Volume 24, Issue 2, Pages 217-229 (February 2016) Suppression of EZH2 Prevents the Shift of Osteoporotic MSC Fate to Adipocyte and Enhances Bone Formation During Osteoporosis  Huan Jing, Li Liao, Yulin An, Xiaoxia Su, Shiyu Liu, Yi Shuai, Xinjing Zhang, Yan Jin  Molecular Therapy  Volume 24, Issue 2, Pages 217-229 (February 2016) DOI: 10.1038/mt.2015.152 Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Expression of EZH2 and H3K27me3 is increased in OVX BMSCs. (a) Representative μCT images and quantitative analysis of trabecular and cortical bone microarchitecture in distal femur from sham and OVX mice. The trabecular bone was assessed by trabecular bone volume and trabecular bone volume/total volume (Tb.BV/TV), whereas the cortical bone was evaluated by cortical volume and %Cortical area (%Ct. Ar). n = 4. (b) H&E staining of the tibia of sham and OVX mice. The area of trabecular bone and adipocytes was analyzed by Image-Pro Plus software. White arrows indicated the adipocytes and black arrows indicated the trabecular bones in bone marrow, respectively. Scale bar: 20 μm. n = 4. (c) Mineralized nodules formed by sham and OVX BMSCs after 14 days of osteogenic-induction were detected with Alizarin Red staining and quantified. Scale bar: 50 μm. n = 4. (d) Lipid droplets formed by sham and OVX BMSCs after 7 days of adipogenic-induction were detected by Oil Red O staining and quantified. Scale bar: 50 μm. n = 4. (e,g) Western blot analysis of EZH2 and H3K27me3 protein accumulation in BMSCs at 0, 3, and 7 days of osteogenic (e) and adipogenic (g) differentiation. β-actin was used as loading control. Representative images of three repeated experiments were shown. (f,h) Real-time RT-PCR analysis of EZH2 mRNA levels in BMSCs at 0, 3, and 7 days of osteogenic (f) and adipogenic (h) differentiation. The values were normalized to β-actin mRNA expression. n = 3. (i,k) Western blot analysis of EZH2 (i) and H3K27me3 (k) protein accumulation in sham and OVX BMSCs. β-actin was used as loading control. Representative images of three repeated experiments were shown. We used 12% percentage of gels to run the protein of EZH2 and H3K27me3. Antibodies of EZH2 (1:1,000) and H3K27me3 (1:4,000) were used. (j) Real-time RT-PCR analysis of EZH2 mRNA levels in sham and OVX BMSCs. The values were normalized to β-actin mRNA expression. n = 3. Data were expressed as means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. Molecular Therapy 2016 24, 217-229DOI: (10.1038/mt.2015.152) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 EZH2 and H3K27me3 enrich in promoter regions of Wnt genes during osteogenesis. (a) Western blot analysis of active β-catenin protein expression in sham and OVX BMSCs. GAPDH was used as loading control. Representative images of three repeated experiments were shown. (b) Real-time RT-PCR analysis of Wnt1, Wnt6, and Wnt10a mRNA expression in sham and OVX BMSCs. The values were normalized to β-actin mRNA levels. n = 3. (c) Binding of EZH2 and mouse IgG to Wnt1, Wnt6, and Wnt10a promoters in BMSCs was analyzed by chromatin immunoprecipitation (ChIP) assays. The promoters of Wnt1, Wnt6, and Wnt10a were ChIP-ed with anti-EZH2 (black bar) or mouse IgG (white bar) control. n = 2. (d) Enrichment of H3K27me3 in promoters of Wnt1, Wnt6, and Wnt10a in BMSCs treated with osteogenic-inducing medium for 0 hours (white bar) and 72 hours (black bar) was evaluated by ChIP assays. The promoters of Wnt1, Wnt6, and Wnt10a were ChIP-ed with anti-H3K27me3 or mouse IgG control. n = 2. Data were expressed as means ± SD. *P < 0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Molecular Therapy 2016 24, 217-229DOI: (10.1038/mt.2015.152) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Knockdown of EZH2 decreases the enrichment of H3K27me3 in Wnt promoters to reactivate Wnt/β-catenin signaling. (a) Western blot analysis of active β-catenin protein expression in BMSCs transfected with Scrsh or EZH2 shRNA after 3 days of osteogenic and adipogenic induction. GAPDH was used as loading control. Representative images of three repeated experiments were shown. (b) Real-time RT-PCR analysis of Wnt1, Wnt6, and Wnt10a mRNA levels in BMSCs transfected with Scrsh or EZH2 shRNA after 3 days of osteogenic and adipogenic induction. The values were normalized to β-actin mRNA levels. n = 3. (c) Binding of EZH2 and mouse IgG to Wnt1, Wnt6, and Wnt10a promoters in BMSCs (P1) transfected with Scrsh or EZH2 shRNA was analyzed by chromatin immunoprecipitation (ChIP) assays. The promoters of Wnt1, Wnt6, and Wnt10a were ChIP-ed with anti-EZH2 or mouse IgG control. n = 2. (d) Enrichment of H3K27me3 in promoters of Wnt1, Wnt6, and Wnt10a in BMSCs (P1) transfected with Scrsh or EZH2 shRNA was analyzed by ChIP assays. The promoters of Wnt1, Wnt6, and Wnt10a were ChIP-ed with anti-H3K27me3 or IgG control. n = 2. Data were expressed as means ± SD, *P < 0.05, **P < 0.01. Molecular Therapy 2016 24, 217-229DOI: (10.1038/mt.2015.152) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Knockdown of EZH2 decreases H3K27me3 levels and restores osteogenic differentiation defect of OVX BMSCs. (a) Alizarin Red staining of OVX BMSCs transfected with Scrsh or EZH2 shRNA after 14 days of osteogenic-induction. Scale bar: 50 μm. n = 3. (b) Real-time RT-PCR analysis of Runx2 and Osterix mRNA levels in OVX BMSCs transfected with Scrsh or EZH2 shRNA after 3 days of osteogenic induction. The values were normalized to β-actin mRNA levels. n = 3. (c) Western blot analysis of H3K27me3 protein expression in OVX BMSCs transfected with Scrsh or EZH2 shRNA after 3 days of osteogenic induction. β-actin was used as loading control. Representative images of three repeated experiments were shown. (d) Oil Red O staining in OVX BMSCs transfected with Scrsh or EZH2 shRNA after 7 days of adipogenic-induction. Scale bar: 50 μm. n = 3. (e) Real-time RT-PCR analysis of peroxisome proliferator-activated receptor-γ (PPARγ) and aP2 mRNA levels in OVX BMSCs transfected with Scrsh or EZH2 shRNA after 3 days of adipogenic induction. The values were normalized to β-actin mRNA levels. n = 3. (f) Western blot analysis of H3K27me3 protein expression in OVX BMSCs transfected with Scrsh or EZH2 shRNA after 3 days of adipogenic induction. β-actin was used as loading control. Representative images of 3 repeated experiments were shown. Data were expressed as means ± SD, *P < 0.05, ***P < 0.001. Molecular Therapy 2016 24, 217-229DOI: (10.1038/mt.2015.152) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 DZNep rescues osteogenic differentiation defect of OVX BMSCs. (a,d) Western blot analysis of H3K27me3 (a) and EZH2 (d) protein levels in OVX BMSCs treated with DMSO or DZNep for 3 days upon osteogenic induction or adipogenic induction. β-actin was used as loading control. Representative images of two repeated experiments were shown. (b) Western blot analysis of EZH2 protein levels in OVX BMSCs treated with DMSO or DZNep. GAPDH was used as loading control. Representative images of three repeated experiments were shown. (c) Real-time RT-PCR analysis of EZH2 mRNA levels in OVX BMSCs treated with DMSO or DZNep. The values were normalized to β-actin mRNA levels. n = 3. (e–g) Real-time RT-PCR analysis of EZH2 (e), Wnt1, Wnt6, and Wnt10a (f,g) mRNA levels in OVX BMSCs treated with DMSO or DZNep after 3 days of osteogenic (f) or adipogenic (g) induction. The values were normalized to β-actin mRNA levels. n = 3. (h,i) Western blot analysis of active β-catenin protein levels in OVX BMSCs treated with DMSO or DZNep after 3 days of osteogenic (h) or adipogenic (i) induction. β-actin was used as loading control. Representative images of three repeated experiments were shown. (j) Alizarin Red staining of OVX BMSCs treated with DMSO or DZNep after 14 days of osteogenic-induction. Scale bar: 50 μm. n = 3. (k) Real-time RT-PCR analysis of Runx2 and ALP mRNA expression in OVX BMSCs treated with DMSO or DZNep after 3 days of osteogenic induction. The values were normalized to β-actin mRNA levels. n = 3. (l) Oil Red O staining of OVX BMSCs treated with DMSO or DZNep after 7 days of adipogenic-induction. Scale bar: 50 μm. n = 3. (m) Real-time RT-PCR analysis of peroxisome proliferator-activated receptor-γ (PPARγ) and aP2 mRNA levels in OVX BMSCs treated with DMSO or DZNep after 3 days of adipogenic induction. The values were normalized to β-actin mRNA levels. n = 3. Data were expressed as means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. Molecular Therapy 2016 24, 217-229DOI: (10.1038/mt.2015.152) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 DZNep administration prevents bone loss and excessive bone marrow fat formation in OVX mice. (a) Representative μCT images and quantitative analysis of trabecular and cortical bone microarchitecture in distal femur of sham and OVX mice treated with DMSO or DZNep. Trabecular bone was evaluated with trabecular bone volume/total volume (Tb.BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th), trabecular separation (Tb. Sp), and Trabecular bone pattern factor, whereas cortical bone was evaluated with cortical volume, cortical thickness (Ct. Th), and %Cortical Area (%Ct. Ar). n = 4. (b) H&E analysis of the tibia of sham and OVX mice treated with DMSO or DZNep. The area of trabecular bone and adipocytes was quantified with Image-Pro Plus software. White arrows indicated adipocytes and black arrows indicated trabecular bone in the bone marrow. Scale bar: 20 μm. n = 4. (c) Immunohistochemical staining of OCN in the tibia of sham and OVX mice treated with DMSO or DZNep. The number of OCN+ osteoblasts was quantified with Image-Pro Plus software. Black arrows indicated the OCN+ osteoblasts. Scale bar: 10 μm. n = 4. (d) Enzyme-linked immunosorbent assay analysis of OCN concentration in serum of sham and OVX mice received either DMSO or DZNep. n = 4. Data were expressed as means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. Molecular Therapy 2016 24, 217-229DOI: (10.1038/mt.2015.152) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 DZNep recovers the osteogenic differentiation defect of OVX BMSCs in vivo. (a) Alizarin Red staining in BMSCs isolated from OVX mice treated with DMSO or DZNep after 14 days of osteogenic-induction. Scale bar: 50 μm. n = 4. (b) Real-time RT-PCR analysis of Runx2, OCN, and ALP mRNA levels in BMSCs isolated from OVX mice received either DMSO or DZNep. The values were normalized to β-actin mRNA levels. n = 4. (c) Real-time RT-PCR analysis of Wnt1, Wnt6, and Wnt10a mRNA expression in BMSCs isolated from OVX mice received either DMSO or DZNep. The values were normalized to β-actin mRNA levels. n = 4. (d,e) Western blot analysis of active β-catenin (d), H3K27me3 and EZH2 (e) protein levels in BMSCs isolated from OVX mice treated with DMSO or DZNep. β-actin was used as loading control. Representative images of four repeated experiments were shown. Data were expressed as means ± SD, *P < 0.05, **P < 0.01. Molecular Therapy 2016 24, 217-229DOI: (10.1038/mt.2015.152) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Proposed mechanism by which DZNep epigenetically reactivates Wnt/β-catenin signaling to promote osteogenic differentiation of osteoporosis BMSCs. During osteoporosis, EZH2 is activated to enhance H3K27me3 signature on promoters of Wnt genes, resulting in suppression of Wnt signaling. Inhibition of Wnt signaling shifts the lineage commitment of BMSCs to adipocytes. DZNep, an inhibitor of histone methylation, effectively suppresses EZH2 function to decrease H3K27me3 levels and reactivate canonical Wnt pathway. DZNep treatment significantly recovers osteogenic differentiation defect of OVX BMSCs, which promotes BMSC-mediated bone formation. Since epigenetic regulation can be maintained persistently, our findings provide a potential BMSC-based strategy for osteoporosis treatment and bone regeneration. Molecular Therapy 2016 24, 217-229DOI: (10.1038/mt.2015.152) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions