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. 2011 Jul 26;108(30):12325-30.
doi: 10.1073/pnas.1102789108. Epub 2011 Jul 11.

Histone H4 Lysine 16 Hypoacetylation Is Associated With Defective DNA Repair and Premature Senescence in Zmpste24-deficient Mice

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Free PMC article

Histone H4 Lysine 16 Hypoacetylation Is Associated With Defective DNA Repair and Premature Senescence in Zmpste24-deficient Mice

Vaidehi Krishnan et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Specific point mutations in lamin A gene have been shown to accelerate aging in humans and mice. Particularly, a de novo mutation at G608G position impairs lamin A processing to produce the mutant protein progerin, which causes the Hutchinson Gilford progeria syndrome. The premature aging phenotype of Hutchinson Gilford progeria syndrome is largely recapitulated in mice deficient for the lamin A-processing enzyme, Zmpste24. We have previously reported that Zmpste24 deficiency results in genomic instability and early cellular senescence due to the delayed recruitment of repair proteins to sites of DNA damage. Here, we further investigate the molecular mechanism underlying delayed DNA damage response and identify a histone acetylation defect in Zmpste24(-/-) mice. Specifically, histone H4 was hypoacetylated at a lysine 16 residue (H4K16), and this defect was attributed to the reduced association of a histone acetyltransferase, Mof, to the nuclear matrix. Given the reversible nature of epigenetic changes, rescue experiments performed either by Mof overexpression or by histone deacetylase inhibition promoted repair protein recruitment to DNA damage sites and substantially ameliorated aging-associated phenotypes, both in vitro and in vivo. The life span of Zmpste24(-/-) mice was also extended with the supplementation of a histone deacetylase inhibitor, sodium butyrate, to drinking water. Consistent with recent data showing age-dependent buildup of unprocessable lamin A in physiological aging, aged wild-type mice also showed hypoacetylation of H4K16. The above results shed light on how chromatin modifications regulate the DNA damage response and suggest that the reversal of epigenetic marks could make an attractive therapeutic target against laminopathy-based progeroid pathologies.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Reduced H4K16 acetylation in Zmpste24−/− cells. (A) Zmpste24+/+ and Zmpste24−/− MEFs at early passage (P3) (Upper panels) or late passage (P6) (Lower panels) were cultured for 2 d. SA β-gal activity was determined. (B and C) Histones were extracted from Zmpste24+/+ and Zmpste24−/− MEFs (P3) and analyzed by Western blotting using the antibodies indicated. (D) Graph shows normalized densitometric values of acetyl H4 and acetyl H4K16 against total H4 for three independently derived lines of MEFs in separate experiments. *P < 0.05 and **P < 0.01 (Student’s t test) when Zmpste24+/+ and Zmpste24−/− MEFs were compared. (E) Immunofluorescence staining of Zmpste24+/+ and Zmpste24−/− MEFs with lamin A/C (green) and anti-acetyl H4K16 (red) antibodies. (F) Histones were extracted from the liver of 1-mo-old mice and analyzed by Western blotting using the antibodies indicated.
Fig. 2.
Fig. 2.
Reduced nuclear matrix association of Mof in Zmpste24−/− MEFs. (A and B) Colocalization of Mof and lamin A in nuclear matrix. (A) Nuclear matrices were prepared from wild-type MEFs and stained with anti-Mof and anti-lamin A/C antibodies. Staining with DAPI indicated efficient digestion of DNA in the isolated nuclear matrix. Control cells (labeled “TOTAL”) were processed directly without pre-extraction and stained with anti-lamin A/C antibody and DAPI. (B) Histogram:immunofluorescence signals collected from 488 channels (lamin A/C) and 594 channels (Mof) were overlaid to depict colocalization. (C) Zmpste24+/+ and Zmpste24−/− MEFs were subjected to subcellular fractionation to yield nucleoplasmic (S2), nuclear matrix + chromatin (P2), soluble chromatin + nucleoplasmic (S2′), nuclear matrix (P2′), and cytosolic (S1) fractions. Equal amounts of protein (20 μg) were resolved and Western-blotted with indicated antibodies. Coomassie brilliant blue (CBB) staining of the gel was performed to ensure equal loading of the fractions. (D) HEK293 cells were transiently transfected with Mof-myc together with FLAG-tagged lamin A or prelamin A for 48 h. As controls, cells were transfected with empty vector, FLAG-tagged lamin A, prelamin A or Mof-myc alone. Immunoprecipitation was performed with FLAG antibody-conjugated agarose beads, and immunoblots were probed with indicated antibodies. Three independent experiments were performed and identical results were obtained.
Fig. 3.
Fig. 3.
Mof overexpression restores 53BP1 recruitment in Zmpste24−/− MEFs. (A) Total cell extracts from Zmpste24−/− MEFs transfected with EGFP or EGFP-Mof were Western-blotted with indicated antibodies. (B and C) EGFP or EGFP-Mof plasmids were transiently transfected into Zmpste24−/− MEFs for 48 h, followed by exposure to 5 Gy of γ-irradiation. Cells were fixed and processed for immunofluorescence at the indicated time points. Immunofluorescence images are represented in B. The number of 53BP1 foci per cell was counted and plotted in C. The controls and irradiated samples were compared using ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; “ns” indicates that the difference has not reached statistical significance.
Fig. 4.
Fig. 4.
Reduced H4K16 acetylation levels and early onset of cellular senescence. (A) Zmpste24+/+ and Zmpste24−/− MEFs were either subjected to Mof siRNA knockdown for 48 h or treated with NaB (2 mM) for 24 h. Total cell extracts were collected, and the effects of Mof siRNA and HDAC inhibition on histone acetylation were analyzed by Western blotting. (B and C) For the experiment described in A, senescent phenotype was studied using SA β-gal assay, and the percentage of senescent cells was quantified. **P < 0.01 and ***P < 0.001 (Student’s t test). (D) Treatment with NaB or TSA rescued early senescence in Zmpste24−/− MEFs, as determined by SA β-gal assay. (E) Quantitative data showing the percentage of senescent cells for the experiment described in D. *P < 0.05 (Student’s t test).
Fig. 5.
Fig. 5.
HDAC inhibition rescues both delayed 53BP1 recruitment and defective DNA repair after γ-irradiation induced DNA damage. (A) Zmpste24+/+ and Zmpste24−/− MEFs were either mock-treated or treated with NaB (0.5 mM) for 2 h and exposed to 5 Gy of γ-irradiation, and immunofluorescence staining was performed using anti-53BP1 after 30 min. The number of 53BP1 foci per cell was quantified and shown in B. (C) Treatment with NaB (2 mM) or TSA (0.2 μM) improved DNA repair in Zmpste24−/− MEFs. Twenty-four hours postirradiation (5 Gy), Zmpste24+/+ and Zmpste24−/− MEFs were fixed and the number of residual γ-H2AX foci per cell was counted. (D) Quantitative data showing the decrease of nonrepairable DNA damage in Zmpste24−/− MEFs treated with NaB (2 mM) or TSA (0.2 μM) for the experiment described in C. NaB- and TSA-treated samples were compared with untreated controls. *P < 0.05 (Student’s t test).
Fig. 6.
Fig. 6.
HDAC inhibition mitigates progeroid phenotypes and extends life span of Zmpste24−/− mice. (A) Photograph of Zmpste24+/+, Zmpste24−/−, and NaB-fed Zmpste24−/− at 5 mo of age. (B) Survival and life span of the NaB-fed Zmpste24−/− mice (n = 20) were compared against the untreated controls (n = 22) using the Kaplan–Meir analysis and found to be statistically significant using the log-rank test (**P < 0.01). Fifty-percent survival was observed at 22 and 26 wk for Zmpste24−/− and NaB-fed Zmpste24−/− mice, respectively. (C) Five-month-old Zmpste24+/+, Zmpste24−/−, and NaB-fed Zmpste24−/− mice (n = 5) were scanned using X-ray for bone density analysis. (D) Bone densities were quantified from X-ray scans using ImageJ software (National Institutes of Health). Fold increase in the bone density of NaB-fed mice relative to the untreated Zmpste24−/− control is shown. *P < 0.05 (Student’s t test). (E) Total cell extracts from bone marrow cells (5-mo-old mice) were resolved and Western-blotted with antibodies indicated. (F) Kidney was excised from 5-mo-old Zmpste24+/+, Zmpste24−/−, and NaB-fed Zmpste24−/− mice, cryosectioned, and stained using a SA β-gal assay kit. Blue cells indicate senescence.

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