performed and hybridizised to a human promoter array. The expression of related transcripts was determined by quantitative SYBR PCR and protein analysis was performed by Western blot and immunohistochemistry. Changes in histone marks, namely H3 K4 trimethylation and H3 K27 trimethylation were analysed by chromatin immunoprecipitation (ChIP). Transient transfections were done in OASF cell cultures with a TBX5 expression vector. cDNA were prepared and used to hybridize in an Affymetrix human microarray. The gene targets were further tested with luciferase reporter assays in HEK293 cells.
Results The promoter array analysis revealed 30 gene promoters which were hypomethylated in late RASF. The TBX5 gene was significantly more methylated in the promoter of OASF than late RASF (OASF 17±4.6 and late RASF 0.6±0.17 % (MeDIP/input), p<0.05, n=4). We further examined the methylation levels of TBX5 promoter in early RA and found that the promoter methylation decreases in early disease stages (OASF 17±3.8, (ReSF) 12±4.2, early RASF 6±3.0 % (MeDIP/input), p<0.05, n=6). TBX5 transcripts were significantly more expressed in late RASF than OASF (late RASF dCt: 11.4 ± 0.5; OASF dCt: 12.7±0.3, p<0.05, n=7). In addition, Western blot showed that the TBX5 protein was expressed in late RASF, but not in OASF. Immunohistochemistry of synovial tissues confirmed that TBX5 is expressed in RA (n=6), but not in OA (n=6). Furthermore, in the TBX5 locus, ChiP revealed that late RASF had more H3 K4 trimethylation– associated with an open chromatin -, than OASF (late RASF H3 K4 trimethylation: 0.67±0.06 ratio to histone 3; OASF H3 K4 trimethylation: 0.02±0.005 ratio to histone 3, p<0.01 n = 4-5). On the other hand, OASF were found to have enrichment for H3 K27 trimethylation – associated with a closed chromatin -, when compared to late RASF (OASF 0.25±0.09; late RASF 0.02±0.001, p<0.01 n = 4-5). Overexpression of TBX5 in OASF revealed 640 genes commonly up regulated from 1.2 to 3-fold. Analysis of these genes by DAVID bioinformatics software identified that the chemokines IL8, CXCL2 and CCL20 were common targets of TBX5 in OASF. The expression of chemokines was significantly upregulated in seven different OASF TBX5 transfected cell cultures (IL8: 2 ± 0.9, CXCL2: 1.97 ± 0.9, CCL20: 2.07 ± 0.8 fold change, p<0.05). Transfection of TBX5 in HEK293 induced the activity of IL8 promoter by 1.6 fold change (p<0.05, n=3).
Conclusions Specific DNA hypomethylation of the TBX5 promoter occurred early in the disease process and is responsible for the intrinsically up regulated TBX5 expression in late RASF. TBX5 appears to be a novel inducer of chemotaxis in the pathogenesis of RA whereby RASF attract inflammatory cells to the synovium.
Acknowledgements IMI BTCure, IAR, Novartis Foundation for Medical and Biological Research.
Disclosure of Interest None Declared