Fig. 4: Reduced methylation of H3K9 increases RhoA activation and drives aging of HSCs.

a, Left: representative images of 3D confocal reconstruction of young, aged and aged + Ri-treated HSCs stained with anti-H3K9me2 antibody (green) and DAPI (gray). Right: representative images of 3D confocal reconstruction of young, aged and aged+Ri with UNC0646 treatment. At least 10 cells were acquired per experiment per condition. Young, aged and aged + Ri data are from eight independent experiments; young UNC0646, aged UNC0646 and aged + Ri UNC0646 data are from four independent experiments. Graph shows H3K9me2 signal volume normalized against DAPI signal volume. Mann−Whitney two-tailed test, total young n = 87, aged n = 79, aged + Ri n = 81; young + UNC n = 36, aged + UNC n = 41, aged + Ri + UNC n = 41, showing mean ± s.e.m., analyzed by Mann−Whitney test, **P < 0.01, *P < 0.05, ****P < 0.0001. b, Experimental setup for retroviral transduction and transplantation of young LSK overexpressing wild-type H3K9 or mutant H3R9 with mCherry as a reporter. Transduced cells were transplanted into lethally irradiated mice. BM of transplanted mice was analyzed by flow cytometry to evaluate the regenerative capacity of transduced HSCs. mCherry⁺ myeloid progenitors (MPs) were sorted to measure H3K9me2 levels. mCherry⁺ HSCs were sorted for the analysis of RhoA activation and nuclear stretching. c, Representative images of sorted MPs transduced with H3K9 and H3R9 stained with anti-H3K9me2 antibody (magenta) and DAPI (blue). The graph shows H3K9me2 signal volume normalized against DAPI signal volume. H3K9 experimental data are from seven independent experiments and six independent experiments from H3R9, showing mean ± s.e.m., analyzed by Mann−Whitney test, two-tailed. Total H3K9 MP n = 43, H3R9 MP n = 39, n = 7 H3K9; n = 6 H3R9, showing mean ± s.e.m., ****P < 0.0001. d, Representative images of 3D confocal reconstruction of HSCs overexpressing H3K9 or H3R9 isolated from transplanted mice H3K9 experimental data from seven independent experiments, and six independent experiments from H3R9 HSCs were stained with anti-Rho-AGTP antibody (red) and DAPI (gray). Showing mean ± s.e.m., analyzed by Mann−Whitney test, two-tailed. Statistics are shown for Rho-AGTP quantification analysis, **P < 0.01, total K9 n = 56, R9 n = 61. For nuclear volume (DAPI volume), unpaired t-test (one-tailed) was used, *P < 0.05, total K9 n = 56, R9 n = 61. e, Gating strategy for the analysis of BM progenitors 12−20 weeks after transplantation. The analysis of the different populations is shown for mice transplanted with LSK transduced with either wild-type H3K9 (n = 7) or H3R9 (n = 6). Mann−Whitney statistics are shown two-tailed, **P < 0.01. f, Engraftment of mCherry−H3K9⁺ or mCherry−H3R9⁺ HSCs in mice. Representative flow cytometry charts for BM engraftment are shown. The box plots display engraftment of mCherry−H3K9⁺ or mCherry−H3R9⁺ HSCs in mice 12−20 weeks after transplantation in BM and peripheral blood (PB). Brown−Forsythe and Welch’s ANOVA tests are shown, *P < 0.05, **P < 0.01. g, Gating strategy for the analysis of B cells (B220⁺), T cells (Cd3⁺) and myeloid cells (Gr1⁺ and Mac1⁺) within the BM 12−24 weeks after transplantation. The analysis of the different populations is shown for mice transplanted with wild-type H3K9 (n = 7) transduced LSK and mutant H3R9 LSK (n = 6). Statistics are B cells, unpaired t-test, two-tailed; T cells, Mann−Whitney test, two-tailed; myeloid cells, Welch’s t-test, two-tailed. *P < 0.05, **P < 0.01. SSC-A, side scatter area. Panel b created with BioRender.