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Originally published In Press as doi:10.1074/jbc.M609041200 on December 11, 2006

J. Biol. Chem., Vol. 282, Issue 7, 4400-4407, February 16, 2007
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Osmotic Stress-dependent Repression Is Mediated by Histone H3 Phosphorylation and Chromatin Structure*

Barbara A. Burkhart, Sarah B. Kennett1, and Trevor K. Archer2

From the Laboratory of Molecular Carcinogenesis, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received for publication, September 22, 2006 , and in revised form, November 24, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Histone H3 phosphorylation has been linked to various environmental stress responses and specific chromatin structure. The role of H3 phosphorylation in the osmotic stress response was investigated on the mouse mammary tumor virus (MMTV) promoter in different chromatin configurations. Hormone-dependent transcription from the MMTV promoter is repressed by osmotic stress when the promoter is integrated and has a normal chromatin structure. However, when the MMTV promoter is transiently transfected, the chromatin structure is less organized, and hormone induction is not affected by osmotic stress. On the integrated MMTV promoter, phosphorylation of histone H3 serine 10 and 28 increases in response to osmotic stress, but the transient promoter shows no change. Hormone-dependent glucocorticoid receptor binding is reduced on the repressed promoter, and elevated H3 phosphorylation is temporally correlated with maximal MMTV repression Additionally, the protein kinase C inhibitor rottlerin, but not other kinase inhibitors, blocks both histone H3 phosphorylation and osmotic repression of MMTV transcription. Glucocorticoid receptor binding is inversely correlated with H3 phosphorylation, suggesting that displacement of the glucocorticoid receptor from the promoter is due to H3 phosphorylation and is the mechanism for the osmotic repression of hormone-dependent transcription.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cells respond to changes in the environment by altering many cellular pathways and functions. Modification of proteins, activation, or repression of genes, growth arrest, or apoptosis may all be affected by environmental stimuli. The responses are specific to the environmental stimulus and cell type and involve independent but overlapping pathways. Modification of chromatin proteins, particularly phosphorylation of histone H3 at serines 10 and 28, is a response to many environmental growth and stress stimuli (1–3).

An increase in histone H3 phosphorylation was first observed in conjunction with chromatin condensation in mitosis, and Aurora B kinase was found to mediate the mitotic H3 phosphorylation (4, 5). Subsequently, a variety of environmental stimuli were found to induce histone H3 phosphorylation in interphase. Growth factors that induce H3 phosphorylation include follicle-stimulating hormone, whose response is mediated by cAMP-dependent protein kinase (6) and epidermal growth factor, whose response requires both ERK3 and RSK (7). Mechanical stressors such as UV-B, UV-C, and heat shock activate p38, ERK, JNK, and MSK, resulting in H3 phosphorylation (8–10). Exposure to the environmental toxin arsenite induces H3 phosphorylation via AKT1, ERK2, and RSK2 (11). Response to the tumor promoter TPA is mediated via PKC and MSK (12, 13), and the response to the antibiotic anisomycin is mediated by p38, JNK, and MSK (14). Environmental stimuli can activate H3 phosphorylation both globally and at specific responsive genes, but these may not be linked because global phosphorylation decreases in some cells, whereas H3 phosphorylation increases at transcriptionally responsive genes (6, 10, 13, 15, 16).

In addition to the various signal transduction kinases, the regulation of H3 phosphorylation is influenced by nucleosomal structure and nucleosome-dependent binding of other chromosomal proteins. In particular, HMGN1 binds specifically to nucleosomes and interacts with histones H1 and H3 (17, 18). HMGN1 is phosphorylated in response to stress stimuli modifying both HMGN1 binding to the nucleosome and chromatin packing, which affects the ability of kinases to phosphorylate histone H3 (19, 20).

Osmotic stress causes the activation of some genes and repression of others. In kidney, hypertonic stress induced genes that promote the accumulation of organic osmolytes (sorbitol, betaine) to reduce loss of water from cells. Osmotic stress causes an increase in chaperones (Hsp70) that protect protein macromolecules against misfolding and aggregation. Hypertonic stress also reduces the level of glucose-regulated proteins (21–24). Proteomics analysis identified a similar number of proteins down-regulated versus up-regulated in osmotic stress (24). The cellular response to osmotic stress involves several signal transduction pathways (25). Important components include the MAP kinases p38, JNK, and ERK, the non-receptor tyrosine kinase Fyn and Syk, the catalytic subunit of cAMP-dependent protein kinase, and both conventional and novel PKCs. These kinases are activated by either hypertonic or hypotonic osmotic stress, although not all kinases are involved in different cell types. Maximal expression may require the convergence of different pathways. Human p38 is a central mediator in response to hypertonicity in most cell types and is essential for activation of some genes (25, 26). JNK is phosphorylated in response to osmotic stress independently of the p38 and ERK pathway and may favor apoptosis (27). In mouse cells, ERK is phosphorylated in response to hypertonic stress independent of p38 and JNK and may promote survival (28, 29). PKC regulates osmotic signaling to ERK and tonicity-responsive enhancer-binding protein expression in mouse cells (28, 30).

This study investigated whether modification of histone H3 phosphorylation is a component of the osmotic stress response and/or required for the response of the MMTV promoter to osmotic stress. The effect of chromatin structure on H3 phosphorylation and osmotic response was investigated by comparing the transiently transfected MMTV promoter with a poorly organized chromatin architecture with the normal nucleosome structure of the integrated promoter (31–33). We found that osmotic stress represses hormone-dependent activation of the integrated MMTV. Histone H3 phosphorylation is chromatin-dependent and inversely correlated with glucocorticoid receptor binding. Finally, both histone H3 phosphorylation and repression are regulated on chromatin by PKC.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Cell Culture—UL3 cells are derived from the human osteosarcoma cell line U2OS by the stable addition of a rat glucocorticoid expression vector and a full-length MMTV promoter regulating a luciferase reporter (34, 35). UL3 cells were maintained in DMEM (H21, Invitrogen), 10% fetal bovine serum, penicillin, and streptomycin at 37 °C and 5% CO2.

Transfection—Cells were transiently transfected with FuGENE reagent (Roche Applied Science) with an efficiency >80% as determined by beta-galactosidase staining of cells transfected with 1 µg of pSport reporter (Clontech) (36). Transient transfections routinely used a total of 0.5 µg of phhCAT in 5 x 105 UL3 cells. The phhCAT plasmid contains 325 bp of proximal MMTV promoter driving a chloramphenicol acetyl transferase (CAT) reporter (S. Nordeen, University of Colorado Medical Center, Denver, CO).

Treatment—Cells were subjected to hypertonic osmotic stress by incubation in 0.2–0.3 M sorbitol in serum-free DMEM for 1–24 h. Cells were treated for hormone induction with 100 nM dexamethasone for 1 h. Hormone was added to sorbitol-containing medium in the last hour of treatment. Cells were pretreated with kinase inhibitors for 30 min before initiation of osmotic stress or hormone treatment. Inhibitors were 10 µM SB203580 for p38, 10 µM UO126 for ERK, 2 µM SP600125 for JNK, and 2 µM rottlerin for PKC (Calbiochem).

Transcription Assay—Following treatment, cells were incubated in DMEM with serum overnight before assaying for luciferase or CAT activity. CAT and/or luciferase activity was measured from the same lysates using CAT and luciferase assay kits (Promega, Madison, WI).

Western Blots—Cells were lysed immediately after stress or hormone treatment in radioimmune precipitation buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 0.1 µg/ml phenylmethylsulfonyl fluoride, and 30 µl/ml aprotinin (Sigma)). Total proteins (10–25 µg) were separated on 4–20% NuPAGE gels (Invitrogen) or 10% SDS-PAGE gels. Proteins were visualized with antibodies to p38 and ERK1 (Upstate%20Biotechnology">Upstate Biotechnology, Lake Placid, NY); phospho-p38, phospho-JNK, and phospho-ERK 1 (Cell Signaling Technology, Danvers, MA); histone H3, phospho-histone H3 Ser-10, Ser-28, and GR (Santa Cruz Biotechnology, Santa Cruz, CA) with horseradish peroxidase-conjugated anti-IgG secondary antibody and chemiluminescence reagent (PerkinElmer Life Sciences).

RNA Induction—Total RNA was purified from cells immediately after treatment using an RNeasy mini kit (Qiagen Sciences, Valencia, CA). First strand cDNA was primed with oligo(dT) and transcribed using a Superscript first strand kit (Invitrogen). Real-time PCR was performed with a Stratagene Mx3000P and SYBR Green detection of products to measure RNA induction (Stratagene, La Jolla, CA). Primers specific for GILZ (sense 5'-GCGTGAGAACACCCTGTTGA-3', and antisense, 5'-GGCTCAGACAGGACTGGAACTT-3'), I{kappa}B (sense, 5'-CCAGGAGTGGGCCATGGAGG-3', and antisense, 5'-GTCTCCCTTCACCTGGCGGATC-3'), EZF (sense, 5'-CGCTCCATTACCAAGAGCTCAT-3', and antisense, 5'-CGATCGTCTTCCCCTCTTTG-3') and GAPDH (sense, 5'-TCGGAGTCAACGGATTTGG-3', and antisense, 5'-GGCAACAATATCCACTTTACCAGAGT-3') coding regions were used.

Chromatin Immunoprecipitation (ChIP) Assay—Chromatin immunoprecipitation assays were performed with a ChIP kit (Upstate%20Biotechnology">Upstate Biotechnology). Immediately following treatment, cells were cross-linked with 1% formaldehyde for 10 min. Complexes were immunoprecipitated with 10 µg of antibodies to pH3 Ser-10, pH3 Ser-28, GR, or rabbit IgG as a nonspecific antibody (Santa Cruz Biotechnology). Complexes were collected using protein A-agarose/DNA beads and washed five times with low salt, high salt, LiCl, and Tris-HCl-EDTA buffers. After reversing cross-links at 65 °C for 4 h, the genomic DNA was purified from eluted complexes with a QIAquick PCR purification kit (Qiagen). MMTV DNA was detected by PCR amplification with primers flanking the hormone-response element (glucocorticoid-response element (GRE)) (sense, 5'-TTAAGTAAGTTTTTGGTTACAAACT-3', and antisense, 5'-TCTGGAAAGTGAAGGATAATGACGA-3') and including ~300 bp of the proximal promoter in the nucleosome B region of the MMTV promoter. The MMTV sense primer and an antisense primer specific to the phhCAT plasmid (5'-TTAGCTTCCTTAGCTCCTGAAAAT-3') were used to detect the transient template. Primers for the GAPDH promoter (sense, 5'-AAAAGCGGGGAGAAAGTAGG-3', and antisense, 5'-CTAGCCTCCCGGGTTTCTCT-3') as the non-linked control and the 3' luciferase gene (sense, 5'-CGTTGTTGTTTTGGAGCAC-3', and antisense, 5'-CTACATTTGGACTTTCCGC-3') as the linked control were used to verify site-specific binding. Total PCR products were quantified from ethidium bromide-stained 4–12% Tris-borate EDTA PAGE gels (Invitrogen) using Alpha Innotech imaging software and verified with real-time PCR (Stratagene).


Figure 1
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  		FIGURE 1.
Osmotic stress and hormone response in UL3 cells. UL3 cells were treated with 0.2 M sorbitol for 3 h and/or treated with dexamethasone (Dex) for 1 h. A, individual proteins were visualized by immunoblot from total cell lysates separated by SDS-PAGE. Results are indicative of those from at least two independent experiments. B, RNA levels of endogenous genes were determined by real time PCR. Error bars represent three or more independent experiments. No, untreated; Sorb, sorbitol.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The osmotic stress response in human UL3 cells was verified by Western blots of p38, JNK, and ERK kinases that are phosphorylated in response to hypertonic stress in other cells. Cells were hypertonically stressed by exposure to either 0.2 M or 0.3 M sorbitol in serum-free DMEM. The response to dexamethasone (10–7 M) in untreated or sorbitol-treated cells was also determined (Fig. 1). Hypertonic stress induced phosphorylation of p38 MAP kinase, whereas total protein levels remain unchanged (Fig. 1A). JNK was also phosphorylated in response osmotic stress. In contrast, ERK1 was not phosphorylated, although hypertonic stress phosphorylates ERK in mouse cells (28, 30). Hormone treatment had no effect on phosphorylation in either untreated or osmotic stressed cells. UL3 cells respond to osmotic stress by activating at least two signal transduction pathways that are responsive to osmotic stress in other cell types. Induction of transcriptional in response to hormone and osmotic stress in UL3 is shown for the endogenous I{kappa}B and EZF genes (Fig. 1B).

Alteration in the phosphorylation of total histone H3 after treatment with osmotic stress or hormones was examined in UL3 cells. The response was analyzed immediately after treatment with dexamethasone for 1 h, sorbitol for 3 h, or sorbitol for 3 h with dexamethasone added in the last hour. No change in global phosphorylation of either H3 Ser-10 or H3 Ser-28 was observed at these treatment times (Fig. 2). No change was found in total glucocorticoid receptor levels in response to osmotic stress either in the presence or in the absence of hormone.

The effect of hypertonic stress on hormone-dependent transcription was studied using the MMTV promoter organized in two distinct chromatin structures. UL3 cells contain a stably integrated MMTV promoter regulating a luciferase reporter. This promoter has a normal chromatin structure including distinct nucleosomes and enhanced transcription factor binding associated with chromatin remodeling (31, 32). Alternatively, an MMTV promoter regulating a CAT reporter that is transiently transfected into cells has a poorly defined chromatin structure lacking clear nucleosomes or evidence of chromatin remodeling. Differences in the activation of the MMTV promoter in these two configurations can be evaluated in the same cell by comparing CAT (transient) and luciferase (integrated) induction. UL3 cells were transiently transfected with phhCAT overnight, osmotically stressed by incubation with sorbitol for 3 h, and then induced with dexamethasone for the final hour. The medium was replaced with DMEM + 10% serum overnight to allow translation of CAT and luciferase transcripts. Hormone-dependent transcription from the luciferase reporter was reduced to 40% of control by osmotic stress, whereas the CAT reporter induction was unaffected by these conditions (Fig. 3). This indicates that a normal chromatin structure or a normal chromatin-dependent mechanism is required for the promoter to respond to hypertonic stress. Moreover, it signifies that osmotic stress is a member of the group of environmental stressors that has a chromatin-dependent component.

Histone H3 phosphorylation at the MMTV promoter was analyzed by ChIP using antibodies directed to histone H3 Ser-10 and 28 with GR antibody and IgG as controls. The relative binding to a promoter fragment surrounding the GRE in nucleosome B of the integrated promoter was examined. Recovery was antibody-dependent; in untreated controls, recovery was ~3.5% of input with anti-Ser-10, 1% with anti-Ser-28, 0.75% with anti-GR, and 0.25% with anti-IgG. The relative change in binding in response to osmotic stress or to hormone treatment is shown with the untreated control set to 100% for each antibody (Fig. 4). Histone H3 Ser-10 phosphorylation at nucleosome B of the MMTV promoter increases following hypertonic stress. Treatment with hormone reduces H3 phosphorylation in both the presence and the absence of osmotic stress. Histone H3 Ser-28 follows a pattern similar to that of Ser-10 phosphorylation. Hormone treatment increases binding of the GR as expected for this positive control. However, osmotic stress reduces GR binding both in the presence and in the absence of hormone. No difference in binding due to osmotic stress or hormone was observed for the IgG precipitated GRE (data not shown).


Figure 2
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  		FIGURE 2.
Effects of osmotic stress or hormone on total histones. A, a representative immunoblot of histone H3 Ser-10 phosphorylation, H3 Ser-28 phosphorylation, and GR levels in total cell lysates from UL3 cells treated as in Fig. 1. Dex, dexamethasone. B, immunoblots were quantified by image analysis from three or more independent experiments. Sorb, sorbitol. Error bars represent three or more independent experiments.

 


Figure 3
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  		FIGURE 3.
Chromatin requirement for hormone–dependent transcription following osmotic stress. A, the hormone-dependent transcription activity from the integrated MMTV promoter is repressed by osmotic stress. MMTV expression was normalized to hormone-induced cells as 100%. The integrated promoter was measured by luciferase activity 24 h after treatment. Error bars represent three or more independent experiments. Basal expression is shown as an inset with scale at 1/1000 of hormone induced. B, hormone-dependent transcription from the transiently transfected MMTV promoter is insensitive to osmotic stress. The transient promoter activity was measured by CAT activity. Standard error was determined from four or more independent experiments.

 
The increase in H3 phosphorylation and the binding of GR are inversely correlated. Osmotic stress-induced phosphorylation reduces binding of GR, and hormone treatment reduces phosphorylation. Hormone-dependent transcription requires GR binding. Therefore, the osmotic repression of hormone-dependent transcription may result from the displacement of GR from the promoter or inhibition of the normal hormone-dependent increase in GR binding by histone H3 phosphorylation.


Figure 4
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  		FIGURE 4.
Histone H3 phosphorylation and GR binding at the integrated MMTV promoter. Histone H3 Ser-10 and Ser-28 phosphorylation and GR binding was measured by ChIP at the nucleosome B region of the MMTV promoter, which includes the hormone response elements. Sorbitol increases H3 phosphorylation, and hormone increases GR binding. Treatment with both sorbitol and hormone reduces the increase in both H3 phosphorylation and GR binding. UN, untreated; Dex, dexamethasone. Error bars represent three or more independent experiments.

 


Figure 5
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  		FIGURE 5.
Temporal correlation of osmotic repression and pH3 phosphorylation on the integrated MMTV promoter. A, UL3 cells were treated for the indicated time with sorbitol and then hormone-stimulated for 1 h, and luciferase measured 24 h after treatment was terminated. Repression was transient with MMTV maximally repressed 3–6 h after initiation of sorbitol treatment. No repression was observed after 24 h of sorbitol treatment. Error bars represent three or more independent experiments. B, ChIP of histone H3 phosphorylation shows an increase after 3 h in sorbitol that returns to untreated levels after 24 h. Dex, dexamethasone.

 
Osmotic repression of the integrated MMTV promoter was examined over a 24-h time course (Fig. 5A). Cells were exposed to sorbitol for 1–24 h with hormone added in the last hour, and then the medium was replaced overnight before luciferase was measured. Osmotic repression of MMTV is a transient effect. Exposure to sorbitol represses the integrated MMTV with maximal effect at 3–6 h. Repression then attenuates to untreated levels by 24 h.

Phosphorylation at the integrated MMTV promoter was determined for 0, 3, and 24 h of exposure to sorbitol (Fig. 5B). Osmotic stress leads to an increase in phosphorylation for both Ser-10 and Ser-28, which is maximal at 3 h and declines to baseline by 24 h. This correlates with the time course of repression of transcription. The time course of phosphorylation is the same in the presence of hormone, although phosphorylation levels are lower with hormone.


Figure 6
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  		FIGURE 6.
The transiently transfected MMTV shows neither osmotic stress repression nor change in H3 phosphorylation. A, sorbitol does not inhibit hormone induction of the transient MMTV. UL3 cells were transiently transfected with the MMTV-CAT construct and treated as in Fig. 5. Transcription was measured by CAT activity. B, H3 phosphorylation is unaffected by sorbitol. ChIP of H3 phosphorylation levels was measured after 3 h of sorbitol treatment. Error bars represent three or more independent experiments. Dex, dexamethasone.

 
A time course of transcription during osmotic stress was also measured on the transient MMTV to determine how the lack of chromatin structure affected repression and H3 phosphorylation. No osmotic repression of the transient MMTV was observed at any time up to 24 h (Fig. 6A). Histone H3 phosphorylation at the transient MMTV was analyzed by amplifying the ChIP products with a CAT-specific primer (Fig. 6B). Although phosphorylated H3 was found associated with the transient promoter, a change in phosphorylation was not observed in response to hypertonic stress.

Inhibitors of several kinases were used to assess the contribution of different signal transduction pathways to osmotic repression and H3 phosphorylation of MMTV. In UL3 cells, p38 and JNK were clearly phosphorylated in response to osmotic stress treatment. However, inhibition with 10 µM SB203580, a p38 inhibitor, or 2 µM SP600125, a JNK inhibitor, had no effect on MMTV induction by hormone or osmotic repression (Fig. 7A). We also tested inhibitors of PKC and ERK because these kinases are regulators of the osmotic stress response in mouse cells (28, 30) and H3 phosphorylation by TPA (13). In UL3 cells, we found no osmotic stress-induced ERK phosphorylation, and inhibition of ERK with 10 µM UO126 failed to affect repression. However, treatment with the PKC inhibitor rottlerin (2 µM) resulted in abrogation of osmotic repression relative to the hormone-treated control. Rottlerin also reduced hormone-dependent activation by ~50%. Rottlerin reduced the effects of both hormone and osmotic stress on MMTV expression, although hormone and stress have opposite effects on transcription.


Figure 7
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  		FIGURE 7.
Inhibition of osmotic stress-associated kinases implicates PKC regulation. A, the effect of inhibition of p38 (10 µM SB203580), ERK (10 µM UO126), JNK (2 µM SP600125), and PKC (2 µM rottlerin) on hormone-dependent transcription and osmotic repression of MMTV from the integrated MMTV. Dex, dexamethasone. Error bars represent three or more independent experiments. The effect of rottlerin on non-hormone-dependent transcription is shown as an inset with 1/1000 scale. B, rottlerin does not affect hormone-dependent transcription from the transient MMTV. CAT activity specific to the transient promoter was measured in the same cells as in A. Sorb, sorbitol.

 
Transcription from a transient MMTV promoter was measured in rottlerin-treated cells to determine whether this compound, which is toxic to UL3 above 10 µM, was non-specifically affecting transcription (Fig. 7B). Rottlerin had no effect on the hormone-dependent activation of the transient MMTV. Thus, rottlerin activity is chromatin-specific, affecting both the efficiency of hormone activation and osmotic repression only on a normal nucleosome-organized promoter.

The effect of rottlerin on H3 phosphorylation in sorbitol-treated cells and on GR binding was examined by ChIP (Fig. 8). Although rottlerin had no effect on phosphorylation of total cellular H3 proteins, rottlerin treatment reduced the increase in H3 phosphorylation in response to sorbitol at Ser-10 on the MMTV promoter. Rottlerin also reduced the hormone-dependent increase in GR binding. On cells treated with a combination of sorbitol and hormone, rottlerin abrogated the decrease in phosphorylation. The overall effect of rottlerin was to inhibit changes in pH3 and GR specifically associated with the integrated MMTV promoter. This suggests that GR binding and H3 phosphorylation on the chromatin promoter have a common chromatin-dependent precursor step that affects the subsequent GR binding or H3 phosphorylation when disrupted by rottlerin.


Figure 8
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  		FIGURE 8.
The effect of PKC inhibition on H3 phosphorylation and GR binding at the integrated MMTV promoter. A, ChIP with pH3 antibodies of cells treated with sorbitol and hormone with or without rottlerin. Rottlerin (2 µM) treatment abrogates the increase in H3 Ser-10 phosphorylation. Un, untreated; Dex, dexamethasone; Sorb, sorbitol. Error bars represent three or more independent experiments. B, ChIP with GR antibodies of the same cells as in A. Rottlerin also abrogates the increase in GR binding in response to hormone.

 


Figure 9
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  		FIGURE 9.
Transcriptional response of endogenous genes to hormone and osmotic stress. A, GILZ, osmotic stress repression of hormone induction. Error bars represent three or more independent experiments. B, I{kappa}B, hormone induction. C, EZF, osmotic stress induction. D, GAPDH, control.

 
The effect of osmotic stress on hormone induction of endogenous genes was evaluated using real-time PCR of RNA levels (Fig. 9). Hormone treatment induces both the I{kappa}B and the GILZ genes. Osmotic stress treatment represses the hormone induction of the GILZ gene to 40% of the hormone-induced control. Thus, the GILZ gene shows the same pattern of osmotic repression as MMTV. However, hormone induction of the I{kappa}B gene is unaffected by osmotic stress in the same cells. EZF is induced by osmotic stress, and hormone treatment has no effect on the osmotic stress induction of EZF. The lack of osmotic stress repression on I{kappa}B or the GAPDH control suggests that osmotic stress repression is targeted to specific genes and is not a non-specific global effect.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Change in histone H3 phosphorylation is a common response to many mitogenic and stress-inducing environmental influences. We have shown here that H3 phosphorylation is also a component of the cellular response to osmotic stress. An increase in histone H3 phosphorylation has previously been associated with specific stress-responsive genes and correlated with gene activation (12, 16, 37). However, we found in the present study that the increase in osmotic stress-dependent H3 phosphorylation at the MMTV promoter is associated with repression of hormone-dependent transcription. Thus, H3 phosphorylation is a component of the osmotic stress response but is not linked specifically to gene activation.

Displacement of several transcription factors is an initial response to osmotic stress at several promoters in yeast (38). HP1 is displaced from chromosomes during mitotic chromatin compaction, but HP1 is retained when phosphorylation of H3 Ser-10 is inhibited (39–41). Hormone-dependent transcription of the MMTV is repressed during mitosis when H3 is highly phosphorylated, and binding of the transcription factor NF1 to the promoter is reduced (42). We observed that H3 phosphorylation of the MMTV promoter and binding of the glucocorticoid receptor GR to the promoter are inversely correlated. Moreover, H3 phosphorylation and repression are temporally correlated. This suggests that the increase in H3 phosphorylation regulates the displacement of GR and represses hormone-dependent transcription.

Comparison of the osmotic response from the integrated versus the transient MMTV promoter shows that both repression and histone H3 phosphorylation require a normal chromatin structure. The transient MMTV promoter is accessible to nuclease digestion in the absence of hormone and binds the GR and other transcription factors more efficiently in the absence of hormone than the normal chromatin of the integrated promoter (32). However, on treatment with hormone, the transient promoter fails to exhibit a chromatin-dependent increase in nuclease hypersensitivity or increase GR binding. We have shown that the transient promoter is similarly insensitive to osmotic stress-induced changes in histone H3 phosphorylation exhibiting no significant change in phosphorylation or repression. The presence of phosphorylated H3 on the transient promoter clearly does not prevent GR binding but may contribute to the lower inducibility of the transient promoter (10-fold) when compared with the integrated promoter (1000-fold).

Rottlerin inhibits several isoforms of PKC and abrogates osmotic stress repression of the MMTV (43). A requirement for PKC signaling has been found for the osmotic response in mouse for both ERK phosphorylation and TonEBP-dependent transcription (28, 30). In mouse cells, osmotic stress increases total PKC activity and induces translocation of PKC{alpha}, PKC{delta}, and PKC{epsilon} from cytosol to the cell membrane (28). Dominant-negative inhibition of PKC{delta} or PKC{epsilon} inhibits ERK phosphorylation but not transcriptional activation (30). Histone H3 phosphorylation and transcription of the low density lipoprotein promoter in response to TPA in human cells is dependent on PKC but not ERK, which suggests that transcription and ERK signaling are independent osmotic responses in human cells (13). In vitro H3 is phosphorylated at Ser-10 by purified PKCbeta or PKC{epsilon} (13), so rottlerin may disrupt the PKC-dependent phosphorylation of H3. However, the effect of rottlerin on hormone-dependent GR binding suggests that PKC regulates a common intermediate and may not specifically phosphorylate H3.

An alternative mechanism for regulating histone H3 phosphorylation in a PKC-dependent manner is chromatin compaction. The HMGN1 protein is a component of normal chromatin structure that binds to nucleosome core particles and modifies chromatin compaction (17–19). The HMGN1-dependent changes in chromatin compaction regulate the access of kinases to histone H3. Like histone H3, HMGN1 is phosphorylated in response to mitogens and stress. However, HMGN1 phosphorylation precedes anisomysin-dependent H3 phosphorylation (19). The lack of HMGN1 in knock-out mice increases the steady state of H3 Ser-10 phosphorylation (19). HMGN1 can be phosphorylated on several residues by different kinases. PKC{alpha} specifically phosphorylates residues in the nucleosome-binding domain of HMGN1 that regulate binding to the nucleosome core (20). HMGN1 bound to the nucleosome can block H3 phosphorylation by RSK or MSK (19). If HMGN1-dependent compaction can modulate the access of kinases to H3, it may also regulate the access of GR to the GRE in hormone-responsive promoters. The hormone-dependent increase in GR binding at MMTV has been attributed to ATP-dependent chromatin remodeling. However, increased GR binding occurs in the absence of Brg1, suggesting that the regulation of GR binding is not solely attributable to that mechanism (44). PKC{alpha} phosphorylates HMGN1 in the nucleosome–binding domain and regulates chromatin compaction, potentially modulating both GR binding and H3 phosphorylation. Rottlerin inhibition of HMGN1 phosphorylation disrupting decompaction could produce the similar expression in dexamethasone + rottlerin-treated and sorbitol + dexamethasone + rottlerin-treated cells. The regulation of chromatin compaction is potentially the common intermediary disrupted by rottlerin.

Osmotic stress activation of several genes does not require normal chromatin. An Sgk-luciferase reporter and a tonicity-responsive enhancer-binding protein-response element luciferase reporter are both activated by osmotic stress when transiently transfected (26, 30). This suggests that chromatin structure and H3 phosphorylation regulate a subset of stress pathways contributing to gene response and that loss of H3 phosphorylation does not necessarily abrogate activation. MSK phosphorylates H3 in response to arsenite and anisomysin, and deletion of Msk inhibits H3 phosphorylation but only partially blocks activation of immediate early genes (16). Osmotic activation of the low density lipoprotein receptor gene has an H3 phosphorylation element that is disrupted by rottlerin, but the low density lipoprotein may be activated without H3 phosphorylation (13). If H3 phosphorylation regulates the displacement of factors that are normally associated with the promoter in the absence of stress, then clearing could facilitate the formation and binding of new transcription complexes at the promoter that contain stress-responsive transcription factors. H3 phosphorylation would then regulate both repression and activation of transcription.


   FOOTNOTES
 
* This research was supported by grants from the Intramural Research Program of the NIEHS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Center for Biologics Evaluation and Research, Interagency Oncology Task Force, Food and Drug Administration and NCI, National Institutes of Health, Bethesda, MD 20892. Back

2 To whom correspondence should be addressed. Tel.: 919-316-4565; Fax: 919-316-4566; E-mail: archer1{at}niehs.nih.gov.

3 The abbreviations used are: ERK, extracellular signal-regulated kinase; RSK, ribosomal S6 kinase; JNK, c-Jun NH2-terminal kinase; MSK, mitogen- and stress-activated protein kinase; MAP, mitogen-activated protein; TPA, 12-O-tetradecanoylphorbol-13-acetate; DMEM, Dulbecco’s modified Eagle’s medium; CAT, chloramphenicol acetyl transferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ChIP, chromatin immunoprecipitation; GR, glucocorticoid receptor; GRE, glucocorticoid-response element; GILZ, glucocorticoid-induced leucine zipper; EZF, epithelial zinc finger; I{kappa}B, inhibitor of NF{kappa}B. Back


   ACKNOWLEDGMENTS
 
We thank Drs. R. DiAugustine and M. Garcia for thoughtful review of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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