Cell cycle distribution of histone modifications in early and late passage cells. (a) Cell cycle distribution of histone modifications in whole cell extracts from PD30 and PD75 HDFs. Shown are cell cycle patterns of modifications involved in transcriptional regulation (H3K4me1/2/3 and H3K27me1/2/3). 75,000 cell equivalents were loaded per lane and histones were visualized by ponceau staining prior to probing with primary antibodies. Hours after G1/S release are indicated and corresponding cell cycle phases have been determined by FACS analysis. Images shown represent western analysis performed, stained and exposed simultaneously to film. Quantification of western analysis can be seen in Fig. S2. Abundance of post-translational modifications was calculated relative to the total amount of that particular protein i.e. the abundance of H3K9me3 was quantified relative to total H3. (b) Shown are cell cycle patterns of modifications and events involved in heterochromatin regulation (H3K9me1/2/3, H4K20me3, H3K9Ac, H3S10ph, HP1α and HP1γ). Western analysis and quantifications were performed as in (A). (c) Cell cycle patterns of H4K5Ac, H4K16Ac, H3K56Ac, H4K20me1/2, H3K79me1/2, SIRT1 and SIRT2. Western analysis and quantifications were performed as in (A). The intensity of signals from westerns of individual modifications does not reflect the levels of each modification in the cell. However, these images reflect the levels of the same individual modification in early and late passage cells. γ-tubulin serves as loading control for SIRT1 and SIRT2.

Telomerase expression resets late passage cells to early passage cells. (a) Expression of H3, H4, SLBP, Asf1, H3K79mes and H4K20mes in synchronized cells from a young (left panel) and old individual (right panel). 75,000 cell equivalents were loaded per lane and equal loading was confirmed by ponceau staining prior to probing with primary antibodies. Hours after G1/S release are indicated and corresponding cell cycle phases have been determined by FACS analysis and indicated. γ-tubulin serves as loading control. (b) SILAC labeling of F9 and F92 HDFs and mass spectroscopy of histone proteins. Shown are percentages of newly synthesized histones (yellow) and histones from previous cell cycle (black). (c) Expression of 53BP1, Rpa32, SLBP, Asf1a/b, H3, H3K79me2, H4, H4K5ac and H4K20me2 in whole cell extracts of PD85 HDFs expressing catalytically active hTERT. 75,000 cell equivalents were loaded per lane and equal loading was confirmed by ponceau staining prior to probing with primary antibodies. Hours after G1/S release are indicated and corresponding cell cycle phases have been determined by FACS analysis and indicated. γ-tubulin serves as loading control.

Altered histone biosynthesis and redistribution of epigenetic marks upon chronic damage and cellular aging. (a) Effects of Bleomycin on histone expression. Early passage IMR90 (PD21) were subjected to 500, 50 or 5ng ml⁻¹ of bleomycin for 6 days. Expression of H3 and H4 is shown. γ-tubulin serves as loading control. (b) Expression of H3 and H4 in early passage (PD30, PD25) and late passage (PD75, PD70) asynchronously growing IMR90 and WI38 fibroblasts. γ-tubulin serves as loading control. (c) Cell cycle expression of H3, H4, SLBP, Asf1a/b and CAF1p150, CAF1p60 and PCNA in PD30 and PD75 HDFs. Hours after G1/S release are indicated and corresponding cell cycle phases have been determined by FACS analysis and indicated. γ-tubulin serves as loading control. (d) Quantifications of cell cycle expression patterns of histones and histone chaperones from PD30 (yellow lines) and PD75 (green lines) HDFs. Shown are cell cycle patterns of H3, H4, Asf1a/b, Caf1p60, CAF1p150 and PCNA. Enrichments were calculated relative to γ-tubulin. Cell cycle phases are indicated on top and hours after release from G1/S on the bottom. Corresponding cell cycle phases have been determined by FACS analysis. The error bars represent the standard deviation of three independent experiments. (e) SILAC labeling of PD30 and PD75 HDFs and mass spectroscopy of histone proteins. Shown are percentages of newly synthesized histones (yellow) and histones from previous cell cycle (black). (f) SILAC labeling of PD30 and PD75 HDFs and mass spectroscopy of histone H3K9 and H4K20 methylation events. Shown are percentages of newly synthesized histone modifications (yellow), modifications from previous cell cycles (black) and those of mixed origin i.e. both old and new methyl groups co-exist forming me2 and/or me3 (grey).

DNA damage accumulation and DDR activation upon cellular aging. Expression of PCNA, Rpa32, Rad51, ATR, Rad17, Rad17-pS645, γ-ATM pS1981, Chk2-pT68, NBS1, NBS1S343ph, 53BP1, p53, p53S15ph, p21 and Cyclins A/B1/D1 in whole cell extracts of PD30 and PD75 HDFs. 75,000 cell equivalents were loaded per lane and equal loading was confirmed by ponceau staining prior to probing with primary antibodies. Hours after G1/S release are indicated and corresponding cell cycle phases have been determined by FACS analysis and indicated. γ-tubulin serves as loading control. Quantification of expression can be found in Fig. S5.

Cellular aging results in an altered chromatin landscape of human telomeres. (a) Synchronized ChIP experiments with chromatin prepared from PD30 (left panels) and PD75 (right panels) HDFs followed by dot-blot southern hybridization with probes to telomeric TTAGGG repeats (upper panels) and sub-telomeric 17P (lower panels). Antibodies used for precipitation are indicated on the left. Hours after G1/S release are indicated and corresponding cell cycle phases have been determined by FACS analysis and indicated. Quantification of ChIP analysis can be seen in Fig. S5.
(b) DNA-IP with anti-BrdU antibodies followed by dot-blot southern hybridization with probes to telomeric TTAGGG repeats, sub-telomeric 17P and DmGbe2 (spiked IP control) in early (PD30) and late passage (PD75) HDFs. Hours after G1/S release and cell cycle phase are indicated above the blots. Quantifications of DNA-IPs from three independent experiments are shown in the lower panel and the error bars represent the standard deviation. The signal at T=0 was deduced from each signal as background. Hours after G1/S release are indicated and corresponding cell cycle phases have been determined by FACS analysis and indicated.
(c) DNA damage signals at short telomeres affect SLBP and in turn H3/H4 biosynthesis. Down regulation of H3/H4 destabilizes Asf1, leading to genome wide epigenetic changes, which amplify the damage signal during many cell cycles and population doublings. Eventually, epigenetic modifications spread into telomeres enabling association of DNA damage and repair proteins with chromosome ends. This self-perpetuating cycle continues until a critical damage threshold is exceeded, prompting exit from the cell cycle.