Induction of Progesterone Target Genes Requires Activation of Erk and Msk Kinases and Phosphorylation of Histone H3

Molecular Cell 24, 367–381, November 3, 2006 ª2006 Elsevier Inc.

DOI 10.1016/j.molcel.2006.10.011

Induction of Progesterone Target Genes Requires Activation of Erk and Msk Kinases and Phosphorylation of Histone H3 Guillermo P. Vicent,1 Cecilia Ballare´,1 A. Silvina Nacht,1 Jaime Clausell,1 Alicia Subtil-Rodrı´guez,1 Ignacio Quiles,1 Albert Jordan,1 and Miguel Beato1,* 1 Centre de Regulacio´ Geno`mica Universitat Pompeu Fabra Parc de Recerca Biome`dica Dr. Aiguader 88 E-08003 Barcelona Spain

Summary How genes are regulated in the context of chromatin is a central question of biology. Steroid hormones control gene expression via interaction of their receptors with target sequences on DNA but can also activate cytoplasmic signaling cascades. Here we report that rapid Erk activation by progestins participates in induction of target genes by preparing the chromatin for transcription. Five minutes after hormone treatment, Erk activation leads to phosphorylation of the progesterone receptor (PR), activation of Msk1, and recruitment of a complex of the three proteins to a nucleosome on the MMTV promoter. Msk1 phosphorylates histone H3, leading to displacement of HP1g and recruitment of Brg1 and RNA polymerase II. Cell-free experiments show a direct interaction between PR, Erk, and Msk1 and support the importance of H3 phosphorylation for nucleosome remodeling. Inhibition of Msk1 activation blocks recruitment of the kinase complex, H3 phosphorylation, and HP1g displacement, thus precluding remodeling and induction of the promoter. Introduction Steroid hormone receptors (SHR) are ligand-dependent sequence-specific transcription factors, which are activated by binding of the corresponding hormones in a process involving homodimerization and binding to hormone-responsive elements (HREs) in regulated genes. SHRs can also activate genes lacking HREs by interaction with other sequence-specific transcription factors bound to their target sequences. But even in genes with HREs, transactivation by SHRs often requires a synergistic interaction with other sequence-specific transcription factors (Beato et al., 1995). How SHRs locate their target sequences in nuclear chromatin is still poorly understood. SHRs can gain access to HREs exposed in the surface of nucleosomes (Pin˜a et al., 1990), but efficient binding of receptors to complex hormone regulatory regions and subsequent transactivation require remodeling of chromatin by ATP-dependent complexes (Muchardt and Yaniv, 1993; Yoshinaga et al., 1992). These complexes use the energy of ATP hydrolysis to make hidden HREs and other cis-regulatory elements accessible for binding of the *Correspondence: [email protected]

cognate factors (Di Croce et al., 1999), which act as nucleation points for recruitment of further coregulators and the basal transcriptional machinery (Kinyamu and Archer, 2004). In addition to these genomic effects, steroid hormones are known to crosstalk to kinase cascades activated by signals impinging on membrane receptors. Estrogens activate the Src/p21ras/Erk and the PI3K/Akt pathways via direct interaction of the estrogen receptor (ER) with c-Src and the regulatory subunit of PI3K, respectively (Castoria et al., 2001; Migliaccio et al., 1996). Progestins can also crosstalk to kinase cascades through an interaction of progesterone receptor (PR) with c-Src (Ballare et al., 2003; Boonyaratanakornkit et al., 2001). However, in breast cancer cells containing ER, the progestin effect on the Src/Erk pathway is mediated by an interaction of two domains of PR with the ligand-binding domain of ER (Ballare et al., 2003), which is activated in the absence of estrogens and triggers activation of the cascades (Migliaccio et al., 1998). Consequently, progestin induction of cell proliferation is inhibited, not only by antiprogestins but also by antiestrogens, as well as by inhibitors of kinase activation (Migliaccio et al., 1998). The ultimate targets of the activated kinase cascades are not known but likely include transcription factors and coregulators (Bjornstrom and Sjoberg, 2005). Traditionally, the nongenomic and genomic actions of steroid hormones have been considered as two independent pathways. However, during activation of immediateearly genes by diverse stimuli, the chromatin over the c-fos and c-jun genes becomes rapidly and transiently hyperacetylated and phosphorylated at histone H3 (Barratt et al., 1994; Mahadevan et al., 1991), and these modifications seem to be required for transcriptional activation (Thomson et al., 1999). Gene activation of certain Drosophila promoters has also been correlated with histone H3 phosphorylation (Labrador and Corces, 2003). These findings suggest a link between signaling-mediated chromatin phosphorylation and gene regulation. To explore the connection between rapid kinase activation, histone modifications, and gene induction by steroid hormones, we have studied progestin activation of the MMTV promoter in the T47D-MTVL breast cancer cell line, which carries an integrated single copy of the MMTV-LTR promoter driving the luciferase gene (Truss et al., 1995). The MMTV promoter is transcriptionally activated by progestins via a hormone-responsive region including a cluster of degenerated HREs and an adjacent site for the ubiquitous transcription factor nuclear factor 1 (NF1) (Beato et al., 1995). This region of the MMTV promoter is wrapped around a positioned nucleosome (Richard-Foy and Hager, 1987), which upon hormone induction becomes hypersensitive to nucleases, (Truss et al., 1995) likely due to the selective displacement of histone H2A and H2B dimers (Vicent et al., 2004). Here we report a direct connection between rapid kinase activation and gene induction by steroid hormones, namely the activation of Erk and Msk1 and their recruitment along with phosphorylated PR (pPR) to the MMTV promoter leading to phosphoacetylation of

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histone H3, displacement of HP1g, and recruitment of ATP-dependent remodeling complexes, coactivators, and RNA polymerase II. Thus, the nongenomic and genomic pathways activated by steroid hormones converge on chromatin to enable gene regulation. Results Progestins Activate Erk Leading to PRB Phosphorylation and Msk1 Activation Progestins stimulate proliferation of the human breast cancer cell line T47D and induce a rapid and transient activation of the Src/p21ras/Erk pathway that requires PR and, unexpectedly, ER (Ballare et al., 2003; Migliaccio et al., 1998). We now analyzed Erk activation in the T47D-MTVL cell line, which carries a chromosomally integrated copy of the MMTV promoter driving the luciferase gene (Truss et al., 1995). Treatment for 5 or 10 min with 10 nM of the PR agonist R5020 enhanced Erk1/2 kinase activity (Figure 1A, upper panel, lanes 1–3), and this activation was inhibited by ICI182780 (ICI), a specific ER antagonist, as well as by PD98059 (PD), a specific inhibitor of mitogen-activated protein kinase kinase 1 (MKK1) (Figure 1A, upper panel, lanes 4 and 5 and 6 and 7, respectively). Erk activation by R5020 was also abrogated by the specific PR antagonist RU 38486 (data not shown). Thus, progestin activation of the Erk pathway in T47DMTVL cells requires both PR and ligand-free ER. We wondered if the rapid and transient progestin activation of Erk1/2 is associated with a change in the phosphorylation status of PR. Treatment of T47DMTVL cells with R5020 resulted in an increased phosphorylation of PRB at S294 (pPRB), which was visible after 5 min but more evident 10 min after hormone addition (Figure 1A, middle panel, lanes 2 and 3). Phosphorylation of PRB was inhibited by ICI as well as by PD (Figure 1A, middle panel, lanes 4 and 5 and 6 and 7, respectively), indicating that the initial progestin activation of the Erk1/2 pathway is needed for PRB phosphorylation at S294. Msk kinases are targets of Erk that have been shown to participate in the rapid activation of immediate-early genes following mitogen stimulation (Soloaga et al., 2003). After progestin treatment for 5 or 10 min, there was an increase in the activated, phosphorylated form of Msk1, which paralleled the increase in pErk and pPRB (Figure 1A, lower panel, lanes 2 and 3). Activation of Msk1 was inhibited by ICI and by PD (Figure 1A, lower panel, lanes 4 and 5 and 6 and 7, respectively) and also by the Msk1 inhibitor H89, which did not influence Erk activation or PR phosphorylation (Figure 1A, lower panel, lanes 8 and 9). Thus, Msk1 activation is located downstream of Erk in progestin signaling. Other kinases that have been shown to participate in mitogenic response to external signals, such as Rsk and p38, are not activated after 5 or 10 min of hormone treatment (see Figure S1 in the Supplemental Data available with this article online). Inhibitors of Erk and Msk Activation Compromise Progestin Induction of MMTV and Other Progesterone Target Genes Progestin treatment of T47D-MTVL cells enhances transcription from the MMTV promoter integrated in chro-

matin (Truss et al., 1995). A gradual increase of MMTV transcripts is observed, starting after 1 hr and reaching around 30-fold increase after 8 hr of hormone induction (Figure 1B). Treatment of the cells with the MKK1 inhibitor PD did not influence basal MMTV transcription (data not shown) but abolished hormone induction (Figure 1B). A similar behavior was observed in cells treated with the estrogen-specific antagonist ICI (Figure 1B). In contrast, wortmannin, a potent inhibitor of the phosphoinositol 3-kinase pathway, did not influence hormonal induction (Figure 1B). Similar negative results were obtained with the selective inhibitor of the p38 MAP kinase, SB 203580 (Figure S2). None of the inhibitors had a significant effect on transcription from the bactin promoter used as control (data not shown). We conclude that activation of the Erk1/2 pathway, likely via an interaction of ligand-activated PR with ligandfree ER, is selectively required for hormone induction of the MMTV promoter. Activation of Msk1 also appears to be required for induction of MMTV transcription by progestins, as induction was inhibited by H89 (Figure 1B). However, H89 also inhibits protein kinase A (Davies et al., 2000). To exclude a participation of protein kinase A on MMTV induction, we used the specific PKA inhibitor RP-cAMP, which binds to the regulatory subunit and precludes dissociation of the catalytic subunit (Dostmann, 1995). This inhibitor had no effect on MMTV induction by progestins (Figure S3), suggesting that the effect of H89 is mediated by an inhibition of Msk1 activation. To obtain independent evidence for this assumption, we transfected T47D-MTVL cells with a dominant-negative mutant of Msk1 along with a GFP expression vector. Hormonal induction of the MMTV promoter was reduced by 50% in the presence of the dominant-negative mutant of Msk1, in good correlation with the percentage of transfected cells (Figure S4). A similar degree of inhibition was observed when a dominant-negative mutant of MEK was transfected in T47D-MTVL prior to hormone treatment (Figure S4). In another set of experiments, we used siRNA against Erk to reduce the levels of Erk protein in T47D-MTVL cells and observed a 52% reduction in the progestin induction of MMTV-luc (Figure 1C). A similar effect at the level of luciferase activity was observed with siRNA against Msk (data not shown), which reduced luciferase mRNA accumulation by 65% (Figure 1D). These results confirm the significance of Erk and Msk activation for the progestin induction of the MMTV promoter. We next asked whether progestin induction of other target genes is dependent on activation of the Erk kinase cascade. We used RT-PCR to study the accumulation of transcripts from several well-known progesterone target genes. After 6 hr of progestin treatment, the induction of EGFR, EGF, and Dusp1 was inhibited by treatment with PD to a level similar to that observed with MMTV-luc (Figure 1E, left panel). Moreover, the transient and rapid induction of c-Fos by progestins was inhibited by PD (Figure 1E, right top panel). In the case of the cyclin D1 gene, the kinase inhibitor caused a delay in progestin induction, which is seen after 2 hr in the control cells but only after 6 hr in the PD-treated cells (Figure 1E, right bottom panel). Thus, cellular genes showing different kinetics of progestin induction

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Figure 1. Activation of Erk Pathway Is Required for Hormone Induction of Progestin Target Genes (A) Cells were cultured in medium deprived of steroids for 48 hr, followed by 24 hr in serum-free conditions, prior to the addition of 10 nM R5020 (R) for 5 or 10 min. When indicated, 1 hr before hormonal induction, cells were treated with 50 mM PD98059 (PD), 10 mM ICI182780 (ICI), or 13.5 mM H89. Cell lysates were analyzed by western blotting with antibodies against phosphorylated Erk1/2 (pErk1/2) or total Erk (upper panel), antibodies against PR phosphorylated at serine 294 (pPR) or total PR (middle panel), or antibodies against phosphorylated Msk1 (pMsk1) or total Msk (lower panel). T0, time zero. (B) Cells cultured as in (A) were incubated for 0, 1, 2, and 8 hr with R5020. When indicated, PD, wortmannin, ICI, or H89 was added 1 hr before induction. Total RNA was prepared, and cDNA was generated and used as a template for PCR with luciferase and b-actin-specific primers. The values represent the mean 6 SD of two experiments performed in duplicate. (C) Cells were transfected with control (C) and Erk siRNAs and incubated with 10 nM R5020 for 24 hr, and the luciferase activity was determined. ERK expression was analyzed by western blotting; tubulin was used as control. The values represent the mean 6 SD of two experiments performed in duplicate. (D) Cells were transfected with control and Msk siRNAs as in (C), and the exposure to 10 nM R5020 was for 8 hr. RNA was measured as described in (B). Msk expression was determined by western blotting. The values represent the mean 6 SD of two experiments performed in duplicate. (E) Cells cultured as in (A) were incubated for 0, 1, 2, and 6 hr, as indicated, with R5020 (R). When indicated, cells were pretreated with PD. Total RNA was prepared, and cDNA was generated and used as a template for PCR with specific primers. Dusp1, dual specificity phosphatase 1.

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Figure 2. Upon Hormone Treatment, PR, Erk1/2, and Msk1 Are Rapidly Recruited to the MMTV Promoter (A) (Upper panel) Schematic representation of nucleosomes over the MMTV-LTR. HREs and the binding site for NF1 are indicated. (Lower panel) Cells cultured as in Figure 1A were incubated for 5 or 10 min with R5020 (+) or vehicle (2) and used for ChIP experiments with a-PR, a-Erk, or a-pErk1/2 and primers corresponding to MMTV nucleosome B (upper panel, 27 cycles) and the b-globin gene (lower panel, 27 cycles). Input material (1%) is shown for comparison (lanes 1–4). (B) Cells were untreated (0) or treated for 5 or 10 min with R5020 and subjected to ChIP assays as described in (A) using a-PR, a-pPR, a-Erk, and a-pErk1/2. The precipitated DNA fragments were subjected to real-time PCR analysis. The values represent the mean 6 SD of three experiments performed in duplicate. (C) Cells were treated with R5020 for 5 min and subjected to ChIP assays as described in (A) using a-PR, a-Erk, and a-pErk1/2. When indicated, 1 hr before hormonal induction, cells were pretreated with PD or ICI. The precipitated DNA fragments were subjected to real-time PCR analysis. The values represent the mean 6 SD of two experiments performed in duplicate. (D) Cells were untreated (0) or treated with R5020 for 5 or 10 min and subjected to ChIP assays as described in (A), using a-pMsk1 and a-Msk1. When indicated, cells were pretreated with PD or H89. The values represent the mean 6 SD of three experiments performed in duplicate.

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are affected by blocking the activation of the Erk1/2 pathway. Upon Hormone Treatment, Erk1/2 and Msk1 Are Rapidly Recruited to the Nucleosome B of the MMTV Promoter Along with Phosphorylated PR Next we used chromatin immunoprecipitation (ChIP) experiments to define the changes taking place at nucleosome B of the MMTV promoter during the initial phase of hormone induction. We consistently observed recruitment of PR to the MMTV promoter after 5 and 10 min of hormone treatment (Figure 2A, lanes 7 and 8, compared to lanes 5 and 6; see also Figure 2B). Using an Erk-specific antibody, we found that, 5 and 10 min after hormone addition, Erk was recruited to the MMTV promoter but not to the b-globin gene (Figure 2A lanes, lanes 11 and 12). The recruited Erk is in the activated form, as demonstrated with antibodies specific for phosphorylated Erk (Figure 2A, lanes 15 and 16; see also Figure 2B). Phosphorylated PRB is also recruited to the MMTV promoter after 5–10 min of progestin treatment but not to the same extent as total PR (Figure 2B), indicating that a fraction of PRB is recruited to the promoter in the nonphosphorylated form. This interpretation is confirmed by the results obtained in the presence of inhibitors. Both the PD and the ER antagonist ICI reduced total PR recruitment to about 50% (Figures 2B and 2C), while they completely blocked pPRB recruitment (data not shown) as well as recruitment of total Erk and pErk (Figures 2B and 2C). We next asked whether Msk1 is also recruited to the MMTV promoter upon induction. ChIP assays with antibodies specific for phosphorylated Msk1 showed that the activated protein accumulates on the MMTV promoter 5 and 10 min after progestin addition (Figure 2D). There was also an increase in total Msk1, indicating that the kinase is recruited to the MMTV promoter following hormone treatment (Figure 2D). The accumulation of pMsk1 on the promoter is inhibited by H89 and by PD (Figure 2D) and thus requires the activation of Erk. Intriguingly, inhibition of Msk1 activation by H89 reduced the recruitment to the MMTV not only of total PR but also of pPR by approximately 50% (Figure 2E). Moreover, inhibition of Msk1 activity completely abolished recruitment of phospho-Erk (Figure 2E). We conclude that although Msk1 activation is downstream to Erk activation and does not participate in progestin activation of Erk, it contributes to the recruitment of the pPR and pErk to the MMTV promoter. We next asked whether the recruitment of kinases is limited to a single nucleosome or extends to the complete promoter and coding region. Using nucleosome resolution ChIP assays, we found that Erk1/2 and Msk1 were bound to the MMTV promoter nucleosome B, but not to a promoter nucleosome further upstream, nor to a nucleosome over the luciferase open reading frame (Figures S5A and S5B, respectively). Thus, the kinases are selectively recruited to the nucleosome encompassing the HREs, supporting a receptor-mediated targeting of the ternary complex.

Activated Erk1/2 and Msk1 Form a Ternary Complex with pPR We next asked whether PR and Erk1/2 form a complex and how this is affected by hormone treatment. Already after 5 min of hormone treatment, PR coprecipitated with Erk (Figure 3A, upper left panel), indicating that both proteins form a complex upon hormone binding to PR. Formation of this complex required Erk activation and ligand-free ER, as it was inhibited by PD and ICI (Figure 3A, upper left panel). The complex contains the activated proteins, as antibodies selective for pPR also precipitated pErk (Figure 3A, upper right panel, lanes 2 and 6). No signal was detected when an unspecific mouse IgG was used for the immunoprecipitation (Figure 3A, upper right panel, lane 9). Thus, a short treatment with progestins induced the formation of a complex between the phosphorylated forms of Erk and PRB. Treatment with PD (Figure 3A, upper right panel, lanes 3 and 7) or with ICI (Figure 3A, upper right panel, lanes 4 and 8) abolished this interaction. Our combined results indicate that upon progestin treatment Erk1/2 is activated via ER and phosphorylates PRB at S294, and both activated proteins form a complex. Next we investigated the interaction of Msk1 with activated PRB upon induction. Ten minutes after progestin addition antibodies to pPRB precipitate Msk1 along with pErk1/2 (Figure 3B, lane 4). Moreover, antibodies to pErk precipitated Msk1 as well as pPRB (Figure 3B, lane 6), and antibodies against Msk1 coprecipitated PR, pPRB, Erk, and pErk (Figure S6). These data are compatible with the three proteins forming a complex 5–10 min after hormone treatment. To demonstrate the formation of a ternary complex, we used sequential immunoprecipitation of crosslinked cell lysates first with an Erk-specific antibody followed, after dissociating the immunoprecipitate at low pH, by a Msk-specific antibody (Figure 3C). No complex was observed prior to hormone treatment (Figure 3C, T0). GAPDH and b-actin were used as a control for unspecific coprecipitation (Figure 3C, bottom). The results show that both PRB and PRA as well as pPRB are part of a ternary complex that includes pErk and pMsk1. To explore whether the interaction between PR, Erk, and Msk is direct, we used the purified recombinant proteins for studies in vitro. The recombinant PR in the activated ligand-bound form was purified from baculovirus-infected insect cells (Di Croce et al., 1999). The purified PRB was phosphorylated at S294, and this phosphorylation was not enhanced by incubation with active Erk (Figure 3D, upper panel). Incubation of this pPR with active Erk and Msk followed by immunoprecipitation with an Erk-specific antibody yielded a precipitate containing all three proteins (Figure 3D, lower panel). When Erk is left out, there is no interaction between PR and Msk1 (Figure S7). These results suggest that the ternary complex can be formed by direct protein-protein interaction between PR and Erk on the one side and between Erk and Msk1 on the other. To obtain independent evidence for the interaction between the activated forms of PR and Msk1, we

(E) Cells untreated (0) or treated for 5 min with R5020 were subjected to ChIP assays using a-pErk, a-PR, and a-pPR. When indicated, cells were pretreated with H89. The precipitated DNA fragments were subjected to real-time PCR analysis. The values represent the mean 6 SD of two experiments performed in duplicate.

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Figure 3. Activated Erk1/2 and Msk1 Form a Complex with pPR (A) Cells cultured as in Figure 1A were incubated for 5 or 10 min with R5020 (R) or vehicle (2). When indicated, cells were pretreated with PD (R+PD) or ICI (R+ICI). (Upper left panel) Cell lysates were immunoprecipitated with an antibody against total Erk; the immunoprecipitates (IP) were analyzed by western blotting with antibodies against Erk and PR. (Upper right panel) Cell lysates were immunoprecipitated with an antibody against PR phosphorylated at S294 (a-pPR) or an antibody against Oct1 as a negative control (IP2). The immunoprecipitates (IP) were analyzed by western blotting with a-pPR and a-pErk1/2. (Lower) Western blot analysis of the inputs (2.5% of cell lysate used for immunoprecipitation) performed with a-pPR and a-PR. (B) Cells were treated for 10 min with R5020 (R) as described in (A), and cell lysates were immunoprecipitated with a-pPR, with a-pErk, or with normal mouse IgG as a negative control (IP2). The IPs were analyzed by western blotting with a-pPR, a-pErk1/2, and a-Msk1. T0, time zero. (C) Cells were treated for 5 and 10 min with R5020 as described in (A), and crosslinked cell lysates were subjected to a sequential coimmunoprecipitation assay as described in Experimental Procedures. Cell lysates were immunoprecipitated with a-Erk antibody eluted with 0.2 M glycine (pH 2.6) and reimmunoprecipitated with antibody against Msk1. The IP were analyzed by western blotting with the indicated antibodies. T0, time zero. (D) Direct interaction of pPR, Msk, and Erk in vitro. (Upper panel) Recombinant PR was incubated with active Erk for 30 min at 30 C in Erk kinase buffer. Kinase reactions were subjected to western blotting with anti-phospho PR or total PR. (Lower panel) For binding of the factors, recombinant PR, active Msk, and active Erk were incubated for 2 hr at 4 C in buffer A (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, plus protease inhibitors). Binding reactions were incubated 2 hr at 4 C with 20 ml protein G/A agarose beads previously coupled with 1.4 mg of the Erk antibody (a-Erk) or buffer (2). The immunoprecipitated proteins (IP) were washed three times with buffer A and eluted by boiling in SDS sample buffer. Input and IPs were analyzed for PR, Erk1/2, and Msk1 by western blot using specific antibodies.

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analyzed their intracellular localization by immunofluorescence. The results confirm activation of PR and Msk1 after hormone treatment. After 5 min of progestin treatment, both proteins exhibited a similar granular distribution within the cell nucleus and colocalized to a large extent, as demonstrated by merging the two fluorescence labels (Figure S8). Thus the two proteins exhibit a distribution pattern compatible with the formation of a complex. Activated Msk1 Phosphorylates Histone H3 at Serine 10 on Nucleosome B of the MMTV Promoter As a possible target for activated Msk1 in chromatin, we investigated the phosphorylation of histone H3 at serine 10 (H3S10p), a hallmark of gene transcription activation during the so-called ‘‘rapid nucleosome response,’’ in which H3S10 phosphorylation is accompanied by acetylation at lysine 14 (H3K14ac) (Cheung et al., 2000; Clayton and Mahadevan, 2003). Cells were treated with R5020, and ChIP assays were performed using antibodies against H3S10p alone or in combination with H3K14ac (H3S10p/K14ac) (Lo et al., 2001). Progestin treatment for 5 or 10 min induced an increase in H3S10p at nucleosome B (Figures 4A and 4B, lanes 3 and 4). Similarly, after 5 min of hormone treatment there was an increase in the amount of H3 exhibiting the combination of S10p and K14ac (Figure 4C, top rows, lanes 7 and 8). To explore whether these modifications extend to adjacent nucleosomes, we used primers specific for nucleosome D and, for sequences over the luciferase open reading frame, sufficiently distant to exclude coprecipitation with nucleosome B (see scheme, Figure 2A). After hormone treatment, antibodies to H3S10p or to H3S10p/ K14ac enriched in the precipitates sequences of nucleosome B (Figure 4C, top row) but did not enrich DNA of nucleosome D or the luciferase open reading frame (Figure 4C, central and lower rows), suggesting that these hormonally induced modifications are confined to nucleosome B. No increase in phosphorylation or acetylation of histone H3 was found in the b-globin gene following treatment with progestins (Figure 4A and data not shown). No change in serine 28 phosphorylation was observed after 5 min of hormone treatment (Figure 4C, lanes 11 and 12). Activation or Erk pathway is required for phosphoracetylation of histone H3. In the presence of the MEK inhibitor PD, hormone treatment did not increase H3S10p (Figure 4A, lanes 7 and 8; for quantification, see Figure 4B) or the combined modification H3S10p/K14ac (data not shown). Phosphorylation of H3 was mediated by crosstalk between PR and ER, as suggested by the inhibitory effect of ICI (Figure 4B). Moreover, the Msk1 inhibitor H89 abolished H3S10 phosphorylation (Figure 4B). Transfection of a siRNA against Msk that reduces the Msk1 levels significantly (see Figure 1D) also reduced H3S10p 5 min after progestin treatment at the nucleosome B of the MMTV promoter (Figure 4D). We interpret these results as an indication that the Msk1 is the kinase responsible for histone H3S10 phosphorylation on the MMTV promoter, although we cannot exclude the possibility that the effect of the Msk1 inhibitor is due to the reduced recruitment of pErk to the promoter. To obtain additional genetic evidence for the participation of the ER-mediated induction of the Erk pathway

in progestin activation of the MMTV promoter and on H3S10p, we constructed T47D-MTLV-derived cell lines expressing FLAG-tagged version of either the WT PRB or a mutant of PRB lacking the ER-interacting motive I (DERID I) and deficient for progestin activation of the Src/Ras/Erk pathway (Ballare et al., 2003). In this cell line, induction of MMTV-luc by progestins was compromised (Figure 4E, upper panel), compatible with a role of Erk activation on MMTV promoter induction. Following hormone treatment, recruitment of the mutant PR to the MMTV promoter was delayed, and there was no phosphorylation of H3S10 or recruitment of Erk up to 10 min after hormone addition (Figure 4E, lower panel). These results support a role of the ER-mediated activation of the Erk pathway in recruitment of kinases to the MMTV promoter and in H3S10 phosphorylation. Hormone Induction Leads to Kinase-Dependent Displacement of HP1g from the Promoter, without Changing H3 Lysine 9 Trimethylation To explore how H3S10 phosphorylation could contribute to activation of the MMTV promoter, we analyzed the role of the binary switch formed by this modification and trimethylation of H3 lysine 9 (H3K9me3). Binding of HP1 to the H3K9me3 has been postulated to be inhibited during M phase of the cell cycle due to phosphorylation of H3S10 by the mitotic Aurora B kinase (Fischle et al., 2005; Hirota et al., 2005). The level of H3K9me3 over the MMTV promoter did not change upon hormone treatment (Figure 5A, lanes 1 and 2; see also Figure 5B), but the level of HP1g was drastically reduced after 5 min of hormone addition (Figure 5A, lanes 5 and 6; see also Figure 5C), in parallel with the increase in H3S10p (Figure 4A). Moreover, while PD did not influence the level of H3K9me3 (Figures 5A and 5B), the depletion of HP1g was blocked by PD as well as by H89 (Figures 5A and 5C). These results suggest that the promoter is in a repressive environment prior to hormone addition and that H3S10 phosphorylation contributes to the displacement of repressive complexes containing HP1g. Therefore, it seems that the combinatorial switch postulated for heterochromatin in mitotic cells is also operative in interphase chromatin in the context of an inducible promoter and provides a possible explanation for the facilitating role of H3S10 phosphorylation by Msk1 in promoter activation. Inhibition of Msk1 Activation Hinders HormoneInduced Recruitment of Chromatin-Remodeling Complexes, Coactivators, and RNA Polymerase II The effect of selectively preventing histone H3 phosphorylation by inhibiting Msk1 activation with H89 was not limited to PR and Erk recruitment but extended to other factors mediating hormone induction. Brg1, the ATPase of the human Swi/Snf complex, was recruited to the MMTV promoter 5 min after progestin treatment, but recruitment was prevented in the presence of PD or H89 (Figure 4F). Similarly, recruitment of the coactivator P/CAF and the loading of RNA polymerase II to the promoter in response to hormone are also inhibited in the presence of PD or H89 (Figure 4F). We tested for the presence of RNA polymerase II within the transcribed region of the MMTV-luc transgene 30 min after hormone treatment when transcriptional

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Figure 4. Hormone Induces Kinase-Dependent Phosphorylation of H3S10, Acetylation of H3K14, and Recruitment of Chromatin-Remodeling Complexes, Coactivators, and RNA Polymerase II (A) Cells cultured as indicated in Figure 1A were incubated for 5 or 10 min with R5020 (R, +) or vehicle (2), harvested, and used for ChIPs experiments with a-H3S10p antibody and primers for the MMTV nucleosome B and the b-globin gene. Input material (1%) is shown for comparison. When indicated, cells were pretreated with PD (lanes 5–8) or vehicle (lanes 1–4). (B) Cells were untreated (0) or treated for 5 min with R5020 (50 ) and subjected to ChIP assays as described in (A) using a-H3S10p antibody. When indicated, cells were pretreated with PD, ICI, or H89. The precipitated DNA fragments were subjected to real-time PCR analysis. The values represent the mean 6 SD of two experiments performed in duplicate. (C) Cells were treated with R5020 for 5 min (R, +) and subjected to ChIP assays as described in (A) using specific antibodies against H3S10p (left panel), H3S10p/K14ac (middle panel), or H3S28p (right panel). PCR analysis was performed with primers corresponding to MMTV nucleosomes B and D as well as to the luciferase coding region (see Figure 2A). (D) Cells were transfected with control and Msk siRNAs, incubated with 10 nM R5020 for 5 min, and subjected to ChIP assays as described in (A) using a-H3S10p antibody. The values represent the mean 6 SD of two experiments performed in duplicate. (E) (Upper panel) MMTV-driven luciferase expression in response to progestin was measured in isogenic T47D-Y (T47D derivative lacking PRB and PRA expression (Sartorius et al., 1994) containing an integrated MMTV-Luc construct (TYML cells) and either the wild-type PRB, the mutant PRBDERIDI (Ballare et al., 2003), or the empty pRAVFlag expression vector (Knuesel et al., 2003). Cells were cultured as indicated in Figure 1A. After 1 day in serum-free conditions, cells were incubated with 10 nM R5020 for 24 hr, and the luciferase activity was determined. The values represent the mean and standard deviation from two experiments performed in duplicate. (Lower panel) TYML cells stably expressing wildtype PRB or mutant PRBDERIDI, tagged with FLAG, were untreated (0) or treated for 5 or 10 min with 10 nM R5020 and subjected to ChIP assays as described above by using specific antibodies against FLAG, H3S10p, or Erk. The precipitated DNA fragments were subjected to PCR analysis

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Figure 5. Hormone Induction Leads to Erk/ Msk1-Dependent Displacement of HP1g from the Promoter without Altering H3K9 Trimethylation (A) Cells cultured as indicated in Figure 1A were treated for 5 min with R5020 (+) and subjected to ChIP assays using a-H3K9me3 (lower left panel) or a-HP1g (lower right panel) antibodies. When indicated, cells were pretreated with PD or H89. The precipitated DNA fragments were subjected to PCR analysis with primers for the MMTV nucleosome B and the b-globin gene. On the upper panel, input material (1%) is shown for comparison. (B) Quantification by real-time PCR of experiments like that shown in (A) with a-H3K9me3 antibody. The values represent the mean 6 SD of two experiments performed in duplicate. (C) Quantification by real-time PCR of experiments like that shown in (A) with a-HP1g antibody. The values represent the mean 6 SD of two experiments performed in duplicate.

induction is clearly detectable (Truss et al., 1995). ChIP experiments showed an increase of both the total and the active phosphorylated form of RNA polymerase over the coding regions that was inhibited by treatment with the kinase inhibitors (Figure S9). We conclude that the rapid hormonal activation of Erk and Msk kinases and H3S10 phosphorylation are required for the transition from a HP1g-containing repressive state to an active state with bound chromatin-remodeling factors and active RNA polymerase II. Nucleosomes Phosphorylated by Msk1 at H3S10 Are a Better Substrate for Cell-Free Transcription and Binding of SWI/SNF To explore the functional significance of H3S10 phosphorylation, we performed transcription assays with MMTV minichromosomes in HeLa cell extracts (Di Croce et al., 1999). Minichromosomes assembled in postblastodermic Drosophila embryo extracts and incubated with recombinant activated-PRB and NF1 support efficient transcription (Figure 6A, lanes 1 and 2), which was reduced by the addition of H89 (Figure 6A, lanes 3 and 4), suggesting a requirement of Msk1 activity in the extract. Consistent with this assumption, ChIP ex-

periments showed that H3 is phosphorylated at serine 10 in the presence of PRB and NF1 over nucleosome B (Figure 6B, top row), but not over a distal nucleosome on vector sequences (Figure 6B, middle row). Minichromosomes assembled with a H3S10A mutant do not support efficient transcription in the presence of PRB and NF1 (Figure 6C, lanes 2 and 4 and lower panel), supporting a key role of H3S10p in remodeling of nucleosome B. Mutation of H3S28 to alanine had no effect on transcription (Figure 6C, lanes 2 and 6 and lower panel), indicating that phosphorylation of H3S28 is not essential for induction in vitro. Moreover, neither H3S10A nor H3S28A mutation had an effect of transcriptional activation by GAL4-VP16 of GAL4-containing minichromosomes (Figure 6D and lower panel), excluding an unspecific effect of the mutant nucleosomes on chromatin transcription. Msk1 or an H89-sensitive kinase is likely responsible for the observed localized phosphorylation of H3S10 over MMTV nucleosome B, as phosphorylation was inhibited by addition of H89 during the in vitro incubation with PR and NF1 (Figure 6E). To delimit the mechanism by which Msk1 H3S10p influences remodeling, we performed histone octamer and nucleosome reconstitution experiments with recombinant histones (Vicent et al.,

with primers corresponding to MMTV nucleosome B and the b-globin gene as a loading control (data not shown). Input material is shown for comparison. (F) Inhibition of Erk or Msk1 blocks rapid recruitment of Brg1, PCAF, and polymerase II. Cells cultured as indicated in Figure 1A were either untreated (0) or treated for 5 min with R5020, harvested, and used for ChIP experiments with specific antibodies against Brg1, PCAF, or RNA polymerase II (Pol II) and primers for the MMTV nucleosome B and the b-globin gene. The precipitated DNA fragments were subjected to real-time PCR analysis. When indicated, cells were pretreated with PD or H89. The values represent the mean 6 SD of two experiments performed in duplicate.

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Figure 6. H3S10 Is Phosphorylated on In Vitro-Assembled Chromatin and Required for Induced Transcription (A) MMTV minichromosomes were incubated with purified recombinant activated PR and NF1 and transcribed in vitro with HeLa nuclear extract. RNA products were visualized by primer extension analysis and sequencing gel electrophoresis. When indicated, 13,5 mM H89 was added. The position of the product from the wild-type MMTV promoter is indicated. (B) MMTV minichromosomes assembled as in (A) were incubated with PR and NF1 and subjected to ChIP assays as described previously (Koop et al., 2003) using a-H3S10p antibody and specific primers for MMTV nucleosome B and for a control nucleosome localized downstream of the MMTV promoter. Input material (1%) is shown for comparison. (C) (Upper panel) MMTV minichromosomes assembled as in (A) with either wild-type H3 or the H3S10A or H3S28A mutant were incubated with PR and NF1, transcribed, and visualized as described in (A). Quantification of RNA transcripts is shown in the lower panel. (D) (Upper panel) In vitro transcription analysis of pG5E4TCAT minichromosomes was carried out as described in (C), except for the utilization of Gal4-VP16 as activator. Quantification of the transcription experiments is shown in the lower panel. (E) MMTV minichromosomes assembled as in (A) were incubated with purified recombinant activated PR and NF1 and subjected to ChIP assays as described in (A). When indicated, 13.5 mM H89 was added. Input material (1%) is shown for comparison. (F) Histone octamers were assembled by salt dialysis either with wild-type histone H3 or with H3S10A and incubated in the presence of purified active Msk1 for 30 min at 30 C as indicated. Kinase reaction was stopped with H89 and the products electrophoresed in 18% SDSPAGE. The separated proteins were transferred to a nitrocellulose membrane for western blotting with antibodies specific for H3 phosphorylated at either serine 10 (H3S10p) or serine 28 (H3S28p). The Coomassie blue-stained gel is shown at the bottom. (G) MMTV nucleosomes phosphorylated at H3 serine 10 bind Swi/Snf preferentially. MMTV mononucleosomes were assembled with either wildtype histone H3 or with the H3S10A mutant and incubated in the presence of active Msk1 for 30 min at 30 C as indicated. Kinase reactions were stopped with H89, and nucleosomes were further incubated with purified Swi/Snf for 20 min at room temperature. Binding of Swi/Snf to nucleosomes was measured by ChIP using SNF2p-specific antibody. The precipitated DNA fragments were subjected to real-time PCR analysis to test for the presence of sequences corresponding to the MMTV promoter. The values represent the mean and standard deviation from two experiments performed in duplicate.

2004). When the octamers were incubated with ATP and Msk1, H3 was selectively phosphorylated at S10 and S28 (Figure 6F). The phosphorylated nucleosomes were bound by purified SWI/SNF more efficiently than unphosphorylated nucleosomes (Figure 6G). Nucleosomes carrying H3S10A did not exhibit enhanced SWI/ SNF binding upon phosphorylation by Msk1 (Figure 6G). As the H3S10A mutant exhibited normal phosphorylation at H3S28 (Figure 6F), this site alone is not sufficient for facilitating SWI/SNF binding. We conclude that phosphorylation of histone H3S10 generates a better platform for SWI/SNF interaction with chromatin, explaining, at least in part, the effect of H89 on Brg1 recruitment observed in cultured cells. Discussion The aim of this work was to explore a possible connection between the rapid signaling by steroid hormones to kinase cascades and their transcriptional effects on the

cell nucleus. We found that the rapid and transient activation of the Erk pathway is required for induction of a number of progesterone target genes in breast cancer cells. One common view is that activated kinases phosphorylate transcription factors or coregulators involved in the genomic effects of the hormones (Bjornstrom and Sjoberg, 2005). Unexpectedly, we found a targeted recruitment to the MMTV HREs of activated Erk and Msk kinases, which play an essential role in preparing the promoter chromatin for gene activation. Kinasemediated phosphorylation of histone H3S10 triggers the shift from a repressive state to an active state by promoting displacement of HP1g. To our knowledge, this is the first report of a localized and transient phosphorylation of histone H3 coupled to a mechanism of promoter activation. Induction of other progesterone target genes is also dependent on Erk activation. Using cDNA microarrays (20,000 array from Agilent) with RNA extracted from cells treated for 6 hr with progestins, we found that 28% of

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the genes induced by progestins in T47D cells exhibit a reduction in hormone induction when activation of Erk is blocked by PD (C.B., B. Min˜ana, M.J. Melia´, and M.B., unpublished data). Among these genes, we found rapidly and transiently induced genes, like c-Fos, as well as genes induced with a more typical kinetic, such as EGF, EGFR, and Dusp-1. Finally, in the case of the Cyclin D1 gene, we found that induction by progestin was delayed and inhibition was only detectable at early time points (2 hr) after hormone induction. It is possible that such a delay takes place in other genes that have therefore escaped detection. Further studies will be necessary to determine whether other kinases are involved in the hormonal induction of genes resistant to inhibition of Erk activation. We have previously reported that 3%–5% of cellular PR is located in the cytoplasm of T47D cells, forming a complex with ERa via an interaction of two domains in the N-terminal half of PRB with the ligand-binding domain of ERa (Ballare et al., 2003). The receptor complex can be activated by progestins binding to PRB, which promotes interaction of ERa with c-Src and activation of the Src/p21ras/Erk pathway (Ballare et al., 2003; Migliaccio et al., 1998). We now show that one of the targets of activated pErk is PRB itself, which already becomes phosphorylated at S294 5 min after progestin treatment. Phosphorylation of PR at S294 has been reported 60 min after progestin treatment but was related to rapid PR degradation by the ubiquitin-proteasome pathway, which is required for accumulation of progestin-induced transcripts (Lange et al., 2000). An unexpected finding in our studies was that already 5 min after progestin addition, Erk is recruited to the MMTV promoter in the active phosphorylated form as part of a complex with pPRB. Compromising Erk activation prevents formation of the complex and reduces but does not eliminate recruitment of PR, pointing to an Erk-independent binding of PR to the MMTV promoter. However, the unphosphorylated PR seems to be unable to orchestrate rapid promoter activation, as blocking the Erk pathway prevents MMTV induction. It is unlikely that the effect of inhibiting Erk activation is due to the inhibition of PRB phosphorylation, as inhibiting Msk1 also blocks MMTV induction but has no influence on PRB phosphorylation at S294. Concomitant with the progestin activation of Erk, we find localized phosphorylation of H3S10, but not H3S28, over the MMTV promoter. Phosphorylation of H3 has been reported to occur during mitosis and in response to various external stimuli in interphase. The kinases responsible for mitotic phosphorylation belong to the Aurora family (Hsu et al., 1988), while Rsk2 and Msk1 have been identified as H3 kinases that are activated upon stimulation of the Ras-MAPK pathway (SassoneCorsi et al., 1999; Thomson et al., 1999). In T47D-MTVL cells, progestins did not enhance Rsk2 activity, while Msk1 was rapidly activated in an Erk-dependent fashion, and we detect the formation of a ternary complex of Msk1 with PRBS294p and pErk. Moreover, a ternary complex can be formed in vitro with recombinant purified pPRB, pErk, and pMsk1 via an interaction of pErk with pPR and pMsk1. ChIP experiments demonstrate the recruitment of the ternary complex to the MMTV promoter shortly after progestin treatment. Recruitment is

limited to the nucleosome containing the HREs, supporting a PR-mediated targeting. Inhibition of the recruitment of the ternary complex pPRB/pErk/pMsk1 by inhibition of either Erk or Msk activation blocks histone H3S10 phosphorylation and the accompanying acetylation at H3K14. Appearance of H3S10p is abrogated by H89, which inhibits Msk kinase but not Rsk2 or Erk (Thomson et al., 1999), as well as by an siRNA directed against Msk1 mRNA. All these results point to Msk1 as the kinase responsible for the rapid phosphorylation of H3S10 in response to progestins. We still do not know the enzyme responsible for acetylation of H3K14. Though we see recruitment of PCAF rapidly after hormone treatment, further experiments are needed to determine its role in acetylation of H3K14. In yeast, the two modifications are coupled, and Gcn5 is the histone acetyltransferase responsible for K14 acetylation (Lo et al., 2001). Similar coupling has been postulated in mammalian cells, although the mechanism is still a matter of debate (Clayton and Mahadevan, 2003; Kouzarides, 2000). Hormone Induction Is Accompanied by a Kinase-Dependent Displacement of HP1g from the MMTV Promoter How could the modification of H3 influence gene induction? One possibility is that it helps ejection of repressive complexes containing members of the HP1 family, which are known to contribute to gene silencing by binding to H3K9me3 (Nakayama et al., 2001; Nielsen et al., 2001; Schotta et al., 2002). After 5 min of progestin treatment, we observed a decrease in HP1g content over the MMTV promoter, without changes in the levels of H3K9me3. Inhibitors of either Erk or Msk activation block the hormonal effect on HP1g levels. These results suggest that, prior to hormone treatment, there is an HP1-dependent repression of the MMTV promoter, which is released by phosphoacetylation of H3 triggered via the hormonal induction of Erk and Msk kinases. This mechanism is reminiscent of that observed during mitosis, which is mediated by the Aurora B-dependent phosporylation of H3S10 and leads to a general displacement of HP1 proteins from heterochromatin (Fischle et al., 2005; Hirota et al., 2005). However, in our study, the displacement of HP1 is rapid and localized to a single promoter region. Erk and Msk Activity Is Required for Recruitment of Chromatin-Remodeling Complexes and RNA Polymerase II to the MMTV Promoter Five minutes after progestin treatment, Brg1- and PCAFcontaining complexes are recruited to the MMTV promoter. Interfering with Erk or Msk activation inhibits recruitment of both types of chromatin-remodeling complexes. Previous reports already suggested a coupling of ATP-dependent remodeling and histone acetylation (DiRenzo et al., 2000). Our results suggest that a similar coupling participates in progestin activation of target promoters and that H3S10, possibly combined with H3K14ac, is a key component of this signaling mechanism. The final outcome of the readout of chromatin modifications triggered by hormones is the recruitment of RNA polymerase II and the initiation of transcription. Already

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5 min after progestin treatment of T47D-MTVL cells, we observed recruitment of RNA polymerase to the MMTV promoter. Recruitment is blocked by inhibitors of either Erk or Msk1, likely accounting for the observed inhibition of RNA accumulation in cells treated with the kinase inhibitors. However, robust hormonal effects on transcript levels are not seen after 5 min but need at least 30 min of hormone exposure (Bartsch et al., 1996). At this time point after progestin treatment, we detect increased levels of total and activated RNA polymerase not only on the promoter region (Vicent et al., 2004) but also over the coding region of the MMTV-luc transgene, likely reflecting actively transcribing RNA polymerase complexes. The levels of RNA polymerase II over the coding region are reduced to the noninduced values by treatment of the cells with inhibitors of either Erk or Msk activation, in good correlation with the effects of these inhibitors on the accumulation of transcripts from the MMTV promoter. Role of H3S10p in Reconstituted Chromatin In MMTV minichromosomes assembled with extracts from Drosophila embryos and activated by PRB and NF1, H3S10 is phosphorylated by a kinase that is inhibited by H89. As in the cell culture experiments, H3S10p is limited to the nucleosome located over the HREs. Inhibition of Msk1 activity or mutation of H3S10 to alanine compromised transactivation of the MMTV promoter, compatible with a requirement for H3S10 phosphorylation. As the synergism between PR and NF1 is mediated by ATP-dependent chromatin remodeling (Di Croce et al., 1999), we hypothesize that H3S10 phosphorylation is required for minichromosome remodeling. Results obtained with histone octamers and nucleosomes assembled in vitro with recombinant histones support this notion, as the purified Swi/Snf complex binds more efficiently to nucleosomes phosphorylated by Msk1 and this preference is abolished by the H3S10A mutation. The results suggest that phosphorylation at H3S10 plays a role in docking of ATP remodeling complexes. This finding is compatible with the observed recruitment of Brg-1 to the MMTV promoter after progestin treatment, which is abolished by blocking Msk1 activity. However, we cannot exclude the possibility that phosphorylation of other targets by Msk1 is also relevant for induction or that other histone modifications, such as acetylation of H3K14 or H4K8 (Agalioti et al., 2002) or methylation of H3R17 (Bauer et al., 2002), are also involved in the induction process. A Model for the Initial Steps of MMTV Promoter Activation The results presented in this paper are compatible with a model in which progestin activation of the PR/ER complex on the cytoplasmic side of the cell membrane triggers ER-mediated activation of the Src/Ras/Erk cascade and accumulation of active Erk in the nucleus (Figure 7). Concomitantly, ligand binding to the complex of PR and Hsp chaperones in the nucleus leads to the formation of PR homodimers that are phosphorylated by activated Erk, which also phosphorylates Msk1. The three activated proteins form a ternary complex that is recruited to the MMTV promoter nucleosome B,

due to the affinity of PR for the exposed HREs (Pin˜a et al., 1990). Once bound to the promoter, Msk1 phosphorylates H3S10, an event possibly coupled to acetylation of H3K14, thus generating a signal that leads to displacement of a repressive complex containing HP1g. Derepression is a requisite for recruitment by PR of ATP-dependent chromatin-remodeling complexes that use the energy of ATP hydrolysis to remove H2A/H2B dimers from nucleosome B, thus allowing binding of further PR molecules, NF1, coactivators, and the basal transcriptional machinery including RNA polymerase II (Vicent et al., 2004). A fraction of PR homodimers is not phosphorylated and binds to the exposed HREs leading to unproductive complexes (Figure 7). We do not know whether the modifications of the H3 tail described here are sufficient to switch the repressive complex into a state competent for activation or whether other modifications are also involved and required. Experimental Procedures Cell Culture and Hormone Treatments T47D-MTVL breast cancer cells carrying one stably integrated copy of the luciferase reporter gene driven by the MMTV promoter (Truss et al., 1995) were routinely grown in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. For the experiments, cells were plated in RPMI medium without phenol red supplemented with 10% dextran-coated charcoal-treated FBS (DCC/FBS), and 48 hr later medium was replaced by fresh medium without serum. After 1 day in serum-free conditions, cells were incubated with R5020 (10 nM) or vehicle (ethanol) for different times at 37 C. When indicated, ICI780 (10 mM), PD (50 mM), H89 (13.5 mM), wortmannin (0.1 mM), SB203580 (10 mM), and rp-cAMP (10 or 100 mM) were also added 1 hr before hormone induction. Cell lines containing either the WT PRB or the PRB DERID-I mutant were generated as previously described (Knuesel et al., 2003). Activation of Kinases and PR Serine 294 Phosphorylation T47D-MTVL cells were incubated 5 or 10 min with R5020. Erk1/2 activation was detected by immunoblotting by using an antibody recognizing the phosphorylated form of Erk1/2 (Cell Signaling) as described (Vallejo et al., 2005). Msk1, Rsk, and p38 activation was detected with antibodies against phospho-Msk1, phospho-Rsk, and phospho-p38MAPK (Cell Signaling). PR serine 294 phosphorylation was analyzed by immunoblotting with anti-PRS294p (Neomarkers). The total proteins were detected with antibodies against Erk-2, Msk1, and PR (Santa Cruz) and anti-a-tubulin (Sigma). Coimmunoprecipitation of Erk1/2, PR, and Msk1 Cells treated as indicated above were lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM MgCl2, and 0.5% Triton X-100 plus protease and phosphatase inhibitors. For immunoprecipitation of Erk, PR, and Msk1, cell extracts (2 mg/ml for each immunoprecipitation) were precleared 2 hr with 40 ml of 50% slurry protein G/A agarose beads (Oncogene) and incubated overnight at 4 C with 60 ml protein G/A agarose beads previously coupled with 4 mg of the corresponding antibodies or an unspecific control antibody. The immunoprecipitated proteins (IP) were eluted by boiling in SDS sample buffer. Inputs and IPs were analyzed for PR, Erk1/2, and Msk1 by western blot using specific antibodies. For sequential coimmunoprecipitation experiments, cells were incubated 5 or 10 min with R5020 and then treated with the membranepermeable protein-crosslinking reagent DTBP (Dimethyl 3.30 dithiobisproprionamidate-2-HCl; Pierce) during 30 min at 37 C. The reaction was stopped with 0.1 M Tris-HCl (pH 7.5), and the cells were lysed and immunoprecipitated with a-Erk antibody as described above. The immunoprecipitated proteins were eluted with 0.2 M glycine (pH 2.6) and reimmunoprecipitated with antibody against Msk1.

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Figure 7. Model for the Initial Steps of MMTV Promoter Induction Progestins (Prog) bind to cytoplasmic PR/ER complexes and activate the Src/Ras/Erk pathway leading to nuclear accumulation of activated Erk. Most of PR is nuclear and complexed to chaperons (Hsps). Upon binding of progestins, PR homodimers dissociate from chaperones, and a fraction of them binds to the exposed HREs on nucleosome B of the repressed MMTV promoter, likely generating a nonproductive complex. Another fraction of PR is phosphorylated by pErk, which also phosphorylates Msk1. A complex of pPR/ pErk/pMsk1 is recruited to the promoter and catalyzes phosphorylation of H3S10 and displacement of a HP1g-containing repressive complex. Subsequently, an ATPdependent chromatin-remodeling complex is recruited to the promoter and catalyzes displacement of H2A/H2B dimers.

Immunofluorescence Assays T47D-MTVL cells were cultured onto coverslips and treated as indicated above. Immunofluorescence assays were performed as previously described (Vallejo et al., 2005) with minor modifications. Briefly, fixation step was done by incubation in 100% methanol for 10 min at 220 C, and pPR (Neomarkers) and pMsk1 antibodies were used to detect the activated proteins. RNA Extraction and RT-PCR Total RNA was prepared from T47D-MTVL cells by using RNeasy kit (Qiagen) as described in the manufacturer’s instructions. The cDNA was generated from 1 mg of total RNA by using Superscript First Strand Synthesis System (Invitrogen). One microliter of cDNA reaction was then used as template for RT-PCR. EGFR, EGF, Dusp 1, Luciferase, b-actin, Fos, and Cyclin D1 gene products were analyzed by PCR. Quantification of Luc and b-actin gene products was performed by real-time PCR. Each value calculated using the standard curve method was corrected by the human b-actin and expressed as relative RNA abundance over time zero. In Vitro Kinase Assays MMTV mononucleosomes were assembled with either wild-type histone H3 or with the H3S10A mutant (Vicent et al., 2004) and incubated in the presence of active Msk1 (Upstate Biotechnologies) for 30 min at 30 C in kinase assay buffer (8 mM MOPS [pH 7], 0.2 mM EDTA, 1 mM EGTA, 100 mM ATP, 5 mM b-glycerol phosphate, 15 mM MgCl2, 0.2 mM sodium orthovanadate, and 0.2 mM dithiothreitol). Kinase reactions were stopped with 13.5 mM H89 and subjected either to ChIP assays or western blotting with anti-pS10H3, antipS28H3 or were stained with Coomassie blue. Chromatin Immunoprecipitation Assays ChIP assays were performed as described (Strutt and Paro, 1999) by using chromatin either from the T47D-MTVL cell line cultured and

treated as described above or in vitro reconstituted MMTV mononucleosomes. The antibodies used for ChIPs assays are listed in Figure S10. Quantification of ChIP was performed by real-time PCR using Roche Lightcycler (Roche). The fold enrichment of target sequence in the immunoprecipitated (IP) compared to input (Ref) fractions was calculated using the comparative Ct (the number of cycles required to reach a threshold concentration) method with the equation 2Ct(IP) 2 Ct(Ref). Each of these values were corrected by the human b-globin gene and referred as relative abundance over time zero. Primer sequences are available on request. RNA Interference Experiments RNA interference experiments to knock down Erk in T47D-MTVL cells were performed using the RNAi human/mouse control kit, including the small RNA duplexes (Qiagen). The siRNAs (100 nM) were transfected using the proper HiperFect reagent ratio (1:8) in RPMI medium. After 48 hr, the medium was replaced by fresh medium without serum. After 1 day in serum-free conditions, cells were incubated with R5020 (10 nM) or vehicle (ethanol) for 8 or 24 hr at 37 C. Msk siRNAs were purchased from Dharmacon and transfected using Lipofectamine 2000 (Invitrogen). The downregulation of Erk and Msk expression was determined by western blotting. Chromatin Reconstitution, Transcription, and Immunoprecipitation Postblastodermic Drosophila melanogaster extracts were used to assembly chromatin as previously described (Krajewski and Becker, 1998). The plasmid pMMTVCAT, used as transcription template, contains the wild-type MMTV promoter from 2640 to +126. In vitro transcription reactions with recombinant human PRB and pig NF1C2 were performed as described (Di Croce et al., 1999). For transcription with GAL4-VP6, the plasmid pG5E4TCAT (Carey et al., 1990) was used as a template. Transcriptions were quantified with Image Gauge package (Fujifilm). For ChIPs experiments, 10 ng of

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DNA of the reconstituted material was incubated with recombinant factors during 30 min and subjected to ChIP assays as previously reported (Koop et al., 2003). Supplemental Data Supplemental Data include ten figures and can be found with this article online at http://www.molecule.org/cgi/content/full/24/3/ 367/DC1/. Acknowledgments We wish to thank S. Dimitrov, Grenoble, France, for recombinant Xenopus laevis H3S10A protein; C. Peterson, Worcester, Massachusetts, for purified SWI/SNF; Weidong Wang, NIH, Bethesda, Maryland, for anti-Brg 1 antibody; B. Gross, IMT Marburg, Germany, for purified PR and NF-1 proteins; B. Min˜ana and L. Sumoy, CRG, for the microarrays experiments; Roser Zaurin, CRG, for designing the model; and Nora Spinedi, CRG, for technical assistance. G.P.V. was a recipient of a fellowship of the Ramo´n y Cajal Programme. The experimental work was supported by grants from the Departament d’Universitats Recerca i Societat de la Informacio (DURSI), Ministerio de Educacio´n y Ciencia (MEC) BMC 2003-02902, and Fondo de Investigacio´n Sanitaria (FIS) PI0411605 and CP04/00087. Received: March 16, 2006 Revised: September 5, 2006 Accepted: October 5, 2006 Published: November 2, 2006 References Agalioti, T., Chen, G., and Thanos, D. (2002). Deciphering the transcriptional histone acetylation code for a human gene. Cell 111, 381–392. Ballare, C., Uhrig, M., Bechtold, T., Sancho, E., Di Domenico, M., Migliaccio, A., Auricchio, F., and Beato, M. (2003). Two domains of the progesterone receptor interact with the estrogen receptor and are required for progesterone activation of the c-Src/Erk pathway in mammalian cells. Mol. Cell. Biol. 23, 1994–2008. 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. Bartsch, J., Truss, M., Bode, J., and Beato, M. (1996). Moderate increase in histone acetylation activates the mouse mammary tumor virus promoter and remodels its ncleosome structure. Proc. Natl. Acad. Sci. USA 93, 10741–10746. Bauer, U.M., Daujat, S., Nielsen, S.J., Nightingale, K., and Kouzarides, T. (2002). Methylation at arginine 17 of histone H3 is linked to gene activation. EMBO Rep. 3, 39–44. Beato, M., Herrlich, P., and Schu¨tz, G. (1995). Steroid hormone receptors: many actors in search of a plot. Cell 83, 851–857. Bjornstrom, L., and Sjoberg, M. (2005). Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. Mol. Endocrinol. 19, 833–842. Boonyaratanakornkit, V., Scott, M.P., Ribon, V., Sherman, L., Anderson, S.M., Maller, J.L., Miller, W.T., and Edwards, D.P. (2001). Progesterone receptor contains a proline-rich motif that directly interacts with SH3 domains and activates c-Src family tyrosine kinases. Mol. Cell 8, 269–280. Carey, M., Lin, Y.S., Green, M.R., and Ptashne, M. (1990). A mechanism for synergistic activation of a mammalian gene by Gal4 derivatives. Nature 345, 361–364. Castoria, G., Migliaccio, A., Bilancio, A., Di Domenico, M., de Falco, A., Lombardi, M., Fiorentino, R., Varricchio, L., Barone, M.V., and Auricchio, F. (2001). PI3-kinase in concert with Src promotes the S-phase entry of oestradiol-stimulated MCF-7 cells. EMBO J. 20, 6050–6059. 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.

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