Fig. 1. Phosphorylation of histone H3 and HMG-14 is blocked by inhibitors of MAP kinase pathways. Wild-type mouse embryonic fibroblasts were serum starved overnight and then treated with 500 ng/ml TSA for 4 h where indicated and/or with 10 µM SB 203580 or 5 µM PD 184352 for 1 h where indicated. Cells were then stimulated with (A) 10 µg/ml anisomycin for 20 min or (B) 400 ng/ml TPA for 20 min. Phosphorylation of histone H3 on Ser10 and Ser28 and of HMG-14 on Ser6 were then examined by immunoblotting. Levels of total ERK1/2 were analysed by immuoblotting from the same lysates.

Fig. 3. (A) Phosphorylation of histone H3 and HMG-14 is absent in MSK1/2 double-knockout cells. Wild-type and MSK1/2 knockout cells and C3H 10T1/2 cells were starved overnight and then labelled with [³²P]phosphate (50 µCi/ml) for 3 h. Cells were then left untreated or stimulated with anisomycin (50 ng/ml) for 30 min. Crude nuclei were prepared and extracted with 1 M NaCl. Salt soluble proteins were run on 15% acid/urea polyacrylamide gels, Coomassie blue stained, dried down and exposed to X-ray film. The Coomassie stained gel is shown on the right and the corresponding autoradiograph on the left. (B) To analyse S6 phosphorylation, the salt-insoluble pellet was resuspended in 8 M urea and samples run on gels similar to (A). The Coomassie stained gel is shown on the right and the corresponding autoradiograph on the left.

Fig. 5. Rescue of histone H3 phosphorylation by transfection of MSK2. MSK1/2 double-knockout cells were transfected with a construct encoding GFP-tagged MSK2. The cells were serum starved and then left unstimulated (A and C) or treated with 10 µg/ml anisomycin for 30 min (B and D). Cells were then fixed, and the expression of MSK2 and phosphorylation of histone H3 on Ser10 (A and B) or Ser28 (C and D) was determined by immunofluorescence. Histone H3 phosphorylation is shown in red, GFP–MSK2 in green and DNA (DAPI) in blue.

Fig. 7. Histone H3 phosphorylation in Coffin–Lowry cells. Wild-type or Coffin–Lowry immortalized human fibroblasts were serum starved overnight and then stimulated with anisomycin (10 µg/ml, 30 min) or TPA (400 ng/ml, 30 min). (A) Cells were lysed, and histone H3 was extracted from the pellet and analysed for the phosphorylation on Ser10 by immunoblotting. The soluble fraction of the cell lysate was analysed by immunoblotting for (B) phosphorylation of CREB and levels of (C) RSK2 and (D) total ERK1/2 (loading control).

Fig. 9. Analysis of c-jun gene modification by ChIP. (A) Schematic representation of the c-jun gene indicating the regions amplified by the primer pairs used in the PCR step of the ChIP assay. (B) Wild-type and MSK1/MSK2 knockout fibroblasts at passage 5 were serum starved overnight and then left unstimulated or stimulated with 25 ng/ml anisomycin for 30 min. Cross-linked chromatin was immunoprecipitated using antibodies that recognize histone H3 that is both phosphorylated on Ser10 and acetylated on Lys9 (pAc H3) or acetylated on Lys9 and Lys14 (Ac H3). The cross-links on the precipitated DNA were then reversed and the DNA analysed for the presence of the c-jun gene sequences by radioactive PCR. Graphical representation of the c-jun gene fragments recovered with the (C) anti-phosphoacetyl histone H3 or (D) anti-acetyl histone H3 antibodies. The data are shown as an average of three (c-jun1) or two (c-jun3) independent experiments, with the PCR step being carried out at least twice for each experiment. (E) Wild-type and MSK1/2 double-knockout cells were serum starved overnight and then left untreated or stimulated with anisomycin (25 ng/ml) for 30 min. Immunoblotting with the anti-phosphoacetyl histone H3 antibody was carried out.

Fig. 2. Mitotic phosphorylation of wild-type and MSK1/MSK2 knockout fibroblasts. Wild-type and MSK1/MSK2 knockout cells growing in serum were fixed and phosphorylation of histone H3 on Ser10 examined by immunofluorescence. DAPI staining of the nucleus is shown in blue, phospho-Ser10 histone H3 staining is shown in red. Phospho histone H3 staining of a cell in mitosis is indicated by an arrow, and histone H3 staining in late G₂ is indicated by an asterisk.

Fig. 4. Phosphorylation of histone H3 and HMG-14 in MSK1/MSK2 knockout cells by anisomycin. (A) Wild-type and MSK1/MSK2 knockout fibroblasts were serum starved overnight and then stimulated with anisomycin (10 µg/ml) for the times indicated. Cells were lysed and analysed for anisomycin-induced phosphorylation of histone H3 on Ser10 and Ser28 and of HMG-14 on Ser6 by immunoblotting. (B) MSK1 and MSK2 were assayed against Crosstide after immunoprecipitation from 0.5 mg of soluble protein lysate from wild-type cells. One unit of activity was defined as the amount of enzyme required to incorporate 1 nmol of phosphate into the substrate in 1 min. Six measurements were taken per time point, and error bars represent the SEM of these values. (C) Wild-type, MSK1, MSK2 and MSK1/MSK2 knockout fibroblasts were serum starved overnight and then stimulated with 10 µg/ml anisomycin for 30 min. Cells were fixed and phosphorylation of histone H3 on Ser10 examined by immunofluorescence. DAPI staining of the nucleus is shown in blue, and phospho-Ser10 histone H3 staining is shown in red. (D) Wild-type, MSK1, MSK2 and MSK1/MSK2 knockout fibroblasts were serum starved overnight, followed by treatment with 500 ng/ml TSA for 4 h where indicated. Cells were then stimulated with anisomycin (10 µg/ml) for 30 min. Cells were lysed and analysed for anisomycin-induced phosphorylation of histone H3 on Ser10 and Ser28 and of HMG-14 on Ser6 by immunoblotting.

Fig. 6. Phosphorylation of histone H3 and HMG-14 in MSK1/MSK2 knockout cells by TPA. (A) Wild-type and MSK1/MSK2 knockout fibroblasts were serum starved overnight and then stimulated with TPA (400 ng/ml) for the times indicated. Cells were lysed and analysed for TPA-induced phosphorylation of histone H3 on Ser10 and Ser28 and of HMG-14 on Ser6 by immunoblotting. (B) MSK1 and MSK2 were assayed against Crosstide after immunoprecipitation from 0.5 mg of soluble protein lysate from wild-type cells. One unit of activity was defined as the amount of enzyme required to incorporate 1 nmol of phosphate into the substrate in 1 min. Six measurements were taken per time point, and error bars represent the SEM of these values. (C) Wild-type, MSK1, MSK2 and MSK1/MSK2 knockout fibroblasts were serum starved overnight and then stimulated with 400 ng/ml TPA for 30 min. Cells were fixed and phosphorylation of histone H3 on Ser10 examined by immunofluorescence. DAPI staining of the nucleus is shown in blue, and phospho-Ser10 histone H3 staining is shown in red. (D) Wild-type, MSK1, MSK2 and MSK1/MSK2 knockout fibroblasts were serum starved overnight, followed by treatment with 500 ng/ml TSA for 4 h where indicated. Cells were then stimulated with TPA (400 ng/ml) for 30 min. Cells were lysed and analysed for anisomycin-induced phosphorylation of histone H3 on Ser10 and Ser28 and of HMG-14 on Ser6 by immunoblotting.

Fig. 8. Analysis of global levels of histone H3 acetylation and methyl ation. Wild-type and MSK1/MSK2 knockout fibroblasts were serum starved overnight and then treated with 500 ng/ml TSA for 4 h where indicated. Cells were then stimulated with anisomycin (10 µg/ml, 60 min) or TPA (400 ng/ml, 30 min). Acetylation and methylation levels were determined using antibodies specific for acetylation on (A) Lys9, Lys14, Lys18 or Lys23 or (B) dimethylation on Lys4 or Lys9. (C) Immunoblotting with an antibody against total histone H3 was used to confirm equal loading of histone H3.

Fig. 10. Induction of c-fos and c-jun mRNA in response to anisomycin and TPA. Wild-type (open circles) and MSK1/MSK2 knockout (closed circles) fibroblasts were serum starved overnight and then stimulated with (A) 25 ng/ml or (B) 10 µg/ml anisomycin or with (C) 400 ng/ml TPA for the times indicated. Total RNA was then isolated from the cells and c-fos and c-jun mRNA levels measured by RNase protection assays. Three measurements were taken per time point, error bars represent the SEM of these values and the results shown are representative of three experiments.