Supplementary Figure S1: Defective heterochromatin repair in HGPS progeroid cells

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Supplementary Figure S1: Defective heterochromatin repair in HGPS progeroid cells Immunofluorescence staining of H3K9me3 and 53BP1 in PH and HGADFN003 (HG003) cells at 24 h after γ-irradiation. Scale bar, 10µm. 1

Supplementary Figure S2: Knocking down or deleting Suv39h1 rescues defective DNA repair in Zmpste24 / cells. (a) H3K9me3 level in wild-type and Zmpste24 / MEFs treated with Suv39h1/2 or scramble sirna determined by Western blotting. (b) 53BP1 foci staining in wild-type and Zmpste24 null MEFs treated with Suv39h1/2 or scramble sirna 24 h after 5 Gy of γ-irradiation. Scale bar, 50 µm. (c) Quantification of 53BP1 foci 24 h after 5 Gy of γ-irradiation in at least 200 cells in wild-type and Zmpste24 / MEFs, with/without Suv39h1/2 sirna. Data represent mean ± s.e.m., n > 200. *P < 0.05, 2-tailed t-test. (d) Representative immunoblot showing downregulated H3K9me3 in Zmpste24 / /Suv39h1 / MEFs. (e) Representative immunofluorescence staining showing 53BP1 foci 24 h after γ-irradiation in Zmpste24 / /Suv39h1 /, Zmpste24 / and wild-type cells. 53BP1 foci per cell were quantified in at least 200 cells. Data represent mean ± s.e.m., n > 200. P < 0.05, 2-tailed t-test, Zmpste24 / /Suv39h1 / Vs Zmpste24 / MEFs. Scale bar, 50 µm. 2

Supplementary Figure S3: SUV39H1 mrna level in progeroid cells (a) Real-time PCR analyses of Suv39h1 mrna level in three lines of Zmpste24 / MEFs compared with wild-type controls. Data represent mean ± s.e.m., n = 3. P > 0.05, 2-tailed t-test. (b) Real-time PCR analyses of SUV39H1 mrna level in HEK293 cells transfected with lamin A, prelamin A or progerin. Data represent mean ± s.e.m., n = 3. P > 0.05, 2-tailed t-test. 3

Supplementary Figure S4: Histone modifications in progeroid cells Representative photos of immunofluorescence staining of H4K20me3 (a), H3K4me3 (b) and H3K9ac (c) in PH and HGADFN003 (HG003) cells at passage 16. Scale bar, 40 µm. 4

Supplementary Figure S5: Levels of histone modifications and Suv39h1 in Lmna null cells (a) Representative photos of immunofluorescence staining of H3K9me3 in Lmna / cells and wild-type controls from littermate. Scale bar, 20 µm. (b-e) Representative photos of immunofluorescence staining of H4K20me3 (b), K3K9me2 (c), H3K4me3 (d) and acetyl H3 (e) in Lmna null and wild-type cells. Scale bar, 5 µm. (f) Real-time PCR analyses of Suv39h1 mrna level in three lines of Lmna null MEFs and wild-type controls. Data represent mean ± s.e.m., n = 3. **P < 0.01, 2-tailed t-test. (g) Representative immunoblots showing upregulated H3K9me3 in HEK293 cells with ectopic lamin A. (h) Representative Western blotting of Suv39h1 in Lmna null and wild-type MEFs with CHX and MG132 treatment (30 µm, 6 h). 5

Supplementary Figure S6: Degradation rate and half-life of GFP-SUV39H1 in HEK293 cells with ectopic A-type lamins. The protein level of GFP-SUV39H1 in HEK293 cells with indicated ectopic A-type lamins was determined at various time points after CHX treatment. Half-life of GFP-SUV39H1 in different HEK293 cell lines is indicated by arrow. Data represent mean ± s.e.m., n = 3. 6

Supplementary Figure S7: Knocking down Suv39h1/2 rescues early senescence in Zmpste24 / MEFs. (a) Senescence-associated β-galactosidase assay in wild-type and Zmpste24 / MEFs treated with Suv39h1/2 or scramble sirna. Scale bar, 200 µm. Levels of H3K9me3 were shown in Fig. S3a. (b) Quantification of senescent cells of at least 200 cells in wild-type or Zmpste24 / MEFs, with Scramble or Suv39h1/2 sirna. Data represent mean ± s.e.m., n > 200.. *P < 0.05, 2-tailed t-test. 7

Supplementary Figure S8: SUV39H1 interacts with lamin B1. GFP-SUV39H1 was ectopically expressed in HEK293 cells. Representative immunoblots showing lamin B1 in the anti GFP immunoprecipitates. Data are representative of three independent experiments. 8

9

Supplementary Figure S9: Schematic model of lamin A in the regulation of H3K9me3 marked heterochromatin. (a) Euchromatin. (b) Lamin A interacts with Suv39h1 thus protecting it from proteasomal degradation. Suv39h1 alone or in cooperation with other co-factors initiates H3K9me3 mark on individual nucleosome. The direct interaction between trimethylated H3K9 and lamin A (ref 34) facilitates recruiting chromatin to the nuclear compartment wherein the lamin A-Suv39h1 complex resides and Suv39h1 trimethylates H3K9 on adjacent nucleosomes thus expanding heterochromatic marks. Increased binding of progerin/prelamin A to Suv39h1 and H3K9me3 enhances the level of Suv39h1 and the recruitment of adjacent nucleosomes, thus increasing H3K9me3-marked heterochromatin in progeria. (c) Elevated heterochromatin marked with H3K9me3 imposes a barrier in DNA damage-induced nucleosomal remodeling, which delays the recruitment of necessary repair proteins and compromises DNA repair, leading to accumulation of unrepaired/irreparable DNA damages in progeria cells. 10

Supplementary Figure S10 Supplementary Figure S10. Full length images of immunoblots. 11

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