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

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

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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

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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. (a) PIM1 colocalizes with MYC in Information, Fig. S9. (c) Endogenous PIM1 forms a complex with MYC–MAX HUVECs on treatment with VEGF-A. Indirect immunofluorescence analysis of in serum-stimulated HEK 293 cells. Western blot analysis of input extracts, HUVECs treated with VEGF-A was performed with antibodies against PIM1 IgG, MAX, MYC or PIM1 immunoprecipitates from serum-induced or (red) and MYC (green). Scale bar, 70 µm. (b) Treatment with VEGF-A induces uninduced cells as indicated. Interacting proteins were revealed with the use interaction of endogenous PIM1 with the MYC–MAX dimer. Cell proteins of anti-MAX, anti-MYC or anti-PIM1 antibodies, respectively. (d) MYC forms a extracted from VEGF-A-treated HUVECs were either immunostained (input) complex with PIM1 through its MBII domain. HEK 293 cells were transiently or subjected to immunoprecipitation (IP) with anti-IgG antibodies (−) or anti- cotransfected with Flag–MYC constructs as indicated and with PIM1. Cell MAX antibodies (+) as indicated. Immunostaining (IB) analysis was performed lysates were immunoprecipitated with anti-IgG, anti-Flag or anti-PIM1 with the use of anti-MAX, anti-MYC or anti-PIM1 antibodies as indicated. antibodies followed by western blot analysis to detect MAX, MYC, PIM1 and Portions (10%) of the total immunoprecipitated protein samples were TRRAP proteins as indicated. The asterisk indicates an non-specific band. loaded as inputs. Uncropped images of blots are shown in Supplementary Uncropped images of blots are shown in Supplementary Fig. S9. induction, both MYC and PIM1 were induced with fast kinetics. and immunoprecipitated with either anti-Flag or anti-PIM1 antibod- Immunoprecipitations with anti-MAX antibodies at various time ies. Western blot analysis showed reciprocal immunoprecipitation of points showed the formation of the MAX–MYC complex from Flag–MYC with PIM1, whereas co-immunoprecipitation was strongly 60 min after stimulation with growth factor, thus corresponding to the reduced between Flag–MYC∆MBII and PIM1, suggesting that the appearance of MYC in the nucleus (Fig. 2b, right panel). Importantly, MBII domain of MYC is required for the formation of a complex with PIM1 co-immunoprecipitated with MYC and MAX, suggesting that PIM1 (Fig. 2d). Endogenous TRRAP associated with MYC but not with a complex containing PIM1 is formed. Endogenous PIM1 was also Flag–MYC∆MBII, as described previously25. In contrast, PIM1 did not found to co-immunoprecipitate with endogenous MYC and MAX co-immunoprecipitate the endogenous TRRAP (Fig. 2d, lanes 5 and from HEK 293 cells after treatment with serum for 120 min (Fig. 2c), 10), suggesting that MYC forms independent complexes with PIM1 or thus demonstrating that a MAX–MYC–PIM1 endogenous complex TRRAP through the MBII domain. is also formed in these cells. As MYC-dependent cell transformation requires the MBII domain PIM1 colocalizes with MYC on the chromatin at actively to bind cofactors either in a TRRAP (transformation/transcription transcribing loci domain-associated protein)-dependent or TRRAP-independent man- To measure PIM1 and MYC association at MYC-target genes, we per- ner24, we tested whether PIM1 interacts with MYC at MBII through formed a nuclear distribution analysis of PIM1 with respect to MYC, TRRAP. Wild-type Flag–MYC or MYC whose MBII domain had been nascent transcripts and chromatin markers. Quantitative analysis of deleted (Flag–MYC∆MBII) was expressed with PIM1 in HEK 293 cells double staining with anti-PIM1 and antibodies against different nuclear

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a MYC H3S10ph H3S10phK14ac H3K9me2 BrUTP b 1 PIM MYC BrUTP Marker ge ge Mer Mer C. points C. points C.plot C.plot

Rr = 0.82 ± 0.06 Rr = 0.85 ± 0.07 Rr = 0.8 ± 0.08 Rr = 0.37 ± 0.03 Rr = 0.77 ± 0.05 Rr = 0.81 ± 0.09 P < 0.0011 P < 0.0014 P < 0.0016 P < 0.002 P < 0.0008 P < 0.0019

Figure 3 PIM1 colocalizes with H3S10ph, active chromatin and MYC. of the correlation plot. The pixels from the colocalization results of seven (a) Immunofluorescence analysis of PIM1 nuclear sublocalization in individual images represented in the correlation plot (c. plot) were quantified

HUVECs treated with VEGF for 60 min. Fixed cells were double-stained by Pearson’s correlation coefficient (Rr) as mean ± s.d.; P values were for PIM1 (red) and one of the indicated markers (green). Scale bar, 8 µm). determined with Student’s t-test. (b) Immunofluorescence analysis of MYC The corresponding pictures were merged and all pixels having the same localization at transcriptional active sites. Cells were labelled with BrUTP position in both images were considered coincident and are represented as and detected with anti-bromodeoxyuridine antibody (green). Double labelling white spots (colocalization points; c. points). The brightness level of each was revealed at transcriptional active sites by staining cells with an anti-MYC pixel is plotted as the x (PIM1) and y (marker) axes of the scatter diagram antibody (red). The colocalization of pixels was performed as in a. markers by sequential laser scanning (Fig. 3) showed a greater degree of of a downstream enhancer that is activated by MYC and AP-1 (refs 27– PIM1 colocalization with MYC, with a Pearson’s correlation coefficient 29). After treatment of HUVECs with VEGF-A, FOSL1 expression was

(Rr) of 0.82 ± 0.06. PIM1 also showed an elevated overlap with phospho- induced with a peak at 60 min (Fig. 4a). To demonstrate a direct binding of rylated H3S10 (H3S10ph) (Rr 0.85 ± 0.07) and H3S10ph acetylated on MYC and PIM1 to the FOSL1 regulatory sequences, chromatin immuno- Lys 14 (H3S10phK14ac), whereas little colocalization could be detected precipitation (ChIP) followed by real-time quantitative polymerase chain for PIM1 and dimethylation of H3K9 (Rr 0.37 ± 0.03), a marker of silent reaction (PCR) analysis was performed at various time points after treat- heterochromatin. Furthermore, we analysed the nuclear distribution of ment with VEGF-A. As shown in Fig. 4b, we generated primers to analyse PIM1 with respect to active transcription sites by in situ transcriptional four FOSL1 regions: a FOSL1 upstream region containing a functional run-on experiments. Double labelling revealed a high degree of overlap SRE and a non-canonical E-box element30,31 (probe A), the downstream between PIM1 and sites of active transcription (Rr 0.77 ± 0.05) with a enhancer containing a canonical E box (probe B), a downstream region pattern resembling that of transcription factories26. A similar pattern of within the first intron (probe C), and a FOSL1 region within the second nuclear localization with respect to nascent transcripts was observed for intron (probe D). Quantitative ChIP analysis with probe B revealed the

MYC (Rr 0.81 ± 0.09). Taken together, these results suggest that, after transient recruitment to the FOSL1 enhancer of MYC and PIM1 at 60 min treatment with growth factor, PIM1 kinase is mostly localized with the after treatment with VEGF-A simultaneously with a marked increase in transcription factor MYC at actively transcribing sites and with phos- H3S10ph signal (Fig. 4c). ChIP with probe B also showed a significant phorylated histones. increase in H3S10phK9ac and H3S10phK14ac, whereas H3K9acK14ac showed elevated levels before induction, suggesting that, at the FOSL1 H3S10 phosphorylation at the FOSL1 enhancer is correlated enhancer, acetylation of H3 precedes binding of MYC and that phospho- with PIM1 association rylation of H3S10 takes place at the acetylated H3. Next we analysed PIM1 association and H3S10 phosphorylation at FOSL1 Analysis of the upstream element (probe A) revealed an association (also known as FRA-1), a well-characterized MYC-target gene27,28. FOSL1 of MYC with this DNA fragment. PIM1 did not associate with this ele- contains an upstream functional serum response element (SRE), which ment, suggesting that the FOSL1 promoter is bound by an alternative is not sufficient to confer promoter activation but requires the activity MYC complex. This FOSL1 upstream element showed an increase in nature cell biology volume 9 | number 8 | AUGUST 2007 935 © 2007 Nature Publishing Group

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FOSL1 transcript 6 FOSL1 a b +1 n 4

Probe A B C D 2 (–590/–330) (+930/+1170) (+2420/+2680) (+4750/+4990) Fold inductio

0 VEGF-A0 15 30 60 90 120 240 min

c Probe A Probe B Probe C Probe D 25 25 20 25 MYC 20 20 15 20 15 15 15 10 10 10 10 ‰ input 5 5 5 5 0 0 0 0 VEGF-A 0 15 30 60 90 120 240 015 30 60 90 120 240 015 30 60 90 120 240 015 30 60 90 120 240 min

10 10 10 10 PIM1 8 8 8 8

t 6 6 6 6 4 4 4 4 ‰ inpu 2 2 2 2 0 0 0 0 VEGF-A 0 15 30 60 90 120 240 015 30 60 90 120 240 015 30 60 90 120 240 015 30 60 90 120 240 min

20 20 25 20 H3S10ph 15 15 20 15

t 15 10 10 10 10

‰ inpu 5 5 5 5 0 0 0 0 VEGF-A 015 30 60 90 120 240 015 30 60 90 120 240 015 30 60 90 120 240 015 30 60 90 120 240 min

20 20 20 20 H3S10phK9ac 15 15 15 15 t 10 10 10 10

‰ inpu 5 5 5 5

0 0 0 0 VEGF-A 015 30 60 90 120 240 015 30 60 90 120 240 015 30 60 90 120 240 015 30 60 90 120 240 min

15 15 15 15 H3S10phK14ac

t 10 10 10 10

‰ inpu 5 5 5 5

0 0 0 0 VEGF-A 0 15 30 60 90 120 240 0 15 30 60 90 120 240 0 15 30 60 90 120 240 0 15 30 60 90 120 240 min

H3K9acK14ac 40 40 40 40

30 30 30 30

20 20 20 20

‰ input 10 10 10 10

0 0 0 0 VEGF-A 015 30 60 90 120 240 015 30 60 90 120 240 015 30 60 90 120 240 015 30 60 90 120 240 min

d FOSL1 transcript e 25 25 25 25 8 H3S10ph 20 20 20 20 6 15 15 15 15 4 10 10 10 10 2 ‰ input 5 Fold induction 5 5 5 0 0 0 0 0 VEGF- A 0 15 30 60 90 120 240 min VEGF-A 015 30 60 90 120 240 015 30 60 90 120 240 015 30 60 90 120 240 015 30 60 90 120 240 min

Figure 4 PIM1 associates with chromatin at the FOSL1 enhancer. (a) FOSL1 in thousandths (‰) of the DNA inputs. Values are mean and s.d. for three is transiently induced by treatment with VEGF-A. Serum-starved HUVECs replicate samples from one representative experiment (n = 3). (d) FOSL1 were treated with VEGF-A, and total RNAs were collected at different time expression in the presence of MSK1/MSK2 inhibitor H89. Serum-starved points. FOSL1 transcript levels were quantified by quantitative RT–PCR HUVECs were treated with VEGF-A in the presence (open bars) or absence and relative values were normalized to the expression of glyceraldehyde- (filled bars) of 10 µM H89, and total RNAs were collected at different time 3-phosphate dehydrogenase (GAPDH). Values are mean and s.d. for three points. FOSL1 transcript levels were quantified by real-time quantitative replicate samples from one representative experiment (n = 3). (b) Schematic RT–PCR and relative values were normalized to GAPDH expression. Values are representation of the FOSL1 gene region including the first two exons (not mean and s.d. for three replicate samples from one representative experiment to scale). Exons are represented as black squares within the gene, and (n = 3). (e) Phosphorylation of H3S10 at the FOSL1 upstream element is the E box is shown as a black circle. The probes used in ChIP analysis are dependent on MSK1/MSK2 kinase activities. H3S10 phosphorylation was indicated. PCR probe intervals are numbered relative to the first nucleotide measured by quantitative ChIP assay on VEGF-A-treated HUVECs. Chromatin of exon 1 (+1). (c) VEGF-A induces PIM1 association with chromatin at the extracts were immunoprecipitated with antibodies as indicated. Real-time FOSL1 enhancer. Protein interactions on FOSL1 chromatin were measured by quantitative PCR measurements of the immunoprecipitated DNA of the quantitative ChIP assay on VEGF-A-treated HUVECs. Chromatin extracts were corresponding gene regions identified as probes A, B, C or D at different immunoprecipitated with antibodies as indicated. Real-time quantitative PCR time points are expressed as thousandths (‰) of the DNA inputs. Values are measurements of the immunoprecipitated DNA of the corresponding gene mean and s.d. for three replicate samples from one representative experiment regions identified as probes A, B, C or D at different time points are expressed (n = 3). Filled bars, control; open bars, H89 treatment.

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a shsP#1 shsPIM1#1 Tet – + – + – + – + Serum – ––+ +– + +

Mr(K) PIM1 30

21 MAX

97 SP1 1 2 3 4 5 6 7 8

b Probe A Probe B Probe C Probe D 25 25 25 25 20 20 20 20 15 15 15 15 H3S10ph 10 10 10 10 ‰ input 5 5 5 5 0 0 0 0 Serum 015 30 60 90 120180 240 015 30 60 90 120180 240 015 30 60 90 120 180 240 015 30 60 90 120 180 240 min

10 10 10 10 8 8 8 8

PIM1 6 6 6 6 4 4 4 4 ‰ input 2 2 2 2 0 0 0 0 Serum 015 30 60 90 120180 240 015 30 60 90 120180 240 015 30 60 90 120 180 240 015 30 60 90 120 180 240 min

25 25 25 25 20 20 20 20 t 15 15 15 15 MYC 10 10 10 10 ‰ inpu 5 5 5 5 0 0 0 0 Serum 015 30 60 90 120180 240 015 30 60 90 120180 240 015 30 60 90 120 180 240 015 30 60 90 120 180 240 min

30 30 30 30

t 20 20 20 20 H3S10phK9ac

‰ inpu 10 10 10 10

0 0 0 0 Serum 015 30 60 90 120180 240 015 30 60 90 120180 240 015 30 60 90 120 180 240 015 30 60 90 120 180 240 min

20 20 20 20

15 15 15 15 t

H3S10phK14ac 10 10 10 10 ‰ inpu 5 5 5 5

0 0 0 0 Serum 015 30 60 90 120 180 240 015 30 60 90 120180 240 015 30 60 90 120 180 240 015 30 60 90 120 180 240 min

45 45 45 45 40 40 40 40 35 35 35 35

t 30 30 30 30 H3K9acK14ac 25 25 25 25 20 20 20 20

‰ inpu 15 15 15 15 10 10 10 10 5 5 5 5 0 0 0 0 Serum 015 30 60 90 120180 240 015 30 60 90 120 180 240 015 30 60 90 120 180 240 015 30 60 90 120 180 240 min

Figure 5 PIM1 mediates H3S10 phosphorylation and contributes to FOSL1 phosphorylation at the FOSL1 enhancer. Probes A, B, C and D are shown transcription. (a) Inducible shRNA expression targeting PIM1. Western schematically in Fig. 4. Time-course analysis by ChIP assay was performed blot analysis of HEK 293 stable clones expressing a control scrambled with the use of antibodies as specified from HEK 293 cells expressing either a shRNA (shsP#1) or PIM1 shRNA (shPIM1#1) inducible by tetracycline. control scrambled shRNA (filled bars) or PIM1 shRNA (open bars). Real-time Immunostaining with antibodies was as indicated. Protein extracts were quantitative PCR measurements of the immunoprecipitated DNA are expressed obtained from untreated or serum-treated cells as indicated, in the presence as thousandths (‰) of the DNA inputs. Values are mean and s.d. for three (Tet +) or absence (Tet −) of tetracycline. (b) PIM1 knockdown alters H3S10 replicate samples from one representative experiment (n = 3).

H3S10 phosphorylation at 15 min after treatment with VEGF-A, which presence of H89, a specific MSK1/MSK2 inhibitor5. Treatment of cells was therefore not dependent on PIM1. As the FOSL1 SRE is constitu- with H89 affected FOSL1 transcription, although a VEGF-A-dependent tively occupied by the ternary complex factor (TCF), which is activated induction was still observed (Fig. 4d). The analysis of H3S10ph signal by the Ras/extracellular signal-regulated kinase (ERK) pathway30, we fur- showed specific inhibition of H3S10 phosphorylation at the FOSL1 ther analysed FOSL1 transcription and H3S10 phosphorylation in the upstream element but not at the enhancer (Fig. 4e), demonstrating

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a b MYC c PIM1 d H3S10ph e H3K9acK14ac f shsM#1 + – + ––+ shsP#1 shMYC#1 25 25 25 25 shMYC#1 – + – + – + Tet – + – + – + – + Flag–MYC –– + + –– – – + + – – + + 20 20 20 20 Serum Flag–MYC∆MBII –– –– + + M (K) r t 15 t 15 t 15 t 15 Mr(K) 66 66 MYC MYC 10 10 10 10 ‰ inpu ‰ inpu ‰ inpu ‰ inpu 21 MAX 21 5 5 5 5 MAX

SP1 0 0 0 0 SP1 97 2 2 2 2 97 ID ID ID ID 1 2 3 4 5 6 7 8 1 2 3 4 5 6 FOSL1 FOSL1 FOSL1 FOSL1

g FOSL1 h ID2

MYC PIM1 H3S10ph H3K9acK14ac 25 15 20 25 15 15 30 30 20 20 15 10 10 10 15 20 15 20 10 10 10 5 5

‰ input 5 ‰ input 10 5 10 5 5

0 0 0 0 0 0 0 0 MBII MBII MBII MBII MBII MBII MBII MBII Mock Mock Mock Mock Mock Mock Mock Mock ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ C C C C C C C C Flag–MYC Flag–MYC Flag–MYC Flag–MYC Flag–MYC Flag–MYC Flag–MYC Flag–MYC F-MY F-MY F-MY F-MY F-MY F-MY F-MY F-MY

Figure 6 PIM1 phosphorylates histone H3 in a MYC-dependent manner. (shsM#1), MYC shRNA (shMYC#1) or the shRNA together with plasmids (a) Inducible MYC gene silencing. HEK 293 cells were transiently transfected expressing either Flag–MYC or Flag–MYC∆MBII as indicated. shMYC#1 with either a control scrambled shRNA (shsM#1) or MYC shRNA (shMYC#1) as recognizes the MYC 3ʹ untranslated region, which is not present in the Flag indicated, by tetracycline treatment. Western blot analysis on protein extracts constructs cotransfected. Protein expression was performed by immunoblotting was performed by immunoblotting, with the use of antibodies recognizing MYC, with antibodies as indicated. (g, h) MYC, but not Flag–MYC∆BMII, rescues the MAX or SP1 as indicated. (b–e) MYC recruits PIM1 on chromatin. Chromatin recruitment of PIM1 to the chromatin. Chromatin samples were obtained from samples were obtained from serum-induced HEK 293 cells expressing the HEK 293 cells expressing the control scrambled shRNA (filled bars), MYC control scrambled shRNA (filled bars) or MYC shRNA (open bars). ChIP assays shRNA (open bars) or the shRNA together with Flag–MYC or Flag–MYC∆MBII were performed with antibodies as indicated. Immunoprecipitated DNA was (F-MYC∆MBII) as indicated. ChIP assays were performed with antibodies analysed by quantitative PCR with primers to amplify the FOSL1 enhancer and as indicated. Immunoprecipitated DNA was analysed by quantitative PCR ID2 upstream MYC-binding sites (−1705/−1460). Real-time quantitative PCR with primers to amplify the FOSL1 enhancer and ID2 upstream MYC-binding measurements of the immunoprecipitated DNA are expressed as thousandths sites (−1705/−1460). Real-time quantitative PCR measurements of the (‰) of the DNA inputs. Values are mean and s.d. for three replicate samples immunoprecipitated DNA are expressed as thousandths (‰) of the DNA inputs. from one representative experiment (n = 3). (f) Western blot analysis of Values are mean and s.d. for three replicate samples from one representative HEK 293 cells transiently transfected with control scrambled shRNA experiment (n = 3). that phosphorylation at the upstream element and at the downstream (see below). Quantitative ChIP analysis (Fig. 5b and see Supplementary enhancer are independent of each other and are mediated by different Information, Fig. S2) showed a peak of H3S10 phosphorylation at the kinases. The acetylation pattern at the FOSL1 upstream element showed FOSL1 enhancer (probe B, as described in Fig. 4b) at 120 min that was a significant increase in H3 phosphorylation and acetylation, with a peak significantly decreased by PIM1 silencing, whereas ChIP analysis at the between 15 and 30 min suggesting that, at this site, H3S10 phosphoryla- FOSL1 upstream element (probe A) showed a faster kinetics of H3S10 tion precedes or is concomitant with H3 acetylation at Lys 9 and Lys 14, phosphorylation that was not affected by PIM1 knockdown. No H3S10 as described previously for immediate early (IE) promoters3,4. phosphorylation was observed at the downstream fragments (probes C The downstream FOSL1 fragments (analysed with probes C and D) and D). We concluded that H3S10 phosphorylation at the FOSL1 enhancer did not reveal MYC or PIM1 binding or an increase in phosphorylated is dependent on PIM1. H3S10, but yielded elevated levels of acetylated H3. These results dem- PIM1 and MYC are found to associate at the FOSL1 enhancer with a onstrate specific recruitment of PIM1 to the FOSL1 enhancer simultane- peak between 90 and 120 min. PIM1 silencing significantly decreased ously with an increase of H3S10 phosphorylation at this site and with PIM1 association at the enhancer without affecting MYC association. transcriptional activation of the gene. The overall pattern of H3 phosphoacetylation at Lys 9 and Lys 14 in HEK 293 cells showed higher levels of H3 acetylation already present H3S10 phosphorylation at FOSL1 enhancer is mediated by PIM1 before H3 phosphorylation at the FOSL1 enhancer (probe B) and PIM1 depletion with small hairpin RNA (shRNA) was peformed to test along the FOSL1 gene (probes C and D), which was not affected by whether PIM1 association with the FOSL1 enhancer was required for PIM1 silencing. H3S10 phosporylation. In HEK 293 cells treated with serum MYC, PIM1 Moreover, expression of the kinase-inactive mutant PIM1-K67M and FOSL1 showed a slower kinetics of induction than HUVECs treated significantly reduced H3S10ph levels at the FOSL1 enhancer (see with VEGF-A, possibly as a result of the different cell types (cell line versus Supplementary Information, Fig. S3) demonstrating that the kinase primary cells). PIM1 silencing strongly inhibited FOSL1 mRNA expression activity of PIM1 is required for H3S10 phosphorylation at this site.

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PIM1 is recruited by MYC at the FOSL1 and ID2 E-box elements serum. MYC silencing affected the expression of 1026 genes, of which MYC silencing was performed to analyse whether its expression is required 818 were upregulated and 208 were downregulated (see Supplementary for the recruitment of PIM1 to the FOSL1 enhancer (Fig. 6a and see Information, Table S1), in line with a previous analysis of MYC-regulated Supplementary Information, Fig. S4). We also analysed the MYC-depend- genes33. Comparison of genes regulated by MYC with those regulated by ent recruitment of PIM1 at the ID2 E box. ID2 is a well-characterized PIM1 showed that the latter contributes to the regulation of 207 of the MYC-regulated gene that, in contrast with FOSL1, contains its functional 1026 MYC-regulated genes. Thus, 20% of MYC-regulated genes are also MYC-binding sites upstream from the promoter28,32. As expected, ChIP regulated by PIM1. Functional characterization of these genes, based on analysis on control cells showed MYC association at the FOSL1 enhancer Gene Ontology categorization through the Panther database (http://www. and the ID2 E box at 120 min after serum treatment, whereas MYC silenc- pantherdb.org), revealed that nucleic acid metabolism, transcriptional reg- ing significantly decreased MYC association at these sites (Fig. 6b and see ulation, RNA processing and oncogenesis are statistically overrepresented Supplementary Information, Fig. S4). ChIP analysis revealed PIM1 associa- (P < 0.05) (see Supplementary Information, Tables S2 and S3). On the tion with both genes in control cells, whereas MYC knockdown significantly basis of this analysis we conclude that PIM1 acts as a cofactor for transcrip- decreased PIM1 association at these E boxes (Fig. 6c and see Supplementary tional regulation in a significant subset of MYC-dependent genes. Information, Fig. S4). Moreover, H3S10 phosphorylation showed a significant decrease in MYC-silenced cells at these sites (Fig. 6d and see Recruitment of PIM1 to chromatin by MYC contributes to cell Supplementary Information, Fig. S4). In contrast, H3K9acK14ac was transformation unaffected by MYC silencing (Fig. 6e and see Supplementary Information, We next investigated the biological relevance of MYC–PIM1 cooperation. Fig. S4), confirming that H3 acetylation is not dependent on MYC binding. First we verified whether PIM1 contributes to the MYC-dependent regu- The reintroduction of wild-type Flag–MYC, but not Flag–MYC∆MBII, in lation of cell cycle progression by silencing PIM1 in P493-6 cells, a cell line MYC-silenced cells restored PIM1 recruitment and H3S10 phosphoryla- that depends on MYC expression for its proliferation27,34,35. Incorporation tion at both sites (Fig. 6f–h and see Supplemenrary Information, Fig. S4). of [3H]thymidine was significantly decreased in PIM1-silenced cells, and These results establish that MYC, through its MBII domain, recruits PIM1 analysis of the cell cycle by fluorescence-activated cell sorting showed that to the E-box elements of these MYC-regulated genes and that its recruit- PIM1 depletion determines a delay in progression of these cells through ment is required for phosphorylating H3S10 at these sites. S phase (see Supplementary Information, Fig. S5). To test whether cooperation of PIM1 with MYC in cell transforma- PIM1 is required for transcriptional activation of FOSL1 and tion and tumour growth is due to the role of PIM1 in the transcriptional ID2 genes activation of MYC-target genes, we performed transformation experi- After treatment of HEK 293 cells with serum, both FOSL1 and ID2 ments in Rat-1 fibroblasts. Overexpression of MYC transforms these mRNA were induced with similar kinetics (Fig. 7b, e), corresponding cells morphologically and induces them to grow in soft agar36. PIM1 to the peak of PIM1 and MYC association and H3S10 phosphorylation silencing strongly inhibited the formation of soft-agar foci induced by in these cells. The same results were obtained by measuring FOSL1 and MYC overexpression, and MYC silencing affected the number of foci ID2 unprocessed heterogeneous nuclear RNA, indicating that increased induced by PIM1 overexpression to a similar extent (Fig. 8a), showing mRNA levels depend on transcriptional activation. PIM1 silencing, that PIM1 is indeed required for MYC-dependent cell transformation. as well as the expression of PIM1-K67M, inhibited the induction of Moreover, overexpression of MYC and PIM1 generates a significantly both FOSL1 and ID2 transcripts (Fig. 7b, e and see Supplementary greater number of colonies in soft agar than cells overexpressing either Information, Fig. S3), showing that PIM1 kinase activity is required for MYC or PIM1 (Fig. 8a, b). full transcriptional activation of both genes. To understand whether PIM1 acts as a MYC cofactor in cell trans- Quantitative ChIP analysis of RNA polymerase II (Pol II) and also formation, we generated chimaeric constructs in which PIM1 was Pol II phosphorylated on Ser 2 (Ser2P) at the C-terminal domain (CTD) directly fused to the N-terminal domain of MYC (PIM1–MYC). We of control cells revealed a significant increase in signal at FOSL1 and found that the PIM1–MYC fusion molecule induced a comparable ID2 promoters and along the genes in response to treatment with serum number of colonies in soft agar relative to the coexpression of MYC (Fig. 7c, f and see Supplementary Information, Fig. S3) mirroring the and PIM1 in the same cells (Fig. 8b), suggesting that PIM1 recruit- increase in transcript levels. PIM1 silencing led to a specific decrease ment to chromatin by MYC is required for cooperation between MYC in Pol II signal at the promoters and within the coding regions. This and PIM1 in cell transformation. Because formation of the MYC com- decrease, however, could not account for the inhibition of the transcrip- plex with PIM1 and its recruitment to MYC-binding sites require tional activity mediated by the PIM1 silencing. By contrast, the decrease the MBII domain (Figs 2 and 6), which is essential for the oncogenic in the elongating Pol II (Pol II-Ser2P) along the gene was more closely activity of MYC, we also fused PIM1 to the transformation-defective correlated with transcript levels of both FOSL1 and ID2, suggesting MYC whose MBII domain had been deleted. We found that the fusion that PIM1 silencing mostly affects the presence of Pol II engaged in PIM1–MYC∆MBII, but not cotransfection of MYC∆MBII with PIM1, transcript elongation. was able to rescue, at least in part, the defective MYC transformation (Fig. 8b). This fusion rescues both transcriptional activity and the PIM1 contributes to the regulation of a subgroup of MYC- ability to phosphorylate H3S10 at its recruiting sites (Fig. 8d, f). In regulated genes contrast, a chimaeric molecule in which PIM1 was fused to a MYC∆C, To understand the extent of PIM1 and MYC cooperation in gene tran- a mutant unable to bind to the DNA, was defective for transforming scription we performed an expression profile analysis of HEK 293 activity and for phosphorylation of H3S10 at the target genes ana- cells silenced either for MYC or PIM1 at 120 min after treatment with lysed (see Supplementary Information, Fig. S6). The transformed foci nature cell biology volume 9 | number 8 | AUGUST 2007 939 © 2007 Nature Publishing Group

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a FOSL1 d ID2 +1 +1

Probe Promoter Coding region Probe Promoter Coding region (–125/+160) (+4750/+4990) (–270/+90) (+1430/+1650)

b Transcript e Transcript

mRNA hnRNA mRNA hnRNA 8 16 16 16

6 n 12 12 12

4 8 8 8

Fold induction 2 4 4 4 Fold inductio Fold induction Fold induction 0 0 0 0 Serum 0 15 30 60 90 120 240 min Serum 015 30 60 90 120 240 min Serum 0 15 30 60 90 120 240 min Serum 0 15 30 60 90 120 240 min

ChIP analysis c f ChIP analysis Promoter Coding region 24 24 Promoter Coding region 20 20

16 t 16 15 15

Pol II t t Pol II 10 10 ‰ input 8 8 ‰ inpu ‰ inpu 5 ‰ inpu 5 0 0 Serum 015 30 60 90 120 180 240 min Serum 015 30 60 90 120 180240 min 0 0 Serum 015 30 60 90 120 180240 min Serum 015 30 60 90 120 180240 min

20 20 20 20

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Ser2P 10 10 Ser2P 10 10 ‰ input ‰ input ‰ input 5 5 ‰ input 5 5

0 0 0 0 Serum 015 30 60 90 120 180 240 min Serum 015 30 60 90 120 180 240 min Serum 015 30 60 90 120 180240 min Serum 015 30 60 90 120180 240 min

Figure 7 PIM1 is required for the transcriptional activation of FOSL1 and scrambled shRNA (shsP#1; filled bars) or PIM1 shRNA (shPIM1#1; open ID2. (a, d) Schematic representation of the FOSL1 (a) and ID2 (d) gene bars) at the FOSL1 promoter or coding region as in a. Real-time quantitative regions (not to scale). Exons are represented as black squares; E boxes are PCR measurements of the immunoprecipitated DNA are expressed as shown as a black circle. The probes used for ChIP analysis are indicated. thousandths (‰) of the DNA inputs. Values are mean and s.d. for three PCR probes are numbered relative to the first nucleotide of exon 1 (+1). replicate samples from one representative experiment (n = 3). (e) Time- (b) Time-course analysis by quantitative real-time RT–PCR of FOSL1 mRNA course analysis by quantitative real time RT–PCR of ID2 mRNA and ID2 and unprocessed heterogeneous nuclear FOSL1 RNA (hnRNA) in HEK 293 hnRNA in HEK 293 cells expressing either a control scrambled shRNA (filled cells expressing either a control scrambled shRNA (shsP#1; filled bars) or bars) or PIM1 shRNA (open bars). RNA levels were measured and normalized PIM1 shRNA (shPIM1#1; open bars) as indicated. Gene-specific RNA levels to GAPDH expression and are represented as fold induction (vertical bars). were measured and normalized to GAPDH expression and are represented as Values are mean and s.d. for three replicate samples from one representative fold induction (vertical bars). Real-time quantitative RT–PCR measurements. experiment (n = 3). (f) Time-course analysis by ChIP assay was performed Values are mean and s.d. for three replicate samples from one representative with antibodies as in c at the ID2 promoter or coding region as in d. Real- experiment (n = 3). (c) Time-course analysis by ChIP assay was performed time quantitative PCR measurements of the immunoprecipitated DNA are with antibodies recognizing Pol II or Pol II phosphorylated in its C-terminal expressed as thousandths (‰) of the DNA inputs. Values are mean and s.d. domain at Ser 2 (Ser2P) from HEK 293 cells expressing either a control for three replicate samples from one representative experiment (n = 3). induced by the PIM1–MYC fusion grew vigorously in soft agar, form- of colonies in soft agar, the transcription of FOSL1 and ID2 and the ing bigger colonies than those induced by kinase-dead PIM1–MYC phosphorylation of H3 at their MYC-binding sites (see Supplementary fusion (PIM1KD–MYC) (see Supplementary Information, Fig. S6). Information, Fig. S8). Finally, we tested whether H3 phosphorylation at Ser 10 is required Taken together, these results show that H3 phosphorylation at MYC- for MYC-dependent transformation by analysing MYC-transforming target genes by PIM1 is required for the cooperative effects of PIM1 and potential in the presence of yellow fluorescent protein (YFP) fused MYC in cell transformation. either to H3 (YFP–H3) or to H3 mutated at Ser 10 (YFP–H3S10A). Both fusion proteins localized together with the endogenous H3S10ph Discussion in the nucleus during interphase and mitosis (see Supplementary This study defines a new role for the serine/threonine kinase PIM1 as Information, Fig. S7) and they did not interfere with cell cycle pro- a MYC-dependent modifier of chromatin. On the basis of biochemical gression (see Supplementary Information, Fig. S7), in agreement with and functional evidence, we show that after stimulation with growth previous experiments with other histone fusion proteins37. We then factor, a MYC–MAX–PIM1 complex formed in the nucleus, recruits performed transformation assays by expressing MYC, PIM1 or PIM1– PIM1 to the chromatin, where it phosphorylates histone H3 at Ser 10. MYC fusion proteins in these cells (see Supplementary Information, This PIM1-dependent nucleosome phosphorylation is required for the Fig. S8). We observed that whereas YFP–H3 did not affect the number transcriptional activation of a subset of MYC-target genes and MYC- of foci, the YFP–H3S10A mutant significantly decreased the number dependent cell transformation.

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a Soft-agar b Soft-agar Constructs colonies SP1 MAX PIM1 MYC Constructs colonies MAX PIM1 MYC Fus. Mock 7 ± 1 Mock 9 7 shsP#3 + MYC 75 ± 6 ± PIM1 + MYC 124 ± 17 shPIM1#3 + MYC 17 ± 3 62 8 shsP#4 + MYC 73 ± 13 PIM1KD + MYC ± shPIM1#4 + MYC 15 ± 4 PIM1–MYC 150 ± 19 PIM1KD–MYC 56 ± 9 Mock 8 ± 1 PIM1 + MYC∆MBII 40 ± 5 65 ± 5 shsM#3 + PIM1 PIM1KD + MYC∆MBII 17 ± 3 shMYC#3 + PIM1 17 ± 4 PIM1–MYC∆MBII 110 ± 6 shsM#4 + PIM1 54 ± 5 PIM1KD–MYC∆MBII 19 ± 4 shMYC#4 + PIM1 12 ± 3 M (K) 21 30 66 97 Mr(K) 97 21 30 66 r c Transcript d Transcript FOSL1 ID2 FOSL1 ID2 10 8 10 10

8 8 8 6 6 6 6 4 4 4 4 2 2 2 Fold induction 2 Fold induction Fold induction Fold induction 0 0 I I I I I I

0 0 C C 1 1 1 1 MBI MBII MBI MBI MBI MBII MBI MBI Mock Mock ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ Mock Mock Mock Mock C PIM1–MY PIM1–MY PIM1 + MYC PIM1 + MYC PIM1KD–MYC PIM1KD–MYC PIM1KD + MYC PIM1KD + MYC shM#3 + PIM shM#4 + PIM shM#3 + PIM shM#4 + PIM shsP#3 + MYC shsP#4 + MYC shsP#3 + MYC shsP#4 + MYC PIM1–MY PIM1– MYC PIM1 + MYC PIM1 + MYC shPIM1#3 + MYC shPIM1#4 + MYC shMYC#3 + PIM1 shMYC#4 + PIM1 shPIM1#3 + MYC shPIM1#4 + MYC shMYC#3 + PIM1 shMYC#4 + PIM1 PIM1KD–MYC PIM1KD–MYC PIM1KD + MYC PIM1KD + MYC

e ChIP analysis f ChIP analysis FOSL1 ID2 FOSL1 ID2 H3S10ph 12 20 H3S10ph 20 20

15 15 15 8

10 10 10

4 ‰ input ‰ input ‰ input 5 5 ‰ input 5

0 0 0 0

20 20 PIM1 PIM1 20 25 20 15 15 15 15 10 10 10 10 ‰ input ‰ input ‰ input 5 ‰ input 5 5 5

0 0 0 0

20 20 20 25 MYC MYC 20 15 15 15 15 10 10 10 10 ‰ input ‰ input ‰ input ‰ input 5 5 5 5 0 0 0 0 I I I I I I I I 1 1 1 1 C C C C C C MBI MBI MBI MBI MBI MBI MBI MBI Mock Mock Mock Mock Mock Mock ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ C C C C C C PIM1–MY PIM1–MY PIM1 + MYC PIM1 + MYC shM#3 + PIM1 shM#4 + PIM1 shM#3 + PIM1 shM#4 + PIM1 PIM1KD–MY PIM1KD–MY shsP#3 + MYC shsP#4 + MYC shsP#3 + MYC shsP#4 + MYC PIM1KD + MY PIM1KD + MY shPIM1#3 + MYC shPIM1#4 + MYC shMYC#3 + PIM shMYC#4 + PIM shPIM1#3 + MYC shPIM1#4 + MYC shMYC#3 + PIM shMYC#4 + PIM PIM1–MY PIM1–MY PIM1 + MYC PIM1 + MYC PIM1KD–MY PIM1KD + MY PIM1KD + MY PIM1KD + MY

Figure 8 PIM1 contributes to MYC-dependent transformation. normalized to GAPDH expression and are represented as fold induction (a, b) Formation of Rat-1 cell-line colonies in soft agar. Rat-1 fibroblasts (vertical bars). Values are mean and s.d. for three replicate samples from were transfected with the constructs indicated. Transforming potential one representative experiment (n = 3). (e, f) ChIP assay performed in Rat- of the transfected constructs was determined by examining the colonies 1 cells with the use of antibodies as indicated. The primers used for the in soft agar formed within two weeks after transfection. Transforming FOSL1 gene correspond to probe B (shown diagrammatically in Fig. 4) potential was estimated by the average number of visible colonies in and for the ID2 MYC-binding sites (shown diagrammatically in Fig. 7). soft agar observed in three plates. A representative experiment is shown. Real-time quantitative PCR measurements of the immunoprecipitated Chimaeric PIM1–MYC fusions are indicated (Fus.). (c, d) Quantitative DNA are expressed as thousandths (‰) of the DNA inputs. Values are real-time RT–PCR of FOSL1 and ID2 mRNA in Rat-1 cells expressing mean and s.d. for three replicate samples from one representative the constructs indicated. Gene-specific RNA levels were measured and experiment (n = 3)

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Immunoprecipitation experiments showed that after stimulation with MYC-dependent transformed colonies in soft agar. Although our exper- growth factor, MYC, in conjunction with MAX, forms a complex with iments do not exclude the possibility that specific PIM1-regulated genes PIM1 in the nucleus. Silencing experiments demonstrated that MYC is cooperate with MYC in cell transformation, our data strongly suggest that required for the recruitment of PIM1 to the chromatin, whereas PIM1 is PIM1-dependent phosphorylation of H3 at MYC-target genes is nec- necessary to phosphorylate H3 at Ser 10 on the nucleosome at E boxes essary to regulate key genes required for MYC-dependent oncogenic to activate transcription. According to the current view, phosphoryla- transformation. In fact, fusion of PIM1 to the transformation-defec- tion at the N-terminal domain of H3 could be required either to loosen tive MYC∆MBII could rescue its transforming potential as well as H3 the interaction between DNA and nucleosome and/or to generate a phosphorylation at MYC-binding sites, whereas PIM1 coexpression platform to recruit additional regulatory factors as described in the with MYC∆MBII did not lead to rescue. ‘histone code’ hypothesis1. PIM1-dependent phosphorylation at the This study therefore provides a molecular mechanism for MYC and FOSL1 and ID2 genes is specific for its recruitment sites and does not PIM1 cooperation in gene regulation and oncogenic transformation. spread over the genes, suggesting that this phosphorylation is necessary Because mutations that alter MYC expression are among the most to activate the enhancer. common found in human and animal cancers44, it is conceivable that It was shown previously that, after treatment with growth factors, H3 inhibiting MYC association with PIM1 and/or inhibiting PIM1 kinase is phosphorylated at Ser 10 with fast kinetics by MSK1/MSK2 (refs 4–7). activity by specific drugs might represent a method for the treatment FOSL1 shows two regions with different phosphorylation kinetics of of cancers in humans. H3S10: a phosphorylation site at the upstream SRE, occurring within Methods 15–30 min as a result of MSK1/MSK2 action, and phosphorylation at the DNA constructs. Human PIM1 and MYC complementary DNA were cloned enhancer that was coincident with the peak of transcriptional activation into pcDNA5/FRT (Invitrogen, Carlsbad, CA, USA) vector under the control of of the gene mediated by PIM1. These two regions behaved differently the CMV/TetO2 tetracycline-inducible promoter. The PIM1-K67M construct with regard to the acetylation of H3 at Lys 9 and Lys 14. Whereas at was generated by introducing a site-specific mutation in the DNA sequence the FOSL1 promoter H3 phosphorylation and acetylation are tightly corresponding to Lys 67 to mutate it into a methionine residue (K67M). The coupled, as shown previously for other IE promoters3,38–40, at the FOSL1 MYC∆MBII construct was obtained by deleting the DNA sequence corresponding to amino-acid residues 119–152. The MYC∆C construct was obtained by enhancer the phosphorylation of H3S10 takes place in a region highly deleting the DNA sequence corresponding to amino-acid residues 353–439. The acetylated at H3 before the binding of MYC and PIM1. This is in agree- PIM1–MYC fusion constructs were obtained by fusing the cDNA corresponding ment with the finding that for recognition by MYC, its target sites must to PIM1 or PIM1-K67M by means of a ten-glycine encoding linker sequence to be highly acetylated in H3 as well as methylated in H3K4/K79 (ref. 41). the cDNA corresponding to MYC, MYC∆MBII or MYC∆C. On the FOSL1 enhancer, the phosphorylation and acetylation of H3 shRNA constructs were obtained by cloning a double-stranded DNA cassette into the pENTR/H1/TO vector, which allows the expression of shRNA under the might have distinct functional roles because PIM1-dependent phospho- control of the tetracycline-inducible H1 promoter TetO2. rylation of H3S10 is transient and limited to the E-box element whereas To generate shRNA cassettes the following oligonucleotides were acetylation at Lys 9 and Lys 14 is more stable and distributed along the designed in accordance with the manufacturer’s protocol. The 21-nucle- gene, suggesting that phosphorylation at these elements is a critical step otide positive strands were the following: PIM1 shRNA#1 (shPIM1#1), 5ʹ-GTCTCTTCAGAGTGTCAGCAC-3ʹ; PIM1 shRNA scrambled con- in transcriptional activation. The fast turnover of H3 phosphorylation trol#1 (shsP#1), 5ʹ-GACTCATCAGTATGTTAGCAT-3ʹ; PIM1 shRNA#2 observed in our experiments, and its temporal coincidence with tran- (shPIM1#2), 5ʹ-GAGGAAGAGAGTATCTATGGG-3ʹ; PIM1 shRNA scramble scriptional activation, might be a general phenomenon because H3 phos- control#2 (shsP#2), 5ʹ-GAAGCCGAGAGTATCTATGTT-3ʹ; PIM1 shRNA#3 phorylation was previously detected during transcriptional activation of (shPIM1#3), 5ʹ-CGAAGAAATCCAGGACCATCC-3ʹ; PIM1 shRNA scrambled heat shock genes in Drosophila and during induction of the RAR-β2 gene control#3 (shsP#3), 5ʹ-AAGTCATCCGAAGACCACAAC-3ʹ; PIM1 shRNA#4 (shPIM1#4), 5ʹ-ACGTGGAGAAGGACCGGATTT-3ʹ; PIM1 shRNA scrambled by retinoic agonists in murine P19 embryonal cells whereas acetylation control #4 (shsP#4), 5ʹ-GATTCGAGGACATGGACTGAG-3ʹ; c-MYC shRNA#1 42,43 was constitutive at these loci . (shMYC#1), 5ʹ-GCAATCACCTATGAACTTGTT-3ʹ; c-MYC shRNA scramble It has been estimated that about 11% of cellular genes present a func- control#1 (shsM#1), 5ʹ-GCATTCACCTAAGAAGTTCTT-3ʹ; c-MYC shRNA#2 tional E box with which MYC can associate on the genome28. MYC (shMYC#2), 5ʹ-GACGACGAGACCTTCATCAAA-3ʹ; c-MYC shRNA2 scramble control #2 (shMYC#2), 5ʹ-AGATCCAACCAGTCAGGACAT-3ʹ; c-MYC silencing demonstrated that MYC recruits PIM1 at specific MYC-bind- shRNA#3 (shMYC#3), 5ʹ-CCTGAGACAGATCAGCAACAA-3ʹ; c-MYC shRNA ing sites, and confocal microscopy showed an elevated degree of nuclear scramble control#3 (shsM#3), 5ʹ-CAACATGGTAACGAACAGCAA-3ʹ; c-MYC colocalization of PIM1 with nascent transcripts and with MYC after shRNA#4 (shMYC#4), 5ʹ-CAGTTGAAACACAAACTTGAA-3ʹ; c-MYC shRNA treatment with growth factor, suggesting that at this stage of the cell scramble control#4 (shsM#4), 5ʹ-AACTAAGGTAACTATGCACAA-3ʹ; c-MYC cycle PIM1 is recruited by MYC to a large number of sites. A gene shRNA#5 (shMYC#5), 5ʹ-CCATAATGTAAACTGCCTCAA-3ʹ; c-MYC shRNA scramble control #5 (shsM#5), 5ʹ-GCAATGACCACCTATTCAATA-3ʹ. expression profile analysis of genes regulated by MYC and PIM1 in cells The recombinant GST–PIM1 construct was obtained by cloning the wild-type treated with serum at 120 min revealed that 207 transcripts correspond- PIM1 and PIM1-K67M cDNA into the bacterial expression vector pGEX-4T; the ing to 20% of MYC-regulated genes also require PIM1 for their regula- fused protein was expressed and purified with a standard protocol. tion. The co-regulation includes genes involved in cell metabolism, The vectors coding for Xenopus laevis histones were expressed and purified as described previously45. Mutant recombinant histones H3 (H3S10A, H3S28A protein synthesis, cycle progression and oncogenesis. A large number of and H3S10A/S28A) were obtained by site-directed mutagenesis as described pre- these genes are transcriptional factors, which suggests that PIM1 partic- viously46. YFP–H3 was kindly provided by M. Bianchi (Milan, Italy) and muta- ipates in MYC-dependent regulatory networks. In agreement with these genized as described previously46. data, cell cycle analysis in PIM1 knockdown cells revealed decreased Cell culture condition. We generated a tetracycline-inducible HEK 293 cell line entry into S phase. Transformation analysis showed a more marked stably expressing wild-type PIM1 or PIM1-K67M with the use of the Flp-T-Rex PIM1 effect because PIM1 silencing strongly inhibited the formation of System from Invitrogen.

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pCDNA5/FRT/TO expression vector containing the gene of interest was about 104 cells in 2 ml of 0.35% low-melting agarose in a 35-mm culture dish inserted into the cells by means of Flp recombinase-mediated DNA recombina- containing 0.7% agarose base. Triplicates were prepared for each tested construct tion at the Flp recombination target (FRT) site. The transfected cells were selected and inspected by eye after staining with crystal violet for colony formation after on the basis of resistance to hygromycin B (Sigma, St Louis, MO, USA), and a incubation at 37 °C for 10–14 days. Efficiency of transfection was measured with pool of clones was isolated. a reporter gene encoding green fluorescent protein, and the levels of proteins were Selected clones were cultivated for two or three weeks in DMEM containing compared by western blotting. 10% fetal bovine serum (FBS; Sigma) and 150 µg ml−1 hygromycin. For time-course experiments, HEK 293 cells were synchronized and then Antibodies. The antibodies used for in this work were purchased from Santa treated with 50% serum shock as described47. Primary HUVECs were grown in Cruz Biotechnology (Santa Cruz, CA, USA) (anti-PIM1 SC-7856 for ChIP and M199 medium supplemented with 15% FBS, brain protein extracts and heparin immunofluorescence experiments; anti-PIM1 SC-13513 for western blot analysis; (50 µg ml−1). The cells were then starved for 24 h in M199 medium containing anti-Myc SC-764 for ChIP and immunofluorescence experiments and anti-Myc 1% FBS and 0.5% BSA and induced with 50 ng ml−1 VEGF-A. SC-40 for western blot analysis; anti-MAX SC-197 and SC-765; anti-SP1 SC-59; The cell line P493-6 (kindly provided by B. Amati, Milan, Italy) was grown in anti-Pol II SC-899), from Covance (Berkeley, CA, USA) (anti-Pol II Ser2 H5), RPMI 1640 containing 10% FCS, 100 U ml−1 penicillin, 100 µg ml−1 streptomycin from Upstate (Charlottesville, VA, USA) (anti-histone H3 06-755; anti-acetyl- and 2 mM l-glutamine. MYC repression was obtained with 0.1 µg ml−1 tetracycline. histone H3 06-599; anti-phospho-(Ser 10)-acetyl (Lys 14)-histone H3 07-081; MYC induction was obtained by washing the cells three times with tetracycline-free anti-phospho-(Ser 10)-histone H3 05-817) and from Abcam (Cambridge, UK) PBS containing 10% FCS, as described previously27. (anti-phospho-(Ser 10)-acetyl (Lys 9)-histone H3 antibody 12181-50).

Immunostaining and colocalization analysis. Colocalization experiments Note: Supplementary Information is available on the Nature Cell Biology website. were performed on fixed cells as described previously11, with a TCS SP2 confocal Acknowledgements microscope (Leica, Wetzlar, Germany). To label nascent RNA, anti-BrUTP (Sigma We thank all members of the laboratory for reagents, helpful suggestions and catalogue no. B2531) was used in permeabilized cells and immunodetected with encouragement; M. Rocchigiani for VEGF-A preparations, B. Grandi for technical 48 small modifications from a previously described protocol . The degree of dou- support, M. Bianchi for the YFP–H3 construct, R. Mantovani for Xenopus laevis ble staining was measured by quantitative analysis of overlapping pixels by using histone constructs, B. Amati for Rat-1 and P493-6 cells, and I. Delany, M. Bianchi Image J version 1.32j software (NIH, Bethesda, MD, USA). All pixels with the same and E. Guccione for critical reading of the manuscript. This work was supported position in both images were considered coincident and the brightness level of each by Associazione Italiana Ricerca sul cancro (AIRC), Ministero Italiano Università e pixel was plotted on the x and y axes of a scatter diagram. Two completely overlap- Ricerca (MIUR), and Fondazione Monte dei Paschi di Siena. ping images would result in a straight diagonal line in the scatter diagram. Author contributions Protein immunoprecipitation, interaction assays, kinase assays and chromatin A.Z. planned and performed the experiments and analysed the data. A.D.R. generated stable clones and performed immunoprecipitation experiments. R.S. immunoprecipitation. Proteins were extracted by using F-buffer (10 mM Tris performed the transformation assays. S.O. planned the experimental design, buffer pH 7.0, 50 mM NaCl, 30 mM Na HPO , 50 mM NaF, 5 µM ZnCl , 1% 2 4 2 analysed the data and wrote the manuscript. Triton-X 100) and treated with 1 U ml−1 micrococcal nuclease (Sigma) at 40 °C for 1 h to obtain the chromatin proteins (chromatin fraction). Competing financial interests To identify interacting protein, 0.5 mg of total protein was incubated overnight The authors declare no competing financial interests. at 4 °C with 3 µg of specific antibody. Immunocomplexes were incubated with Protein-A-conjugated or Protein-G-conjugated magnetic beads (Dynal, Oslo, Published online at http://www.nature.com/naturecellbiology/ Reprints and permissions information is available online at http://npg.nature.com/ Norway) for 30 min at 4 °C. Samples were washed four times with F buffer and reprintsandpermissions/ twice with F-buffer containing 0.15 M NaCl, for 20 min at 4 °C. Proteins were eluted by boiling the samples and analysed by western blotting. 1. Strahl, B. D. & Allis, C. D. The language of covalent histone modifications. Nature 403, The GST interaction assay was performed by incubating 1 mg of protein extracts 41–45 (2000). 2. Berger, S. L. Histone modifications in transcriptional regulation. Curr. Opin. Genet. (derived from chromatin fractions) with recombinant proteins GST, GST–PIM1 Dev. 12, 142–148 (2002). wild-type (WT) and GST–PIM1-K67M bound to Glutathione-Sepharose resin 3. Cheung, P., Allis, C. D. & Sassone-Corsi, P. Signaling to chromatin through histone (Amersham, Piscataway, NJ, USA). To allow specific interactions, proteins were modifications. Cell 103, 263–271 (2000). incubated in interaction buffer (20 mM Tris buffer pH 8.0, 200 mM KCl, 10 mM 4. Nowak, S. J. & Corces, V. G. Phosphorylation of histone H3: a balancing act between chromosome condensation and transcriptional activation. Trends Genet. 20, 214–220 dithiothreitol, 0.1 mM EGTA, 20% glycerol and inhibitor protease cocktail) for 2 h (2004). at 4 °C in the presence of 21-residue peptides. Unbound proteins were eliminated 5. Thomson, S., Mahadevan, L. C. & Clayton, A. L. MAP kinase-mediated signalling to by three washes in the same buffer followed by protein equilibration in kinase nucleosomes and immediate-early gene induction. Semin. Cell Dev. Biol. 10, 205–214 (1999). buffer (30 mM Tris buffer, 10 mM dithiothreitol; 0.1 mM EGTA, 2 mM MnCl2, 6. Sassone-Corsi, P. et al. Requirement of Rsk-2 for epidermal growth factor-activated 10% glycerol). Kinase assay was performed by incubating samples in the same phosphorylation of histone H3. Science 285, 886–891 (1999). buffer in the presence of 4 pmol of [γ-32P]ATP for 2 min at 30 °C. The reaction was 7. Soloaga, A. et al. MSK2 and MSK1 mediate the mitogen- and stress-induced phospho- stopped by the addition of 10 mM EDTA and samples were washed six times with rylation of histone H3 and HMG-14. EMBO J. 22, 2788–2797 (2003). 8. Yamamoto, Y., Verma, U. N., Prajapati, S., Kwak, Y. T. & Gaynor, R. B. Histone H3 kinase buffer containing 10 mM EDTA and 200 mM KCl. Phosphorylated proteins phosphorylation by IKK-α is critical for cytokine-induced gene expression. Nature 423, were separated by SDS–PAGE and revealed by autoradiography. 655–659 (2003). The reconstitution of nucleosome core particles was performed as described49. 9. Anest, V. et al. A nucleosomal function for IκB kinase-α in NF-κB-dependent gene ChIP assays were performed as described by Upstate Protocols (http://www. expression. Nature 423, 659–663 (2003). 10. Cuypers, H. T. et al. Murine leukemia virus-induced T-cell lymphomagenesis: integration upstate.com). DNA was analysed by quantitative real-time PCR with the use of proviruses in a distinct chromosomal region. Cell 37, 141–150 (1984). of Light Cycler DNA Amplification Kit SYBR Green I (Roche, Mannheim, 11. Zippo, A., De Robertis, A., Bardelli, M., Galvagni, F. & Oliviero, S. Identification of Germany). All experiment values were normalized to those obtained with a non- Flk-1-target genes in vasculogenesis: Pim-1 is required for endothelial and mural cell immune serum and divided by the input, using the procedure described previ- differentiation in vitro. Blood 103, 4536–4544 (2004). 12. van Lohuizen, M. et al. Predisposition to lymphomagenesis in pim-1 transgenic mice: 50 ously . Experiments on inhibition by MSK1/MSK2 were performed by incubating cooperation with c-myc and N-myc in murine leukemia virus-induced tumors. Cell 56, cells with H89 (Calbiochem, San Diego, CA, USA) as described previously5. 673–682 (1989). Quantitative RT–PCR analysis was performed as described previously11. 13. van Lohuizen, M. et al. Identification of cooperating oncogenes in E µ-myc transgenic mice by provirus tagging. Cell 65, 737–752 (1991). 14. Verbeek, S. et al. Mice bearing the E µ-myc and E µ-pim-1 transgenes develop pre-B- Cell transformation and colony formation in soft agarose. The analysis of cell cell leukemia prenatally. Mol. Cell. Biol. 11, 1176–1179 (1991). transformation and anchorage-independent growth were performed as described 15. Wang, Z. et al. Pim-1: a serine/threonine kinase with a role in cell survival, proliferation, previously36. Cells transfected with the constructs indicated were suspended at differentiation and tumorigenesis. J. Vet. Sci. 2, 167–179 (2001).

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A., Cano, E. & Mahadevan, L. C. Mitogen-stimulated phos- 20. Murphy, M. J., Wilson, A. & Trumpp, A. More than just proliferation: Myc function in phorylation of histone H3 is targeted to a small hyperacetylation-sensitive fraction. stem cells. Trends Cell Biol. 15, 128–137 (2005). Proc. Natl Acad. Sci. USA 91, 4781–4785 (1994). 21. Grandori, C. et al. c-Myc binds to human ribosomal DNA and stimulates transcription 39. Cheung, P. et al. Synergistic coupling of histone H3 phosphorylation and acetyla- of rRNA genes by RNA polymerase I. Nature Cell Biol. 7, 311–318 (2005). tion in response to epidermal growth factor stimulation. Mol. Cell 5, 905–915 22. Arabi, A. et al. c-Myc associates with ribosomal DNA and activates RNA polymerase I (2000). transcription. Nature Cell Biol. 7, 303–310 (2005). 40. Clayton, A. L., Rose, S., Barratt, M. J. & Mahadevan, L. C. Phosphoacetylation of 23. 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Active genes dynamically colocalize to shared sites of ongoing linked to retinoic acid receptor beta promoter activation. EMBO Rep. 3, 335–340 transcription. Nature Genet. 36, 1065–1071 (2004). (2002). 27. Schuhmacher, M. et al. The transcriptional program of a human B cell line in response 44. Cole, M. D. & McMahon, S. B. The Myc oncoprotein: a critical evaluation of transactiva- to Myc. Nucleic Acids Res. 29, 397–406 (2001). 28. Fernandez, P. C. et al. Genomic targets of the human c-Myc protein. Genes Dev. 17, tion and target gene regulation. Oncogene 18, 2916–2924 (1999). 1115–1129 (2003). 45. Caretti, G., Motta, M. C. & Mantovani, R. NF-Y associates with H3-H4 tetramers and 29. Bergers, G., Graninger, P., Braselmann, S., Wrighton, C. & Busslinger, M. Transcriptional octamers by multiple mechanisms. Mol. Cell. Biol. 19, 8591–8603 (1999). activation of the Fra-1 gene by AP-1 is mediated by regulatory sequences in the first 46. 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VEGF-A 0 20 40 60 120240 min DAPI PECAM1 PIM1 PIM1 PECAM1

b VEGF-A 0 20 40 60 90 120 240 min interferential Overlay H3S1Oph

c VEGF-A 0 20 40 60 90 120 240 min

17 kDA H3S10ph

17 kDA Histones

Figure S1. VEGF treatment induces PIM1 expression and translocation in the Immunostaining with antibodies recognising H3S10ph on HUVEC treated nucleus and H3S10 phosphorylation. (a) PIM1 localization in HUVEC upon with VEGF-A at the times indicated. Interferential picture of the same field VEGF-A induction. Indirect immunofluorescence analysis of HUVEC treated and merge are shown. (Scale bar = 74 µm). (c) Upper panel, Western blot with VEGF at the times indicated. Cytoplasmatic membranes and nuclei were analysis of H3S10ph with cell extracts from HUVEC treated. Western blot stained with anti-PECAM1 antibody (green), and DAPI (blue) respectively. analysis on acidic-protein extracts was performed by immunoblotting using PIM1 was immunostained with anti-PIM1 antibody (red). PECAM1 and antibodies to reveal H3S10ph from HUVEC treated with VEGF-A at the time PIM1 co-localization are shown in the enlarged merging pictures (Scale points indicated. Lower panel, Coomassie staining of total histones. bar = 48µm). (b) H3S10 phosphorylation upon VEGF-A treatment. A representative experiment is shown.

WWW.NATURE.COM/NATURECELLBIOLOGY 1 SUPPLEMENTARY INFORMATION

a shsP#2 shPIM1#2 b FOSL1 Tet - + - + - + - + +1 Serum - - + + - - + + 30 kDa PIM1 21 kDa MAX Probe A B 97 kDa SP1 1 2 3 4 5 6 7 8 (-590/-330) (+930/+1170)

cd Probe A ( 30 min) Probe B ( 120 min)

H3S10ph PIM1 MYCH3S10phK14ac H3S10phK9ac H3K9acK14ac H3S10ph PIM1MYC H3S10phK14ac H3S10phK9ac H3K9acK14ac 25 12 20 25 30 35 25 12 20 25 30 35 shsP#2 shsP#2 30 10 25 30 10 25 shPIM1#2 20 20 shPIM1#2 20 20 15 25 15 25 8 20 8 20 15 15 20 15 15 20 6 10 15 6 10 15 15 15 Input 10 10 10 10 4 10 Input 4 10 ‰ 10 10

5 ‰ 5 5 5 5 5 5 2 5 5 2 5

0 0 0 0 0 0 0 0 0 0 0 0

ef shsP#1 shPIM1#1

12 Serum 0 15 30 60 90 120 240 0 15 30 60 90 120 240 min shsP#1 10 shPIM1#1 17 kDA 8 H3S10ph 6 4 17 kDA Histones 2 H3S10ph relative value 0 Serum 0 15 30 60 90 120 240 min

Figure S2. PIM1 mediates H3S10 phosphorylation. (a) Western blot analysis three replicate samples from one representative experiment (n = 3). (d) of 293 stable clones expressing a control scrambled shRNA (shsP#2) or PIM1 knockdown affects H3S10 phosphorylation at the FOSL1 enhancer. PIM1 shRNA (shPIM1#2) inducible by tetracycline by immunostaining using ChIP assay was performed by using antibodies as in panel c from 293 cells antibodies recognising PIM1, MAX, or SP1. Protein extracts were obtained expressing either a control scrambled shRNA (shsP#2) or PIM1 shRNA from not stimulated or serum-stimulated cells as indicated, in presence (Tet (shPIM1 #2) at 120 minutes after serum treatment. Real-time quantitative +) or absence (Tet -) for shRNA expression. (b) Schematic representation PCR measurements of the immunoprecipitated DNA are expressed as of the FOSL1 gene region (not in scale). Exons are represented as black 1/1000 (‰) of the DNA inputs. The values represent the mean s. d. of three squares within the gene and the E box is figured as a black spot. The probes replicate samples from one representative experiment (n = 3). (e) PIM1 used in ChIP analysis are indicated. PCR probes intervals are numbered knockdown affects H3S10 phosphorylation in 293 cells treated with serum. relatively to the fist nucleotide of exon 1 (+1). (c) PIM1 knockdown does Upper panel, Western blot analysis on acidic-protein extracts was performed not alter H3S10 phosphorylation at the FOSL1 promoter. ChIP assay by immunoblotting using antibodies to reveal H3S10ph from 293 treated was performed by using antibodies recognising H3S10ph, PIM1, MYC, with serum either expressing a scrambled shRNA (shsP#1) or a PIM1 shRNA phosphoacetylated H3 or acetylated H3 as specified from 293 cells (shPIM1#1) at the time indicated. Lower panel, Coomassie staining of total expressing either a control scrambled shRNA (shsP#2) or PIM1 shRNA histones. A representative experiment is shown. (f) Densitometric analysis. (shPIM1#2) at 30 minutes after serum treatment. Real-time quantitative The values represent the mean s. d. of three independent experiments (n = PCR measurements of the immunoprecipitated DNA are expressed as 3; P<0.01). 1/1000 (‰) of the DNA inputs. The values represent the mean s. d. of

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a b FOSL1 Probe B (120 min.)

PIM1 WT PIM1-K67M Tet - + - + - + - + H3S10ph PIM1 MYC H3S10phK14ac H3S10phK9ac H3K9acK14ac 25 25 Serum - - + + - - + + 25 25 25 25 PIM1 WT 20 20 20 20 20 20 PIM1-K67M 30 kDA 15 15 15 15 15 15 21 kDA 10 10 10 Input 10 10 10 ‰ 5 5 97 kDA 5 5 5 5 0 0 1 2 3 4 5 6 7 8 0 0 0 0 Tet - - - - + ++ + Tet - - - - + ++ + Tet - - - - + + + + Tet - - - - + ++ + Tet - - - - + ++ + Tet - - - - + + + + Serum - -+ + - - + + Serum - -+ + - - + + Serum - -+ + - - + + Serum - -+ + - - + + Serum - -+ + - - + + Serum - -+ + - - + +

FOSL1 ID2 FOSL1 ID2 c transcript 120 min transcript 120 min ChIP 120 min ChIP 120 min mRNA hnRNA mRNA hnRNA 12 12 14 14 Pol IISer2P Pol II Ser2P 18 18 18 18 shsP#2 10 10 12 12 16 16 16 16 14 14 14 shPIM1#2 10 10 14 8 8 12 12 12 12 8 8 6 6 10 10 10 10 6 6 8 8 8 8 4 4 6 6 6 6 4 4 ‰ Input ‰ Input

Fold induction Fold induction 4 4 4 4 2 2 2 2 2 2 2 2 0 0 0 0 0 0 0 0

FOSL1 ID2 FOSL1 ID2 d transcript 120 min. transcript 120 min. ChIP analysis 120 min. ChIP analysis 120 min. mRNA hnRNA mRNA hnRNA Pol II Ser2P Pol II Ser2P 10 14 10 14 25 25 25 25 PIM1 WT 12 12 8 8 20 20 20 20 PIM1-K67M 10 10 6 15 15 15 15 6 8 8

6 4 6 4 10 10 10 10 ‰ Input 4 4 ‰ Input Fold induction Fold induction 2 2 5 5 5 5 2 2

0 0 0 0 0 0 0 0

Figure S3. PIM1 kinase activity is required for H3S10 phosphorylation and Polimerase II (Pol II) or RNA Polimerase II phosphorylated in its C terminal for transcriptional activation. (a) Western blot analysis of 293 stable clones domain at Serine 2 (Ser2P) from 293 cells expressing either a control expressing, under the control of tetracycline, either wild type PIM1 or PIM1- scrambled shRNA or PIM1 shRNA at the FOSL1 promoter or coding region. K67M mutant as indicated. Protein extracts were obtained from untreated Real-time quantitative PCR measurements of the immunoprecipitated DNA or serum-treated 293 cells as indicated, in presence (Tet +) or absence (Tet are expressed as 1/1000 (‰) of the DNA inputs. The values represent the -) for the expression of PIM1 constructs. (b) The expression of PIM1 kinase mean s. d. of three replicate samples from one representative experiment inactive affects H3S10 phosphorylation at the FOSL1 enhancer. ChIP assay (n = 3). (d) Left panels. Quantitative real time RT-PCR of ID2 mRNA and was performed by using antibodies indicated from 293 cells expressing ID2 hnRNA in 293 cells expressing either the wild type PIM1 or the either the wild type PIM1 or the PIM1-K67M. Real-time quantitative PCR PIM1-K67M mutant as indicated. Gene specific RNA levels were measured measurements of the immunoprecipitated DNA and expressed as 1/1000 and normalized on GAPDH expression and represented as fold induction (‰) of the DNA inputs. The values represent the mean s. d. of three (vertical bars) by real-time quantitative RT-PCR measurements. Right panels. replicate samples from one representative experiment (n = 3). (c) Left The values represent the mean s. d. of three replicate samples from one panels. Quantitative real time RT-PCR of FOSL1 mRNA and unprocessed representative experiment (n = 3). ChIP assay performed by using antibodies heterogeneous nuclear FOSL1 RNA (hnRNA) in 293 cells expressing recognising RNA Polimerase II (Pol II) or RNA Polimerase II phosphorylated either a control scrambled shRNA (shsP#2) or PIM1 shRNA (shPIM1#2) in its C terminal domain at Serine 2 (Ser2P) from 293 cells expressing as indicated. Gene specific RNA levels were measured and normalized on either the wild type PIM1 or the PIM1-K67M mutant as indicated. Real- GAPDH expression and represented as fold induction (vertical bars) by real- time quantitative PCR measurements of the immunoprecipitated DNA are time quantitative RT-PCR measurements. The values represent the mean s. expressed as 1/1000 (‰) of the DNA inputs. The values represent the mean d. of three replicate samples from one representative experiment (n = 3). s. d. of three replicate samples from one representative experiment (n = 3). Right panels. ChIP assay performed by using antibodies recognising RNA

CORRIGENDUM A labelling error for Tetracycline induction in panel b of Figure S3 has been corrected on 3rd November 2007.

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a bc de f MYC PIM1 H3S10ph H3K9acK14ac 25 12 16 30 shsM#5 + - + - + - shMYC#5 14 - + - + - + 10 25 shsM #2 shMYC #2 20 MYCWT - - + + - - 12 Tet - + - + - + - + Δ 8 20 MYC MBII - - - - + + Serum - - + + - - + + 15 10 6 8 15 66 kDA Input Input 66 kDA Input MYC

MYC Input 10 6 ‰

‰ 4 ‰ 10 ‰ 21 kDA MAX 4 5 MAX 2 5 21 kDA 97 kDA SP1 2 shsM #2 0 0 0 0 shMYC #2 97 kDA 1 2 3 4 5 6 7 8 SP1 ID2 ID2 ID2 ID2 1 2 3 4 5 6 FOSL1 FOSL1 FOSL1 FOSL1 gh FOSL1 ID2

MYC PIM1 H3S10ph H3K9acK14ac MYC PIM1 H3S10ph H3K9acK14ac 25 15 20 35 25 15 20 35 shsM #5 30 30 20 20 shMYC #5 15 15 25 25 10 10 15 20 15 20 10 10 15 15 Input

10 Input 10 5 5

‰ 10 10 5 ‰ 5 5 5 5 5

0 0 0 0 0 0 0 0 MBII MBII MBII MBII MBII MBII MBII MBII Δ Δ Δ Δ Δ Δ Δ Δ MOCK MOCK MOCK MOCK MOCK MOCK MOCK MOCK FLAG-MYC FLAG-MYC FLAG-MYC FLAG-MYC FLAG-MYC FLAG-MYC FLAG-MYC FLAG-MYC F-MYC F-MYC F-MYC F-MYC F-MYC F-MYC F-MYC F-MYC

Figure S4. MYC recruits PIM1 to phosphorylates histone H3 at FOSL1 MYC gene silencing by transient transfection with either a control scrambled and ID2 regulatory elements. (a) Inducible MYC gene silencing. 293 were shRNA (shsM#5) or MYC shRNA (shMYC#5) as indicated. shMYC#5 transiently transfected with either a control scrambled shRNA (shsM#2) recognizes MYC 3’ untranslated sequence which is not present in the or MYC shRNA (shMYC#2) as indicated by tetracycline treatment. Western FLAG constructs co-transfected. (g, h) MYC but not MYC_BMII rescues the blot analysis on protein extracts was performed by immunoblotting, recruiting of PIM1 to the chromatin. Chromatin samples were obtained from using antibodies recognising MYC, MAX, or SP1 as indicated. (b-e) 293 cells expressing the control scrambled shRNA (shsM#5), MYC shRNA MYC recruits PIM1 on chromatin. Chromatin samples were obtained (shMYC#5), or the shRNA together with FLAG-MYC or FLAG-MYC_MBII from serum-induced 293 cells expressing the control scrambled shRNA as indicated. ChIP assays were performed using antibodies as indicated. scM #2 or MYC shRNA #2. A ChIP assay was performed using antibodies shMYC#5 recognizes MYC 3’ untranslated region which is not present in the as indicated. Immunoprecipitated DNA was analyzed by quantitative PCR FLAG constructs co-transfected. Immunoprecipitated DNA was analyzed with primers to amplify the FOSL1 enhancer and ID2 upstream MYC-bindig by quantitative PCR with primers to amplify the FOSL1 enhancer and ID2 sites (-1705/-1460). Real-time quantitative PCR measurements of the upstream MYC-binding sites (-1705/-1460). Real-time quantitative PCR immunoprecipitated DNA are expressed as 1/1000 (‰) of the DNA inputs. measurements of the immunoprecipitated DNA areexpressed as 1/1000 (‰) The values represent the mean s. d. of three replicate samples from one of the DNA inputs. The values represent the mean s. d. of three replicate representative experiment (n = 3). (f) Western blot analysis on protein samples from one representative experiment (n = 3). extracts was performed by immunoblotting using antibodies as indicated.

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a b shsP#2 shPIM1#2 contr. shsP#1shPIM1#1 Tet + - + - Tet + - + - 25 30 shsP#1 shsP#2 PIM1 20 shPIM1#1 PIM1 25 shPIM1#2

15 20 MYC MYC 15 10

MAX (Fold induction) MAX 10 5 (Fold induction) 5 [3H]Thymidine incorporation 0 SP1

SP1 [3H]Thymidine incorporation 0 24 48 72 96 hours 24 48 72 96 hours 1 2 3 4 1 2 3 4 c -Tet: 0h -Tet: 12h -Tet: 24h

shsP#1 shPIM1#1 shsP#1 shPIM1#1 shsP#1 shPIM1#1 Ap 0.2% Ap 0.2% Ap 0.3% Ap 0.1% Ap 0.1% Ap 0.7% G1 89.3% G1 90.8% G1 84.6% G1 90.2% G1 73.9% G1 82.8% S 3.2% S 2.2% S 4.3% S 2.3% S 14.4% S 6.2% G2/M 7.2% G2/M 6.7% G2/M 10.7% G2/M 7.2% G2/M 11.6% G2/M 10.2%

shsP#2 shPIM1#2 shsP#2 shPIM1#2 shsP#2 shPIM1#2 Ap 0.3% Ap 0.3% Ap 0.3% Ap 0.1% AP 0.1% Ap 0.7% G1 89.9% G1 90.3% G1 82.8% G1 89.8% G1 75.4% G1 84.9% S 2.2% S 2.1% S 5.6% S 2.3% S 12.4% S 5.6% G2/M 7.4% G2/M 7.3% G2/M 11.3% G2/M 7.7% G2/M 12.1% G2/M 8.6%

Figure S5. Cell cycle progression of P 493-6 cells. (a, b) Left panels. Tymidine incorporation was measured either in P493-6 expressing either Western blot analysis of MYC and PIM1 expression in P493-6 transiently scrambled shRNA or PIM1 shRNA as indicated. The values represent the transfected either with control scrambled shRNA or PIM1 shRNA as mean s. d. of three independent experiments (n = 3; P<0.02). (c) Cell cycle indicated in the presence or absence of tetracycline. Immunoblotting were analysis by propidium iodide incorporation in P493-6 either expressing performed with antibodies as indicated. Right panels. MYC expression scrambled shRNA or PIM1 shRNA as indicated. was induced in P493-6 cells arrested in G1 by tetracycline washing and

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Soft agar colonies Averafge size (μm) a PIM1 MYC e 1 313 1 MBII bHLH-LZ 439 ± ± PIM1 + MYC 132 12 311 58 transcript K67M 70 ± 9 240 ± 51 PIM1kD + MYC FOSL1 ID2 PIM1-MYC 145 ± 8 503 ± 102 14 14 ± ± 12 12 PIM1kD-MYC 73 7 244 38 10 10 118 152 8 8 PIM1-MYCΔMBII 95 ± 9 344 ± 63 6 6 PIM1kD-MYCΔMBII 11 ± 1 124 ± 19 4 4

353 2 2 Δ 13 ± 1 164 ± 24 Fold induction Fold induction PIM1-MYC C 0 0 C C C Δ 8 ± ± C Δ PIM1kD-MYC C 1 114 35 Δ Δ Δ MBII MBII MBII MBII Δ Δ Δ Δ control control PIM1-MYC PIM1-MYC PIM1 + MYC PIM1 + MYC PIM1kD-MYC PIM1kD-MYC PIM1-MYC PIM1-MYC C MBII PIM1kD + MYC Δ PIM1kD + MYC Δ MBII C PIM1kD-MYC PIM1kD-MYC PIM1-MYC PIM1-MYC Δ Δ PIM1kD-MYC PIM1kD-MYC

b control PIM1 + MYC PIM1kD + MYC PIM1-MYC PIM1kD-MYC PIM1-MYC PIM1kD-MYC PIM1-MYC PIM1kD-MYC 97 kDA PIM1 f ChIP analysis MYC 66 kDA

PIM1 FOSL1 ID2 30 kDA H3S10ph 30 30 25 25 21 kDA MAX 20 20 1 2 3 4 5 6 7 8 9 15 15 10 10 ‰ Input 5 5 0 0 c 30 30 PIM1 25 25 20 20 PIM1 + MYC PIM1kD + MYC PIM1-MYC PIM1kD-MYC 15 15 10 10 5 ‰ Input5 ‰ Input 0 0

30 30 PIM1-MYCΔMBII PIM1kD-MYCΔMBII PIM1-MYCΔC PIM1kD-MYCΔC MYC 25 25 20 20 15 15 10 10 ‰ Input ‰ Input

5 ‰ Input 5 d 0 0 C C C C Δ Δ Δ Δ Δ Δ MBII MBII MBII PIM1 + MYC PIM1-MYC PIM1-MYC MBII PIM1-MYC C MBII Δ Δ Δ Δ control control PIM1-MYC PIM1-MYC PIM1 + MYC PIM1 + MYC PIM1kD-MYC PIM1kD-MYC PIM1-MYC PIM1-MYC PIM1kD + MYC PIM1kD + MYC PIM1kD-MYC PIM1kD-MYC PIM1-MYC PIM1-MYC PIM1kD-MYC PIM1kD-MYC PIM1kD + MYC PIM1kD-MYC PIM1kD-MYCΔMBII PIM1kD-MYCΔC Fig. S6

Figure S6. Soft agar colony formation of Rat-1 cell lines. (a) Rat-1 is shown. Scale bar = 200 µm. (e) Quantitative real time RT-PCR of fibroblasts were transfected with the constructs indicated which are FOSL1 and ID2 mRNA in exponentially growing Rat-1 cells expressing represented schematically. Numbers refer to the amino acid of PIM1 and the constructs indicated. Gene specific RNA levels were measured and MYC proteins. MYC MBII domain was indicated by dashed box. A black normalized on GAPDH expression and represented as fold induction (vertical dot indicates PIM1 carrying the single amino acid substitution K67M. bars) by real-time quantitative RT-PCR measurements. The values represent Transforming potential of the transfected constructs was determined by the mean s. d. of three replicate samples from one representative experiment examining the soft agar colonies formed within two weeks post-transfection. (n = 3). (f) ChIP assay performed in Rat-1 cells by using antibodies as The average size of soft agar colonies observed in three plates were indicated. The primers used for the FOSL1 gene correspond to probe B measured and determined as mean ± SD of 10 colonies per sample. (b) shown in Figure 4 and for the ID2 MYC binding sites as in Figure 7. Real- Western blot analysis of cells transfected with the constructs indicated. (c) time quantitative PCR measurements of the immunoprecipitated DNA are Plates from cells transfected with each chimeric construct are shown. (d) expressed as 1/1000 (‰) of the DNA inputs. The values represent the mean A representative field of cells transfected with each chimaeric construct s. d. of three replicate samples from one representative experiment (n = 3).

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a H3S10 H3S10 YFP-H3 YFP-H3S10A * * MERGE MERGE

MOCKYFP-H3 YFP-H3S10A

Ap 0.1 % Ap 0.1 % Ap 0.1 % G1 56.0 % G1 51.7 % G1 50.7 % S 21.7% S 19.3 % S 20.6 % G2/M 22.3 % G2/M 29.0 % G2/M 28.8 %

Figure S7. YFP-H3 expression in Rat-1 cell lines. (a) YFP-H3 is incorporated Mitotic (asterics) and interphasic (arrow) nuclei are shown. Scale bar=15μm in the chromatin. Rat-1 fibroblasts were transfected with the DNA constructs (b) Cell cycle analysis of proliferating Rat-1 fibroblasts expressing either coding for either YFP-H3 or YFP-H3S10A as indicated. Immunofluorescence YFP-H3 or YFP-H3S10A. Rat-1 fibroblasts were transfected with either analysis of YFP-H3 or YFP-H3S10A nuclear sub-localization in proliferating YFP-H3 or YFP-H3S10A and cell cycle phase distribution was determined by Rat-1 fibroblasts. Fixed cells were stained for H3S10ph (red) and the level FACS analysis to compare mock with transfected cells. of co-localization with YFP-H3 or YFP-H3S10A (yellow) was determined.

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Figure S8. Soft agar colony formation of Rat-1 cell lines. (a-b) Western specific RNA levels were measured and normalized on GAPDH expression blot analysis on protein extracts obtained from Rat-1 cells transfected and represented as fold induction (vertical bars) by real-time quantitative with the constructs indicated. Protein expression level was determined by RT-PCR measurements. The values represent the mean s. d. of three immunoblotting using antibodies as indicated. (c) Rat-1 fibroblasts were replicate samples from one representative experiment (n = 3). (e) ChIP assay transfected with the constructs indicated. Transforming potential of the performed in Rat-1 cells by using antibodies as indicated. The primers used transfected constructs was determined by examining the soft agar colonies for the FOSL1 gene correspond to probe B shown in Figure 4 and for the ID2 formed within two weeks post-transfection. Transforming potential was MYC binding sites as in Figure 7. Real-time quantitative PCR measurements estimated as the average number of visible soft agar colonies observed in of the immunoprecipitated DNA areexpressed as 1/1000 (‰) of the DNA three plates. (d) Quantitative real time RT-PCR of FOSL1 and ID2 mRNA in inputs. The values represent the mean s. d. of three replicate samples from exponentially growing Rat-1 cells expressing the constructs indicated. Gene one representative experiment (n = 3).

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a b PIM1

32 32P-H3 P-H3

GST-PIM1 PIM1

H3 GST

histones

PIM1 MYC PIM1 MYC PIM1

MAX

Figure S9. Full scans of key gels and Westerns. (a) Full scans relative to panel b (right panel). The middle panel is a shorter exposure of the same Figure 1 panel a. (b) Full scans relative to Figure 1 panel c. (c) Full scans filter showed in the upper panel. relative to Figure 2 panel b (left panel). (d) Full scans relative to Figure 2

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TABLE SI-SIII Gene expression profiling. The gene expression analysis was performed by hybridizing RNA samples to the Whole Human Genome Oligo Microarray from Agilent Technologies. Samples were obtaind from 293 cells, trteated with serum for 120 minutes, expressing either two independent MYC shRNA (shMYC#1 and shMYC#2) or their relative scrambled shRNA (shsM#1 and shsM#2) for MYC expression analysis; two independent PIM1 shRNA (shPIM1#1 and shPIM1#2) or their relative scrambled shRNA (shsP#1 and shsP#2) for PIM1 expression analysis. RNA preparation, labelling hybridization scanning and feature extraction were performed by the UHN Microarray Center, Toronto, Canada (www.microarrays.ca) following the protocols suggested by Agilent Technologies (www.agilent.com/chem/dnamanuals-protocols).

Data analysis was performed by using GeneSpring software (Agilent Technologies). Filtering was performed across all arrays to remove spots that were at or below the background levels. Arrays were then examined individually and filtered to create lists of genes for those features that had a fold change of greater than or equal to 2 between the two channels. These lists were combined in Venn diagrams to create lists of genes that were common between pairs of samples. A list of genes demonstrating all of the changing features across the 4 arrays was used to create a cluster using a Pearson Correlation and average linkage. Gene clustering analysis was performed with PANTHER gene expression data analysis (www.pantherdb.org). The microarray data are deposited in Gene expression omnibus (GEO www.ncbi.nlm.nih.gov/geo) with accession number GSE7960.

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Table SI. Gene expression analysis

Categories UP DOWN TOTAL

MYC regulated genes 818 208 1026 PIM1 regulated genes 482 435 917 MYC and PIM1 regulated genes 155 52 207

Table SII. Panther gene cluster

MYC AND PIM1 NCBI: H. sapiens genes (REF) MYC REGULATED GENES PIM1 REGULATED GENES REGULATED GENES

Biological Process #of genes P value #of genes P value # P value

Nucleoside, nucleotide and nucleic acid metabolism 38 1.52E-07 148 3.52E-19 115 1.04E-13 Biological process unclassified 22 4.01E-06 152 6.20E-11 110 8.42E-13 mRNA transcription 22 2.31E-03 87 2.25E-10 77 3.29E-11 Oncogenesis 8 2.97E-02 15 1.00E00 17 7.17E-02 Pre-mRNA processing 7 3.79E-02 16 5.55E-02 16 4.78E-03 Developmental processes 19 5.43E-02 65 2.70E-02 63 1.55E-04 mRNA splicing 6 6.47E-02 14 3.14E-02 13 1.33E-02 Amino acid activation 3 8.78E-02 4 1.00E00 5 6.51E-02 mRNA transcription regulation 16 8.90E-02 68 4.27E-08 59 3.62E-08 Cell cycle 11 1.19E-01 43 1.66E-04 30 4.25E-02 Cell cycle control 7 3.09E-01 24 1.07E-03 15 6.11E-01 Cell proliferation and differentiation 9 9.70E-01 32 3.70E-01 27 3.54E-01

Molecular Function

Nucleic acid binding 37 6.27E-09 131 5.76E-18 104 7.60E-14 Molecular function unclassified 20 1.85E-06 132 5.68E-15 92 1.32E-17 Transcription factor 26 1.04E-05 89 2.87E-10 80 1.02E-11 Other transcription factor 10 4.35E-04 26 3.92E-06 22 1.94E-05 Other RNA-binding protein 6 2.96E-02 15 2.07E-03 9 7.77E-01 mRNA processing factor 5 7.07E-02 11 4.17E-02 13 2.17E-04 Aminoacyl-tRNA synthetase 3 1.04E-01 4 1.00E00 5 7.04E-02 Synthetase 4 1.28E-01 5 1.00E00 6 8.64E-01 Synthase and synthetase 4 3.84E-01 8 1.00E00 7 1.00E+00 Transferase 0 6.35E-01 13 1.00E00 10 1.00E+00

Table SIII. MYC and PIM1 regulated genes

Common Description GO biological process GO molecular function EXPRESSION

GO:0003676(nucleic acid Homo sapiens mRNA for KIAA2025 binding);GO:0004842(ubiquitin-protein ligase AB095945 GO:0016567(protein ubiquitination) UP protein. [AB095945] activity);GO:0008270(zinc ion binding);GO:0046872(metal ion binding) Homo sapiens metallothionein 2A GO:0005507(copper ion NM_005953 GO:0006878(copper ion homeostasis) UP (MT2A), mRNA [NM_005953] binding);GO:0046872(metal ion binding) Homo sapiens RNA binding motif GO:0000166(nucleotide NM_014892 protein 16 (RBM16), mRNA UP binding);GO:0003723(RNA binding) [NM_014892] GO:0001558(regulation of cell growth);GO:0006350(transcription);GO: Homo sapiens inhibitor of growth GO:0005515(protein 0006355(regulation of transcription, NM_019071 family, member 3 (ING3), transcript binding);GO:0008270(zinc ion UP DNA- variant 1, mRNA [NM_019071] binding);GO:0046872(metal ion binding) dependent);GO:0016568(chromatin modification) GO:0003779(actin binding);GO:0005509(calcium ion Homo sapiens actinin, alpha 2 NM_001103 binding);GO:0005515(protein UP (ACTN2), mRNA [NM_001103] binding);GO:0008307(structural constituent of muscle) GO:0006917(induction of apoptosis);GO:0007049(cell cycle);GO:0042981(regulation of Homo sapiens B-cell CLL/lymphoma apoptosis);GO:0043123(positive GO:0004871(signal transducer NM_003921 UP 10 (BCL10), mRNA [NM_003921] regulation of I-kappaB kinase/NF- activity);GO:0005515(protein binding) kappaB cascade);GO:0045786(negative regulation of progression through cell cycle)

GO:0000122(negative regulation of GO:0004861(cyclin-dependent protein kinase Homo sapiens hexamethylene bis- transcription from RNA polymerase II inhibitor activity);GO:0005515(protein NM_006460 acetamide inducible 1 (HEXIM1), promoter);GO:0045736(negative binding);GO:0016564(transcriptional UP mRNA [NM_006460] regulation of cyclin dependent protein repressor activity);GO:0017069(snRNA kinase activity) binding) Homo sapiens vasodilator- stimulated phosphoprotein (VASP), NM_003370 GO:0006928(cell motility) GO:0003779(actin binding) UP transcript variant 1, mRNA [NM_003370] Homo sapiens hypothetical protein NM_018382 FLJ11292 (FLJ11292), mRNA UP [NM_018382] GO:0001501(skeletal development);GO:0001541(ovarian GO:0005125(cytokine follicle activity);GO:0005160(transforming growth Homo sapiens inhibin, beta A development);GO:0006917(induction of factor beta receptor (activin A, activin AB alpha apoptosis);GO:0006952(defense NM_002192 binding);GO:0005179(hormone UP polypeptide) (INHBA), mRNA response);GO:0007050(cell cycle activity);GO:0005515(protein [NM_002192] arrest);GO:0007166(cell surface binding);GO:0008083(growth factor receptor linked signal activity);GO:0017106(activin inhibitor activity) transduction);GO:0007267(cell-cell signa GO:0007169(transmembrane receptor Homo sapiens abl-interactor 1 protein tyrosine kinase signaling NM_005470 (ABI1), transcript variant 1, mRNA GO:0008092(cytoskeletal protein binding) UP pathway);GO:0008285(negative [NM_005470] regulation of cell proliferation) GO:0006171(cAMP biosynthesis);GO:0006701(progesteron e biosynthesis);GO:0007165(signal Homo sapiens adrenomedullin transduction);GO:0007267(cell-cell NM_001124 GO:0005179(hormone activity) UP (ADM), mRNA [NM_001124] signaling);GO:0007565(pregnancy);GO: 0007588(excretion);GO:0008015(circula tion);GO:0009611(response to wounding) GO:0007267(cell-cell Homo sapiens sprouty homolog 2 signaling);GO:0007275(development);G NM_005842 (Drosophila) (SPRY2), mRNA UP O:0009887(organogenesis);GO:000996 [NM_005842] 6(regulation of signal transduction)

GO:0000074(regulation of progression through cell Homo sapiens enhancer of cycle);GO:0006139(nucleobase, NM_004450 rudimentary homolog (Drosophila) GO:0005554(molecular function unknown) UP nucleoside, nucleotide and nucleic acid (ERH), mRNA [NM_004450] metabolism);GO:0006213(pyrimidine nucleoside metabolism)

Homo sapiens slit-like 2 (Drosophila) NM_138440 UP (SLITL2), mRNA [NM_138440] GO:0004722(protein serine/threonine GO:0000074(regulation of progression phosphatase activity);GO:0004725(protein Homo sapiens dual specificity through cell tyrosine phosphatase NM_001946 phosphatase 6 (DUSP6), transcript cycle);GO:0000188(inactivation of UP activity);GO:0016787(hydrolase variant 1, mRNA [NM_001946] MAPK activity);GO:0006470(protein activity);GO:0017017(MAP kinase amino acid dephosphorylation) phosphatase activity) Homo sapiens ribosomal protein GO:0003735(structural constituent of NM_000998 L37a (RPL37A), mRNA GO:0006412(protein biosynthesis) UP ribosome);GO:0046872(metal ion binding) [NM_000998] Homo sapiens inhibitor of DNA binding 2, dominant negative helix- GO:0007275(development);GO:004544 NM_002166 GO:0030528(transcription regulator activity) UP loop-helix protein (ID2), mRNA 9(regulation of transcription) [NM_002166] GO:0006334(nucleosome Homo sapiens H2A histone family, assembly);GO:0007001(chromosome NM_012412 member V (H2AFV), transcript GO:0003677(DNA binding) UP organization and biogenesis (sensu variant 1, mRNA [NM_012412] Eukaryota)) Homo sapiens RNA binding motif GO:0000166(nucleotide NM_0010174 (RNP1, RRM) protein 3 (RBM3), GO:0006396(RNA processing) binding);GO:0003676(nucleic acid UP transcript variant 2, mRNA 30 binding);GO:0003723(RNA binding) [NM_001017430] GO:0006350(transcription);GO:0006355 (regulation of transcription, DNA- GO:0003714(transcription corepressor Homo sapiens ring finger protein 12 dependent);GO:0016481(negative activity);GO:0004842(ubiquitin-protein ligase NM_016120 (RNF12), transcript variant 1, mRNA UP regulation of activity);GO:0008270(zinc ion [NM_016120] transcription);GO:0016567(protein binding);GO:0046872(metal ion binding) ubiquitination) Homo sapiens FOS-like antigen 1 GO:0006357(regulation of transcription NM_005438 GO:0003700(transcription factor activity) UP (FOSL1), mRNA [NM_005438] from RNA polymerase II promoter);

Homo sapiens clone HQ0457 AF090927 PRO0457 mRNA, complete cds. UP [AF090927] GO:0008115(sarcosine oxidase Homo sapiens pipecolic acid GO:0006118(electron activity);GO:0016491(oxidoreductase NM_016518 oxidase (PIPOX), mRNA transport);GO:0046653(tetrahydrofolate UP activity);GO:0050031(L-pipecolate oxidase [NM_016518] metabolism) activity) Homo sapiens DAZ associated GO:0007283(spermatogenesis);GO:003 GO:0000166(nucleotide NM_170711 protein 1 (DAZAP1), transcript UP 0154(cell differentiation) binding);GO:0003723(RNA binding) variant 1, mRNA [NM_170711] GO:0003700(transcription factor Homo sapiens GA binding protein GO:0006350(transcription);GO:0006355 activity);GO:0003702(RNA polymerase II transcription factor, alpha subunit (regulation of transcription, DNA- NM_002040 transcription factor UP 60kDa (GABPA), mRNA dependent);GO:0006366(transcription activity);GO:0003713(transcription coactivator [NM_002040] from RNA polymerase II promoter) activity);GO:0005515(protein binding) Homo sapiens zinc finger protein GO:0006350(transcription);GO:0006355 GO:0003677(DNA binding);GO:0008270(zinc NM_201269 644 (ZNF644), transcript variant 1, (regulation of transcription, DNA- UP ion binding);GO:0046872(metal ion binding) mRNA [NM_201269] dependent) Homo sapiens phorbol-12-myristate- NM_021127 13-acetate-induced protein 1 UP (PMAIP1), mRNA [NM_021127] GO:0006350(transcription);GO:0006355 Homo sapiens period homolog 2 (regulation of transcription, DNA- NM_022817 (Drosophila) (PER2), transcript dependent);GO:0007165(signal GO:0004871(signal transducer activity) UP variant 1, mRNA [NM_022817] transduction);GO:0007623(circadian rhythm) Homo sapiens kinesin family GO:0000166(nucleotide GO:0007018(microtubule-based NM_182903 member 9 (KIF9), transcript variant binding);GO:0003777(microtubule motor UP movement) 3, mRNA [NM_182903] activity);GO:0005524(ATP binding) Homo sapiens adenosylmethionine GO:0006597(spermine GO:0004014(adenosylmethionine NM_001634 decarboxylase 1 (AMD1), mRNA biosynthesis);GO:0008295(spermidine decarboxylase activity);GO:0016829(lyase UP [NM_001634] biosynthesis) activity) Homo sapiens histone 1, H4c NM_003542 UP (HIST1H4C), mRNA [NM_003542]

GO:0000079(regulation of cyclin dependent protein kinase Homo sapiens SERTA domain activity);GO:0006350(transcription);GO: NM_013376 containing 1 (SERTAD1), mRNA UP 0006355(regulation of transcription, [NM_013376] DNA-dependent);GO:0008284(positive regulation of cell proliferation) Homo sapiens cyclin J (CCNJ), GO:0000074(regulation of progression NM_019084 UP mRNA [NM_019084] through cell cycle) Homo sapiens metallothionein 2A, ENST000002 mRNA (cDNA clone MGC:12397 GO:0005507(copper ion GO:0006878(copper ion homeostasis) UP 45185 IMAGE:4051220), complete cds. binding);GO:0046872(metal ion binding) [BC007034] Homo sapiens RAD23 homolog B GO:0006289(nucleotide-excision NM_002874 (S. cerevisiae) (RAD23B), mRNA repair);GO:0006464(protein GO:0003697(single-stranded DNA binding) UP [NM_002874] modification) Homo sapiens DEAD/H (Asp-Glu- GO:0004386(helicase NM_012141 Ala-Asp/His) box polypeptide 26 activity);GO:0004888(transmembrane UP (DDX26), mRNA [NM_012141] receptor activity) GO:0006355(regulation of transcription, Homo sapiens distal-less homeo box DNA- NM_004405 GO:0003700(transcription factor activity) UP 2 (DLX2), mRNA [NM_004405] dependent);GO:0007275(development); GO:0007420(brain development) GO:0006355(regulation of transcription, Homo sapiens distal-less homeo box DNA- NM_004405 GO:0003700(transcription factor activity) UP 2 (DLX2), mRNA [NM_004405] dependent);GO:0007275(development); GO:0007420(brain development) GO:0006376(mRNA splice site Homo sapiens CUG triplet repeat, selection);GO:0006397(mRNA GO:0000166(nucleotide RNA binding protein 1 (CUGBP1), processing);GO:0007281(germ cell binding);GO:0000900(translation repressor NM_198700 UP transcript variant 2, mRNA development);GO:0009790(embryonic activity, nucleic acid [NM_198700] development);GO:0016246(RNA binding);GO:0042835(BRE binding) interference) Homo sapiens RAB11 family NM_0010022 interacting protein 1 (class I) UP 33 (RAB11FIP1), transcript variant 2, mRNA [NM_001002233] GO:0008289(lipid Homo sapiens START domain GO:0006694(steroid binding);GO:0015485(cholesterol NM_139164 containing 4, sterol regulated biosynthesis);GO:0006869(lipid UP binding);GO:0017127(cholesterol transporter (STARD4), mRNA [NM_139164] transport) activity)

Homo sapiens hypothetical protein NM_173822 MGC39518 (MGC39518), mRNA UP [NM_173822] Homo sapiens zinc finger protein GO:0006355(regulation of transcription, GO:0003676(nucleic acid NM_153231 UP 550 (ZNF550), mRNA [NM_153231] DNA-dependent) binding);GO:0008270(zinc ion binding) Homo sapiens CCAAT/enhancer GO:0006350(transcription);GO:0006355 NM_001806 binding protein (C/EBP), gamma (regulation of transcription, DNA- GO:0003677(DNA binding) UP (CEBPG), mRNA [NM_001806] dependent) GO:0006350(transcription);GO:0006357 GO:0003700(transcription factor Homo sapiens jun D proto-oncogene NM_005354 (regulation of transcription from RNA activity);GO:0003702(RNA polymerase II UP (JUND), mRNA [NM_005354] polymerase II promoter) transcription factor activity) GO:0006813(potassium ion GO:0003677(DNA Homo sapiens Zic family member 2 transport);GO:0007399(neurogenesis); binding);GO:0005249(voltage-gated NM_007129 (odd-paired homolog, Drosophila) GO:0007420(brain UP potassium channel activity);GO:0008270(zinc (ZIC2), mRNA [NM_007129] development);GO:0030154(cell ion binding);GO:0046872(metal ion binding) differentiation) GO:0000166(nucleotide binding);GO:0003725(double-stranded RNA Homo sapiens Dicer1, Dcr-1 GO:0006396(RNA binding);GO:0004519(endonuclease homolog (Drosophila) (DICER1), processing);GO:0030423(RNA activity);GO:0004525(ribonuclease III NM_177438 UP transcript variant 1, mRNA interference, targeting of mRNA for activity);GO:0005524(ATP [NM_177438] destruction) binding);GO:0008026(ATP-dependent helicase activity);GO:0016787(hydrolase activity) GO:0000166(nucleotide binding);GO:0003676(nucleic acid binding);GO:0005524(ATP Homo sapiens dJ467N11.1 protein NM_022091 binding);GO:0008026(ATP-dependent UP (DJ467N11.1), mRNA [NM_022091] helicase activity);GO:0016787(hydrolase activity);GO:0017111(nucleoside- triphosphatase activity) Homo sapiens microtubule-actin GO:0003779(actin crosslinking factor 1 (MACF1), binding);GO:0005509(calcium ion NM_033044 GO:0007050(cell cycle arrest) UP transcript variant 2, mRNA binding);GO:0008017(microtubule [NM_033044] binding);GO:0051015(actin filament binding) GO:0006334(nucleosome Homo sapiens histone 1, H4d assembly);GO:0007001(chromosome NM_003539 GO:0003677(DNA binding) UP (HIST1H4D), mRNA [NM_003539] organization and biogenesis (sensu Eukaryota))

Homo sapiens cDNA FLJ39417 fis, AK096736 UP clone PLACE6016942. [AK096736] GO:0006350(transcription);GO:0006355 Homo sapiens elongation factor, (regulation of transcription, DNA- GO:0003702(RNA polymerase II transcription NM_012081 RNA polymerase II, 2 (ELL2), mRNA dependent);GO:0006368(RNA UP factor activity) [NM_012081] elongation from RNA polymerase II promoter) ENST000003 Homo sapiens mRNA for FLJ00074 UP 04963 protein, partial cds. [AK024480] Homo sapiens hypothetical protein NM_194285 FLJ39441 (FLJ39441), mRNA UP [NM_194285] GO:0000074(regulation of progression through cell Homo sapiens FBJ murine cycle);GO:0000122(negative regulation GO:0003677(DNA osteosarcoma viral oncogene of transcription from RNA polymerase II NM_006732 binding);GO:0008134(transcription factor UP homolog B (FOSB), mRNA promoter);GO:0006355(regulation of binding) [NM_006732] transcription, DNA- dependent);GO:0007275(development); GO:0007610(behavior)

GO:0006118(electron transport);GO:0006350(transcription);G O:0006355(regulation of transcription, GO:0000166(nucleotide DNA-dependent);GO:0006397(mRNA binding);GO:0000287(magnesium ion processing);GO:0006468(protein amino binding);GO:0004674(protein serine/threonine Homo sapiens endoplasmic acid kinase activity);GO:0004713(protein-tyrosine reticulum to nucleus signalling 1 NM_152461 phosphorylation);GO:0006915(apoptosi kinase activity);GO:0005524(ATP UP (ERN1), transcript variant 2, mRNA s);GO:0006917(induction of binding);GO:0016740(transferase [NM_152461] apoptosis);GO:0006986(response to activity);GO:0016787(hydrolase unfolded protein);GO:0006987(unfolded activity);GO:0016891(endoribonuclease protein response, activation of signaling activity, producing 5'-phosphomonoesters) protein activity);GO:0007050(cell cycle arrest)

Homo sapiens LQFBS-1 mRNA, AF062385 UP complete cds. [AF062385]

A_23_P46070 UP GO:0006350(transcription);GO:0006355 (regulation of transcription, DNA- Homo sapiens early growth GO:0003700(transcription factor dependent);GO:0007420(brain response 2 (Krox-20 homolog, activity);GO:0005515(protein NM_000399 development);GO:0007422(peripheral UP Drosophila) (EGR2), mRNA binding);GO:0008270(zinc ion nervous system [NM_000399] binding);GO:0046872(metal ion binding) development);GO:0007638(mechanose nsory behavior) Homo sapiens butyrophilin, subfamily 2, member A1 (BTN2A1), NM_078476 UP transcript variant 2, mRNA [NM_078476] Homo sapiens hypothetical protein GO:0006355(regulation of transcription, NM_032753 MGC15631 (MGC15631), mRNA GO:0003700(transcription factor activity) UP DNA-dependent) [NM_032753] GO:0006350(transcription);GO:0006357 GO:0003700(transcription factor Homo sapiens metal-regulatory (regulation of transcription from RNA activity);GO:0003713(transcription coactivator NM_005955 transcription factor 1 (MTF1), mRNA polymerase II UP activity);GO:0008270(zinc ion [NM_005955] promoter);GO:0010038(response to binding);GO:0046872(metal ion binding) metal ion) Homo sapiens neuronal pentraxin II GO:0005509(calcium ion NM_002523 GO:0007268(synaptic transmission) UP (NPTX2), mRNA [NM_002523] binding);GO:0005529(sugar binding) Homo sapiens serine/arginine NM_005839 repetitive matrix 1 (SRRM1), mRNA UP [NM_005839] GO:0006350(transcription);GO:0006355 Homo sapiens transducin (beta)-like (regulation of transcription, DNA- NM_024665 1X-linked receptor 1 (TBL1XR1), UP dependent);GO:0006512(ubiquitin mRNA [NM_024665] cycle) Homo sapiens htra2-beta-2 mRNA, GO:0000398(nuclear mRNA splicing, GO:0000166(nucleotide U87836 UP complete cds. [U87836] via spliceosome) binding);GO:0003723(RNA binding) Q82VR6 (Q82VR6) Phosphate THC2310678 transport system permease protein, UP partial (6%) [THC2310678] GO:0000166(nucleotide GO:0007018(microtubule-based Homo sapiens tubulin alpha 6 binding);GO:0003924(GTPase NM_032704 movement);GO:0051258(protein UP (TUBA6), mRNA [NM_032704] activity);GO:0005198(structural molecule polymerization) activity);GO:0005525(GTP binding)

Homo sapiens hypothetical protein NM_018195 FLJ10726 (FLJ10726), mRNA UP [NM_018195] Homo sapiens apoptosis related AF144054 protein APR-4 mRNA, partial cds. UP [AF144054] A_24_P22943 UP 8 Homo sapiens mesoderm induction early response 1 homolog (Xenopus NM_020948 GO:0003677(DNA binding) UP laevis) (MIER1), mRNA [NM_020948] GO:0000185(activation of MAPKKK Homo sapiens growth arrest and activity);GO:0006915(apoptosis);GO:00 NM_015675 DNA-damage-inducible, beta UP 06950(response to (GADD45B), mRNA [NM_015675] stress);GO:0030154(cell differentiation)

A_24_P25040 UP Homo sapiens tribbles homolog 1 GO:0006468(protein amino acid GO:0004672(protein kinase NM_025195 (Drosophila) (TRIB1), mRNA phosphorylation);GO:0008283(cell UP activity);GO:0005524(ATP binding) [NM_025195] proliferation) Homo sapiens neurofilament 3 GO:0005200(structural constituent of NM_005382 (150kDa medium) (NEF3), mRNA UP cytoskeleton) [NM_005382] Homo sapiens SM-11044 binding GO:0005215(transporter NM_020123 protein (SMBP), mRNA GO:0006810(transport) UP activity);GO:0005488(binding) [NM_020123] A_24_P32930 UP

GO:0003755(peptidyl-prolyl cis-trans GO:0006457(protein isomerase Homo sapiens RAN binding protein NM_006267 folding);GO:0006606(protein-nucleus activity);GO:0005488(binding);GO:0008536(R UP 2 (RANBP2), mRNA [NM_006267] import);GO:0006810(transport) an GTPase binding);GO:0016853(isomerase activity);GO:0046872(metal ion binding)

Homo sapiens solute carrier family 25 (mitochondrial carrier; phosphate GO:0003774(motor NM_0010066 carrier), member 25 (SLC25A25), GO:0006810(transport);GO:0007517(m activity);GO:0005488(binding);GO:0005509(c UP nuclear gene encoding mitochondrial uscle development) 41 alcium ion binding) protein, transcript variant 2, mRNA [NM_001006641] Homo sapiens metallothionein 2A GO:0005507(copper ion NM_005953 GO:0006878(copper ion homeostasis) UP (MT2A), mRNA [NM_005953] binding);GO:0046872(metal ion binding) GO:0006935(chemotaxis);GO:0006954( inflammatory response);GO:0006968(cellular defense response);GO:0007155(cell adhesion);GO:0007165(signal GO:0001584(rhodopsin-like receptor Homo sapiens chemokine (C-C transduction);GO:0007186(G-protein activity);GO:0004872(receptor NM_001837 motif) receptor 3 (CCR3), transcript UP coupled receptor protein signaling activity);GO:0016493(C-C chemokine variant 1, mRNA [NM_001837] pathway);GO:0007188(G-protein receptor activity) signaling, coupled to cAMP nucleotide second messenger);GO:0007204(elevation of cytoplasmic calcium ion concentration) GO:0006869(lipid Homo sapiens GGA binding partner transport);GO:0015031(protein NM_018318 GO:0008289(lipid binding) UP (FLJ11088), mRNA [NM_018318] transport);GO:0042157(lipoprotein metabolism) GO:0006091(generation of precursor metabolites and energy);GO:0007186(G- protein coupled receptor protein Homo sapiens gastric inhibitory GO:0004930(G-protein coupled receptor signaling NM_000164 polypeptide receptor (GIPR), mRNA activity);GO:0016519(gastric inhibitory UP pathway);GO:0007190(adenylate [NM_000164] peptide receptor activity) cyclase activation);GO:0007584(response to nutrients) Homo sapiens RUN and FYVE NM_017987 domain containing 2 (RUFY2), GO:0008270(zinc ion binding) UP mRNA [NM_017987] GO:0000166(nucleotide Homo sapiens eukaryotic translation GO:0006412(protein binding);GO:0003743(translation initiation NM_001969 initiation factor 5 (EIF5), transcript biosynthesis);GO:0006446(regulation of UP factor activity);GO:0003924(GTPase variant 1, mRNA [NM_001969] translational initiation) activity);GO:0005525(GTP binding)

GO:0006355(regulation of transcription, Homo sapiens distal-less homeo box DNA- NM_004405 GO:0003700(transcription factor activity) UP 2 (DLX2), mRNA [NM_004405] dependent);GO:0007275(development); GO:0007420(brain development) AI955728 wt37g09.x1 NCI_CGAP_Pan1 Homo sapiens AI955728 UP cDNA clone IMAGE:2509696 3', mRNA sequence [AI955728] THC2273623 UP

Homo sapiens chromosome 3 open NM_174908 reading frame 6 (C3orf6), transcript UP variant 1, mRNA [NM_174908] Homo sapiens mRNA for splicing GO:0000398(nuclear mRNA splicing, GO:0000166(nucleotide AB209694 factor, arginine/serine-rich 12 variant UP via spliceosome) binding);GO:0003676(nucleic acid binding) protein. [AB209694] Homo sapiens chromosome 6 open reading frame 216 (C6orf216), GO:0004553(hydrolase activity, hydrolyzing O- NM_206910 GO:0005975(carbohydrate metabolism) UP transcript variant 3, mRNA glycosyl compounds) [NM_206910] Homo sapiens, clone BC000206 IMAGE:3350658, mRNA. UP [BC000206] Homo sapiens glycosyltransferase GO:0009103(lipopolysaccharide NM_015101 25 domain containing 2 (GLT25D2), GO:0016740(transferase activity) UP biosynthesis) mRNA [NM_015101] PREDICTED: Homo sapiens similar XM_496579 to RIKEN cDNA E030024N20 gene UP (LOC440892), mRNA [XM_496579]

Homo sapiens cDNA FLJ13497 fis, AK023559 UP clone PLACE1004518. [AK023559]

Homo sapiens cDNA FLJ12277 fis, AK022339 UP clone MAMMA1001711. [AK022339]

GO:0006350(transcription);GO:0006355 (regulation of transcription, DNA- Homo sapiens SMAD, mothers dependent);GO:0007165(signal GO:0003700(transcription factor against DPP homolog 1 (Drosophila) transduction);GO:0007179(transforming activity);GO:0005057(receptor signaling NM_005900 UP (SMAD1), transcript variant 1, growth factor beta receptor signaling protein activity);GO:0016563(transcriptional mRNA [NM_005900] pathway);GO:0009880(embryonic activator activity) pattern specification);GO:0030509(BMP signaling pathway)

THC2442931 UP

THC2309459 UP Homo sapiens ribosomal protein GO:0003735(structural constituent of NM_005617 S14 (RPS14), transcript variant 3, GO:0006412(protein biosynthesis) UP ribosome) mRNA [NM_005617] A_24_P84268 UP Homo sapiens ribosomal protein AY090769 S18/S6-like mRNA, complete UP sequence. [AY090769] qh07g09.x1 Soares_NFL_T_GBC_S1 Homo sapiens cDNA clone IMAGE:1844032 3' similar to AI216457 UP SW:RL39_HUMAN P02404 60S RIBOSOMAL PROTEIN L39. [2] SW:RL39_HUMAN ;, mRNA sequence [AI216457] GO:0001558(regulation of cell growth);GO:0006952(defense Homo sapiens suppressor of response);GO:0007242(intracellular NM_004232 cytokine signaling 6 (SOCS6), UP signaling cascade);GO:0007259(JAK- mRNA [NM_004232] STAT cascade);GO:0009968(negative regulation of signal transduction)

CB133893 K-EST0185101 L14ChoiCK0 Homo sapiens cDNA THC2334681 UP clone L14ChoiCK0-9-E10 5', mRNA sequence [CB133893] Homo sapiens hairy and enhancer of GO:0006355(regulation of transcription, GO:0003677(DNA NM_005524 split 1, (Drosophila) (HES1), mRNA DNA- binding);GO:0030528(transcription regulator UP [NM_005524] dependent);GO:0007399(neurogenesis) activity) A_24_P94007 UP 9 T28605 EST48922 Human Spleen Homo sapiens cDNA 5' end similar THC2395355 to proto-oncogene junD UP (GB:X51346) (HT:1498), mRNA sequence [T28605] A_32_P10480 UP 9 A_32_P10646 UP 6 CF528315 UI-1-BC0-aen-f-04-0- UI.s1 NCI_CGAP_Pl1 Homo CF528315 sapiens cDNA clone UI-1-BC0-aen-f- UP 04-0-UI 3', mRNA sequence [CF528315] THC2309459 UP Homo sapiens COMM domain NM_203495 containing 6 (COMMD6), transcript UP variant 2, mRNA [NM_203495] BOVPOUBA polyubiquitin {Bos THC2274051 taurus;} , partial (52%) UP [THC2274051] Homo sapiens splicing factor, GO:0000398(nuclear mRNA splicing, GO:0000166(nucleotide NM_006275 arginine/serine-rich 6 (SFRS6), via spliceosome);GO:0006376(mRNA UP binding);GO:0003723(RNA binding) mRNA [NM_006275] splice site selection) A_32_P11855 UP 6 A_32_P11895 UP 9

Homo sapiens mRNA; cDNA BX647641 DKFZp451L096 (from clone GO:0006118(electron transport) GO:0009055(electron carrier activity) UP DKFZp451L096). [BX647641] A_32_P12745 UP 4 AW804491 QV0-UM0093-170400- 191-d05 UM0093 Homo sapiens AW804491 UP cDNA, mRNA sequence [AW804491] CD607715 56025722H1 FLP Homo CD607715 sapiens cDNA, mRNA sequence UP [CD607715] ALU2_HUMAN (P39189) Alu subfamily SB sequence THC2406576 UP contamination warning entry, partial (4%) [THC2406576] W86899 zh60b12.s1 Soares_fetal_liver_spleen_1NFLS_ S1 Homo sapiens cDNA clone W86899 IMAGE:416447 3' similar to UP gb:L05091 40S RIBOSOMAL PROTEIN S28 (HUMAN);, mRNA sequence [W86899] BF511442 UI-H-BI4-app-d-02-0- UI.s1 NCI_CGAP_Sub8 Homo BF511442 sapiens cDNA clone UP IMAGE:3088155 3', mRNA sequence [BF511442] BQ000122 UI-H-DP0-avs-b-08-0- UI.s1 NCI_CGAP_Fs1 Homo BQ000122 sapiens cDNA clone UP IMAGE:5883559 3', mRNA sequence [BQ000122] BM982926 UI-CF-EN1-acs-n-01-0- UI.s1 UI-CF-EN1 Homo sapiens BM982926 UP cDNA clone UI-CF-EN1-acs-n-01-0- UI 3', mRNA sequence [BM982926]

CF143262 UI-HF-BR0p-aqt-a-11-0- UI.r1 NIH_MGC_52 Homo sapiens CF143262 UP cDNA clone IMAGE:3101469 5', mRNA sequence [CF143262] BM664367 UI-E-DW0-agh-p-04-0- UI.s1 UI-E-DW0 Homo sapiens BM664367 UP cDNA clone UI-E-DW0-agh-p-04-0- UI 3', mRNA sequence [BM664367] A_32_P16757 UP 7 A_32_P17134 UP 8 Homo sapiens clone FLB0708 AF113008 UP mRNA sequence. [AF113008] A_32_P17709 UP 7 Homo sapiens mRNA; cDNA BX641027 DKFZp686O10247 (from clone UP DKFZp686O10247). [BX641027] CHR90108 Chromosome 9 exon II T12588 Homo sapiens cDNA clone P94_53 UP 5' and 3', mRNA sequence [T12588] A_32_P19641 UP 1 Homo sapiens cDNA clone BC094794 UP IMAGE:30520069. [BC094794] GO:0005216(ion channel Homo sapiens clone FLB9630 activity);GO:0005249(voltage-gated AF130091 PRO2603 mRNA, complete cds. GO:0006813(potassium ion transport) UP potassium channel [AF130091] activity);GO:0005515(protein binding) BE924986 MR1-AN0036-280800- BE924986 002-f08 AN0036 Homo sapiens UP cDNA, mRNA sequence [BE924986] Homo sapiens, clone GO:0006450(regulation of translational GO:0017068(glutamyl-tRNA(Gln) BC034962 IMAGE:4822098, mRNA, partial cds. UP fidelity) amidotransferase activity) [BC034962]

BU734416 UI-E-CI1-agf-a-07-0- UI.s1 UI-E-CI1 Homo sapiens cDNA BU734416 UP clone UI-E-CI1-agf-a-07-0-UI 3', mRNA sequence [BU734416] AA399656 zt86b05.s1 Soares_testis_NHT Homo sapiens cDNA clone IMAGE:729201 3' AA399656 similar to gb:M38188 OVARIAN UP GRANULOSA CELL 13.0 KD PROTEIN HGR74 (HUMAN);, mRNA sequence [AA399656] AV702101 ADB Homo sapiens AV702101 cDNA clone ADBCGB06 5', mRNA UP sequence [AV702101] A_32_P40611 UP

Homo sapiens cDNA FLJ16526 fis, GO:0003676(nucleic acid AK131416 UP clone OCBBF2006987. [AK131416] binding);GO:0046872(metal ion binding) PR18_HUMAN (Q99633) Pre-mRNA splicing factor 18 (PRP18 homolog) THC2430346 UP (hPRP18), partial (31%) [THC2430346] UI-H-DH0-aul-h-03-0-UI.s1 GO:0000166(nucleotide NCI_CGAP_DH0 Homo sapiens BM994378 GO:0006412(protein biosynthesis) binding);GO:0003735(structural constituent of UP cDNA clone IMAGE:5871026 3', ribosome);GO:0019843(rRNA binding) mRNA sequence [BM994378] Homo sapiens mRNA, clone: AB188491 UP TH020D07. [AB188491] Homo sapiens hypothetical LOC400590, mRNA (cDNA clone BC062632 UP MGC:70830 IMAGE:5248762), complete cds. [BC062632] BM504117 ih21d06.x1 Human insulinoma Homo sapiens cDNA 3' similar to SW:HMG1_CRIGR BM504117 UP P07156 HIGH MOBILITY GROUP PROTEIN HMG1 ;, mRNA sequence [BM504117]

BX362492 Homo sapiens HELA CELLS COT 25-NORMALIZED BX362492 Homo sapiens cDNA clone UP CS0DK001YC23 3-PRIME, mRNA sequence [BX362492] CD513837 AGENCOURT_14375404 CD513837 NIH_MGC_179 Homo sapiens UP cDNA clone IMAGE:30392937 5', mRNA sequence [CD513837] Homo sapiens hypothetical protein NM_152344 FLJ30656 (FLJ30656), mRNA UP [NM_152344] N28017 yx50d12.r1 Soares melanocyte 2NbHM Homo sapiens cDNA clone IMAGE:265175 5' N28017 similar to gb:D10522 Human mRNA UP for 80K-L protein, complete cds. (HUMAN);contains Alu repetitive element;, mRNA sequence [N28017] Homo sapiens basic, immunoglobulin-like variable motif GO:0003697(single-stranded DNA NM_017693 GO:0006289(nucleotide-excision repair) DOWN containing (BIVM), mRNA binding);GO:0004519(endonuclease activity) [NM_017693] Homo sapiens speckle-type POZ NM_0010072 protein (SPOP), transcript variant 1, GO:0006397(mRNA processing) GO:0005515(protein binding) DOWN 26 mRNA [NM_001007226] GO:0000166(nucleotide binding);GO:0003677(DNA Homo sapiens F-box protein, GO:0006281(DNA binding);GO:0004003(ATP-dependent DNA NM_178150 helicase, 18 (FBXO18), transcript DOWN repair);GO:0006512(ubiquitin cycle) helicase activity);GO:0004386(helicase variant 2, mRNA [NM_178150] activity);GO:0005524(ATP binding);GO:0016787(hydrolase activity) Homo sapiens MSTP146 (MST146) AF178574 DOWN mRNA, complete cds. [AF178574]

GO:0006468(protein amino acid phosphorylation);GO:0007049(cell cycle);GO:0007051(spindle organization Homo sapiens BUB1 budding and GO:0000166(nucleotide uninhibited by benzimidazoles 1 biogenesis);GO:0007067(mitosis);GO:0 binding);GO:0004674(protein serine/threonine NM_001211 DOWN homolog beta (yeast) (BUB1B), 007093(mitotic kinase activity);GO:0005524(ATP mRNA [NM_001211] checkpoint);GO:0008283(cell binding);GO:0016740(transferase activity) proliferation);GO:0048015(phosphoinosi tide-mediated signaling);GO:0051301(cell division) GO:0000166(nucleotide Homo sapiens phenylalanine-tRNA GO:0006412(protein binding);GO:0004826(phenylalanine-tRNA NM_005687 synthetase-like, beta subunit biosynthesis);GO:0006432(phenylalanyl- DOWN ligase activity);GO:0005524(ATP (FARSLB), mRNA [NM_005687] tRNA aminoacylation) binding);GO:0016874(ligase activity) GO:0000166(nucleotide Homo sapiens ADP-ribosylation GO:0006886(intracellular protein binding);GO:0003924(GTPase NM_001658 factor 1 (ARF1), transcript variant 4, transport);GO:0007264(small GTPase DOWN activity);GO:0005057(receptor signaling mRNA [NM_001658] mediated signal transduction) protein activity);GO:0005525(GTP binding) Homo sapiens adducin 3 (gamma) GO:0005200(structural constituent of NM_016824 (ADD3), transcript variant 1, mRNA cytoskeleton);GO:0005516(calmodulin DOWN [NM_016824] binding) Homo sapiens SAC1 suppressor of NM_014016 actin mutations 1-like (yeast) DOWN (SACM1L), mRNA [NM_014016] GO:0003677(DNA binding);GO:0008270(zinc Homo sapiens zinc finger protein 6 ion binding);GO:0030528(transcription NM_021998 (CMPX1) (ZNF6), mRNA GO:0045449(regulation of transcription) DOWN regulator activity);GO:0046872(metal ion [NM_021998] binding)

Homo sapiens PAI-1 mRNA binding NM_0010180 protein (PAI-RBP1), transcript GO:0003723(RNA binding) DOWN 67 variant 1, mRNA [NM_001018067]

GO:0003677(DNA binding);GO:0008270(zinc Homo sapiens zinc finger protein 6 ion binding);GO:0030528(transcription NM_021998 (CMPX1) (ZNF6), mRNA GO:0045449(regulation of transcription) DOWN regulator activity);GO:0046872(metal ion [NM_021998] binding) Homo sapiens hypothetical protein NM_015383 DJ328E19.C1.1 (DJ328E19.C1.1), DOWN mRNA [NM_015383]

Homo sapiens zinc finger protein GO:0003676(nucleic acid NM_014897 DOWN 652 (ZNF652), mRNA [NM_014897] binding);GO:0008270(zinc ion binding) Homo sapiens pumilio homolog 2 GO:0003723(RNA NM_015317 (Drosophila) (PUM2), mRNA GO:0006445(regulation of translation) DOWN binding);GO:0005488(binding) [NM_015317] GO:0004601(peroxidase Homo sapiens peroxiredoxin 3 activity);GO:0008785(alkyl hydroperoxide (PRDX3), nuclear gene encoding GO:0051092(activation of NF-kappaB NM_006793 reductase DOWN mitochondrial protein, transcript transcription factor) activity);GO:0016491(oxidoreductase variant 1, mRNA [NM_006793] activity);GO:0019901(protein kinase binding)

GO:0006355(regulation of transcription, DNA-dependent);GO:0007387(anterior compartment specification);GO:0007388(posterior Homo sapiens pre-B-cell leukemia compartment GO:0003700(transcription factor NM_006195 transcription factor 3 (PBX3), mRNA DOWN specification);GO:0009790(embryonic activity);GO:0005515(protein binding) [NM_006195] development);GO:0030902(hindbrain development);GO:0045898(regulation of transcriptional preinitiation complex formation)

GO:0000166(nucleotide binding);GO:0004672(protein kinase GO:0006468(protein amino acid activity);GO:0004674(protein serine/threonine Homo sapiens discoidin domain phosphorylation);GO:0007155(cell kinase activity);GO:0004713(protein-tyrosine receptor family, member 1 (DDR1), NM_013994 adhesion);GO:0007169(transmembrane kinase activity);GO:0004714(transmembrane DOWN transcript variant 3, mRNA receptor protein tyrosine kinase receptor protein tyrosine kinase [NM_013994] signaling pathway) activity);GO:0004872(receptor activity);GO:0005524(ATP binding);GO:0016740(transferase activity)

Homo sapiens enthoprotin (ENTH), NM_014666 GO:0006897(endocytosis) GO:0008289(lipid binding) DOWN mRNA [NM_014666] A_24_P13539 DOWN 1

Homo sapiens muskelin 1, GO:0006928(cell intracellular mediator containing motility);GO:0007160(cell-matrix NM_013255 DOWN kelch motifs (MKLN1), mRNA adhesion);GO:0007165(signal [NM_013255] transduction) GO:0000049(tRNA GO:0006412(protein binding);GO:0000166(nucleotide Homo sapiens alanyl-tRNA biosynthesis);GO:0006419(alanyl-tRNA binding);GO:0003676(nucleic acid NM_001605 synthetase (AARS), mRNA DOWN aminoacylation);GO:0008033(tRNA binding);GO:0004813(alanine-tRNA ligase [NM_001605] processing) activity);GO:0005524(ATP binding);GO:0016874(ligase activity) Homo sapiens putative NM_020432 homeodomain transcription factor 2 DOWN (PHTF2), mRNA [NM_020432] A_24_P19617 DOWN 5 Homo sapiens leucine rich repeat NM_020710 containing 47 (LRRC47), mRNA DOWN [NM_020710] A_24_P20397 DOWN 6 A_24_P20448 DOWN 4 ENST000003 DOWN 16369 A_24_P22147 DOWN 5 ENST000003 DOWN 26920 Homo sapiens heterogeneous NM_0010117 nuclear ribonucleoprotein A1-like GO:0000166(nucleotide DOWN 24 (LOC144983), transcript variant 1, binding);GO:0003676(nucleic acid binding) mRNA [NM_001011724] full-length cDNA clone CS0DJ006YC05 of T cells (Jurkat CR622342 DOWN cell line) Cot 10-normalized of Homo sapiens (human). [CR622342]

Homo sapiens hypothetical protein FLJ20719, mRNA (cDNA clone BC034418 DOWN MGC:34369 IMAGE:3896523), complete cds. [BC034418] ENST000002 DOWN 59575 ENST000003 DOWN 31523 ENST000003 DOWN 18909 full-length cDNA clone ENST000003 CS0DA010YB10 of Neuroblastoma DOWN 30752 of Homo sapiens (human). [CR596550] A_24_P49311 DOWN 6 CB131079 K-EST0181079 L13SNU387 Homo sapiens cDNA CB131079 DOWN clone L13SNU387-14-G04 5', mRNA sequence [CB131079] Homo sapiens phosphoglycerate GO:0004618(phosphoglycerate kinase GO:0006096(glycolysis);GO:0016310(p NM_000291 kinase 1 (PGK1), mRNA activity);GO:0005524(ATP DOWN hosphorylation) [NM_000291] binding);GO:0016740(transferase activity) A_24_P67432 DOWN GO:0006412(protein Homo sapiens eukaryotic translation GO:0003746(translation elongation factor biosynthesis);GO:0006414(translational NM_001402 elongation factor 1 alpha 1 activity);GO:0003924(GTPase DOWN elongation);GO:0006445(regulation of (EEF1A1), mRNA [NM_001402] activity);GO:0005525(GTP binding) translation) A_24_P85330 DOWN 2 Homo sapiens adducin 3 (gamma) GO:0005200(structural constituent of NM_016824 (ADD3), transcript variant 1, mRNA cytoskeleton);GO:0005516(calmodulin DOWN [NM_016824] binding) ALU8_HUMAN (P39195) Alu subfamily SX sequence THC2313538 DOWN contamination warning entry, partial (3%) [THC2313538]

AA353695 EST62114 Jurkat T-cells AA353695 V Homo sapiens cDNA 5' end, DOWN mRNA sequence [AA353695] Homo sapiens heterogeneous NM_0010117 nuclear ribonucleoprotein A1-like GO:0000166(nucleotide DOWN 24 (LOC144983), transcript variant 1, binding);GO:0003676(nucleic acid binding) mRNA [NM_001011724] Homo sapiens heterogeneous GO:0006397(mRNA GO:0000166(nucleotide nuclear ribonucleoprotein A1 NM_002136 processing);GO:0006406(mRNA- binding);GO:0003676(nucleic acid DOWN (HNRPA1), transcript variant 1, nucleus export);GO:0006810(transport) binding);GO:0003723(RNA binding) mRNA [NM_002136] PREDICTED: Homo sapiens similar to ATPase, H+ transporting, lysosomal 31kD, V1 subunit E isoform 1; V-ATPase, subunit E; XM_497670 H(+)-transporting two-sector DOWN ATPase, 31kDa subunit; H+- transporting ATP synthase chain E, vacuolar; vacuolar proton pump, 31- kd s... GO:0006412(protein Homo sapiens eukaryotic translation GO:0003746(translation elongation factor biosynthesis);GO:0006414(translational NM_001402 elongation factor 1 alpha 1 activity);GO:0003924(GTPase DOWN elongation);GO:0006445(regulation of (EEF1A1), mRNA [NM_001402] activity);GO:0005525(GTP binding) translation) Homo sapiens cDNA FLJ40901 fis, AK098220 DOWN clone UTERU2003704. [AK098220] GO:0006412(protein GO:0000166(nucleotide Homo sapiens glutaminyl-tRNA biosynthesis);GO:0006424(glutamyl- binding);GO:0004818(glutamate-tRNA ligase NM_005051 synthetase (QARS), mRNA tRNA activity);GO:0004819(glutamine-tRNA ligase DOWN [NM_005051] aminoacylation);GO:0006425(glutaminyl-activity);GO:0005524(ATP tRNA aminoacylation) binding);GO:0016874(ligase activity)