Epigenetic regulator UHRF1 suppressively orchestrates multiple pathogenesis in rheumatoid arthritis

34 Rheumatoid arthritis (RA) is characterized by chronic synovial inflammation with 35 aberrant epigenetic alterations, eventually leading to joint destruction. However, 36 epigenetic regulatory mechanisms underlying RA pathogenesis remain largely 37 unknown. Here, we showed that Ubiquitin-like containing PHD and RING finger 38 domains 1 (UHRF1) , a key molecule involved in maintenance of DNA methylation 39 during cell division, is a central epigenetic regulator that orchestrates the suppression 40 of expression of multiple factors that exacerbate RA. We found that murine arthritis 41 tissue and human RA tissue, particularly synovial fibroblasts (SF), exhibit remarkable 42 up-regulation of expression of Uhrf1. SF-specific Uhrf1 conditional knockout mice 43 showed more severe arthritic phenotypes and apoptosis-resistant SF. Integrative 44 analysis of the transcriptome and methylome showed that expression of several 45 cytokines including Ccl20 was up-regulated in Uhrf1-deficient SF. In RA patients, 46 disease activity scores, CCL20 expression, Th17 accumulation and apoptosis 47 resistance were negatively correlated with UHRF1 expression in synovium. Finally, 48 stabilization of UHRF1 by Ryuvidine administration diminished disease pathogenesis in 49 arthritis model mice. Our results demonstrated that UHRF1 expressed in SF can 50 contribute to suppression of multiple pathogenic events associated with RA such as 51 Th17 recruitment, SF apoptosis and bone destruction, suggesting that targeting 52 UHRF1 could represent a novel therapeutic strategy for RA. 53


122
Morphological analyses showed that hyperplasia of synovium as well as cartilage and 123 bone destruction were also more severe in Uhrf1 ΔCol6a1 mice than in Uhrf1 fl/fl mice 124 (Fig.2c,d,. Given the detectable presence of Uhrf1 125 expression in SM and a previous report that Tnf-α expression is regulated by Uhrf1 in

139
Collectively, these data demonstrate that Uhrf1 expressed in SF, but not in SM, plays a  To translate our findings to human RA pathogenesis, we next examined the 203 significance of UHRF1 in RA patients. We collected synovium specimens from patients 204 with OA or RA. RT-qPCR analysis revealed that UHRF1 mRNA expression levels were 205 significantly elevated in RA synovium relative to those for OA, although the expression 206 level was highly variable among the RA patients (Fig.4a) Fig.9h). To confirm that accumulation of Th17 cells is regulated by UHRF1 228 expression in human SF, we then investigated the correlation between the frequency of 229 Th17 cells and UHRF1 mRNA expression in SF in synovium samples from the same 230 patients. We found that the Th17 frequency was indeed negatively correlated with UHRF1 mRNA expression levels in human SF (Fig.4f). In addition, consecutive 232 knockdown of UHRF1 mRNA resulted in the resistance to FAS-induced apoptosis in 233 RASF (Extended Data Fig.10a-c), similarly to that seen for murine SF lacking Uhrf1 234 (Fig.2f) (Fig.4g). We also administered Ryuvidine to STA model 250 mice (Fig.4h). Immunofluorescent staining of tissue samples showed that sustainable 251 Uhrf1 expression was achieved by Ryuvidine treatment in vivo (Fig.4i). Ryuvidine 252 treatment alleviated arthritis phenotypes in STA model mice compared to mice treated with DMSO (Fig.4j, k) and also reduced histological pathology and Ccl20 serum levels 254 (Fig.4l, m). These results collectively indicate that stabilization of Uhrf1 protein is a 255 potential therapeutic strategy for RA.

258
Epigenetic alterations have potential to be a mechanism that promotes RA 259 heterogeneity and treatments that target proteins involved in epigenetic changes could 260 be a novel therapeutic strategy for patients with RA, particularly those who do not 261 respond to current treatments 38,39 . In this study, we identified UHRF1 as a central patients with RA showed that CCL20 is a common UHRF1 target gene among 266 cytokine-and RA-related genes. However, a role for other genes (CSF3, TNFSF11, 267 CCL5, TNFRSF9, IL2RB, IL12RB1 and ACP5) was not validated in RASF (Data not 268 shown). These data indicate that regulation of the expression of specific gene(s) by 269 UHRF1 is dependent on species and/or arthritis types. Intriguingly, expression levels of 270 UHRF1, but not DNMTs, were diverse and negatively correlated with disease scores in 271 RA patients, suggesting that UHRF1 could also serve as a biomarker for disease 272 severity and/or one of the criteria for RA heterogeneity. Our finding that Ryuvidine 273 treatment ameliorated arthritis provides support for the ability of UHRF1 stabilization to 274 inhibit expression of multiple exacerbating factors in RA. These findings could contribute to a basis for exploration of novel alternative therapeutic approaches, 276 especially for those patients who do not respond to existing treatments.

279
The primary antibodies used in this study include: mouse monoclonal antibody against

396
After washing, secondary antibodies were incubated with 5 μg/mL DAPI for 30 min at 397 room temperature.

431
To t a l R N A w a s e x t r a c t e d w i t h I s o g e n ( N i p p o n g e n e , J a p a n ) a n d R N e a s y s p i n c o l u m n 432 kits (Qiagen, USA). First-strand cDNA was synthesized from the total RNA using

649
Distribution of Uhrf1-mediated methylated DNA annotated using given intervals and scores with genome features by CEAS (cis-regulatory element annotation system). h.

651
Venn diagram to compare Uhrf1-mediated methylated DNA loci between SF and 652 chondrocytes using MBD-seq data from this study and from a public database           Relative mRNA expression  Com emen and coag lation cascades Cyto ine-cyto ine rece tor interaction h matoid arthritis F signaling thway         Fig. 8: GO analysis of genes that are directly regulated by Uhrf1 in SF. a Resources. Enriched pathways are illustrated by gene counts and P values. b, c. Representative mRNA expression of genes included in the (b) KEGG pathways "Rheumatoid arthritis" and "Cytokine-cytokine receptor interaction" and (c) GO biological process "Negative regulation of apoptotic process" in SF from Uhrf1 and Uhrf1 mice (n=3) as measured by RT-qPCR. Mean±SD is shown. * and ** indicate P<0.05 and P<0.01 versus Uhrf1 , respectively, by unpaired t-test. d. Venn diagram for 89 down-regulated genes in Uhrf1 SF and 39 genes having Uhrf1-mediated methylated DNA peaks within the gene body. e. KEGG pathway enriched pathways are illustrated by gene counts and P values.   Fig. 9: UHRF1 expression level is negatively correlated with RA severity. a. DNMTs mRNA expression in OA (n=32) and RA (n=26) synovium measured by RT-qPCR. Mean±SD is shown. N.S.; not significant versus OA by unpaired t-test. b UHRF1 mRNA expression in RA synovium and TJC28, SDAI (n=19-20). c-e c) DNMT1, (d) DNMT3A and (e) DNMT3B mRNA expression in RA synovium and TJC28, SJC28, SDAI, MMP3 and CRP (n=19-20). f. UHRF1 and CCL20 N.S.; not P g. CCL20 and UHRF1 mRNA expression in OA (n= 28) and RA synovium (n=19). Among genes involved in maintaining DNA methylation, UHRF1 mRNA expression level is elevated as a whole and levels are more diverse in RA synovium than in OA synovium. Insufficient UHRF1 expression levels that arise in response to as yet unknown effects could accelerate progression of RA pathogenesis. In contrast, increased levels of UHRF1 can reduce mRNA expression levels of genes that encode multiple RA-exacerbating factors such as RA-related, cytokine-related and anti-apoptosis-related genes by altering DNA methylation. These results suggest that UHRF1 stabilization could be a new strategy for RA therapeutic treatment. Figure 1 Up-regulation of the epigenetic regulator Uhrf1 in arthritis tissue. Please see .pdf for full caption.

Figure 2
Speci c Uhrf1 depletion in synovial broblasts exacerbates arthritis pathogenesis. Please see .pdf for full caption.  Uhrf1 stabilization attenuates arthritis pathogenesis. Please see .pdf for full caption.