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PIM1-Dependent Phosphorylation of Histone H3 at Serine 10 Is Required for MYC-Dependent Transcriptional Activation and Oncogenic Transformation

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PIM1-Dependent Phosphorylation of Histone H3 at Serine 10 Is Required for MYC-Dependent Transcriptional Activation and Oncogenic Transformation ARTICLES PIM1-dependent phosphorylation of histone H3 at serine 10 is required for MYC-dependent transcriptional activation and oncogenic transformation Alessio Zippo1, Alessandra De Robertis1, Riccardo Serafini1 and Salvatore Oliviero1,2 The serine/threonine kinase human Pim1 (hereafter PIM1) cooperates with human c-Myc (hereafter MYC) in cell cycle progression and tumorigenesis. However, the nature of this cooperation is still unknown. Here we show that, after stimulation with growth factor, PIM1 forms a complex with the dimer of MYC with MAX (Myc-associated factor X) via the MYC BoxII (MBII) domain. MYC recruits PIM1 to the E boxes of the MYC-target genes FOSL1 (FRA-1) and ID2, and PIM1 phosphorylates serine 10 of histone H3 (H3S10) on the nucleosome at the MYC-binding sites, contributing to their transcriptional activation. MYC and PIM1 colocalize at sites of active transcription, and expression profile analysis revealed that PIM1 contributes to the regulation of 20% of the MYC-regulated genes. Moreover, PIM1-dependent H3S10 phosphorylation contributes to MYC transforming capacity. These results establish a new function for PIM1 as a MYC cofactor that phosphorylates the chromatin at MYC-target loci and suggest that nucleosome phosphorylation, at E boxes, contributes to MYC-dependent transcriptional activation and cellular transformation. Chromatin structure has a crucial function in eukaryotic gene that Pim1 is overexpressed in Myc-driven prostate tumours16. However, transcription. Several transcription activators have been shown the nature of the cooperation of c-Myc and Pim1 in cell transformation to recruit chromatin-modifying activities, which mediate either is still unknown. covalent modifications of histones or ATP-dependent mobiliza- MYC codes for a basic helix–loop–helix leucine zipper transcription tion of nucleosomes. Covalent modifications of histones including factor that binds to the E box (CACGTG) when dimerized with MAX acetylation, methylation, phosphorylation and ubiquitination have and regulates the transcription of distinct genes involved in cell cycle been associated with gene regulation1,2. Phosphorylation of H3S10 progression, apoptosis, cell growth, and differentiation17–23. The DNA- has been connected with transcriptional activation in different binding domain of MYC is at the carboxy terminus. The MBII domain organisms and with chromosome condensation during mitosis3,4. from the amino-terminal region that is essential for MYC-dependent In mammalian cells, stimulation with growth factors induces rapid cell transformation has been shown to recruit multiprotein enzymatic phosphorylation of histone H3 at Ser 10 (H3S10) at c-Jun and c-Fos complexes to the MYC-activated genes24. promoters mediated by mitogen- and stress-activated protein kinase To understand the function of PIM1, we investigated the molecular (MSK)1, MSK2 and RSK2 kinases4–7. In response to inflammatory connection between PIM1 and MYC. We propose that the recruitment cytokines, IκB kinase-α (IKK-α) phosphorylates H3S10 at NF-κB- of PIM1 to the chromatin by MYC contributes to the transcriptional responsive promoters8,9. activation of target genes for MYC by phosphorylating H3S10 at the In this study we analysed the function of PIM1, a constitutively E-box element and suggest that this cooperation is relevant for MYC- active serine/threonine kinase. Pim1 was first identified as a com- dependent tumour formation. mon insertion site in T-cell lymphomas induced by Moloney murine leukaemia virus (MMLV)10. RNA interference (RNAi) experiments ResULts demonstrated that PIM1 is essential for the cell cycle progression of PIM1 phosphorylates the nucleosome at H3S10 human umbilical-vein endothelial cells (HUVECs) and for the differ- PIM1 messenger RNA is induced with fast kinetics in HUVECs treated entiation of endothelial precursors in vitro11. Myc and Pim1 have been with vascular endothelial growth factor (VEGF)-A11. On treatment shown to cooperate in cell growth and transformation. Overexpression with VEGF-A, PIM1 is induced and is localized in HUVEC nuclei of Pim1 in transgenic mice demonstrated its cooperation with c-Myc within 40 min (see Supplementary Information, Fig. S1). As PIM1 in cell transformation12–15, and microarray expression profiling showed is a serine/threonine kinase with putative consensus sites present on 1Dipartimento di Biologia Molecolare Università di Siena, Via Fiorentina 1, 53100 Siena, Italy. 2Correspondence should be addressed to S.O. (e-mail: [email protected]) Received 12 February 2007; accepted 27 June 2007; published online 22 July 2007; DOI: 10.1038/ncb1618 932 NATURE CELL BIOLOGY VOLUME 9 | NUMBER 8 | AUGUST 2007 © 2007 Nature Publishing Group ARTICLES a Competitor – unr.H3 H2B GST + –– + –– + – – + – – PIM1 WT – + – – + – – + – – + – PIM1 K67M – – + – – + – – + – – + A 8 M (K) 2 r /S A A A 21 0 8 0 KA: 32P-H3 T b W S1 S2 S1 14 GST–PIM1 WT +– + – +–– + GST–PIM1 K67M ––+ +– + – + Mr(K) 97 21 32 66 GST–PIM1 KA: P-H3 45 66 Kinase 30 GST 21 Histones 14 21 1 2 3 4 5 6 7 8 Histones 14 1 2 3 4 5 6 7 8 9 10 11 12 c WT K67M IP: PIM1 – + – + Input Mr(K) M 7 30 IB: PIM1 T W K6 Mr(K) 21 30 IB: PIM1 IB: H3 21 IB: H3 21 KA: 32P-H3 1 2 1 2 3 4 Figure 1 PIM1 phosphorylates H3S10 on the nucleosome. (a) PIM1 histones. Nucleosomes were assembled containing, respectively, the histone phosphorylates histone H3 on nucleosomes from chromatin fraction. H3 wild type (lanes 1 and 2), the mutant H3S10A (lanes 3 and 4), the mutant GST-tagged recombinant proteins, as indicated, were incubated with purified H3S28A (lanes 5 and 6) or the double mutant H3S10A/S28A (lanes 7 and nucleosomes from HEK 293 cells and the kinase assays (KA) were performed 8). Histone H3 autoradiography resulting from [γ-32P]ATP incorporation (top) in the presence of [γ-32P]ATP. GST–PIM1 interactions with nucleosomes is shown. The input kinases and nucleosomes were revealed by Coomassie were performed in the absence (−) or presence of a 20-fold molar excess of staining (middle and bottom). (c) PIM1 interacts with histone H3 in vivo. unrelated (unr.), H3 or H2B peptides as indicated. GST-tagged recombinant PIM1 was immunoprecipitated from HEK 293 stable clones expressing the proteins and nucleosomes were revealed by Coomassie staining. Histone inducible PIM1 (WT) or the kinase-inactive mutant PIM1-K67M (K67M) as phosphorylation was revealed by autoradiography. The N-terminal domain of indicated. PIM1 and H3 were revealed by immunoblotting (IB) with specific H3, but not that of H2B, competes for PIM1 association with the nucleosome antibodies. A kinase reaction was performed by incubating the inhibiting the kinase reaction. Uncropped images of blots are shown in PIM1/H3 co-immunoprecipitate in the presence of [γ-32P]ATP. Histone Supplementary Information, Fig. S9. (b) PIM1 phosphorylates histone H3 phosphorylation was revealed by autoradiography. Portions (2%) of the at Ser 10. Recombinant GST–PIM1 or the PIM1 kinase-inactive mutant total immunoprecipitated protein samples were loaded as input (bottom). were incubated with nucleosomes reconstituted in vitro from recombinant Uncropped images of blots are shown in Supplementary Information, Fig. S9. histones, its nuclear localization could account for the increase in H3 assembled with the H3S10A mutant or the double mutant (Fig. 1b), phosphorylation observed in these cells between 60 and 90 min after demonstrating that PIM1 phosphorylates H3 at Ser 10. Histone H3 treatment with VEGF-A (see Supplementary Information, Fig. S1). We co-immunoprecipitated with transiently expressed PIM1 or the found that PIM1 could directly phosphorylate nucleosomes in vitro by kinase-inactive mutant, suggesting that PIM1 associates in vivo with incubating the recombinant glutathione S-transferase (GST)–PIM1 the nucleosome (Fig. 1c). Incubation of the immunoprecipitate in the protein with chromatin fractions obtained from HEK 293 cells in the presence of [γ-32P]ATP showed that the wild-type PIM1, but not the presence of [γ-32P]ATP. This analysis revealed that a single protein, kinase-inactive mutant PIM1-K67M, phosphorylated H3. We conclude with a molecular mass corresponding to H3, was phosphorylated by that PIM1 associates in vitro and in vivo with H3 and directly phos- wild-type GST–PIM1 but not by the kinase-inactive mutant GST– phorylates H3S10 on the nucleosome. PIM1-K67M (Fig. 1a, lanes 1–3). Specific inhibition of nucleosome phosphorylation was observed in the presence of a peptide derived PIM1 forms a complex with MYC and MAX from the N terminus of H3 but not with a peptide derived from the Treatment with VEGF-A induces colocalization of MYC and PIM1 N terminus of H2B or with an unrelated peptide (Fig. 1a, lanes 4–12). in the cell nucleus at 60 min, decreasing thereafter (Fig. 2a). We PIM1 could phosphorylate reconstituted nucleosomes containing tested whether PIM1 co-immunoprecipitates with MYC. A time- either recombinant wild-type H3 or H3S28A, but not nucleosomes course analysis in HUVECs treated with VEGF-A showed that, on NATURE CELL BIOLOGY VOLUME 9 | NUMBER 8 | AUGUST 2007 933 © 2007 Nature Publishing Group ARTICLES a VEGF-A 0 20 40 60 120 240 min 1 PIM MYC ge Mer b Input IP c IP VEGF-A 0 30 60 90 120 240 0 30 60 90 120 240 Input IgG MAX PIM1 MYC – + – + – + – + – + – + IP: MAX Serum – + – + – + – + – + Mr(K) Mr(K) 30 IB: PIM1 30 IB: PIM1 66 IB: MYC 66 IB: MYC 21 IB: MAX 21 IB: MAX 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 17 18 1 2 3 4 5 6 7 8 9 10 d Flag–MYC Flag–MYC∆MBII t t G G G G Inpu Ig IP: Flag Ig IP: PIM1 Inpu Ig IP: Flag Ig IP: PIM1 Mr(K) IB: TRRAP 250 66 IB: MYC 30 IB: PIM1 21 IB: MAX 1 2 3 4 5 6 7 8 9 10 Figure 2 PIM1 forms a complex with MYC.
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