J Biol Chem, Vol. 274, Issue 35, 24914-24920, August 27, 1999

Increased Ser-10 Phosphorylation of Histone H3 in Mitogen-stimulated and Oncogene-transformed Mouse Fibroblasts^*

Deborah N. Chadee^§, Michael J. Hendzel^¶, Cheryl P. Tylipski^§, C. David Allis^**, David P. Bazett-Jones^¶, Jim A. Wright^§, and James R. Davie^§

From the  Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, R3E 0V9 Canada, the ^§ Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Manitoba, R3E 0W3 Canada, the ^** Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908, and the ^¶ Department of Cell Biology and Anatomy, Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1 Canada

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

When the Ras mitogen-activated protein kinase (MAPK) signaling pathway of quiescent cells is stimulated with growth factors or phorbol esters, the early response genes c-fos and c-myc are rapidly induced, and concurrently there is a rapid phosphorylation of histone H3. Using an antibody specific for phosphorylated Ser-10 of H3, we show that Ser-10 of H3 is phosphorylated, and we provide direct evidence that phosphorylated H3 is associated with c-fos and c-myc genes in stimulated cells. H3 phosphorylation may contribute to proto-oncogene induction by modulating chromatin structure and releasing blocks in elongation. Previously we reported that persistent stimulation of the Ras-MAPK signaling pathway in oncogene-transformed cells resulted in increased amounts of phosphorylated histone H1. Here we show that phosphorylated H3 is elevated in the oncogene-transformed mouse fibroblasts. Further we show that induction of ras expression results in a rapid increase in H3 phosphorylation. H3 phosphatase, identified as PP1, activities in ras-transformed and parental fibroblast cells were similar, suggesting that elevated H3 kinase activity was responsible for the increased level of phosphorylated H3 in the oncogene-transformed cells. Elevated levels of phosphorylated H1 and H3 may be responsible for the less condensed chromatin structure and aberrant gene expression observed in the oncogene-transformed cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Histone H1 and the N-terminal tail of H3 have key roles in the folding and inter-association of the chromatin fiber (1-5). Modification of the N- and C-terminal tails of H1 by phosphorylation or the N-terminal tail of H3 by acetylation and/or phosphorylation could destabilize higher order chromatin structure (6, 7). Myc- and ras-transformed and Rb-deficient fibroblasts have a more decondensed chromatin structure than parental cells (8-10). A general feature of these oncogene-transformed and Rb-deficient cells is increased H1 phosphorylation. H1 phosphorylation may relax chromatin by interfering with its action in chromatin folding and intermolecular fiber-fiber interactions (3).

Continuous stimulation of the Ras mitogen-activated protein kinase (MAPK)¹ signaling pathway in mouse fibroblasts transformed with oncogenes ras, fes, mos, and c-myc elevates the level of phosphorylated H1 (9, 11). Activation of the Ras-MAPK signaling pathway of quiescent fibroblasts treated with growth factors or phorbol esters results in the phosphorylation of H3 (12-14). Thus, persistent activation of the Ras-MAPK pathway in oncogene-transformed cells may also increase the level of phosphorylated H3, contributing to the destabilization of the higher order compaction of chromatin.

The N-terminal domain of H3 can be phosphorylated on Ser-10 and/or Ser-28 (15, 16); however, the site of mitogen-induced phosphorylation remains unknown. Phosphorylation of H3 happens concurrently with the transcriptional activation of the immediate early response genes, e.g. c-fos (12, 13). However, inhibition of transcription does not prevent mitogen-activated H3 phosphorylation (12). It was proposed that phosphorylated H3 (pH3) is associated with the immediate early genes and may be a prerequisite to the expression of these genes (13). Unlike the extensive mitosis-specific phosphorylation, which occurs on all H3 molecules (17), this mitogen-stimulated phosphorylation is targeted to a small, hyperacetylation-sensitive nucleosomal fraction (18).

To study H3 phosphorylation in growth factor- or phorbol ester-stimulated quiescent and oncogene-transformed mouse fibroblasts, we used an antibody that specifically recognizes phospho-Ser-10 of H3. We show that mitogenic stimulation, oncogene transformation, or induction of oncogenic ras expression is accompanied with increases in Ser-10 phosphorylation of H3. We provide direct evidence that pH3 is associated with the induced c-fos and c-myc genes in mitogen-stimulated fibroblasts. The elevated levels of phosphorylated H3 in ras-transformed cells were not a consequence of a decrease in the activity of PP1, which was identified as the H3 phosphatase.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Lines and Culture Conditions-- The cell line Ciras-3 was derived from 10T1/2 cells by transfection with the T-24 Ha-ras oncogene (19). The NIH-3T3 mouse fibroblast cell lines transformed with human c-myc, v-mos, and v-fes were NIH/myc1, Mos 1, Fes 1, and NIH/9IV#5, respectively (20). The 2H1 cell line was derived from 10T1/2 cells by transfection with the T-24 Ha-ras oncogene under the control of a metallothionein promoter (21). Cell lines were grown in plastic tissue culture plates in a humidified atmosphere containing 7% CO₂ in medium supplemented with penicillin G (100 units/ml) and streptomycin sulfate (100 µg/ml). Cell lines were grown in -minimal essential medium plus 10% fetal bovine serum (Intergen, Purchase, NY). Cells were plated in 15 ml of the above medium at 5 × 10⁵ cells per 150-mm diameter plastic tissue culture dish and grown for 72 h. The proportion of cells in the different cell cycle phases was determined by flow cytometry (9).

Manipulation of Cell Lines-- 10T1/2 cells were grown as described above and then serum starved in medium containing 0.5% fetal bovine serum for 24 h. The cells were then either untreated or treated with 50 ng/ml epidermal growth factor (EGF) or 100 nM TPA for 5 or 30 min. 2H1 cells (21) were grown as described above, and then the medium was changed to fresh medium for the control cells or medium containing 100 µM ZnSO₄ (to induce the ras oncogene) for 24 h.

After treatment, the medium was removed and the monolayer was washed twice with phosphate-buffered saline, pH 7.3, and trypsinized. The cells were collected by centrifugation and stored at 80 °C.

Isolation of Histones-- For the isolation of histones, approximately 4 × 10⁷ to 1 × 10⁸ cells were used. Cells were homogenized in 5 ml of nuclear preparation buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1.5 mM MgCl₂, 0.65% Nonidet P-40, and 1 mM PMSF) in the presence of protein phosphatase inhibitors (10 mM NaF, 1 mM sodium orthovanadate, 25 mM -glycerophosphate). For some experiments, the protein phosphatase inhibitors were omitted; we did not observe changes in the relative levels of pH3 in samples isolated with and without these inhibitors. Nuclei were recovered by centrifugation at 1500 × g for 10 min. All centrifugations were carried out at 4 °C. Nuclei were resuspended in 3 ml of RSB buffer (10 mM Tris-HCl, pH 7.6, 3 mM MgCl₂, 10 mM NaCl, 1 mM PMSF, and protein phosphatase inhibitors). Nuclei were extracted with 0.4 N H₂SO₄ to isolate total histones. The samples were precipitated with trichloroacetic acid and then resuspended in double distilled H₂O.

Fractionation of Chromatin-- Chromatin was fractionated by a procedure described previously (22). Ciras-3 mouse fibroblasts (1.35 × 10⁸ cells) resuspended in RSB buffer were homogenized several times and then passed through a syringe with a 22-gauge needle. Nuclei were collected by centrifugation. Nuclei were resuspended in 5 ml of RSB buffer to 10 A₂₆₀ units/ml. CaCl₂ and micrococcal nuclease (Worthington Biochemical Corporation) were added to 1 mM and 15 A₂₆₀ units/ml, respectively. Digestion at 37 °C was for 7.5 min and then stopped by addition of EGTA to 10 mM. The nuclei were collected by centrifugation. The supernatant, which did not contain chromatin fragments, was discarded. The nuclei were sequentially incubated at 0 °C for 30 min with buffer (50 mM Tris-HCl, pH 7.2, 2 mM MgCl₂, 25 mM KCl, 250 mM sucrose, 10 mM EDTA, 1% thiodiglycol, 1 mM PMSF, 25 mM -glycerophosphate, 10 mM sodium fluoride, and 1 mM sodium orthovanadate) that had an increasing concentration of sodium chloride (0.05, 0.1, 0.2, and 0.4 M). The supernatants SS0.05, SS0.10, SS0.20, and SS0.40 were collected following centrifugation. The residual nuclear material (P) remaining after the final extraction was saved. Proteins and DNA fragments were isolated and resolved on SDS-15% polyacrylamide gels and 1% agarose gels, respectively, as described previously (22, 23).

Electrophoresis and Western Blotting-- Proteins were analyzed by SDS-15% polyacrylamide gels. The proteins were visualized by Coomassie Blue staining or transferred to nitrocellulose membranes (9). The anti-pH3 was isolated as described (17). The membrane containing the histones was immunochemically stained with anti-pH3 and horseradish peroxidase-conjugated goat anti-rabbit antibody (Bio-Rad) using the ECL (Enhanced Chemiluminescence) detection (Amersham Pharmacia Biotech). The relative amount of pH3 in each sample was determined by scanning the stained gel and the autoradiogram obtained from Western blots using a PDI 325IE (PDI, Huntington Station, New York) and performing densitometric analysis using the Imagemaster 1-D software (Amersham Pharmacia Biotech, Uppsala, Sweden). The loading of each H3 sample was corrected by dividing the value obtained for pH3 by the value obtained for the amount of total H3 (from the Coomassie Blue stained gel). The relative increase in pH3 in each of the preparations isolated from the oncogene-transformed cell lines was divided by the value obtained for the parental cell line as described previously (9).

To determine specificity of the antibody, total cellular protein from Ciras-3 cells resolved on an SDS-15% polyacrylamide gel was transferred to a membrane, which was subsequently immunochemically stained with anti-pH3 antibody. Only one band corresponding to pH3 was detected on the immunoblot (results not shown). This result is in agreement with previously published results (17). The anti-pH3 antibody will detect pH3 in uncondensed and condensed (e.g. centromeric heterochromatin) chromatin regions (17). Centromeric heterochromatin is associated with unacetylated histones.

Indirect Immunofluorescence Analysis-- Fixation and staining of cells was carried out as described (17). In addition, cells were co-stained with DAPI. Digital optical sectioning and deconvolution microscopy of nuclei was performed as described (24).

CHIP Assays-- 10T1/2 cells were serum starved and either not treated or stimulated with TPA for 30 min. The cells were then treated with 1% formaldehyde for 8 min (final concentration in the medium) to cross-link histones to DNA. The cells were collected, washed twice in phosphate-buffered saline, pH 7.4, and then washed for 10 min in solution 1 (0.25% (v/v) Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 7.5) and 10 min in solution 2 (0.2 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 7.5). The pellet was resuspended in lysis buffer (150 mM NaCl, 25 mM Tris-Cl, pH 7.5, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate) and sonicated for 4 min in 15-s bursts. DNA fragments isolated from the lysate are referred to as input. Typically 90% of the nuclear DNA is present in the lysate. The lysate was diluted 10-fold in lysis buffer, and anti-pH3 antibody was added and incubated at 4 °C overnight. Immunoprecipitated complexes were collected by adding protein A-Sepharose beads for 1 h at 4 °C. Immunoprecipitates were washed once with RIPA (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 0.1% SDS, 0.5% sodium deoxycholate, 1.0% Nonidet P-40), once in high salt wash (500 mM NaCl, 1.0% Nonidet P-40, 0.1% SDS, 50 mM Tris-Cl, pH 8.0), once in LiCl wash (250 mM LiCl, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 50 mM Tris-Cl, pH 8.0) and twice in TE buffer (10 mM Tris-Cl, pH 8.0, 1 mM EDTA). The beads were then treated with RNase (50 µg/ml) for 30 min at 37 °C and then proteinase K overnight. The cross-links were reversed by heating the sample at 65 °C for 6 h, and the DNA was extracted with phenol/chloroform and was ethanol-precipitated. The DNA was then labeled using the DIG DNA nonradioactive labeling system (Boehringer Mannheim). The various genes of interest were slotted onto a nylon membrane and probed with the immunoprecipitated or input labeled DNA. Hybridizations were done at 68 °C overnight. The development times ranged from 4 to 8 h. Note that the development times for blots probed with immunoprecipitated DNA from nonstimulated and stimulated cells were identical. The same protocol was applied for blots probed with input DNA from nonstimulated and stimulated cells.

The four cloned DNA sequences used were immunoglobulin  matrix attachment region (IG MAR), c-fos, c-myc, and prolactin. All clones are mouse genomic sequences except for the prolactin clone, which has a mouse cDNA insert. For IG MAR, a 2.85-kb BamHI-HindIII fragment from the recombinant plasmid pG19/45 was isolated (25). A 2.6-kb BglI fragment from the recombinant pSVfos plasmid carrying mouse genomic c-fos was isolated (26). A 2.1-kb PvuII fragment from the mouse genomic c-myc DNA sequence containing a segments of exon 2 and extending into exon 3 was isolated (27). A 0.8-kb mouse prolactin cDNA insert which is subcloned into the Gem 2 plasmid was isolated (28).

Phosphatase Assay-- A cell pellet containing 4 × 10⁶ 10T1/2 or Ciras-3 cells were lysed in Nonidet P-40 buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1.5 mM MgCl₂, 0.65% Nonidet-P40, and 1 mM PMSF). The cellular extract was centrifuged at 10,000 × g for 10 min and the pellet discarded. Okadaic acid was added (at a final concentration of either 2 nM or 100 nM) to 20 µg of cellular-extracted protein in a 50-µl volume of Nonidet P-40 buffer. Five µg of total histone (isolated by sulfuric acid extraction of 10T1/2 cells that were treated with 0.06 µg/ml colcemid in media for 16 h) was added. The reaction was allowed to proceed at 37 °C for 30 or 60 min. The reaction was stopped by the addition of SDS loading buffer. For the zero min time point, the reaction was immediately stopped by the addition of SDS loading buffer. For comparison of phosphatase activity from 10T1/2 and Ciras-3 cell extracts, the extracts were incubated with phosphorylated H3 for 15, 30, 45, and 60 min at 37 °C. The proteins were separated by SDS-polyacrylamide gel electrophoresis, and Western blotting with the anti-pH3 antibody was performed. To determine the level of phosphorylated H3 in each sample, densitometric analysis of the Western blots was performed. The assays were typically done in triplicate.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Induction of H3 Phosphorylation in Serum-starved 10T1/2 Cells in Response to Growth Factors and Phorbol Esters-- To determine whether Ser-10 of H3 is phosphorylated in response to mitogen stimulation, we used an antibody that was generated against Ser-10 pH3; henceforth called anti-pH3 antibody. Serum-starved 10T1/2 cells were treated with either EGF or TPA for 5 or 30 min, and the level of pH3 was analyzed in immunoblotting experiments. We observed a 7.1-10.3-fold increase in the level of pH3 in the EGF- or TPA-treated cells in comparison with the untreated 10T1/2 cells (Fig. 1). These results show that Ser-10 of H3 is phosphorylated in response to EGF or TPA stimulation.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Effect of EGF and TPA treatment on H3 phosphorylation in quiescent 10T1/2 mouse fibroblasts. Total histone was extracted from 10T1/2 cells that were serum starved and then untreated (lane 1) or treated with 50 ng/ml EGF for 5 min (lane 2), 50 ng/ml EGF for 30 min (lane 3), 100 nM TPA for 5 min (lane 4), or 100 nM TPA for 30 min (lane 5). The histone sample (10 µg) was electrophoretically resolved on a 12.5% SDS-polyacrylamide gel, transferred to membranes, and immunochemically stained with anti-pH3. Panel A shows a Coomassie Blue-stained gel. Panel B shows the immunochemically stained membrane.

Immunolocalization of pH3 in 10T1/2 Cells Treated with TPA-- In murine cell lines, AT-rich centromeric heterochromatin can be seen as regions of intense DAPI staining. These regions colocalize with the domains of intense H3 phosphorylation observed in G₂/M phase cells, reflecting G₂ phosphorylation of centromeric heterochromatin (17). In TPA-induced 10T1/2 cells, a second pattern is seen. The pH3 was located in numerous small foci scattered throughout all interphase nuclei (Fig. 2, panel A). The pH3 foci shown in Fig. 2 were found not to colocalize with centromeric heterochromatin in three-dimension reconstructions.² In the vast majority of the cells, all of the foci were located outside of condensed regions of chromatin (Fig. 2, panel B). This observation is consistent with the hypothesis that pH3 of mitogen-stimulated cells is associated with less condensed chromatin.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Localization of pH3 in nuclei of TPA-induced 10T1/2 mouse fibroblasts. Cells were co-stained with anti-pH3 and DAPI. Digital optical sections of 0.3 mm were obtained and false colored red (phosphorylated H3, panel A) and blue (DAPI, panel C), and a composite image was produced using Adobe Photoshop (panel B). The arrows indicate regions of centromeric heterochromatin. The bar represents 10 µm.

Stimulated c-fos Gene Is Associated with pH3-- The CHIP (chromatin immunoprecipitation) assay was used to test directly if pH3 was associated with immediate early genes, the expression of which is stimulated in mitogen-stimulated cells (12, 29). Fig. 3 shows that pH3 of TPA-stimulated cells was associated with c-fos and c-myc DNA sequences (coding regions). In three separate experiments, the c-fos DNA probe reproducibly gave a stronger signal than did the c-myc DNA probe. Note, however, that both probes generated a comparable signal when hybridized to the input DNA (from TPA-treated cells). In contrast to expressed genes, transcriptionally inactive DNA sequences (immunoglobulin  MAR DNA and prolactin DNA) did not hybridize to the DNA bound to pH3. As expected, the CHIP assay with chromatin from serum-starved cells, which had low levels of pH3, failed to show an association of pH3 with any of these DNA sequences. These observations provide direct evidence that pH3 of TPA-treated cells is associated with the induced c-myc and c-fos genes.

View larger version (75K): [in this window] [in a new window]   Fig. 3.   Association of pH3 with mitogen-induced c-fos and c-myc genes. Quiescent 10T1/2 cells were stimulated with TPA (100 nM) for 30 min or unstimulated. DNA fragments associated with pH3 were isolated by immunoprecipitation with anti-pH3, labeled with DIG, and hybridized to a slot blot containing 0.5-µg DNA inserts of the c-myc, c-fos, prolactin, and IG MAR (IGK) genes (lanes 3 and 4). In lanes 1 and 2, the blot was hybridized with labeled DNA isolated from the total chromatin sample prior to immunoprecipitation (input fraction).

Ras-transformed Fibroblasts Have Elevated Levels of pH3-- We have shown previously that the levels of phosphorylated H1 were elevated in ras-transformed mouse fibroblasts in which the Ras-MAPK signaling pathway is persistently activated (9). In this study, we investigated whether persistent activation of the Ras-MAPK signaling pathway would elevate the steady state levels of pH3 in ras-transformed Ciras-3 cells. In immunoblotting experiments with anti-pH3 antibody, we observed that the level of pH3 was elevated in Ciras-3 cells (Fig. 4B, compare lane 2 with lane 1). As H3 phosphorylation at Ser-10 is high during mitosis (17), it was necessary to compare the cell cycle distribution of the Ciras-3 and 10T1/2 cells. Using flow cytometric analysis of the cell populations, we found that the distributions of cells in different phases of the cell cycle were not significantly different (Table I). We conclude that the observed differences in pH3 levels are not simply because of a higher number of cells in G₂/M phase from one cell line.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Phosphorylated H3 of parental and ras-transformed mouse fibroblasts. Total histone was extracted from parental 10T1/2 cells (lane 1) and Ciras-3 cells (lane 2). The total histone sample (10 µg) was electrophoretically resolved on a 12.5% SDS-polyacrylamide gel, transferred to membranes, and immunochemically stained with anti-pH3. Panel A shows a Coomassie Blue-stained gel. Panel B shows the immunochemically stained membrane.

To provide further evidence that elevated pH3 in Ciras-3 cells was not a mitosis-related event, we studied the distribution of pH3 in Ciras-3 cells by indirect immunofluorescence. The distribution of pH3 in Ciras-3 was similar to that of the TPA-stimulated 10T1/2 parental cells (compare Fig. 5 with Fig. 2). Most pH3 did not colocalize with regions of intense DAPI staining in Ciras-3 nuclei, with the exception of nuclei in a minor population of G₂ phase cells. For the G₂ phase cells, we did observe pH3 associated with centromeric heterochromatin (see arrows in Fig. 5A), in agreement with previous results (17). These observations suggest that pH3 of most ras-transformed mouse fibroblasts is associated with relaxed chromatin regions.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Relative organization of pH3 and condensed chromatin in ras-transformed Ciras-3 mouse fibroblasts. Cells were co-stained with anti-pH3 and DAPI. Digital optical sections of 0.3 mm were obtained and false colored red (phosphorylated H3, panel A) and blue (DAPI, panel C), and a composite image was produced using Adobe Photoshop (panel B). The arrows indicate regions of centromeric heterochromatin in a G2 phase cell. The bar represents 10 µm.

Phosphorylated H3 Is Associated with Micrococcal Nuclease-sensitive Chromatin in Ciras-3 Cells-- Nuclei isolated from Ciras-3 cells were digested with micrococcal nuclease, and the chromatin fragments were sequentially solubilized with buffers of increasing ionic strength. As the ionic strength of the extracting buffer increased so did the lengths of the chromatin fragments, in agreement with our previous results (22) (Fig. 6A). Fractions SS 0.05, SS 0.10, and SS 0.20 accounted for approximately 30% of the chromatin, with 70% of the chromatin being present in fractions SS 0.40 and the residual nuclear pellet (P). Histones isolated from these chromatin fractions were electrophoretically resolved on SDS-polyacrylamide gels and transferred to nitrocellulose membranes, which were stained with India ink (Fig. 6B). Fig. 6C shows that pH3 levels were higher in the chromatin fractions SS 0.05, SS 0.10, and SS 0.20 than in fractions SS 0.40 and P. This result provides evidence that the pH3 observed outside of condensed chromatin regions (Fig. 5) is associated with less condensed regions of chromatin in Ciras-3 cells.

View larger version (49K): [in this window] [in a new window]   Fig. 6.   Fractionation of Ciras-3 chromatin. Ciras-3 chromatin was fractionated as described under "Materials and Methods," yielding chromatin fractions SS 0.05 (15%), SS 0.10 (7%), SS 0.20 (9%), SS0.40 (22%), and residual nuclear material P (47%). The values given in the parentheses indicate the percentage of chromatin in that fraction. DNA fragments (5 µg) isolated from these fractions were electrophoretically resolved in 1% agarose gels, which were stained with ethidium bromide (panel A). Lane M contains HindIII-digested lambda DNA fragments. Histones (5 µg) isolated from the chromatin fractions were resolved on 15% SDS-polyacrylamide gels and transferred to nitrocellulose membranes, which were stained with India ink (panel B). The membrane was immunochemically stained with anti-pH3 (panel C).

Ras-transformed and Parental Mouse Fibroblasts Have Similar H3 Phosphatase Activities-- Increased phosphorylation of H3 in the ras-transformed cells may be a consequence of increased H3 kinase activity and/or decreased H3 phosphatase activity. Histones incubated with cellular extracts isolated from 10T1/2 and Ciras-3 cells were electrophoretically resolved on SDS gels and then transferred to nitrocellulose. Western blot analysis with anti-pH3 antibodies determined the amount of pH3 remaining following incubation with the cellular extracts, providing a measure of the H3 phosphatase activity. Fig. 7 shows that the H3 phosphatase activities were similar in the cellular extracts.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Comparison of H3 dephosphorylation in lysates from parental and ras-transformed cells. Twenty µg of cellular-extracted protein from 10T1/2 (2.9 × 104 cell equivalents) or Ciras-3 (3.5 × 104 cell equivalents) were incubated with 5 µg of total histone substrate for 0, 15, 30, 45, and 60 min at 37 °C. The reaction was stopped, and the proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and immunochemically stained with anti-pH3. The percent of pH3 remaining after each time period was determined by densitometric analysis of the Western blots as described under "Experimental Procedures." Inspection of the India ink-stained immunoblot showed that the protein loads in each lane were equivalent and that protein degradation was not occurring (not shown).

The activities of the two major protein phosphatases, PP1 and PP2A, in mammalian cells can be distinguished by their sensitivity to okadaic acid (30). PP2A activity is completely inhibited at 1 nM okadaic acid, whereas 50% inhibition of PP1 activity is observed at 10-15 nM okadaic acid. A cellular extract from 10T1/2 cells was incubated with histones and 2 or 100 nM okadaic acid and incubated for various times. Fig. 8 shows that for control and 2 nM okadaic acid dephosphorylation of pH3, 29 and 39%, respectively, of the pH3 remained after 60 min. However, when 100 nM okadaic acid was added to the cellular extract, dephosphorylation of pH3 was not observed following 60 min of incubation. These observations suggest that PP1 is the major H3 phosphatase.

View larger version (48K): [in this window] [in a new window]   Fig. 8.   Inhibition of H3 dephosphorylation by okadaic acid in 10T1/2 cell extracts. Cell extracts were incubated in the presence of 0, 2, or 100 nM okadaic acid and 5 µg of total histone as substrate. The reaction mixture was incubated for 0, 30, or 60 min at 37 °C. The reaction was stopped, and the proteins were separated on an SDS-polyacrylamide gel, transferred to membranes, and immunochemically stained with anti-pH3. The percent of pH3 remaining after each time period was determined by densitometric analysis of the Western blots as described under "Experimental Procedures."

The Level of pH3 Is Increased upon Induction of the ras Oncogene in 2H1 Cells-- To test if altered levels of pH3 are an early event in cellular transformation, which occurs upon expression of oncogenic ras, we used the mouse fibroblast cell line, 2H1, which is a 10T1/2 cell line transfected with an inducible-ras oncogene (21). In these cells, ras oncogene expression is controlled by a metallothionein promoter, which can be induced by treating the cells with 100 µM ZnSO₄. Fig. 9 shows the levels of Ras and pH3 at different times after the induction of the ras oncogene. One h after the addition of zinc, Ras levels were increased severalfold, diminishing over 18 h. The amount of pH3 increased rapidly for 2 h and then increased at a much slower rate. These observations show the rapidity and sustained phosphorylation of H3 in response to the expression of the oncoprotein Ras. Treatment of parental 10T1/2 cells with 100 µM ZnSO₄ for 0, 8, or 24 h did not result in increased levels of phosphorylated H3, and therefore phosphorylation of H3 was not affected by ZnSO₄ treatment alone (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 9.   Effect of induction of the ras-oncogene on pH3 levels. Total histone was extracted from 2H1 cells that were treated with 100 µM ZnSO4 for 0, 1, 2, 4, 8, or 16 h. The relative levels of pH3 and Ras from each sample were determined by densitometric analysis of Western blots with anti-pH3 as described under "Experimental Procedures."

Phosphorylation of H3 in Cell Lines Transformed with Oncogenes Encoding Protein Kinases-- Previously we reported that mouse fibroblasts transformed with oncogenes (e.g. fes, mos, c-myc) whose products stimulate the Ras-MAPK signaling pathway have elevated levels of phosphorylated H1 (9, 11, 31, 32). The relative level of pH3 in the control NIH-3T3 cells was compared with the pH3 level in the oncogene-transformed cells. There was an increase in the level of pH3 in the fes-, mos-, and myc-transformed cells in comparison with the NIH-3T3 parental cells (Fig. 10). Table I shows that the cell cycle distributions of parental and oncogene-transformed NIH-3T3 mouse fibroblasts were closely matched. These results show that cells transformed with various different oncogenes have increased levels of pH3.

View larger version (41K): [in this window] [in a new window]   Fig. 10.   Phosphorylated H3 of parental and oncogene-transformed mouse fibroblasts. Total histone was isolated from parental NIH-3T3 (lane 1) and NIH-3T3 cells transformed with v-fes (lane 2), v-mos (lane 3), and c-myc (lane 4). The total histone sample (10 µg) was electrophoretically resolved on a 12.5% SDS-polyacrylamide gel, transferred to membranes, and immunochemically stained with anti-pH3. Panel A shows a Coomassie Blue-stained gel. Panel B shows the immunochemically stained membrane.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we show that activation of Ras-MAPK pathway in quiescent murine fibroblasts treated with phorbol esters or growth factors results in the phosphorylation of Ser-10 of H3. We demonstrate that pH3 is located with chromatin that is not highly condensed, and we provide direct evidence that pH3 is associated with the induced c-fos and c-myc genes.

The c-fos gene is transcribed in quiescent cells; however, elongation of the gene is blocked approximately 100 nucleotides from the site of initiation (33). Stimulation of the Ras-MAPK pathway results in the release of this block in elongation. It is possible that phosphorylation of H3 associated with the c-fos gene allows the chromatin fiber to be less compact, favouring elongation. Consistent with this hypothesis, the c-fos chromatin becomes more DNase I sensitive following activation of the Ras-MAPK pathway (34). Similarly, phosphorylation of H3 may release the block within the c-myc gene (35). Increased c-myc expression is seen later than that of c-fos (29, 36), concurrent with the induced expression of these genes is the appearance of unfolded nucleosomes along the coding region of the c-fos and c-myc genes (29). Dynamically acetylated H3 is the target of mitogen-stimulated phosphorylation (18). As the H3 tail contributes to the folding and inter-association of chromatin fibers, modification of the H3 tail by acetylation and phosphorylation may destabilize higher order compaction of the chromatin fiber and contribute to maintaining the unfolded structure of the transcribing nucleosome (2, 6, 37-41).

Although the H3 that is the target of mitogen-stimulated phosphorylation is dynamically acetylated, it should be noted that acetylation does not predispose H3 to phosphorylation (18). Further, the anti-pH3 antibody will not bind to hyperacetylated H3 unless it is phosphorylated (42). In stimulated mouse fibroblasts, most newly phosphorylated H3 is non-, mono-, and diacetylated (18).

Murine fibroblasts transformed with ras have elevated levels of Ser-10 pH3. Increased levels of pH3 are seen soon after Ras expression is elevated. Either an increased H3 kinase and/or decreased H3 phosphatase activity would result in an elevated level of pH3. The activity of PP1, which our studies indicate is the H3 phosphatase, is regulated by cyclin-dependent kinases (cdc2/cyclin A; cdc2/cyclin B) (43). Increased activity of these kinases in the ras-transformed cells may decrease the activity of PP1. However, we found that the activities of H3 phosphatase in parental and ras-transformed mouse fibroblasts were similar. We conclude that the increased levels of pH3 in the ras-transformed cells are due to increased activity of the H3 kinase.

An increased level of pH3 is also observed in cells transformed with fes, mos, and myc oncogenes. Several of these oncogenes code for serine/threonine or tyrosine kinases that act upon the Ras-MAPK signaling pathway (31). Mammalian cells transformed with c-myc have a stimulated Ras-MAPK pathway (11). Further, we found that the MAPK (ERK-1 and -2) activity was higher in cellular extracts from the ras-, fes-, mos-, and myc-oncogene transformed cells than in the parental cellular extracts (data not shown). By persistent activation of the Ras-MAPK signaling pathway, the phosphorylated isoforms of H3 would remain elevated, resulting in the destabilization of chromatin observed in oncogene-transformed cells (8, 9, 44). Consistent with this idea is the observation that pH3 of ras-transformed cells is associated with less condensed chromatin regions. The increased steady state levels of pH3 may result in deregulation of transcription at the level of transcriptional elongation, resulting in aberrant gene expression observed in cancer cells (45-47).

	FOOTNOTES

^* This work was supported by the Cancer Research Society, Inc. (to J. A. W., J .R. D., and D. P. B.-J.), by a Medical Research Council operating grant (to D. P. B.-J.), and by Public Health Service Grant GM 40922 from the National Institutes of Health (to C. D. A.). The awards of a Manitoba Health Research Council Studentship (to D. N. C.), a Medical Research Council of Canada Senior Scientist (to J. R. D.), and a National Cancer Institute of Canada Terry Fox Senior Scientist (to J. A. W.) are also gratefully acknowledged.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a Postdoctoral Fellowship from the Medical Research Council of Canada and the Alberta Heritage Foundation for Medical Research.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, Winnipeg, MB, Canada R3E 0W3. Tel.: 204-787-2391; Fax: 204-787-2190; E-mail: Davie@cc.umanitoba.ca.

² M. J. Hendzel, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: MAPK, mitogen-activated protein kinase; pH3, phosphorylated H3; EGF, epidermal growth factor; TPA, 12-O-tetradecanoylphorbol 13-acetate; PMSF, phenylmethylsulfonyl fluoride; IG MAR, immunoglobulin  matrix attachment region; kb, kilobase; DAPI, diamidinophenolindole.

	REFERENCES

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