Figure S1.
UK Biobank cohort and mouse models of CHIP. (A) Cohort selection and exclusion criteria from 200,000 individuals from the UK Biobank who had whole-exome sequencing performed. (B) Forest plot of the β-estimates for eBMD generated from multivariate linear regression model using CHIP mutations stratified by VAF ≥10% or <10%, and adjusted for age ≥65 yr, sex, BMI <18.5 kg/m2 (underweight), BMI >30 kg/m2 (obese), prior or current smoking status, self-reported history of steroid use, and rheumatoid arthritis diagnosis. The horizontal lines represent Wald 95% CIs. (C) Analysis of cells reconstituted after BM transplantation. BM from mouse with constitutive expression of GFP under a CAG promoter (JAX 024858) was transplanted into WT recipient mice. BM was analyzed at 8 wk after transplantation for CD45+ and GFP+. Most of the GFP positivity is retained in the CD45+ hematopoietic population. On the right is a quantification of GFP percentage in the hematopoietic (CD45+) and nonhematopoietic (CD45−) populations. n = 5. (D) Influence of Dnmt3a KO on bone mass in a nontransplant setting. Female WT (Vav1-Cre) or Dnmt3a KO (Dnmt3afl/flVav1-Cre) mice were aged 33–34 wk and analyzed on µCT for the femoral mid-shaft Ct Ar. n = 3–7. (E and F) Tibias of transplanted WT or Dnmt3a R878H mice were stained with Goldener’s Trichrome stain 20 wk after transplantation. n = 6. Samples were analyzed in a blinded fashion for Ob.N/BS (E) and Ob.S/BS (F) by the Center for Skeletal Research at Massachusetts General Hospital. (G) Procollagen 1 N-terminal propeptide (P1NP) serum levels in mice transplanted with WT or Dnmt3a KO BM into 8– to 10-wk-old Ldlr KO mice fed an HFD. ELISA analysis of serum obtained at 4 mo after transplantation. n = 11–14. (H) Representative images of F-actin rings marking osteoclasts shown by phalloidin immunofluorescence staining. White bar represents 100 µm. Green represents Alex Fluor 488–phalloidin. Blue represents DAPI staining. (I) Quantification of osteoclast differentiation from whole BM of 12-wk-old Dnmt3a KO and WT female mice. n = 5. (J) Correlation between TRAP quantification for osteoclasts and F-actin ring staining with simultaneous staining for TRAP and F-actin rings. (K) Immunoblot (IB) for Dnmt3a in RAW264.7 cells with heterozygous (+/−) or homozygous (−/−) frame-shift mutations in Dnmt3a. (L and M) Quantification of number of osteoclast-like cells from osteoclast differentiation of Dnmt3a +/+, +/−, −/− RAW264.7 cells. (L and M) Osteoclast differentiation was performed using vehicle treatment (L; n = 7) or LPS (M; 0.1 ng/ml; n = 6). (N and O) Quantitative hydroxyapatite resorption assay using osteoclast differentiation of Dnmt3a +/+, +/−, and −/− RAW264.7 cells. Osteoclast differentiation was performed using vehicle treatment (N; n = 7) or LPS (O; 0.1 ng/ml; n = 7). Resorbed areas were distinguished by image thresholding using ImageJ software. Each data plot represents average resorbed areas across all nonoverlapping images taken in a well on day 7 of differentiation. Error bars represent SEM. (P) Quantification of donor CD11b+ in the Dnmt3a KO and WT BM of transplants into Ldlr−/− recipient mice fed a HFD. n = 10–14. (Q) Quantification of donor CD11b+ in the Dnmt3a KO and WT BM of transplants into WT mice fed a ND. n = 7–10. (R) Quantification of donor CD11b+ in the Dnmt3a R878H and WT BM of transplants into WT mice fed a ND. n = 10–15. (S) Quantification of CD11b−/lowCD115+Ly6Chi osteoclast precursors in the Dnmt3a KO and WT BM of transplants into WT mice fed a ND. n = 7–19. (T) Quantification of CD11b−/lowCD115+Ly6Chi osteoclast precursors in the Dnmt3a R878H and WT BM of transplants into WT mice fed a ND. n = 10–15. (U–W) RNA sequencing of unstimulated BMDMs derived from WT or Dnmt3a−/− mice. n = 4. (U) Differentially expressed genes from all genes. (V) Cytokine pathway enrichment analysis using gene set variation analysis (Hänzelmann et al., 2013). Highlighted pathways indicate FDR < 0.05. (W) Significant differentially expressed cytokines/chemokines genes. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. All error bars represent SD unless specified. Source data are available for this figure: SourceData FS1.