Figure 1. Dnmt3b expression in murine and human knee joint cartilage. (A) Representative images

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1 Figure Legends Figure. expression in murine and human knee joint cartilage. () Representative images showing that Dnmta is not expressed in chondrocytes from mo W articular cartilage [Dnmta expression in pancreas tissue (positive control)], while robust expression of in chondrocytes of mo W articular cartilage and lower expression in underlying growth plate cartilage (n=); the magnified images of the dashed and solid boxed area of articular cartilage in separate panels; () Representative images showing expression in mo (n=) versus 7 mo (n=) W murine knee articular cartilage; () Representative images showing expression in wk old murine articular cartilage following MLI surgery (n=) or cartilage from sham control knees (n=); (D) Representative images showing expression in wk old murine articular cartilage in mice fed a high fat diet (HFD) (n=) or controls (trl) fed a normal diet (n=). (E) wo representative images showing DNM expression in human healthy articular cartilage (n=) or osteoarthritic cartilage tissue sections (n=7). (F) Reduced DNM expression in human primary O chondrocytes compared to healthy chondrocytes (n=). () Induction of human primary chondrocytes (n = ) with IL-β results in decreased expression of DNM mrn and protein levels. ll scale bars, µm. Figure S. expression in embryonic and adult murine knee joint cartilage. Immunohistochemical staining shows protein expression in chondrocytes from 57L/ W murine articular cartilage knee joints at embryonic time point E8.5 and post-natal time points P7, P8 and P5 (n=)., hypertrophic chondrocyte;, bone marrow cells. ll scale bars, µm. Figure S. ltered anabolic and catabolic gene expression in IL-β-treated human primary chondrocytes. Reduced OL and increased MMP expression following IL-β treatment (8 h) of human primary articular chondrocytes isolated from total knee replacement surgeries (n=). Figure S. IL-β regulation of is mediated in part by NF-κ. () Sequence alignment and conservation of an NF-κ binding site (red) in the promoter region of the gene of different 8

2 species. () Reduced luciferase expression in IL-β stimulated ( h) chondrogenic murine D-5 cells transfected with a luciferase reporter plasmid containing the promoter sequence when compared to control (trl) untreated cells. Reduced luciferase activity is attenuated when D-5 cells were transfected with a reporter plasmid containing a mutated NF-κ binding site () (n=). (, D) Pull-down of genomic DN with an NF-κ antibody (hip assay) shows interaction of NF-κ with its binding site in the promoter region by qpr () and semi-quantitative gel electrophoresis (D) utilizing specific primers to amplify the promoter region containing the NF-κ binding site (n=). (E, F) mrn and protein levels were reduced in murine W primary articular chondrocytes (extracted from mo murine knee joints) following IL-β induction (8 h) (n=). Figure S. blation of in articular chondrocytes in vitro alters cell homeostasis. Reduced expression of () mrn and () protein following transfection with nm sirn for 8 h in murine primary chondrocytes isolated from mo W mice (n=). () sirn treatment resulted in increased expression of the catabolic/hypertrophic chondrocyte markers ola, Runx, and Mmp, and decreased expression of the anabolic marker, ola in murine primary chondrocytes (n=). (D) Representative images showing reduced expression resulted in increased alkaline phosphatase (LP) activity in murine primary chondrocytes, but not to the extent resulting from MP- treatment (positive control) (n=). (E, F) sirn treatment affected the balance of F-β and MP signaling as shown by decreased phospho(p)-smad expression and increased p-smad/5 expression in murine primary chondrocytes, respectively (n=). Figure. loss-of-function mice develop accelerated O. () Representative alcian blue / hematoxylin / orange (H/O) staining of tissue sections of knee joints harvested from 5 mo and 8 mo loss-of-function () mice and re + control (trl) mice (n=5). he magnified images of the boxed regions are shown respectively. () ORSI scoring system was used to quantify the H/O stained tissue sections (n=5). () rticular cartilage area was quantified by histomorphometry (n=5). (D) 9

3 Representative micro images of knee joints from 8 mo and trl mice (n=5). (E) Subchondral bone volume and (F) subchondral bone trabecular connective density were calculated from the micro images (n=5). rrows in 5 mo old mice show areas of proteoglycan loss and cartilage fibrillation, respectively. rrows in 8 mo old mice show osteophyte formation and an area of proteoglycan loss in articular cartilage, respectively. Yellow arrows in micro images denote osteophyte formation. Scale bars, µm. Figure S5. blation of in articular chondrocytes in vivo. () Representative fluorescence microscopy shows recombination efficiency in mo gcre ER ; m/m mice followed by tamoxifen injection for 5 days (n=). () protein knock-down in mo articular chondrocytes of mice compared to re + control (trl) littermates (n=). Scale bars, µm. Figure S. Expression of Dnmt as well as anabolic and catabolic genes in cartilage tissue. () qpr analysis of Dnmts and ets in mo articular cartilage isolated from and re+ control (trl) mice (n=). () et activity analysis in chondrocytes from mo or trl mice (n=). ()ola (anabolic) and ola, Runx and Mmp (catabolic / hypertrophic) marker expression in articular chondrocytes from mo and re + control (trl) mice (n=). Figure S7. nalysis of apoptosis and reactive oxygen species in cartilage. () Representative UNEL staining and analysis of knee joint articular cartilage tissue sections from 5 mo and re+ control (trl) mice by fluorescence microscopy (n=5); () Quantification of apoptotic cell numbers from the UNEL-stained fluorescent images (n=5). () Reactive oxygen species (ROS) analysis of mo articular chondrocytes (i.e. chondrocytes from f/f mice treated with d5-re for 8 h) compared to control (trl) chondrocytes ( chondrocytes treated with d5-fp for 8 h) (n=). Figure. ltered epigenomic and transcriptomic signatures in chondrocytes. gdn and RN isolated from control and cells for RN-seq and methyl-seq analysis (n=). ()

4 P plot of samples based on RN-seq data; () Heatmap display of top 5 significantly differentially expressed genes between and control; () Word cloud representing gene frequency of enriched function categories from all differentially expressed genes; (D) lobal methylation distribution of and control samples showing there is no global difference; (E) Methylation difference of DMR versus genome as background; (F) Significant overlap of genes nearby DMRs and differentially expressed genes, p-values calculated by hypergeometric test. Figure S8. nalysis of RN-seq and methyl-seq data. () Heatmap of sample-to-sample distances using the rlog-transformed values showing more similarity of RN-seq signal observed in each group; () n M-plot of gene expression changes. he log fold change for a particular comparison is plotted on the y-axis and the average of the counts normalized by size factor is shown on the x-axis. Each gene is represented with a dot. enes with an adjusted p value below a threshold (here., the default) are shown in red; () Enrichment of differentially expressed genes show enriched function related to cell cycle process, bone development etc; (D) enes from differentially expressed list are related with F/MP pathway network, green indicates down-regulation and red indicates up-regulation; (E) enomic feature distributions of DMRs; (F) Enriched transcription factor binding sites found in DMRs using Homer software. n=. Figure. Mitochondria function and cellular homeostasis in chondrocytes. Primary articular chondrocytes were isolated from mo f/f mice and infected with d5-re ( ) or d5- FP (trl) for 8 h. () Mitochondrial respiration was measured by the Seahorse XF Extracellular Flux nalyzer. asal respiration and maximal respiration, as measured by the oxygen consumption rate (OR) are shown (n=8). () metabolite (succinate, fumarate) and NDH analysis by HPL-MS (n=). () Mitochondrial metabolism analysis was measured in mo W cells treated with either mm diethyl succinate or vehicle for 8 h by the Seahorse XF Extracellular Flux nalyzer (n=8). (D) Mitochondrial respiration analysis in MP--treated mo W chondrocytes in the presence or absence antimycin + rotenone for 8 h (n=8).

5 Figure S9. hondrocyte gene expression in cells. Primary articular chondrocytes were isolated from mo f/f mice and infected with d5-re ( ) or d5-fp (trl) for 8 h. Expression of anabolic (ola) or catabolic/hypertrophic genes (ola, Runx, Mmp) is shown (n=). Figure S. hondrocyte gene expression under succinate treatment. Primary articular chondrocytes from mo W mice were treated with mm diethyl succinate or vehicle for 8 h. () ellular succinate levels in murine chondrocytes (n=). () hondrocyte gene expression in response to succinate treatment was analyzed by qpr (n=). Figure S. hondrocyte gene expression under antimycin and rotenone (&R) treatment. Primary articular chondrocytes form mo W mice were treated with. µμ antimycin and rotenone for 8 h. () Effect of antimycin and rotenone treatment on chondrocyte proliferation and apoptosis (n=). () nalysis of chondrocyte gene expression in response to MP- treatment + / - antimycin + rotenone (n=). Figure 5. gain-of-function mice are protected from cartilage degeneration following surgical induction of O. MLI or sham surgeries were performed on gain-of-function (OF) mice or re + control (trl) mice. () lcian blue / hematoxylin / orange stained sections of trl or OF knee joints weeks following sham surgery. Representative images of histological sections from trl or OF mice at 8 wk or wk following MLI surgery (n=5). he magnified images of the boxed regions are shown respectively. () Quantitation of histological assessment by ORSI scoring (n=5). () Histomorphometric analysis of trl or OF cartilage (n=5). Scale bars, µm. Figure. Mitochondria function and cellular homeostasis in OF chondrocytes. () ene expression in chondrocytes isolated from wk trl or OF chondrocytes (n=). () Mitochondrial respiration in primary articular chondrocytes isolated from mo olare; Rosa-rt f/+ ;

6 -tg mice, treated with vehicle (trl) or doxycycline ( OF) for 8 h (n=). Mitochondrial respiration was measured by the Seahorse XF Extracellular Flux nalyzer. Figure S. eneration of gain-of-function (OF) transgenic mice. () Schematic representation of the strategy utilized to generate doxycycline (DOX)-inducible over-expression in murine cartilage tissue. () enotyping strategy, utilizing three different primer pairs, to confirm recombination. Mouse line #9 was used for breeding and subsequent experimental analyses. Figure S. Over-expression of in articular chondrocytes in vivo. () Representative fluorescence microscopy shows recombination efficiency in wk olare; Rosa-rt f/+ ; HFP mice (n=). Scale bar, µm. () onfirmation of protein over-expression in primary articular chondrocytes from wk OF or re + control (trl) mice by Western blotting using antibodies (n=).

7 Figure S E8.5 P7 P8 P5

8 Figure S Relative ene Expression..8. OL trl IL-β MMP trl IL-β

9 Figure S Mouse -8-5 Human -9 - Dog Horse -9 - Promoter ctivity.5.5 trl IL-β IL-β -pl -pl m-pl Signal relative to input.9.. Positive ontrol Negative ontrol nti-nfκ 5bp bp D mrn expression..8. E trl IL-β F β ctin trl IL-β : Input : Positive control : Negative control : nti-nfκ

10 Figure S Relative ene Expression..8. trl sirn β-ctin trl sirn Relative ene Expression..8. trl ola sirn ola...8 trl sirn.8.. trl Runx sirn Mmp.5.5 trl sirn D trl sirn MP E trl sirn F trl sirn psmad psmad/5 Smad Smad/5 β-ctin β-ctin

11 Figure S5 m/m gcreer;m/m trl β-ctin

12 Figure S Relative ene Expression..8. Dnmt trl..8. Dnmta trl.5.5 et trl..8. et trl.5.5 et trl E activity (normalized to trl).5.5 trl Relative ene Expression..8. ola trl 8 ola trl Runx trl.5.5 Mmp trl

13 Figure S7 trl % UNEL Positive ells 5 5 trl ROS (relative to trl) trl

14 Fig. S8... log fold change.... trl trl trl e+ e+ e+ e+ mean expression 8 E Enrichment of differentially expressed genes % cell cycle process 8% mitotic cell cycle skeletal system development ossification bone development % regulation of osteoblast 9% differentiation synapse organization regulation of ossification Intron Sox bits Nfatc p=e- bits RNN p=e-5 bits Inflammatory response Pax:Fkhr p=e- lp Foxk bits SMD Sox p=e-7 9 MMP Promoter hondrogenesis related p=e- 5 RUNX 5 bits Foxc SMD SMURF bits SMD7 p=e-5 SMURF Intergenic FoxO-related mor pathway, energy metabolism Smad/-Smad Exon F Smad/ D 8 8 -log P-value osteoblast differentiation

15 Figure S9 Relative ene Expression.5.5 ola trl ola 8 trl Runx trl Mmp trl

16 Figure S Succinate onc (ng/ml) Veh Succinate Relative ene Expression..8. ola ola Runx Mmp Veh Succinate Veh Succinate Veh Succinate Veh Succinate

17 Figure S Proliferation (% of trl)..8. poptosis (% of trl)..8. Veh &R Veh &R Relative ene Expression..8. ola ola Runx Mmp Veh &R Veh &R Veh &R Veh &R

18 Figure S loxp loxp Rosa SOP rt + ola-re Rosa rt rt teto + DOX teto b c d b c d Primers b, b Primers c, c Primers d, d Kb 5bp Kb 5bp Kb 5bp

19 Figure S olre;rosa-rt f/+ ;HFP trl OF β-ctin