Promoter chromatin remodeling of immediate-early genes is mediated through H3 phosphorylation at either serine 28 or 10 by the MSK1 multi-protein complex

Generation and maintenance of MSK1 stable knockdown mouse fibroblasts

Empty GIPZ lentiviral vector and the GIPZ Lentiviral shRNAmir clones for mouse MSK1 (clone V2LMM_54318- SENSE 2240, Thermo Scientific Open Biosystems, USA) were obtained from the Biomedical Functionality Resource at University of Manitoba. To produce replication-incompetent lentiviral vector particles for mouse fibroblasts transduction, we transfected the GIPZ vector, the packaging vector and the VSV-G env-expressing plasmid into HEK293T cells using the BD CalPhos™ Mammalian Transfection kit, as described previously (32). To generate stable MSK1 knockdown 10T1/2 mouse fibroblasts, we seeded 5 × 10⁴ cells in a 24-well plate 24 h before transduction. Medium was aspirated and replaced with 250 µl of the lentiviral supernatant (at 1 × 10⁶ TU) and 8 μg/ml of Polybrene. After incubation for 2 h at 37°C in 5% CO₂, the culture medium that contained the lentiviral vectors was removed. The transduced cells were cultured in 1 ml of fresh media for 3 days before they were selected for puromycin resistance (at a final concentration of 8 μg/ml). Freshly prepared selective media was added every 2–3 days and the cells were monitored for cell survival by light microscopy, and/or for the TurboGFP expression by flow cytometry using BD FACSCalibur. After 10 days, puromycin-resistant cells were expanded in puromycin-containing media until the desired confluency was reached.

ChIP/reChIP assay

10T1/2 cells were treated with 1% formaldehyde for 10 min at room temperature. After harvesting, the cells were pelleted and resuspended in cell lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM NaF, 1.0 µg/ml leupeptin, 1 µg/ml aprotinin and 25 mM β-glycerophosphate). After 10 min rotation at 4°C the cellular material was spun at 2000 × g for 10 min to obtain the nuclei. The nuclear pellet was resuspended in MNase digestion buffer (10 nM Tris–HCl pH 7.5, 0.25 M sucrose, 75 mM NaCl, plus above indicated phosphatase/protease inhibitors) and A₂₆₀ was measured. In order to obtain ∼150 bp DNA fragments, 2.5 U of MNase per A₂₆₀ of nuclear suspension were added in the presence of 3 mM CaCl₂ and incubated at 37°C for 20 min. The MNase reaction was stopped by the addition of EDTA pH 8.0 (5 mM final concentration). In order to release nuclear material, the samples were adjusted to 0.5% SDS and rotated for 1 h at room temperature. Insoluble material was pelleted at 2000 × g for 10 min and the soluble material was diluted to 0.1% SDS with radio immuno precipitation assay (RIPA) buffer along with above-mentioned phosphatase/protease inhibitors. Twelve A₂₆₀ of protein G-Sepharose (Pierce) pre-cleared 10T1/2 lysate was incubated with 12 µl of anti-MSK1 (Sigma, M5437) or anti-H3S10ph (Santa Cruz, sc-8656-R), anti-H3S28ph (Milipore, 07–145), anti-H3K9acK14ac (Milipore, 06–599), anti-14-3-3ε (Santa Cruz, sc-1020), anti-14-3-3ζ (Santa Cruz, sc-1019), anti-BRG1 (Milipore, 07–478), anti-PCAF (AbCam, ab12188-50), anti-JUN (Santa Cruz, sc-1694), or anti-RNAPII S5ph (AbCam, ab5131-50) overnight at 4°C. Magnetic protein G Dynabeads (Invitrogen) were added for 2 h at 4°C. The specificity of the antibodies against H3S10ph and H3S28ph was determined in several ways. In immunoblot experiments, both antibodies only detected H3. Using commercial (Abcam) H3 peptides with S10ph or S28ph, these antibodies only recognized one or the other peptide. With regards to acetylation, we previously reported that these antibodies detected S10ph or S28ph when H3 was hyperacetylated (9). With regards to the MSK1 antibody, in immunoblot experiments, we found that the antibody only detected one band. We have also shown that in MSK1 knockdown experiments, the MSK1 signals detected in immunoblot experiments, were greatly diminished. For reChIP assays, after the elution of first ChIP, the samples were diluted 10 times with dilution buffer (15 mM Tris–HCl pH 8.1, 1% Triton X-100, 1 mM EDTA, 150 mM NaCl) and subjected to the ChIP procedure again. Negative control included performing ChIP/reChIP assays without adding antibody. DNA–protein fragments were processed as previously described (33). Input and ChIP/reChIP DNAs were quantified using PicoGreen assay. Equal amounts of input, ChIP (0.1 ng) or reChIP (0.05 ng) DNA were used to perform SYBR Green real time PCR on iCycler IQ5 (BioRad). The enrichment was calculated as follows: Fold enrichment = R^{(Ct input – Ct ChIP)}, where R is the rate of amplification. Values for each time point were normalized to time 0 values. Primer sequences were designed using Oligo5 software and are shown in Supplementary Table S1.

Preparation of cell extracts and immunoprecipitations

Cells were harvested and lysed in 400 µl of ice-cold Nonidet P40 (NP-40) buffer (150 mM NaCl, 50 mM Tris–HCl pH 8.0, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM NaF, 1.0 µg/ml leupeptin, 1.0 µg/ml aprotinin, 25 mM β-glycerophosphate). Cell extracts were subjected to centrifugation at 11 950 × g for 10 min at 4°C, and the supernatant was saved. Protein concentration of the supernatant was determined using the BCA protein assay (BioRad). For immunoprecipitations in HEK293 cells, 500 µg of total cell extract were incubated with 20 µg of anti-MSK1 antibodies overnight at 4°C. Twenty microliters of protein G-Sepharose beads were added the following day and incubated for 2 h at 4°C. The beads were washed with ice-cold NP-40 buffer two times. Input (20 µg), bound, un-bound and non-specific (no antibody IP) fractions were resolved on SDS–10%-PAGE and immunoblotted with indicated antibodies. In some instances, non-specific antibodies (anti-FLAG) were used in control immunoprecipitations. For immunoprecipitations in cycling 10T1/2 cells, 20 µg of anti-MSK1 antibodies were incubated with 1 mg of total cell extract overnight at 4°C. Further processing of the samples was as described above. For sequential immunoprecipitations, 30 min TPA-treated 10T1/2 cells were treated with membrane-permeable protein cross-linking reagent dimethyl 3.3′-dithiobisproprionamidate-2-HCl (DTBP, 2 mM, Pierce) for 30 min at 37°C. The reaction was stopped with ice-cold 0.1 M Tris–HCl (pH 7.5) and the cells were lysed and incubated with anti-MSK1 antibodies as described above. The immunoprecipitated proteins were eluted with 0.2 M glycine (pH 2.6) for 10 min at room temperature and re-immunoprecipitated with antibodies against BRG1.

Recruitment of 14-3-3 proteins and chromatin remodelers/modifiers to regulatory regions of induced genes in response to ERK-MAPK signaling

As 14-3-3 isoforms were reported to be effectors of H3 phosphorylation at inducible genes (29), we performed ChIP assays to determine the distribution of 14-3-3ε or 14-3-3ζ along the Jun, Cox-2 and Fosl1 genes before and after their induction with TPA. Figure 2 shows that, upon TPA-stimulation of quiescent 10T1/2 cells, 14-3-3ε and 14-3-3ζ associated with the same regulatory regions of the Jun, Cox-2 and Fosl1 genes as did H3S10ph and H3S28ph. BRG1, the ATPase subunit of the SWI/SNF remodeling complex, and PCAF, an H3 K-acetyltransferase (also known as KAT2B) also associated with the same regulatory regions. In the case of the Cox-2 gene, BRG1 and PCAF, to a lesser extent, associated with the 5′ distal region (−493) upon TPA stimulation, while the TPA-induced association of BRG1 with the 5′ proximal region (−111) was weaker, and TPA-induced binding of PCAF to the −111 region was negligible (Figure 2). Yet, TPA-induced H3 acetylation was evident at both regulatory regions (−493 and −111) of the Cox-2 gene (Figure 1B).

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Figure 2.

TPA-induced recruitment of 14-3-3 proteins and chromatin remodelers/modifiers to Jun, Cox-2 and Fosl1 regulatory regions. ChIP experiments were performed using antibodies against 14-3-3ε, 14-3-3ζ, BRG1 or PCAF on formaldehyde-crosslinked mononucleosomes prepared from serum-starved 10T1/2 cells treated with TPA for 0, 15 or 30 min. Enrichment values, obtained by real-time quantitative PCR assays on equal amounts of input and immunoprecipitated DNA, are the mean of three independent experiments, and the error bars represent the standard deviation.

14-3-3-Mediated interactions between MSK1 and chromatin remodelers/modifiers at regulatory regions of induced genes in response to ERK-MAPK signaling

The function of 14-3-3 proteins in IE gene expression is currently unknown. Since they exist as homodimers and heterodimers, it is possible that 14-3-3 proteins act as scaffolds to anchor proteins at the promoter of IE genes (39). For example, one 14-3-3 site would bind to H3S10ph or H3S28ph, while the other site would bind to a component of a nucleosome remodeling complex. To test this hypothesis, we checked if MSK1 could be co-immunoprecipitated with antibodies raised against 14-3-3. Figure 3A shows that MSK1 was present in the anti-14-3-3ζ immunoprecipitate from a 10T1/2 cell lysate. Reciprocally, anti-MSK1 antibodies co-precipitated BRG1 and 14-3-3ζ, while anti-BRG1 antibodies co-precipitated MSK1 and 14-3-3ζ (Figure 3A). Thus, in 10T1/2 cells, 14-3-3ζ is associated with BRG1 and MSK1. These interactions were also detected in HEK293 cells, where 14-3-3 proteins and BRG1 were co-immunoprecipitated with antibodies against MSK1 (Figure 3A). PCAF and NF-κB p65 transcription factor, but not histone deacetylase 1 (HDAC1), were also co-immunoprecipitated with anti-MSK1 antibodies (Figure 3A). To determine if a ternary complex is formed between MSK1, 14-3-3 and BRG1, cell lysates from TPA-stimulated 10T1/2 cells were subjected to sequential immunoprecipitations first with antibodies against MSK1, and second with antibodies against BRG1. Figure 3A shows 14-3-3ζ is part of a complex including MSK1 and BRG1. To determine if these interactions occur at the promoter region of IE genes, re-ChIP assays were performed. Formaldehyde-crosslinked mononucleosomes from 10T1/2 cells treated or not for 30 min with TPA were subjected to the ChIP procedure with anti-MSK1 antibodies and again precipitated using anti-H3S10ph antibodies. Figure 3B shows that the MSK1-H3S10ph re-ChIP assay resulted in a marked increase in DNA precipitation of the proximal regulatory regions from the Jun (−146), Cox-2 (−111) and Fosl1 (−187) genes when cells were treated with TPA. Conversely, the H3S10ph–H3S28ph re-ChIP assay did not result in a significant TPA-induced increase of immunoprecipitation of the proximal regulatory regions from the Jun and Fosl1 genes, and only showed a slight increase in the case of Cox-2. These data demonstrate that, upon TPA stimulation, MSK1 and H3S10ph are found together on the same proximal regulatory region from the Jun, Cox-2 and Fosl1 genes, while a nucleosome from the Jun or Fosl1 promoter region has either H3S10ph or H3S28ph. In the case of the Cox-2 promoter region, a low level of nucleosomes including both H3S10ph and H3S28ph was detected. Other re-ChIP experiments performed on the same crosslinked mononucleosome preparations demonstrated that following TPA treatment of 10T1/2 cells, there was an increase in co-occupancy of the proximal regulatory regions from the Jun, Cox-2 and Fosl1 genes by MSK1 and 14-3-3ζ, 14-3-3ζ and BRG1, MSK1 and BRG1, as well as MSK1 and PCAF (Figure 3B). On the other hand, a TPA-induced increase in co-occupancy of the proximal regulatory region by 14-3-3ζ and 14-3-3ε was detected for the Jun and Fosl1 genes, but not for the Cox-2 gene (Figure 3B).

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Figure 3.

MSK1, 14-3-3 proteins and BRG1 complex formation and TPA-induced co-occupancy of MSK1, 14-3-3 proteins and chromatin remodelers/modifiers to Jun, Cox-2 and Fosl1 regulatory regions. (A) Aliquots of 1 mg 10T 1/2 (left panel) or 500 μg HEK293 (middle panel) total cell extracts were incubated with anti-14-3-3ζ, anti-MSK1 or anti-BRG1 antibodies. Alternatively, 1 mg total cell extract from serum-starved 10T1/2 cells stimulated with TPA for 30 min and treated with DTBP was sequentially incubated with anti-MSK1 and anti-BRG1 antibodies (right panel). The whole ‘Bound’ and ‘Non-specific’ fractions and equivalent volumes of ‘Input’ and ‘Un-bound’ fractions, corresponding to 25 μg of total cell extracts were resolved on SDS–10%-PAGE and immunoblotted with indicated antibodies (IB). (B) Re-ChIP experiments were performed on formaldehyde-crosslinked mononucleosomes prepared from serum-starved 10T1/2 cells treated with TPA for 0 or 30 min. The antibodies were used as indicated. Equal amounts of input and immunoprecipitated DNA were quantified by real-time quantitative PCR. The enrichment values of the 5′ proximal sequences of Jun (−146), Cox-2 (−111) and Fosl1 (−187) genes are the mean of three independent experiments, and the error bars represent the standard deviation.

While it is well known that H3S10ph is associated with the induction of IE genes in different cell types, so far there has only been one study reporting the association of H3S28ph with the Jun IE gene promoter region (12,40). To elaborate on the contribution of H3S28ph in the remodeling of IE gene promoter regions, we performed sequential ChIP assays to determine whether there would be co-recruitment of H3S28ph and 14-3-3ε, 14-3-3ζ or BRG1 to the proximal regulatory regions from the Jun (−146), Cox-2 (−111) and Fosl1 (−187) genes when cells were treated with TPA. Figure 3B shows that 14-3-3ε, 14-3-3ζ and BRG1 were associated with H3S28ph at the promoter region of these IE genes upon TPA induction. Figure 3B also demonstrates an association of 14-3-3ε with H3S10ph. These data demonstrate that either MSK-induced phosphorylation of H3 S10 or S28 of IE gene promoter region nucleosomes is involved in the events leading to the chromatin remodeling of IE gene promoter regions.

Inhibition by MSK inhibitor H89 of IE gene transcriptional induction in response to ERK-MAPK signaling

At a concentration of 10 μM, the MSK inhibitor H89 has been shown to selectively inhibit the nucleosomal response (41). Supplementary Figure S1A shows that the TPA-induced expression of the Jun, Cox-2 and Fosl1 genes was abolished when quiescent 10T1/2 cells were exposed to H89 prior to TPA treatment. Conversely, the GAPDH expression levels were not affected by H89 pre-treatment [data not shown and (7)]. As H89 is an equally potent inhibitor of another H3 kinase, protein kinase A (PKA) (42), we analyzed the effects of Rp-cAMP, a specific PKA inhibitor, on the induction of IE genes. We found that prior exposure of quiescent 10T1/2 cells to Rp-cAMP did not affect the TPA-induced transcription of IE genes (Supplementary Figure S2), demonstrating that the effects of H89 were due to the inhibition of MSK. As revealed by ChIP assays, RNA polymerase II phosphorylated at serine 5 (RNAPII S5ph), the initiation-engaged form of RNAPII (43), was not associated with the promoter region of Jun, Cox-2 and Fosl1, upon TPA stimulation of H89 pre-treated 10T1/2 cells (Supplementary Figure S1B). These results suggest that the formation of an initiation complex at the 5′ end of IE genes requires a chromatin remodeling step which is dependent on the MSK-mediated nucleosomal response. To test this hypothesis, we analyzed by ChIP assays the effect of H89 treatment of quiescent 10T1/2 cells on the TPA-induced MSK1 recruitment, nucleosomal response and 14-3-3ε, 14-3-3ζ, BRG1 and PCAF binding to the upstream regulatory regions of the Jun, Cox-2 and Fosl1 genes. Results for the proximal 5′ regions of the three genes [Jun (−146), Cox-2 (−111) and Fosl1 (−187)] demonstrated that the TPA-induced MSK1 recruitment and ensuing H3 S10 and S28 phosphorylation were abolished, when 10T1/2 cells were exposed to H89 before TPA treatment (Figure 4A). Similarly, TPA-induced binding of 14-3-3ε, 14-3-3ζ, BRG1 and PCAF, as well as TPA-induced H3 acetylation at the 5′ proximal region of the three genes [Jun (−146), Cox-2 (−111) and Fosl1 (−187)] were markedly reduced following H89 pre-treatment of cells (Figure 4A). Although results on BRG1 and PCAF occupancy of the Cox-2 promoter region (−111) were less clear, TPA-induced H3 acetylation was reduced by prior exposure of 10T1/2 cells to H89 (Figure 4A). Analysis of the immunoprecipitated DNA for the 5′ distal regulatory regions of the three genes [Jun (−711), Cox-2 (−493) and Fosl1 (−1113)] showed that TPA-induced MSK1 recruitment, H3 phosphoacetylation and binding of 14-3-3ε, 14-3-3ζ, BRG1 and PCAF were similarly affected at these upstream regulatory regions by H89 pre-treatment of cells (Supplementary Figure S3).

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Figure 4.

Inhibition by H89 of TPA-induced nucleosomal response and chromatin remodeler/modifier recruitment to the Jun, Cox-2 and Fosl1 5′ proximal regulatory regions. (A) Serum-starved 10T1/2 cells were pre-treated or not with H89 prior to TPA stimulation for 0, 15 or 30 min. Formaldehyde crosslinked mononucleosomes were prepared and used in ChIP assays with antibodies against MSK1, H3S10ph, H3S28ph, 14-3-3ε, 14-3-3ζ, BRG1, PCAF or H3K9acK14ac. Equal amounts of input and immunoprecipitated DNAs were quantified by real-time quantitative PCR. The enrichment values of the 5′ proximal sequences of Jun (−146), Cox-2 (−111) and Fosl1 (−187) genes are the mean of three independent experiments, and the error bars represent the standard deviation. (B) Serum-starved 10T1/2 cells were pre-treated with H89 prior to TPA stimulation for 0 or 30 min. Aliquots of 1 mg total cell extract were incubated with anti-MSK1 antibodies. The whole ‘Bound’ and ‘Non-specific’ fractions and equivalent volumes of ‘Input’ and ‘Un-bound’ fractions, corresponding to 25 μg of total cell extracts were resolved on SDS–10%-PAGE and immunoblotted with indicated antibodies (IB).

The lack of MSK1 recruitment to the promoter regions of IE genes following H89 pre-treatment of cells suggests that the recruitment of MSK1 is dependent on its kinase activity, which is required to activate the complex such that it is recruited to the IE gene promoter regions. An alternative explanation could be that H89 interferes with the formation of the MSK1–14-3-3–SWI/SNF complex. To explore this latter possibility, we tested the ability of anti-MSK1 antibodies to co-immunoprecipitate 14-3-3ζ or BRG1 when quiescent 10T1/2 cells were exposed to H89 prior to TPA treatment. Figure 4B shows that BRG1 and 14-3-3ζ formed complexes with MSK1 in 10T1/2 cells pre-treated with H89 and TPA-stimulated for 0 or 30 min. Thus, H89 did not prevent the formation of MSK1 complexes, and its negative effect on promoter region remodeling and IE gene induction is most likely due to the inhibition of MSK1 kinase activity.

The AP-1 transcription factor is a homodimer or heterodimer consisting of proteins belonging to the JUN, FOS (including FRA) and ATF families, and is involved in the regulation of IE genes, including genes coding for its components. We investigated the TPA-induced association of JUN with the regulatory regions of the Jun, Cox-2 and Fosl1 by ChIP assay. Figure 5 shows that, upon TPA stimulation, JUN binds to the 5′ proximal, but not distal, regulatory regions of Jun, Cox-2 and Folsl1, as well as the regulatory region located in the first intron of Folsl1 (989). These results are in agreement with the mapping of known JUN and AP-1 binding sites and TPA-responsive element (TRE). Moreover, Figure 5 shows that, when 10T1/2 cells were treated with H89 prior to TPA stimulation, JUN association with its DNA binding sites was abolished for each of the three genes. This indicates that the binding of JUN to its cognate DNA sequences in the three investigated genes was dependent on the TPA-induced nucleosomal response.

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Figure 5.

Inhibition by H89 of TPA-induced JUN recruitment to the Jun, Cox-2 and Fosl1 regulatory regions. Serum-starved 10T1/2 cells were pre-treated or not with H89 prior to TPA stimulation for 0, 15 or 30 min. Formaldehyde crosslinked mononucleosomes were prepared and used in ChIP assays with anti-JUN antibodies. Enrichment values, obtained by real-time quantitative PCR assays on equal amounts of input and immunoprecipitated DNAs, are the mean of three independent experiments, and the error bars represent the standard deviation.

The authors thank Geneviève Delcuve for manuscript preparation.