1 Nuclear Signalling Laboratory, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK
2 Aichi Cancer Centre Research Instititute, 1-1 Kanokoden, Chikusa-ku, Nagoya, Aichi 46 8681, Japan
3 MRC Protein Phosphorylation Unit, School of Life Sciences, Dundee, DD1 5EH, UK
4 Chromatin and Gene Expression, Institute of Biomedical Research, University of Birmingham, Vincent Drive, Edgbaston, Birmingham, B15 2TT, UK
5 The Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK
* Author for correspondence (e-mail: louiscm{at}bioch.ox.ac.uk)
Accepted 10 March 2005
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Summary |
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Key words: MAP kinases, MSK, Histone H3 phosphorylation, Gene induction
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Introduction |
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H3 has also been shown to become phosphorylated on S10 on condensed mitotic chromosomes (Gurley et al., 1978; Hendzel et al., 1997
; Paulson and Taylor, 1982
) (reviewed in Prigent and Dimitrov, 2003
). Unlike MAP kinase-mediated inducible events described above, mitotic H3 modification involves the majority of H3 in the nucleus (Gurley et al., 1978
) and appears more persistent, commencing in pericentric heterochromatin during late G2 phase, becoming widespread by prophase, and persisting until telophase (Hendzel et al., 1997
). Recently, the characterisation of monoclonal antibodies raised against mitotic chromosomal proteins resulted in phosphorylated S28 of H3 (phosphoS28-H3) being identified as a new chromosomal phospho-epitope. The independent generation of antibodies specific for phosphoS28-H3 confirm its presence on mitotic chromosomes (Goto et al., 1999
). Mitotic phosphorylation of S10 and S28 on the H3 tail is proposed to be mediated by Aurora B kinase (Crosio et al., 2002
; Goto et al., 2002
; Hauf et al., 2003
), although the phosphorylation of S28 occurs later than that of S10, only becoming detectable at prophase.
With the availability of these antibodies, S28 has now also been shown to be a second site of MAP kinase-mediated inducible phosphorylation. In quiescent JB6 fibroblasts, phosphorylation of S28 (along with that of S10) was rapidly induced in response to UV-B irradiation (Zhong et al., 2001a; Zhong et al., 2001b
). In vitro kinase assays and inhibitor studies implicate MAP kinases and the downstream MSKs in this response (Zhong et al., 2001a
; Zhong et al., 2001b
). Most recently, we found S28-H3 phosphorylation in quiescent fibroblasts after stimulating them with several agents that activate MAP kinase signalling (Soloaga et al., 2003
). Finally, embryonic fibroblasts of double-knockout mice that lack both MSK1 and MSK2, have been used to prove that these kinases are responsible for inducible phosphorylation of both S10 and S28 on histone H3 (Soloaga et al., 2003
).
These studies suggest a scenario whereby upon activation, MSKs phosphorylate both S10 and S28 on the same subfraction of histone H3, namely that associated with nucleosomes at IE genes. However, whereas the association of phosphoS10-H3 with inducible IE genes has been readily demonstrated using ChIP assays (Thomson et al., 2004), similar studies using antibodies against phosphorylated S28 show little association with these genes (M.H.D. and L.C.M., unpublished observation), raising the possibility that phosphoS28-H3 is predominantly located elsewhere. Here, we have rigorously analysed the appearance, acetylation characteristics and colocalisation of phosphoS10-H3 and phosphoS28-H3. Although their physiological characteristics in vivo are very similar and MSK can quantitatively phosphorylate both residues on the same H3 tail on reconstituted nucleosomes in vitro, these two phosphorylation events are associated with different sets of crosslinked nucleosomes and localise to different chromatin loci in vivo. These studies reveal an exquisite level of targeting S10 and S28 phosphorylation in the intact mouse nucleus. Several aspects of this targeting in the intact mouse nucleus were explored using transfection-based approaches, and are discussed below (see Discussion).
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Materials and Methods |
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Acid-soluble proteins were prepared as described in Clayton et al. (Clayton et al., 2000). Alternatively, total soluble proteins were obtained by scraping cells into lysis buffer [50 mM Tris-HCl pH 7.5, 0.1% (v/v) Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.27 M sucrose, 10 mM sodium butyrate, 20 mM sodium ß-glycerophosphate, 100 µM sodium orthovanadate, 1 µM microcystin-LR), lysing them on ice for 5 minutes and spinning out insoluble components at 16,000 g. To extract cyclic AMP response element binding protein (CREB), cells were extracted with high-salt lysis buffer as described (Rutault et al., 2001
).
Antibodies
Preparation, affinity-purification and characterisation of anti-phosphoS10-H3occ., anti-phosphoacetyl-H3, anti-phosphoHMG-14 and anti-HMG-14 have been described in detail previously (Clayton et al., 2000; Thomson et al., 1999
). Anti-phosphoS28-H3 was kindly supplied by Hidemasa Goto and Masaki Inagaki (Aichi Cancer Research Centre, Nagoya, Japan). Mouse anti-GFP was obtained from Molecular Probes, and rabbit anti-GFP from Roche. Anti-MSK1 was kindly provided by J.S.C. Arthur (University of Dundee, UK). Anti-phosphoCREB was obtained from UBI and anti-ERK from Zymed Laboratories.
Generation of a GFP-MSK1-expressing stable cell line
Exponentially growing C3H 10T cells (250 µl, containing 1.5x107 cells) were electroporated (Easyject, Flowgen single pulse: 230 V, 1500 µF, giving a pulse time of 60-70 mseconds) with 5 µg of GFP-MSK1-neomycin resistance plasmid. Following 24 hours growth, stable transfectants were selected by addition of G418 Geneticin (1 mg/ml; Roche). Fresh G418 was added each day until single colonies formed (10-14 days). Colonies were picked and several clones were screened for GFP expression by western blotting.
Western blotting
Samples were run on 15% SDS-PAGE or 15% or 20% acid-urea gels and transferred to PVDF membranes (Millipore) as described in Clayton et al. (Clayton et al., 2000). Alternatively, to enhance phosphorylation-dependent retardation, proteins were electrophoresed in modified SDS-PAGE conditions (Leevers and Marshall, 1992
). Membranes were probed with anti-phosphoS10-H3 (1:2000 serum), anti-phosphoS10-H3occ. (1:2000 affinity-purified antibody), anti-phosphoS28-H3 (1:500), anti-phosphoacetyl-H3 (1:2000 serum), anti-phosphoHMG-14 (1:400 affinity-purified antibody), anti-HMG-14 (1:1000 affinity-purified antibody), anti-MSK1 (1:1000), anti-GFP (1:500) or anti-phosphoCREB (1:2500 affinity purified antibody).
In vitro kinase assays
Recombinant Drosophila histone H3 and reconstituted octamers were kindly provided by Karl Nightingale (University of Birmingham, UK). Radioactive kinase reactions were carried out in 50 µl kinase assay buffer (50 mM Tris-HCl pH 7.5, 0.03% (v/v) Brij-35, 0.1 mM EGTA, 0.1% (v/v) ß-mercaptoethanol, 10 µM PKI peptide, 10 mM MgCl2, 0.4 mg/ml BSA, 100 µM ATP and 10 µCi [32P]ATP (3000 Ci/mmol). Labelling (30°C, 30 minutes) was started by adding 103 units of recombinant MSK1 and terminated in 25% (w/v) trichloroacetic acid to precipitate protein (on ice, 1 hour). Samples were centrifuged (30 minutes, 16,000 g 4°C), pellets washed three times with 1 ml cold acetone and air-dried. Pellets were resuspended in 8 M urea and separated by SDS-PAGE. Gels were dried under vacuum and autoradiographed.
Non-isotopic assays were conducted as above, except that [32P]ATP was omitted from the reaction. For acid-urea gels, proteins were TCA precipitated as described above, and resuspended in acid-urea loading buffer (Clayton et al., 2000
). For SDS-PAGE, reactions were terminated by adding Laemmli loading buffer and boiling for 5 minutes.
Chromatin preparation and immunodepletion
Formaldehyde-crosslinked chromatin fragments were prepared and immunodepletions conducted essentially as described (Clayton et al., 2000; Thomson et al., 2001
). Briefly, C3H 10T
cells were crosslinked with formaldehyde (10 minutes, room temperature, final concentration 1%), nuclei were isolated, resuspended and sonicated for 5 minutes (Sonics Vibracell VC130 sonicator). SDS was added to 1% final concentration and samples were rotated at room temperature for one hour. Insoluble material was removed by centrifugation (16,000 g) and soluble chromatin fractions were sonicated for a further 10 minutes to generate an average fragment size of 300-400 base pairs. Fragment size was verified by electrophoresis in TAE-agarose gels. Soluble chromatin samples were diluted 10-fold to reduce the SDS concentration to 0.1%, and chromatin re-concentrated using Vivaspin 50 concentrators (Sartorius). Chromatin fractions were adjusted to RIPA buffer and aliquoted.
For anti-phosphoacetyl-H3 and anti-phosphoS10-H3occ. immunodepletions, 50 µg affinity-purified antibody was added to chromatin aliquots, and incubated at 4°C for 2 hours. Protein A/G-agarose, pre-adsorbed with 10 µg sonicated herring sperm DNA and 10 µg BSA, was then added and samples rotated for a further 2 hours at 4°C. For anti-phosphoS28-H3 immunodepletions, 500 µl antibody solution was incubated with protein G-agarose beads for 2 hours at 4°C. The beads were then pelleted and the supernatant discarded. Chromatin solution was added to the antibody-bound beads, and rotated for a further 2 hours at 4°C. Samples were briefly spun (1 minute, 3000 rpm in a tabletop centrifuge) to pellet beads, and the unbound, immunodepleted material was removed. Immunodepleted and input fractions were adjusted to SDS-PAGE sample buffer and heated to 95°C to reverse cross-links.
Immunofluorescence
Cells were grown on glass coverslips to subconfluence, quiesced and stimulated as described above. Coverslips were washed three times with cold PBS, and incubated with fixative [PBS containing 3% (v/v) paraformaldehyde and 2% (w/v) sucrose] for 20 minutes at room temperature. Cells were washed again with PBS and incubated with cold permeabilisation solution [PBS containing 0.5% (v/v) Triton X-100] for 5 minutes at 4°C, before washing again three times with PBS. All subsequent steps were performed at room temperature. Non-specific binding sites were blocked by incubating the coverslips with blocking solution [PBS containing 10% (v/v) FCS and 0.02% (v/v) Tween-20] for 1 hour. Coverslips were incubated with primary antibodies, diluted in blocking buffer (1:100 for anti-phosphoS28-H3; 1:2000 for anti-phosphoS10-H3 and 1:100 for anti-GFP), for 1 hour and washed with PBS five times for a total duration of at least 30 minutes. Incubations with 1:100 dilutions of secondary antibodies were performed as for the primary antibody. Species-specific secondary antibodies were purchased either as Cy3-conjugates, or as conjugated proteins which were subsequently coupled to Alexa-488 using the Alexa-488 protein labelling kit (Molecular Probes). Coverslips were washed as for the primary antibody, and mounted on glass slides in Vectashield mounting medium (Vector Laboratories), which contained 1 µg/ml 4',6-diamidino-2-phenylindole dihydrochloride (DAPI).
Conventional microscopy
Cells were examined with an Axioplan 2 epifluorescence light microscope (Zeiss), using 40x (Neofluor, 0.75 NA) and 20x (Plan-Neofluor, 0.5 NA) objective lenses. Digital images were collected with a 12-bit peltier-cooled CCD (Diagnostic Instruments) camera using SPOT camera software, and flat-field correction was applied. Microscope settings and exposure times for each channel were kept constant within a given set of samples, but were optimised for each experiment.
Confocal microscopy and image analysis
Cells were examined on a Radiance 2000 laser scanning confocal microscope (BioRad) with LaserSharp 2000 software, which was mounted on a Nikon TE300 inverted microscope equipped with 60x PlanApo oil-immersion objective lens (1.4 NA). Images were acquired by sequential excitation at 488 nm (Argon ion, for Alexa-488 and GFP) and 543 nm (Green HeNe, for Cy3) laser lines. Data were collected by Kalman-averaging over 5-8 images. Microscope settings were kept constant in each experiment. Images were saved as BioRad files, viewed with Confocal Assistant Software version 4.02 (© T. Brelje, 1996), and compiled using Adobe Photoshop.
Cross correlation function (CCF) analysis was performed as described (van Steensel et al., 1996). Red- and green-channel confocal images were combined into a dual-colour image using Adobe Photoshop and imported into MetaMorph software (Universal Imaging Corporation). Lines were drawn through the nucleoplasm and red- and green-channel line-scan intensity profiles obtained. CCF analysis was performed in Microsoft Excel by shifting the intensity profiles of the red and green channels with respect to one another [increasing or decreasing the shift along the x-axis (
X) by one voxel at a time] to a maximum value of 20 voxels in either direction. At each
X-point, the correlation between the red and green intensities was determined according to Pearson's correlation coefficient (RP). For each examined nucleus, CCF analysis was performed for three independent line-scans. RP-values were averaged over the three line-scans and a graph of mean RP against
X was plotted.
Immunoprecipitation and assay of protein kinase activity
Cells were lysed in 50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM sodium pyrophosphate, 0.27 M sucrose, 1 µM microcystin-LR, 1% (v/v) Triton X-100, 0.1% (v/v) 2-mercaptoethanol, plus protease inhibitors. Lysates were cleared by centrifugation (16,000 g at 4°C), and insoluble material was discarded. Immunoprecipitation was performed essentially as described (Williams et al., 2000), except that 4 µg anti-GFP antibody was used for each immunoprecipitation. Immunoprecipitates were assayed for activity against Crosstide (peptide GRPRTSSFAEG) as described (Williams et al., 2000
).
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Results |
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Confluent, quiescent cells were treated with EGF (Fig. 1A, lanes 2-6), sub-inhibitory anisomycin (sAn) (Fig. 1A, lanes 7-11) and TPA (Fig. 1A, lanes 12-16), agents previously shown to differentially activate mammalian MAP kinase pathways, induce IE genes and stimulate H3-S10 phosphorylation (Cano et al., 1996; Cano et al., 1995
; Cano et al., 1994
; Edwards and Mahadevan, 1992
; Hazzalin et al., 1996
; Mahadevan et al., 1991
). H3-S28 phosphorylation was monitored by western blotting and compared with profiles of H3-S10 phosphorylation and phosphoacetylation (Fig. 1A). Treatment with all three stimuli results in the rapid induction of H3-S28 phosphorylation (Fig. 1A, panel i). The intensity, latency and duration of the responses vary between stimuli. However, for each stimulus, profiles of H3-S28 phosphorylation are very similar to those for H3-S10 phosphorylation and phosphoacetylation (Fig. 1A, panels ii, iii). These data indicate that the rapidly inducible phosphorylation of histone H3 initially demonstrated by [32P]phosphate labelling studies (Mahadevan et al., 1991
) is actually composed of the phosphorylation at two sites in the H3 tail, S10 and S28, although it does not address the issue of whether both sites become phosphorylated on the same H3 tail.
PhosphoS28-H3 is targeted to the TSA-hypersensitive fraction of histone H3
We have shown that a striking characteristic of S10-phosphorylated histone H3 is its extreme susceptibility to TSA-induced hyperacetylation. This is clearly detectable by shift of the phospho-epitope up the H3 `ladder' on acid-urea gels, where each shift corresponds to an additional modification event (Clayton et al., 2000). Further, TSA-induced H3-K9 acetylation of phosphoS10-H3 tails creates a new combined epitope specifically recognised by our phosphoacetyl antibody. By contrast, TSA-induced H3-K14 acetylation occludes recognition by our original anti-phosphoS10occ. antibody, evident from the diminished signal on western blots (Fig. 1B,C, panels iii), The new anti-phosphoS10 antibody is not affected in this way (Fig. 1B,C, panels iv).
S28-phosphorylated histone H3 is susceptible to TSA-induced hyperacetylation in two ways. First, TSA pretreatment increases the amount of phosphorylation detected with this antibody (Fig. 1C). We have recently shown a similar result using a different antibody (Soloaga et al., 2003), indicating that this is a genuine increase and not a quirk of the antibody with respect to TSA-induced acetylation of adjacent residues. Secondly, on acid-urea gels (Fig. 1B), phosphoS28-H3 tails are clearly shown to be hyperacetylated upon TSA-treatment, migrating markedly higher up the H3 ladder similar to the phosphoS10-H3 tails detected with anti-phosphoS10- or anti-phosphoacetyl-H3 antibodies (Fig. 1B, panels iv and ii respectively). This is consistent with our original [32P]phosphate metabolic labelling studies which showed that all radiolabelled histone H3 in these cells is hypersensitive to butryate-induced acetylation (Barratt et al., 1994
).
MSK1 phosphorylates histone H3 in octamers on both S10 and S28 in vitro
Because S10- and S28-phosphorylated H3 show similar profiles of appearance, are both subject to TSA-induced hyperacetylation and are both phosphorylated by the same kinase we asked whether both sites can be efficiently phosphorylated on the same H3 tail. Recombinant MSK1 (Deak et al., 1998) was incubated with a mixture of histones extracted from C3H 10T
cells, with purified free recombinant histone H3 or with octamers reconstituted from recombinant histones (H2A/H2B/H3/H4) in the presence of [
32P]ATP and phosphorylation monitored by autoradiography (Fig. 2A). Histone H3 extracted from C3H 10T
cells would have pre-existing mammalian histone modifications, whereas recombinant histones are free of these. In all cases, MSK1 clearly phosphorylates histone H3. Furthermore, stronger H3 phosphorylation is seen within reconstituted recombinant octamers compared to free recombinant H3 or acid extracts (Fig. 2A, lanes 2 and 6). Site-specificity of H3 phosphorylation was examined by performing identical in vitro reactions using unlabelled ATP and analysing reaction products by western blotting (Fig. 2B). For all three substrate types, both H3-S10 and H3-S28 become readily phosphorylated by MSK1 in vitro.
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PhosphoS10-H3 and phosphoS28-H3 are associated with different chromatin fragments
Their similar profiles of appearance in vivo, their common sensitivity to TSA-induced acetylation and the finding that MSK1 very efficiently phosphorylates both sites on the same tail in vitro raises the possiblity that both S10 and S28 become phosphorylated on the same H3 tails in vivo. This possibility was tested by immunodepleting solubilised crosslinked chromatin fragments of one phospho-epitope and asking whether the second phospho-epitope was also co-depleted or whether it remained in the unbound fraction. Cells were formaldehyde-crosslinked, nuclei isolated and chromatin fragments of an average size of 300-400 bp (encompassing 2-3 nucleosomes) released by sonication. Chromatin preparations from control and sAn-stimulated cells were immunodepleted with anti-phosphoS10occ., anti-phosphoacetyl- and anti-phosphoS28-H3 (Fig. 3A), and the loss other epitopes in the unbound fraction was examined by western blotting (Fig. 3A).
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The input chromatin used for immunoprecipitations shows clear induction of all three epitopes following stimulation (Fig. 3A, lanes 1-2, 9-10). In each case, mock immunodepletions in which antibodies were omitted showed no change in the amount of any of the epitopes present in the unbound fraction (Fig. 3A, compare lanes 3-4 with 1-2). Each of the three antibodies immunodepleted its cognate epitope with high efficiency (Fig. 3A; for anti-phosphoS10occ. panel i, compare lanes 5-6 with 1-2; for anti-phosphoacetyl H3 panel ii, compare lanes 7-8 with 1-2; for anti-phosphoS28 panel iii, compare lanes 11-12 with 9-10). Consistent with these modifications being present on a tiny fraction of histone H3, no effect on the total stainable amount of histone in the unbound fraction was detected (data not shown).
Remarkably, for each antibody, immunodepletion did not markedly reduce the amount of the other epitopes in the supernatant, indicating that these epitopes are located on different chromatin fragments. This is the expected result for nucleosomes recognised by anti-phosphoacetyl and anti-phosphoS10occ. because these antibodies recognise mutually exclusive populations of H3. Anti-phosphoS10occ. does not recognise phosphoacetyl-H3 due to occluding acetylation at Lys14 (Clayton et al., 2000) and conversely, the anti-phosphoacetyl antibody does not recognise H3 reactive to anti-phosphoS10occ. because the latter antibody recognises unacetylated phospho-H3 (evident from acid-urea gels, Fig. 1).
Most significantly, depletion with the anti-phosphoacetyl- or anti-phosphoS10occ. antibodies does not lead to any obvious depletion of the phosphoS28 epitope (Fig. 3A, panel iii, compare lanes 5-8 with 1-2). Similarly, depletion with anti-phosphoS28 does not affect the amount of phosphoacetyl-H3 or phosphoS10-H3 epitopes in the supernatant (Fig. 3A, panels i-ii, compare lanes 11-12 with 9-10). This surprising result suggests that phosphorylation of S28 is associated with different chromatin fragments from S10 phosphorylation and phosphoacetylation. Because the chromatin fragments used are 300-400 bp in length (2-3 nucleosomes), this raises the possibility that H3 phosphorylated at S28 has a distinct spatial distribution from that phosphorylated at S10 in the mouse nucleus. This would be remarkable, given that both are phosphorylated by the same kinase.
PhosphoS10- and phosphoS28-H3 are associated with different subnuclear foci
To directly address the relative spatial localisation of chromatin carrying S10- and S28-phosphorylation, a dual-staining immunofluorescence approach coupled with high resolution confocal microscopy was implemented. Control or sAn-stimulated C3H 10T cells were fixed and subjected to co-staining with the anti-phosphoS10- and anti-phosphoS28-H3 antibodies. Preliminary peptide-competition experiments demonstrated that both antibodies maintain strict site-specificity in immunofluorescence (M. Dyson, Site-specificity and targeting of histone H3 phosphorylation concomitant with Immediate-Early gene induction, PhD thesis; Oxford University, 2004), and the use of the non-occluded anti-phosphoS10 antibody ensured that all acetylated forms of phosphoS10-H3 were detectable. Without stimulation, nuclear staining with either of these antibodies is low. Following sAn-treatment, many nucleoplasmic foci of anti-phosphoS10- and anti-phosphoS28-H3 staining are observed in confocal sections (Fig. 3B). Remarkably, these foci generally do not colocalise and the two modifications are largely associated with different subnuclear locations (Fig. 3B, merged images). This is confirmed by examination of line-scan intensity profiles taken through each image, in which the peaks of intensity in each channel do not generally coincide (Fig. 3C).
CCF analysis (van Steensel et al., 1996) was also employed to test colocalisation of these two modifications (Fig. 3D). In CCF analysis, green and red line-scan profiles are shifted with respect to one another in a stepwise manner, one voxel at a time (
X), and the correlation (RP) at each point is recorded. A positive correlation between two signals is signified by a peak in the graph of
X against RP at
X=0, whereas two signals which are not correlated generate a flat graph with no peak at
X=0. Analysis of the anti-phosphoS10- and anti-phosphoS28-H3 staining patterns by this method gives a flat graph, indicating that any colocalisation between the two signals is no more than what would be expected if the two were randomly distributed with respect to one another (Fig. 3D, solid line). By contrast, similar analysis of nuclei in which anti-phosphoS10-H3 staining is detected by secondary antibodies that fluoresce in both red and green channels, which would be expected to show perfect colocalisation, produces a graph with a clear peak at
X=0 (Fig. 3D, broken line). Taken together, the data from immunodepletion experiments and confocal microscopy provide strong evidence that S10 and S28 phosphorylation are targeted to different populations of histone H3, and must be associated with different loci. Notice that, although limited data is presented here, these phenomena have been exhaustively analysed and confirmed elsewhere (M. Dyson, Site-specificity and targeting of histone H3 phosphorylation concomitant with Immediate-Early gene induction, PhD thesis; Oxford University, 2004)
Overexpression of GFP-tagged MSK1 in C3H 10T cells
A possible mechanism for strict targeting of S10 or S28 phosphorylation in the mouse nucleus is by restricting activation or localisation of the kinase to particular subnuclear regions or loci. To examine this, a C3H 10T cell line stably overexpressing GFP-MSK1 was established (Fig. 4). Immunofluorescence with anti-GFP demonstrates a high level of ectopic protein expression, and like its endogenous counterpart, the fusion protein is exclusively nuclear (Fig. 4A). Comparison of ectopic and endogenous protein bands in western blots indicates that the GFP-tagged protein is overexpressed to high levels (Fig. 4B).
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Finally, proof that the ectopically expressed GFP-MSK1 does have efficient enzymatic activity was obtained by in vitro kinase assay after recovering the kinase with anti-GFP-antibodies from these cells (Fig. 4D). Wild-type cells showed no anti-GFP-precipitated kinase activity. Likewise, kinase activity was low in quiescent cells expressing GFP-MSK1, but stimulation with either TPA (20 minutes) or sAn (30 minutes) led to an induction of kinase activity in the immunoprecipitates. The relative activity under these stimulation conditions compared well with the magnitude of the gel-shift (compare Fig. 4D, bars 5 and 6 with Fig. 4C, lanes 17 and 15, respectively). These data confirm that the overexpressed GFP-tagged kinase remains regulated, and that the GFP fusion does not compromise kinase activation or activity. This cell line was used to test aspects of possible mechanisms underlying the targeting of H3-S10 and H3-S28 phosphorylation to distinct loci in the nucleus.
Effect of GFP-MSK1 overexpression on substrate phosphorylation
If the exquisite targeting of H3 phosphorylation seen in vivo was owing to restricted localisation of MSK, its overexpression would result in breakdown of targeting and more H3 phosphorylation. Acid-extracts from wild-type and GFP-MSK1-overexpressing cells were separated by SDS-PAGE and examined for the various phospho-histone H3 epitopes by western blotting (Fig. 5A). Despite the presence of high levels of GFP-kinase, fully activatable by either TPA or EGF, overexpression had little effect on the amount of histone H3 which became S10-phosphorylated (Fig. 5A, panels i,ii), phosphoacetylated (Fig. 5A, panel iii) or S28-phosphorylated (Fig. 5A, panel iv).
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In addition to histone H3, the nucleosome-binding high-mobility group protein HMGN1 (previously known as HMG-14) and the transcription factors CREB and ATF1 are well-established MSK substrates (Wiggin et al., 2002). It was of interest to determine whether GFP-MSK1 overexpression had any effect on phosphorylation of these proteins. Phosphorylation of HMGN1 was analysed by western blotting of acid extracts with an anti-phosphoS6-HMGN1 antibody (Fig. 5A, panel v), or by monitoring the phosphorylation-dependent electrophoretic mobility retardation of this protein on acid-urea gels (Fig. 5B, panel i). Both of these approaches revealed that overexpression of GFP-MSK1 had little effect on the amount of HMGN1 that became phosphorylated. Similarly, western blots of nuclear extracts with an antibody specific for phospho-CREB and phospho-ATF1 revealed that overexpression had little effect on the phosphorylated proportions of these proteins after stimulation (Fig. 5C). Therefore, despite its clear activation, overexpression of GFP-MSK1 does not cause increase the amount of phosphorylation of its substrates. This result suggests that the action of this kinase is restricted in a way that prevents the deregulation of phosphorylation, even upon overexpression. This is in contrast to the many overexpression experiments with kinases that produce high levels and often inappropriate substrate phosphorylation.
Relationship between localisation of GFP-MSK1 and foci of histone phosphorylation in the nucleus
A possible explanation why the overexpression of GFP-MSK1 had no effect on substrate phosphorylation, might be that ectopic kinase was sequestered to particular subnuclear sites, thus physically restricting access of the kinase to its substrate. This might be particularly applicable for histone H3, which is known to be relatively immobile within the nucleus (Kimura and Cook, 2001). Wild-type or GFP-MSK1-overexpressing cells were stimulated as required and examined by confocal immunofluorescence microscopy to compare the location of the ectopic kinase to that of H3-phosphorylation foci in fixed nuclei (Fig. 6). Immunofluorescence directed against GFP showed widespread distribution of ectopic kinase throughout the nucleus (Fig. 6B). This distribution is in stark contrast with the pattern of anti-phosphoS28-H3 staining in the same nucleus, which remains restricted to foci even in the presence of overexpressed kinase (Fig. 6A,B). Notice that, the 20-minute TPA treatment used here results in almost quantitative kinase activation (Fig. 4C). It was possible to stain for GFP and phosphoS28-H3 simultaneously in the same cell because the antibodies against them were raised in different species (rabbit and rat, respectively); however, this simultaneous co-staining was not possible with phosphoS10-H3, these antibodies were also raised in rabbits. Nevertheless, it was still possible to compare the distribution of GFP and phosphoS10 more generally in different preparations (Fig. 6C).
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Discussion |
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This observation raises several issues of targeting. First, despite the complete activation of MSKs and the massive amount of potential substrate, the kinase only phosphorylates a minute defined fraction. This cannot be explained by limitations in the amount of kinase, because overexpression of MSK1 that is demonstrably highly active does not lead to greatly enhanced phosphorylation of histone H3. The targeting must therefore be explained either by the specific deployment of the kinase, for example by complexation with other proteins, or by tightly restricted availability of substrate. Alternatively, this phenomenon may be explained by prevalent phosphatase activity, which is discussed further below. Second, on any particular H3 tail to which MSKs are directed, the kinase selects either S10 or S28 for phosphorylation, not both. This selectivity is not an intrinsic property of this kinase-substrate relationship because, in vitro, using reconstituted recombinant octamers, MSK1 phosphorylates both sites on the same tail extremely efficiently. The distinct targeting of the two modifications can be explained by two possible mechanisms. One possibility is that availability of site on an H3 tail is controlled, for example by prior modification of sites adjacent to S10 or S28 that prevent or enhance phosphorylation. Another option is a spatial restriction of the kinase in vivo to one or the other site at these loci where the kinase and the H3 tail are in a tight complex with other proteins such that only one site, S10 or S28, is accessible to the kinase within each complex. The latter model would predict that the kinase exists in different complexed forms at these distinct loci. These possibilities are discussed further below.
Targeting based on properties of the histone substrate
This postulates some conformational property of the chromatin fibre, complexation of the H3 tail with other proteins or pre-existing modifications of these tails that renders site-specific modification more favourable. Although MSK1 very efficiently phosphorylates H3 in octamers, there is evidence that this ability is suppressed when DNA is also present to constitute nucleosomes (Zhang et al., 2004). Because virtually all H3 in the nucleus is nucleosomal, this may explain the lack of effect of overexpression of MSK1 in vivo. Presentation of the substrate is an important factor in histone-modification enzyme-assays. Enhanced histone acetyltransferase (HAT) activity of Gcn5-containing SAGA complex toward phosphorylated H3-tail peptides in vitro is lost when the substrate is presented as a nucleosomal array (Shogren-Knaak et al., 2003
). Furthermore, some modifications impact positively or negatively upon deposition of other modifications. Histone H3-K9 and H3-K4 methylation events are inhibitory to one another in vitro (Nishioka et al., 2002
), which may underpin their anti-correlation in vivo (Litt et al., 2001
; Noma et al., 2001
), whereas Rad6-mediated ubiquitination of H2B-K123 is required for both Set1-mediated H3-K4 methylation and H3-K79 methlyation by Dot1 (Briggs et al., 2002
; Sun and Allis, 2002
). The selective enhancement of S28 phosphorylation (but not that of S10) by TSA pretreatment could suggest a link between acetylation and S28 phosphorylation although, in C3H 10T
cells, TSA alone does not activate MAP kinase signalling (C. A. Hazzalin and L.C.M., unpublished observation) nor induce H3 phosphorylation at either site.
Targeting based on complexation of the kinase
The ability of associated accessory proteins to modulate the activity and site-specificity of histone-modifying enzymes is well-established. Incorporation into multi-subunit complexes promotes nucleosome-specific HAT or histone methyltransferase (HMT) activities of Gcn5 and E(Z), respectively (Grant et al., 1997; Muller et al., 2002
), whereas association of ESET with the mAM protein specifically enhances its ability to tri-methylate H3-K9 (Wang et al., 2003
). Furthermore, in chromatin, HMT activity of Ezh2 is directed towards either H3-K27 or H1-K26, depending upon which isoform of Eed protein it is complexed with (Kuzmichev et al., 2004
). Modulation of MSK activity by interactions within different complexes provides an attractive mechanism to target site-specific modifications to particular loci and to allow MSK to overcome its reported poor activity on nucleosomal H3 (Zhang et al., 2004
).
Targeting through differential phosphatase activity
It is possible that targeting is also regulated by the action of phosphatases that selectively reverse phosphorylation. In Drosophila, targeting of heat shock-induced histone H3-phosphorylation to responsive loci is partly mediated by inhibiting PP2A phosphatase at responsive loci (Nowak et al., 2003) (reviewed in Dyson et al., 2005
). There is also evidence that temporal differences in Aurora-B-mediated histone H3-S10 and H3-S28 phosphorylation during entry into mitosis are caused by PP1 phosphatase acting on S28 (but not S10) during late G2 phase (Goto et al., 2002
). S28 later becomes stably phosphorylated at the onset of M phase owing to inactivation of PP1 by Cdc2 kinase. The inabililty of GFP-MSK1 overexpression to deregulate histone phosphorylation might be because of dominant nuclear phosphatase activity directed towards H3. However, treatment of wild-type or GFP-MSK1-overexpressing cells with phosphatase inhibitors has failed to produce widespread, deregulated histone H3 phosphorylation (Mahadevan et al., 1991
) (M.H.D. and L.C.M., data not shown).
Functional implications of differential site-specific H3 phosphorylation
The molecular function of histone H3 phosphorylation during IE gene induction is unclear, although induction is attenuated under conditions where H3 phosphorylation is chemically or genetically ablated (Soloaga et al., 2003; Thomson et al., 1999
). It is clear that S10 phosphorylation is associated with Fos and Jun, but the genetic associations of S28 phosphorylation are largely unknown, and the subject of current study. Identification of two sites of phosphorylation on the H3 tail might have suggested that these act in concert, analogous to the proposed interaction between the double bromodomain of TAFII250 and the di-acetylated H3 tail (Agalioti et al., 2002
; Jacobson et al., 2000
). This is impossible if the two sites occur on different H3 tails. It may be that differentially phosphorylated H3 tails function as binding platforms for distinct factors whose recruitment imposes a different function locally within chromatin. Resolution of these issues awaits the determination of the molecular function and associations of differentially phosphorylated histone H3.
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Acknowledgments |
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Footnotes |
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Note added in proof Dunn and Davie report distinct localisation of S10- and S28-phosphorylated histone H3 in ras-transformed fibroblasts (Dunn and Davie, 2005).
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References |
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Agalioti, T., Chen, G. and Thanos, D. (2002). Deciphering the transcriptional histone acetylation code for a human gene. Cell 111, 381-392.[CrossRef][Medline]
Barratt, M. J., Hazzalin, C. A., Cano, E. and Mahadevan, L. C. (1994). Mitogen-stimulated phosphorylation of histone H3 is targeted to a small hyperacetylation-sensitive fraction. Proc. Natl. Acad. Sci. USA 91, 4781-4785.
Briggs, S. D., Xiao, T., Sun, Z. W., Caldwell, J. A., Shabanowitz, J., Hunt, D. F., Allis, C. D. and Strahl, B. D. (2002). Gene silencing: Trans-histone regulatory pathway in chromatin. Nature 418, 498.[CrossRef][Medline]
Cano, E., Hazzalin, C. A. and Mahadevan, L. C. (1994). Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun. Mol. Cell Biol. 14, 7352-7362.[Abstract]
Cano, E., Hazzalin, C. A., Kardalinou, E., Buckle, R. S. and Mahadevan, L. C. (1995). Neither ERK nor JNK/SAPK MAP kinase subtypes are essential for histone H3/HMG-14 phosphorylation or c-fos and c-jun induction. J. Cell Sci. 108, 3599-3609.
Cano, E., Doza, Y. N., Ben-Levy, R., Cohen, P. and Mahadevan, L. C. (1996). Identification of anisomycin-activated kinases p45 and p55 in murine cells as MAPKAP kinase-2. Oncogene 12, 805-812.[Medline]
Cheung, P., Tanner, K. G., Cheung, W. L., Sassone-Corsi, P., Denu, J. M. and Allis, C. D. (2000). Synergistic coupling of histone H3 phosphorylation and acetylation in response to epidermal growth factor stimulation. Mol. Cell 5, 905-915.[CrossRef][Medline]
Clayton, A. L. and Mahadevan, L. C. (2003). MAP kinase-mediated phosphoacetylation of histone H3 and inducible gene regulation. FEBS Lett. 546, 51-58.[CrossRef][Medline]
Clayton, A. L., Rose, S., Barratt, M. J. and Mahadevan, L. C. (2000). Phosphoacetylation of histone H3 on c-fos- and c-jun-associated nucleosomes upon gene activation. EMBO J. 19, 3714-3726.
Crosio, C., Fimia, G. M., Loury, R., Kimura, M., Okano, Y., Zhou, H., Sen, S., Allis, C. D. and Sassone-Corsi, P. (2002). Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian Aurora kinases. Mol. Cell Biol. 22, 874-885.
Deak, M., Clifton, A. D., Lucocq, L. M. and Alessi, D. R. (1998). Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB. EMBO J. 17, 4426-4441.
Dunn, K. L. and Davie, J. R. (2005). Stimulation of the Ras-MAPK pathway leads to independent phosphorylation of histone H3 on serine 10 and 28. Oncogene doi: 10.1038/sj.onc.1208521.[CrossRef]
Dyson M. H., Thomson, S. and Mahadevan, L. C. (2005). Heatshock, histone H3 phosphorylation and the Cell Cycle. Cell Cycle 4, 13-17.[Medline]
Edwards, D. R. and Mahadevan, L. C. (1992). Protein synthesis inhibitors differentially superinduce c-fos and c-jun by three distinct mechanisms: lack of evidence for labile repressors. EMBO J. 11, 2415-2424.[Abstract]
Goto, H., Tomono, Y., Ajiro, K., Kosako, H., Fujita, M., Sakurai, M., Okawa, K., Iwamatsu, A., Okigaki, T., Takahashi, T. et al. (1999). Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation. J. Biol. Chem. 274, 25543-25549.
Goto, H., Yasui, Y., Nigg, E. A. and Inagaki, M. (2002). Aurora-B phosphorylates Histone H3 at serine28 with regard to the mitotic chromosome condensation. Genes Cells 7, 11-17.
Grant, P. A., Duggan, L., Cote, J., Roberts, S. M., Brownell, J. E., Candau, R., Ohba, R., Owen-Hughes, T., Allis, C. D., Winston, F. et al. (1997). Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex. Genes Dev. 11, 1640-1650.[Abstract]
Gurley, L. R., D'Anna, J. A., Barham, S. S., Deaven, L. L. and Tobey, R. A. (1978). Histone phosphorylation and chromatin structure during mitosis in Chinese hamster cells. Eur. J. Biochem. 84, 1-15.[CrossRef][Medline]
Hauf, S., Cole, R. W., LaTerra, S., Zimmer, C., Schnapp, G., Walter, R., Heckel, A., van Meel, J., Rieder, C. L. and Peters, J. M. (2003). The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint. J. Cell Biol. 161, 281-294.
Hazzalin, C. A., Cano, E., Cuenda, A., Barratt, M. J., Cohen, P. and Mahadevan, L. C. (1996). p38/RK is essential for stress-induced nuclear responses: JNK/SAPKs and c-Jun/ATF-2 phosphorylation are insufficient. Curr. Biol. 6, 1028-1031.[CrossRef][Medline]
Hendzel, M. J., Wei, Y., Mancini, M. A., van Hooser, A., Ranalli, T., Brinkley, B. R., Bazett-Jones, D. P. and Allis, C. D. (1997). Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348-360.[CrossRef][Medline]
Jacobson, R. H., Ladurner, A. G., King, D. S. and Tjian, R. (2000). Structure and function of a human TAFII250 double bromodomain module. Science 288, 1422-1425.
Kimura, H. and Cook, P. R. (2001). Kinetics of core histones in living human cells: little exchange of H3 and H4 and some rapid exchange of H2B. J. Cell Biol. 153, 1341-1353.
Kuzmichev, A., Jenuwein, T., Tempst, P. and Reinberg, D. (2004). Different EZH2-containing complexes target methylation of histone H1 or nucleosomal histone H3. Mol. Cell 14, 183-193.[CrossRef][Medline]
Leevers, S. J. and Marshall, C. J. (1992). Activation of extracellular signal-regulated kinase, ERK2, by p21ras oncoprotein. EMBO J. 11, 569-574.[Abstract]
Litt, M. D., Simpson, M., Gaszner, M., Allis, C. D. and Felsenfeld, G. (2001). Correlation between histone lysine methylation and developmental changes at the chicken beta-globin locus. Science 293, 2453-2455.
Mahadevan, L. C., Willis, A. C. and Barratt, M. J. (1991). Rapid histone H3 phosphorylation in response to growth factors, phorbol esters, okadaic acid, and protein synthesis inhibitors. Cell 65, 775-783.[CrossRef][Medline]
Muller, J., Hart, C. M., Francis, N. J., Vargas, M. L., Sengupta, A., Wild, B., Miller, E. L., O'Connor, M. B., Kingston, R. E. and Simon, J. A. (2002). Histone methyltransferase activity of a Drosophila Polycomb group repressor complex. Cell 111, 197-208.[CrossRef][Medline]
Nishioka, K., Chuikov, S., Sarma, K., Erdjument-Bromage, H., Allis, C. D., Tempst, P. and Reinberg, D. (2002). Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding histone tail modifications required for heterochromatin formation. Genes Dev. 16, 479-489.
Noma, K., Allis, C. D. and Grewal, S. I. (2001). Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries. Science 293, 1150-1155.
Nowak, S. J., Pai, C. Y. and Corces, V. G. (2003). Protein phosphatase 2A activity affects histone H3 phosphorylation and transcription in Drosophila melanogaster. Mol. Cell Biol. 23, 6129-6138.
Paulson, J. R. and Taylor, S. S. (1982). Phosphorylation of histones 1 and 3 and nonhistone high mobility group 14 by an endogenous kinase in HeLa metaphase chromosomes. J. Biol. Chem. 257, 6064-6072.
Pierrat, B., Correia, J. S., Mary, J. L., Tomas-Zuber, M. and Lesslauer, W. (1998). RSK-B, a novel ribosomal S6 kinase family member, is a CREB kinase under dominant control of p38alpha mitogen-activated protein kinase (p38alphaMAPK). J. Biol. Chem. 273, 29661-29671.
Prigent, C. and Dimitrov, S. (2003). Phosphorylation of serine 10 in histone H3, what for? J. Cell Sci. 116, 3677-3685.
Rutault, K., Hazzalin, C. A. and Mahadevan, L. C. (2001). Combinations of ERK and p38 MAPK inhibitors ablate tumor necrosis factor-alpha (TNF-alpha) mRNA induction. Evidence for selective destabilization of TNF-alpha transcripts. J. Biol. Chem. 276, 6666-6674.
Shogren-Knaak, M. A., Fry, C. J. and Petersen, C. L. (2003). A native peptide ligation strategy for deciphering nucleosomal histone modifications, J. Biol. Chem. 278, 15744-15748.
Soloaga, A., Thomson, S., Wiggin, G. R., Rampersaud, N., Dyson, M. H., Hazzalin, C. A., Mahadevan, L. C. and Arthur, J. S. (2003). MSK2 and MSK1 mediate the mitogen- and stress-induced phosphorylation of histone H3 and HMG-14. EMBO J. 22, 2788-2797.
Sun, Z. W. and Allis, C. D. (2002). Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature 418, 104-108.[CrossRef][Medline]
Thomson, S., Clayton, A. L., Hazzalin, C. A., Rose, S., Barratt, M. J. and Mahadevan, L. C. (1999). The nucleosomal response associated with immediate-early gene induction is mediated via alternative MAP kinase cascades: MSK1 as a potential histone H3/HMG-14 kinase. EMBO J. 18, 4779-4793.
Thomson, S., Clayton, A. L. and Mahadevan, L. C. (2001). Independent dynamic regulation of histone phosphorylation and acetylation during immediate-early gene induction. Mol. Cell 8, 1231-1241.[CrossRef][Medline]
Thomson, S., Hollis, A., Hazzalin, C. A. and Mahadevan, L. C. (2004) Distinct stimulus-specific histone modifications at Hsp70 chromatin targeted by the transcription factor Heat Shock Factor 1. Mol. Cell 15, 585-594.[CrossRef][Medline]
van Steensel, B., van Binnendijk, E. P., Hornsby, C. D., van der Voort, H. T., Krozowski, Z. S., de Kloet, E. R. and van Driel, R. (1996). Partial colocalization of glucocorticoid and mineralocorticoid receptors in discrete compartments in nuclei of rat hippocampus neurons. J. Cell Sci. 109, 787-792.
Wang, H., An, W., Cao, R., Xia, L., Erdjument-Bromage, H., Chatton, B., Tempst, P., Roeder, R. G. and Zhang, Y. (2003). mAM facilitates conversion by ESET of dimethyl to trimethyl lysine 9 of histone H3 to cause transcriptional repression. Mol. Cell. 12, 475-487.[CrossRef][Medline]
Wiggin, G. R., Soloaga, A., Foster, J. M., Murray-Tait, V., Cohen, P. and Arthur, J. S. (2002). MSK1 and MSK2 are required for the mitogen- and stress-induced phosphorylation of CREB and ATF1 in fibroblasts. Mol. Cell Biol. 22, 2871-2881.
Williams, M. R., Arthur, J. S., Balendran, A., van der Kaay, J., Poli, V., Cohen, P. and Alessi, D. R. (2000). The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells. Curr. Biol. 10, 439-448.[CrossRef][Medline]
Zhang, Y., Griffin, K., Mondal, N. and Parvin, J. D. (2004). Phosphorylation of histone H2A inhibits transcription on chromatin templates. J. Biol. Chem. 279, 21866-21872.
Zhong, S., Jansen, C., She, Q. B., Goto, H., Inagaki, M., Bode, A. M., Ma, W. Y. and Dong, Z. (2001a). Ultraviolet B-induced phosphorylation of histone H3 at serine 28 is mediated by MSK1. J. Biol. Chem. 276, 33213-33219.
Zhong, S., Zhang, Y., Jansen, C., Goto, H., Inagaki, M. and Dong, Z. (2001b). MAP kinases mediate UVB-induced phosphorylation of histone H3 at serine 28. J. Biol. Chem. 276, 12932-12937.
Zhong, S., Goto, H., Inagaki, M. and Dong, Z. (2003). Phosphorylation at serine 28 and acetylation at lysine 9 of histone H3 induced by trichostatin A. Oncogene 22, 5291-5297.[CrossRef][Medline]
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