Nucleophosmin Interacts with HEXIM1 and Regulates RNA Polymerase II Transcription

doi:10.1016/j.jmb.2008.02.055

J. Mol. Biol. (2008) 378, 302–317

Available online at www.sciencedirect.com

Nucleophosmin Interacts with HEXIM1 and Regulates RNA Polymerase II Transcription Meera Gurumurthy 1 , Chuan Hao Tan 1 , Raymond Ng 1 , Lisa Zeiger 1,5 , Joanne Lau 1 , Jialing Lee 1 , Anwesha Dey 2 , Robin Philp 3 , Qintong Li 4 , Tit Meng Lim 6 , David H. Price 4 , David P. Lane 2 and Sheng-Hao Chao 1 ⁎ 1

Expression Engineering Group, Bioprocessing Technology Institute, 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore 2

Department of Cell Cycle Control, Institute of Molecular and Cell Biology, Proteos, Singapore 138668, Singapore 3

Proteomics Group, Bioprocessing Technology Institute, 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore 4 Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA 5

Fachhochschule Esslingen, Hochschule für Technik, University of Applied Sciences, Kanalstraße 33, D-73728 Esslingen, Germany

Hexamethylene bis-acetamide-inducible protein 1 (HEXIM1) was identified earlier as an inhibitor of positive transcription elongation factor b (P-TEFb), which is a key transcriptional regulator of RNA polymerase II (Pol II). Studies show that more than half of P-TEFb in cells is associated with HEXIM1, which results in the inactivation of P-TEFb. Here, we identify a nucleolar protein, nucleophosmin (NPM), as a HEXIM1-binding protein. NPM binds to HEXIM1 in vitro and in vivo, and functions as a negative regulator of HEXIM1. Over-expression of NPM leads to proteasomemediated degradation of HEXIM1, resulting in activation of P-TEFbdependent transcription. In contrast, an increase in HEXIM1 protein levels and a decrease in transcription are detected when NPM is knocked down. We show that a cytoplasmic mutant of NPM, NPMc+, associates with and sequesters HEXIM1 in the cytoplasm resulting in higher RNA Pol II transcription. Correspondingly, cytoplasmic localization of endogenous HEXIM1 is detected in an acute myeloid leukemia (AML) cell line containing the NPMc+ mutation, suggesting the physiological importance of HEXIM1-NPMc+ interaction. Over-expression of NPM has been detected in tumors of various histological origins and our results may provide a possible molecular mechanism for the proto-oncogenic function of NPM. Furthermore, considering that 35% of AML patients are diagnosed with NPMc+ mutation, our findings suggest that in some cases of AML, RNA Pol II transcription may be disregulated by the malfunction of NPM and the mislocation of HEXIM1. © 2008 Elsevier Ltd. All rights reserved.

6 Department of Biological Sciences, National University of Singapore, Science Drive 4, Blk S1A #05-36, Singapore 117543, Singapore

*Corresponding author. E-mail address: [email protected]. Abbreviations used: AML, acute myeloid leukemia; AR, acidic region; BR, basic region; Cdk9, cyclin-dependent kinase 9; CMV, cytomegalovirus; Co-IP, Co-immunoprecipitation; DAPI, 4′,6-diamidino-2-phenylindole; DD, dimerization domain; DRB, 5, 6-dichloro-1-β-D-ribofuranosylbenzimidazole; ERα, estrogen receptor α; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; HEXIM1, hexamethylene bis-acetamide inducible protein 1; HIV, human immunodeficiency virus; HTLV, human T-cell leukemia virus; ID, inhibitory domain; LTR, long terminal repeat; Luc, luciferase; MIE, major immediate-early; MLV, murine leukemia virus; NLS, nuclear localization signal; NPM, nucleophosmin; P-TEFb, positive transcription elongation factor b; RNA Pol II, RNA polymerase II; snRNA, small nuclear RNA; YFP, yellow fluorescent protein. 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.

303

Nucleophosmin is a HEXIM1-binding Protein

Received 10 November 2007; received in revised form 18 February 2008; accepted 25 February 2008 Available online 4 March 2008 Edited by J. Karn

Keywords: P-TEFb; HEXIM1; NPM; AML; RNA polymerase II

Introduction Positive transcription elongation factor b (P-TEFb), a protein complex composed of cyclin-dependent kinase 9 (Cdk9) and a cyclin partner T1, T2, or K, has an essential role in the regulation of RNA polymerase II (Pol II) transcription elongation.1–3 P-TEFb phosphorylates serine 2 residues in the heptad repeats (i.e., Tyr-Ser-Pro-Thr-Ser-Pro-Ser) of the carboxylterminal domain of the largest subunit of RNA Pol II and the subunits of the negative factors, including 5,6-dichloro-1-β- D -ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF),4 and negative elongation factor (NELF).5 P-TEFb phosphorylation events result in the transition from abortive to productive elongation and generation of full-length transcripts.6,7 Treatment with P-TEFb inhibitors, such as DRB or flavopiridol, blocks 70–80% of total RNA Pol II transcription,7,8 indicating the importance of PTEFb in transcriptional regulation. P-TEFb is also a required cellular factor for human immunodeficiency virus (HIV) Tat transactivation, a critical phase of the HIV life-cycle that controls viral transcription.9 Mancebo et al. carried out a compound screen to identify inhibitors of HIV Tat transcriptional activation. After testing more than 100,000 chemicals, all the compounds identified, including DRB, were found to inhibit P-TEFb.10 Flavopiridol, the most selective and potent P-TEFb inhibitor,8,11 is the first Cdk inhibitor that has entered clinical trials as a potential cancer therapeutic.12 As expected, flavopiridol blocks HIV transcription and replication effectively in vitro and in vivo.11 Other Cdk inhibitors, including purvalanol A, olomoucine, and roscovitine (or CYC202), have been shown to block HIV basal and Tat-activated transcription through the inhibition of P-TEFb.13 These results suggest that P-TEFb represents an attractive target for the development of novel anti-HIV therapy. P-TEFb is found in two forms in cells. The free, active form (composed of Cdk9 and cyclin T) associates with many other factors, which leads to recruitment to active genes.7 The second form is a larger, inactive P-TEFb complex containing Cdk9, cyclin T, hexamethylene bis-acetamide inducible protein 1 (HEXIM1), and a small nuclear RNA (snRNA), 7SK.14–17 While the free form is generally considered the active form, P-TEFb in the large form is modified by T-loop phosphorylation and is potentially active.18,19 In fact, the HIV transactivator Tat was shown to acquire P-TEFb from the large inactive

form when expressed in human cells.20,21 A HEXIM1 homolog, HEXIM2, was identified by sequence analysis22,23 and, like HEXIM1, inhibits P-TEFb in the presence of 7SK.22,23 HEXIM2 was found to functionally compensate for the loss of HEXIM1 after siRNA-mediated knockdown of HEXIM1 by forming a 7SK/HEXIM2/P-TEFb complex.22,23 HEXIM1 was first cloned from vascular smooth muscle cells treated with hexamethylene bis-acetamide, a compound that suppresses the proliferation of vascular smooth muscle cells.24 Recent studies suggest that HEXIM1 has a role in cancers, acquired immunodeficiency syndrome, cardiac hypertrophy, and inflammation through the inhibition of RNA Pol II transcription.25 The N terminus of HEXIM1, amino acids 1–150, has been characterized as a selfinhibitory domain (ID). Deletion of the ID enhances the inhibitory effects of HEXIM1 on P-TEFb activity and RNA Pol II transcription.17,18 The region between amino acids 150 and 180 of HEXIM1, which includes a stretch of basic residues, is referred to as the basic region (BR). The BR contains the binding motif for 7SK snRNA, KHRR (152–155). When the KHRR sequence is replaced by ILAA, the mutant HEXIM1 protein fails to interact with 7SK snRNA and the formation of the large P-TEFb complex is disrupted.26 The BR also contains the nuclear localization signal (NLS).26,27 Furthermore, the fluorescent protein-tagged BR chimera demonstrates a predominantly nucleolar localization.27 The PYNT motif (202–205) and the C-terminal domain (274– 359) of HEXIM1 are involved in P-TEFb binding through the interaction with cyclin T1.15,26 The acidic region (AR, 210–250) of HEXIM1 has been shown to interact with the adjacent BR in the absence of 7SK snRNA.27 HEXIM1 exhibits P-TEFb inhibition properties only when associated with 7SK snRNA while neither 7SK snRNA nor HEXIM1 alone demonstrate any inhibitory effects on P-TEFb activity.17,26 Since the P-TEFb binding motif is located between the BR and AR, it has been suggested that the BR–AR interaction establishes an auto-inhibitory conformation that prevents the association between HEXIM1 and P-TEFb. It has been proposed that only after 7SK snRNA binds to the BR, the PYNT motif becomes accessible to P-TEFb.27 HEXIM1 can form a homodimer or a heterodimer with HEXIM2 in vitro and in vivo through the dimerization domain (DD) at the C terminus of HEXIM1.18,28,29 In cells, more than half of P-TEFb is associated with the 7SK snRNA-bound HEXIM1, resulting in the inactivation of P-TEFb. 14–16,22 Several HEXIM1-

304 binding proteins have been identified, including glucocorticoid receptor (GR), NF-κB, N-CoR, and estrogen receptor α (ERα).30–33 The association between HEXIM1 and ERα, a critical factor involved in the growth of breast cancer, suggests the possible connection between cancers and P-TEFb/HEXIM1.25 Nucleophosmin (NPM, also known as B23, numatrin, or NO38) is a predominantly nucleolar phosphoprotein that has an important role in ribosome biogenesis and the regulation of cell growth and proliferation.34 Most importantly, NPM is a crucial gene implicated in cancers.34 NPM is one of the most frequently mutated genes in acute myeloid leukemia (AML), with 35% of AML patients carrying NPMc+, the cytoplasmic-mislocated mutant form of NPM.35 NPM is also an important regulator in the p53 signaling pathway.36–39 NPM binds to ARF, HDM2, and p53, resulting in the stability and activation of p53.36,38,39 These studies suggest that NPM is a tumor-suppressor gene, which is further supported by genetic knockout studies.40 In contrast to its tumor suppressor properties, over-expression of NPM has been detected in tumors of various histological origins.34 NPM over-expression has been shown to inhibit programmed cell death and increase cell growth and proliferation, suggesting that NPM functions as a proto-oncogene.34 All these studies suggest that NPM has an important role in tumorigenesis, either as an oncogene, a tumor suppressor, or both. In this study, we identify NPM as a HEXIM1-binding protein. Our study suggests a functional correlation between NPMc+ and subcellular localization of HEXIM1 in AML, suggesting the physiological importance of the HEXIM1–NPMc+ interaction.

Results NPM interacts with HEXIM1 To identify cellular proteins that interact with HEXIM1, we first expressed a histidine-tagged HEXIM1 (HEXIM1-His) protein in Escherichia coli BL21(DE3) cells and purified the protein using nickel resin. The nickel-bound protein was incubated with pre-cleared HeLa cell lysates (see Materials and Methods). The resin was eluted with 250 mM imidazole and the HEXIM1-His and putative HEXIM1-binding proteins, were analyzed by SDSPAGE (Supplementary Data Fig. 1A and B). The identities of the putative HEXIM1-binding proteins were determined by mass spectrometry. NPM, one of the candidate proteins, was chosen for further investigation (Supplementary Data Fig. 1B). Co-IP was carried out using HeLa cell lysates to examine the interaction between endogenous HEXIM1 and NPM in mammalian cells. As shown in Fig. 1a, both NPM and Cdk9 were co-immunoprecipitated by anti-HEXIM1 antibody. Our results agree with previous finding that Cdk9 associates with HEXIM1 in a large, inactive P-TEFb complex.15

Nucleophosmin is a HEXIM1-binding Protein

Fig. 1. Endogenous interaction between NPM and HEXIM1. (a) Co-IP was performed using cell lysates prepared from HeLa cells and the indicated antibodies. Normal IgG (IgG) was used as a negative control. The immunoprecipitated complexes were analyzed by Western blotting. (b) MCF7 cell lysates were pre-incubated with RNase followed by Co-IP and Western blotting analyses.

However, when anti-NPM antibody was used, HEXIM1, but not Cdk9, was precipitated (Fig. 1a). Correspondingly, NPM was not present in the immunoprecipitated complexes using anti-Cdk9 antibody. These results confirm the association between NPM and HEXIM1 in cells and suggest that NPM binds to HEXIM1 in the absence of P-TEFb (Fig. 1a). The requirement of 7SK snRNA for the association between NPM and HEXIM1 was investigated next. MCF7 cell lysates treated with RNase were analyzed by Co-IP. Treatment with RNase resulted in a significant decrease in HEXIM1-Cdk9 interaction as reported previously (Fig. 1b).15 In contrast, a slight increase in HEXIM1-NPM interaction was detected after treatment with RNase (Fig. 1b). These results suggest that 7SK snRNA is not required for the association between HEXIM1 and NPM. To determine which domain of HEXIM1 is required for NPM-HEXIM1 interaction, FLAG-tagged fulllength HEXIM1 and deletion mutants of HEXIM1 protein were tested for their binding to endogenous NPM (Fig. 2a and b). Cell lysates prepared from HEK293 cells transfected with the FLAG-tagged constructs were utilized for Co-IP. Co-IP was performed using anti-FLAG antibody and the immunoprecipitates were analyzed by Western blotting using anti-NPM antibody. HEXIM1(WT), HEXIM1

Nucleophosmin is a HEXIM1-binding Protein

(120–359), and HEXIM1(1–180) interacted with NPM and the strongest binding was detected between HEXIM1(1–180) and NPM (Fig. 2b). In contrast, HEXIM1(180–359), HEXIM1(1–150) and HEXIM1 (ΔBR) failed to bind NPM, suggesting that the basic region is required for HEXIM1 binding (Fig. 2b). This result was further confirmed by co-immunoprecipitating BR-yellow fluorescent protein (YFP) with antiNPM antibody (Fig. 2c). In this Co-IP, HEXIM1-YFP was used as a positive control to show the association between the YFP-tagged full-length HEXIM1 and NPM, while no interaction between NPM and the negative control, YFP, was detected (Fig. 2c). The results of the RNase assay indicated that 7SK snRNA might not be essential for HEXIM1-NPM interaction (Fig. 1b). We next wished to assess if the 7SK snRNA binding motif, KHRR (amino acids 152–156, located

305 within the BR), was involved in NPM binding. It has been shown that the HEXIM1 ILAA mutant, in which KHRR residues are replaced with ILAA, does not interact with 7SK snRNA.26 In our experiment, NPM was found to co-precipitate with the HEXIM1 ILAA mutant (Fig. 2d), suggesting that the 7SK motif was not required for NPM-HEXIM1 interaction. Domains of NPM involved in HEXIM1 binding were investigated next by generating five Myctagged NPM proteins, wild-type NPM, NPM(1– 117), NPM(120–294), NPM(1–191), and NPM(191– 294) (Fig. 2e). We first examined the expression of the Myc-tagged NPM proteins. It has been shown that NPM exists as a hexamer and/or larger oligomer and the N terminus of NPM (i.e., amino acids 1–117) is required for the formation of oligomers.41,42 Oligomers of NPM were detected in the NPM(WT), NPM

Fig. 2. Domain analysis. (a) Diagram of HEXIM1 deletions. The HEXIM1 constructs were FLAG- or YFP-tagged. NPM binding data are summarized on the right. ID, inhibitory domain; BR, basic region; NLS, nuclear localization signal; AR, acidic region; DD, dimerization domain. (b) HEK293 cells were transiently transfected with the indicated FLAG-tagged HEXIM1 plasmids. Lysates of the transfected cells were precipitated with anti-FLAG antibody. The precipitated protein complexes were analyzed by immunoblotting using anti-NPM antibody. (c) HeLa cells were transiently transfected with YFP, BR-YFP, and HEXIM1-YFP plasmids. Co-IP was carried out using the cell lysates of the transfected cells and antiNPM antibody. The immunoprecipitated complexes were analyzed by Western blotting using anti-YFP antibody. HC, the heavy chain of anti-NPM antibody. (d) HEK293 cells were transfected with FLAG-HEXIM1 WT or FLAG-HEXIM1 ILAA plasmids. IP and Western blotting were performed as described for b. (e) A diagram of NPM deletions. The NPM constructs were Myc-tagged. HEXIM1 binding data are summarized on the right. OD, oligomerization domain; AD, acidic domain; NBD, nucleic acid binding domain. (f) 293T cells were transiently transfected with an indicated plasmid. The expression of Myc-tagged NPM proteins was determined by Western blotting. (g) Lysates of the transfected 293T cells were precipitated with anti-HEXIM1 antibody. The precipitated protein complexes were analyzed by immunoblotting using anti-Myc antibody.

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Nucleophosmin is a HEXIM1-binding Protein

Fig. 2 (legend on previous page)

(1–117), and NPM(1–191) expressing cells (Fig. 2f, oligomers) while no oligomer was observed in cells transfected with NPM(120–294) and NPM(191–294)expressing cells (Fig. 2f). Co-IP was performed using anti-HEXIM1 antibody and the immunoprecipitates were analyzed by Western blotting using anti-Myc antibody. We found that NPM(WT), NPM(1–191), and NPM(191–294) interacted with HEXIM1 and detected the strongest binding between NPM(WT)HEXIM1 and NPM(191–294)-HEXIM1 (Fig. 2g, short exposure). Weak interactions between NPM(1–117), NPM(120–294), and HEXIM1 were also detected (Fig. 2g, long exposure). Our results suggest that the C-terminal region of NPM (i.e., amino acids 191–294) may be required for HEXIM1 binding while the middle region of NPM (amino acids 120–191) may interfere the NPM-HEXIM1 interaction (Fig. 2g, compare NPM(120–294) and NPM(191–294)). The involvement of the N terminus of NPM (amino acids 1–117) in the interaction is not clear, since NPM(1– 117) and NPM(1–191) can potentially form heterooligomers with endogenous NPM through the oligomerization domain (Fig. 2e and f). We used immunofluorescence to examine the subcellular localization of HEXIM1 and NPM. HEXIM1 was expressed uniformly throughout the nuclei, and NPM was localized in the nucleoli (Fig. 3a). The BR of

HEXIM1 was identified as the NPM-binding domain in our Co-IP experiments (Fig. 2b and c). Cells transiently transfected with the YFP-BR construct demonstrated nucleolar localization as described (Fig. 3b).27 We found that endogenous NPM and YFP-BR co-localized in nucleoli (Fig. 3b), which was consistent with the interaction between the BR and NPM. NPM is a negative regulator of HEXIM1 Due to the important role of HEXIM1 in P-TEFb regulation, we next examined the effect of NPM over-expression on RNA Pol II transcription. Cellbased assays were performed using various luciferase reporters. Expression of luciferase was driven by different P-TEFb-dependent promoters, including human cytomegalovirus (CMV) major immediateearly (MIE) promoter, Moloney murine leukemia virus (MLV) long terminal repeat (LTR), human immunodeficiency virus (HIV) LTR, and human Tcell leukemia virus (HTLV) LTR to examine RNA Pol II transcription in general (Fig. 4a).11,43,44 Effects of NPM over-expression on HIV Tat and HTLV Tax transactivation, which are highly sensitive to P-TEFb activity,11,44 were also examined. Up to a fourfold increase in luciferase expression was observed in

Nucleophosmin is a HEXIM1-binding Protein

307

Fig. 3. Sub-cellular localization of NPM and HEXIM1 in cells. (a) HeLa cells were immunostained with anti-HEXIM1 (green) and anti-NPM (red) antibodies. Nuclei were visualized by DAPI. (b) The BR-YFP (green) expressed in HeLa cells co-localized with endogenous NPM (red) as shown in the overlay image (yellow).

different promoters and much higher activation was detected in HIV Tat- and HTLV Tax-activated transcription (eightfold and 12-fold, respectively) (Fig. 4a). To further investigate the effect of NPM on the luciferase expression in these cell-based assays, RNA isolated from the cells transfected with HIVLTR-Luc, Tat and NPM expression vectors were analyzed by quantitative RT-PCR. A sixfold increase (6.02 ± 0.20-fold) in luciferase mRNA was detected in the cells over-expressing NPM, suggesting that NPM could regulate the expression of luciferase at the transcriptional level (data not shown). Effects of NPM over-expression on endogenous P-TEFbdependent genes were investigated next. RNAs isolated from the cells transfected with an NPM expression vector or an empty plasmid (used as a control) were analyzed by quantitative RT-PCR. Up to threefold increases in P-TEFb-dependent genes, including Mcl-1, cyclin D1, and Pbx1,43,45,46 were detected when NPM was over-expressed (Fig. 4b). No significant change was observed at the RNA levels of histone H2b,47 which is known to be a PTEFb-independent gene (Fig. 4b).47 The repressive effect of HEXIM1 on P-TEFbdependent transcription, including HIV transcription, has been reported.17 The influence of NPM on HEXIM1-mediated transcriptional inhibition was examined next using cell-based luciferase assay. More than 50% of HIV Tat-dependent transcription was inhibited when HEXIM1 was overexpressed (Fig. 4c). However, the transcriptional repression caused by HEXIM1 was abolished when NPM was co-expressed (Fig. 4c). Similar results were obtained in an identical set of experiments to determine the effect of NPM on HEXIM1-repressed HTLV Tax transactivation (Fig. 4d). To further investigate if the activation of transcription was regulated through the direct interaction between HEXIM1 and NPM, the effects of NPM

deletion mutants on HIV Tat and HTLV Tax transactivation were examined using a cell-based luciferase assay. NPM(120–294) and NPM(191–294), containing the HEXIM1-binding domain (i.e., amino acids 191–294) (Fig. 2e–g), showed comparable stimulating effects with wild-type NPM (Fig. 4e and f). Little or no effect was caused by NPM(1–117) or NPM(1–191), which lack the HEXIM1-binding motif (Fig. 4e and f). Taken together with the study of the NPM domains involved in HEXIM1 binding (Fig. 2e–g), our data suggests that NPM up-regulates the P-TEFb-mediated transcription through the direct interaction with HEXIM1. We next determined the effect of NPM overexpression on the localization and expression of HEXIM1. In the cells expressing hemagglutinin (HA)-NPM, a portion of endogenous HEXIM1 was found to be tethered in nucleoli, probably caused by the HA–NPM–HEXIM1 interaction (Fig. 5a). This observation was consistent with the results of Co-IP showing the association between endogenous HEXIM1 and NPM (Fig. 1). The intriguing effects of NPM over-expression on P-TEFb-mediated transcription led us to further investigate the regulatory mechanism of NPM over-expression on HEXIM1 protein levels. Our data demonstrated that overexpression of NPM could activate transcription without having any detectable effect on the protein levels of HEXIM1 (data not shown). However, in some cases we found that when levels of HA-NPM overexpression surpassed a certain threshold, a decrease in HEXIM1 protein was detected while no effect on Cdk9 was observed (Fig. 5b). Similar results on the decreased levels of HEXIM1 protein were obtained when untagged wild-type NPM was ectopically expressed in cells (data not shown). Recently, it was reported that NPM acts as a transcriptional repressor of certain retinoic-acid-responsive genes,48 raising the possibility that expression of the HEXIM1 gene might

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Nucleophosmin is a HEXIM1-binding Protein

Fig. 4. Effects of NPM on P-TEFb-mediated transcription. (a) HeLa cells were transiently co-transfected with a NPM expression plasmid (fourfold increment) and an indicated luciferase reporter, including CMV-Luc (CMV), MLV-LTR-Luc (MLV), HIV-LTR-Luc (HIV), and HTLV-LTR-Luc (HTLV). To examine the effects of NPM on HIV Tat (HIV + Tat) and HTLV Tax (HTLV + Tax) transactivation, a Tat- and a Tax-producing plasmid were included, respectively. The mock plasmid, pcDNA6, was used as a negative control. Total amount of transfected plasmid was kept constant by adjusting with the mock vector. (b) HEK293 cells were transiently transfected with a mock or NPM expression plasmids. RNAs isolated from the transfected cells were analyzed by quantitative RT-PCR to examine the expression levels of P-TEFb-dependent (i.e., Mcl-1, cyclin D1, and Pbx1) and -independent genes (i.e., histone H2b). Effects of NPM on HEXIM1-repressed HIV Tat (c) and HTLV Tax (d) transactivation were examined in the transfected HeLa cells. The NPM expression plasmid was titrated at fivefold increment. Effects of NPM mutants on HIV Tat (e) and HTLV Tax (f) transactivation were examined in the transfected 293T cells. The NPM mutants used in the assay included NPM (1–191), NPM (191–294), and NPM (120–294) (i.e. 1–191, 191–294, and 120–294, respectively). Wild-type NPM (WT) was used as a control.

be regulated by NPM at the transcriptional level. RNAs isolated from the cells transfected with different amounts of NPM expression plasmid were

analyzed by quantitative RT-PCR. No significant change in HEXIM1 transcripts was detected by quantitative RT-PCR when NPM was over-expressed

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Nucleophosmin is a HEXIM1-binding Protein

Fig. 5. Effects of NPM over-expression on the expression level and sub-cellular localization of HEXIM1. (a) HeLa cells were transiently transfected with a HA-NPM expression vector and examined by immunofluorescence using anti-HA and anti-HEXIM1 antibodies. Increased nucleolar HA-NPM-HEXIM1 interaction was observed in the HA-NPM expressed cells (white arrows). (b) HEK293 cells were transiently transfected with a mock or a HA-NPM expression plasmid. The protein levels of HEXIM1, NPM, HA-NPM, and Cdk9 were analyzed by Western blotting. Actin was used as the loading control. The protein levels of HEXIM1 were presented in percentage after normalization of the quantified data with the loading control. (c) HEK293 cells were transiently transfected with a mock or a HA-NPM expression plasmid and treated with or without 10 μM MG132. The quantification of HEXIM1 protein is presented as a percentage after normalizing with the loading control.

(Supplementary Data Fig. 2). Because of this, the possible involvement of proteasome in the regulation of HEXIM1 protein was examined next. In the presence of MG132, a proteasome inhibitor, the negative effect of NPM over-expression on HEXIM1 protein was abolished (Fig. 5c). These results suggested that high-level expression of NPM could result in instability of HEXIM1 through proteasomemediated degradation. Over-expression studies were complemented with siRNA-mediated knockdown assays to confirm the effects of NPM on transcription and on levels of HEXIM1. Two siRNAs targeting NPM (i.e., NPM-1 and NPM-7) were utilized in the cell-based luciferase assay to determine their effects on P-TEFb-dependent reporters, including CMV-Luc, MLV-LTR-Luc, and HIV-LTR-Luc ± Tat (Fig. 6a). The siRNA against GL2 firefly luciferase was used as a positive control. Both NPM siRNAs resulted in significant decreases in all reporters examined (Fig. 6a) without causing any significant cytotoxic effects in the transfected cells (data not shown). The effectiveness of the NPM siRNAs on NPM protein synthesis was confirmed by Western blotting (Fig. 6b), indicating that the influence of the siRNAs on transcription was indeed NPMspecific. The NPM-7 siRNA, which was more effective

in reducing NPM protein than the NPM-1 siRNA, caused an increase in the protein levels of HEXIM1 (Fig. 6b). These results suggest that increased levels of HEXIM1 protein could be detected only when NPM was knocked down below a certain threshold. In agreement with the effectiveness of the siRNAs, a greater inhibitory effect on the luciferase assays was detected in the NPM-7 siRNA transfected cells (Fig. 6a). NPMc+ sequesters HEXIM1 in cytoplasm Approximately one-third of AML patients are characterized by the presence of the cytoplasmic mutant of NPM, NPMc+.35 The effects of NPMc+ on transcription were investigated next using a cellbased luciferase assay. Cells were co-transfected with a reporter vector (HIV-LTR-Luc or HTLV-LTR-Luc) and an expression plasmid to over-express wild-type NPM or NPMc+. To examine the influence of wildtype NPM and NPMc+ on HIV Tat and HTLV Tax transactivation, a Tat- and a Tax-producing plasmid was included, respectively (Fig. 7a). Compared to wild-type NPM, NPMc+ demonstrated stronger activation of HIV and HTLV transcription, including both basal and Tat-/Tax-activated transcription (Fig.

310

Fig. 6. Repression of HIV basal and Tat-dependent transcription by NPM knockdown. (a) HEK293 cells were transiently co-transfected with a luciferase reporter (i.e., CMV-Luc, MLV-LTR-Luc, and HIV-LTR-Luc) and an indicated siRNA (i.e., control, GL2, NPM-1 and NPM-7). A Tat expression vector was included to examine the effects of the siRNAs on Tat transactivation (HIV + Tat). The siRNA against luciferase (i.e., GL2 siRNA) served as a positive control while the non-specific siRNA (i.e., con) was used as a negative control. (b) Effects of the NPM siRNAs on the protein syntheses of NPM and HEXIM1 were examined by Western blotting. Actin was used as the loading control.

7a). Similar activating effects of NPMc+ were also detected in human CMV MIE and Moloney MLV promoters (data not shown). To further assess the interaction between NPMc+ and HEXIM1, we performed Co-IP using the cell lysates prepared from enhanced green fluorescent protein (EGFP), EGFP-NPM, or EGFP-NPMc+ transfected cells. EGFP and EGFP-NPM were used as negative and positive controls, respectively. The results showed that anti-HEXIM1 antibody precipitated EGFP-NPM and EGFP-NPMc+, but not EGFP, confirming the interaction between HEXIM1 and NPMc+ (Fig. 7b). However, NPMc+ is a cytoplasmic protein, while HEXIM1 is mainly localized within nuclei. In the domain study, we identified the BR of HEXIM1 as the NPM binding region (Fig. 2b and c). We next investigated the influence of NPMc+ on subcellular localization of the BR using the YFP-BR construct. Expression of NPMc+ was visualized using anti-NPM antibody that recognizes both NPM and NPMc+. Cytoplasmic localization of the

Nucleophosmin is a HEXIM1-binding Protein

BR was detected in the NPMc+ and YFP-BR coexpressing cell (Fig. 7c, white arrows) while nuclear/ nucleolar localization of the BR was found in the BRtransfected cell (Fig. 7c, red arrows). The influence of EGFP-NPMc+ on endogenous HEXIM1 was also examined. Cytoplasmic localization of HEXIM1 was clearly detected in EGFP-NPMc+ expressing cells, while typical nuclear localization of HEXIM1 was observed in the cells that were not transfected with EGFP-NPMc+ (Fig. 7d). We also analyzed the cytosol of EGFP-NPMc+ transfected cells by Western blot and confirmed the cytoplasmic presence of endogenous HEXIM1 (data not shown). Additional Co-IP performed using the cytosol of EGFP-NPMc+ expressing cells showed precipitation of EGFPNPMc+ with HEXIM1 (data not shown). These findings demonstrate that NPMc+ sequesters a portion of HEXIM1 protein in the cytoplasm, which may help to explain the activating effect of NPMc+ on RNA Pol II transcription (Fig. 7a). Of greatest importance would be the examination of the sub-cellular localization of HEXIM1 in NPMc+ AML cells. An AML cell line, AML3, has been characterized to contain a TCTG duplication at exon-12 of NPM, resulting in the generation of NPMc+ mutant protein.35,49 AML3 cells were coimmunostained with anti-HEXIM1 and anti-NPM antibodies. Another AML cell line, AML2, which contains the wild-type NPM,49 was used as a control. Compared with AML2 cells, a significant increase in cytoplasmic localization of endogenous HEXIM1 was observed in AML3 cells (Fig. 8). Transcription of PTEFb-dependent genes (including Mcl-1, cyclin D1, and Pbx1) in AML2 and AML3 cells were measured using quantitative RT-PCR. Histone H2b, whose transcription is not regulated by P-TEFb, was used as a negative control. Compared to AML2 cells, twoto ninefold higher expression levels of Mcl-1, cyclin D1, and Pbx1 were detected in AML3 (Fig. 9). A similar mRNA level of histone H2b was observed in both cell lines (Fig. 9). These results suggest that NPMc+ contributes to the mislocalization of HEXIM1 in the cytoplasm of the NPMc+ AML cells, resulting in higher P-TEFb activity.

Discussion In this study, we identified NPM as a novel HEXIM1-binding protein. HEXIM1 associated with NPM in vitro and in vivo in a 7SK snRNA-independent manner (Figs. 1–3). NPM functioned as a negative regulator of HEXIM1. Over-expression of NPM decreased HEXIM1 protein levels through proteasome-dependent degradation and up-regulated PTEFb-dependent transcription (Figs. 4 and 5). The opposite effects on HEXIM1 levels and transcription were detected when NPM was knocked down (Fig. 6). In addition, over-expression of NPM reduced HEXIM1-mediated transcriptional repression while the HEXIM1 binding-deficient NPM mutants had little or no effect on P-TEFb-dependent expression, suggesting a possible correlation between NPM and

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HEXIM1 on transcriptional regulation (Fig. 4c–f). Importantly, NPMc+, the cytoplasmic mutant of NPM, sequestered a portion of HEXIM1 in the cytoplasm, resulting in the activation of RNA Pol II transcription (Fig. 7). Most interestingly, in an NPMc+ AML cell line, we detected cytoplasmic sequestration

of HEXIM1 (Fig. 8) and higher expression levels of PTEFb-dependent genes (Fig. 9), suggesting the physiological importance of the association between NPMc+ and HEXIM1. This supports a potential role of HEXIM1/P-TEFb in the tumorigenesis of NPMc+ AML.

Fig. 7 (legend on next page)

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Fig. 8. Cytoplasmic localization of HEXIM1 in the NPMc+ mutant cell line, AML3. AML2 (with wild type NPM) and AML3 (with NPMc+ mutation) were immunostained with anti-HEXIM1 (green) and anti-NPM (red) antibodies. Nuclei were visualized by DAPI.

Co-IP analysis showed that a small portion of NPM interacts with HEXIM1 (Fig. 1a). Indeed, we are surprised to find that such a weak NPM–HEXIM1 interaction could result in such profound influence on HEXIM1-/P-TEFb-mediated transcription (Fig. 4). Study of NPM mutants demonstrates the functional correlation between HEXIM1 and NPM on RNA Pol II transcription (Fig. 4e and f). Weak interactions between NPM-p53 and NPM-HDM2 have been described. Similarly, in spite of the weak interactions, NPM was shown to have important roles in p53- and HDM2-dependent pathways.36,38 Several HEXIM1-binding proteins (i.e., cyclin T1, glucocorticoid receptor, NF-κB, and ERα) have been described.26,30–32 Besides cyclin T1, which binds to the PYNT motif (amino acids 202–205), all the other proteins interact with HEXIM1 through the BR. We show here that NPM binds to the BR as well. We find that HEXIM1(1–180) has the strongest binding affinity to NPM (Fig. 2a). Among the four BR-containing HEXIM1 proteins (i.e., WT, 120–359, 1–180, and ILAA), HEXIM1(1–180) is the only one that does not contain the AR and DD (Fig. 2a). There are two

possible explanations for this result: (i) In the wildtype HEXIM1 protein, the AR interacts with the adjacent BR in the absence of 7SK snRNA.27 Since HEXIM1(1–180) does not contain the AR, the BR is more accessible for NPM binding. (ii) HEXIM1(1–180) cannot form the homodimer due to lack of the DD, suggesting that NPM may interact only with the monomer, but not the dimer of HEXIM1. Our results suggest that NPM could regulate RNA Pol II transcription through the interaction with HEXIM1. Without causing any detectable effect on HEXIM1 protein, over-expression of NPM resulted in an increase in the amounts of HEXIM1 being sequestered by NPM in nucleoli (Fig. 5a; data not shown). As shown in Fig. 1a, the NPM-bound HEXIM1 failed to associate with P-TEFb. Therefore, we hypothesized that the increased NPM-HEXIM1 interaction may influence the equilibrium between small and large P-TEFb complexes and affect P-TEFbdependent RNA Pol II transcription. As shown in the NPM over-expression assays, P-TEFb-dependent transcription was activated when NPM was overexpressed (Fig. 4), while a corresponding transcrip-

Fig. 7. NPMc+ activates RNA Pol II transcription and sequesters HEXIM1 in cytoplasm. (a) HEK293 cells were transiently transfected with a reporter vector (HIV-LTR-Luc or HTLV-LTR-Luc) and an indicated expression plasmid. To examine the effects on HIV Tat (HIV + Tat) and HTLV Tax (HTLV + Tax) transactivation, a Tat- and a Tax-producing plasmid was included, respectively. The pcDNA6 (i.e., mock) and NPM coding (i.e., NPM WT) plasmids were used as controls. (b) Co-IP was performed using cell lysates of HEK293 cells transfected with the EGFP, EGFP-NPM, or EGFPNPMc+ plasmids, respectively. The lysates were then precipitated with anti-HEXIM1 antiserum. The precipitated protein complexes were analyzed by immunoblotting using anti-GFP antibody. (c) HeLa cells were co-transfected with NPMc+ (red) and YFP-BR (green) expression plasmids. Cells were immunostained with anti-NPM antibody which recognizes both NPM and NPMc+ (red). The NPMc+/YFP-BR co-expressing cell is indicated by white arrows. The singly YFP-BR expressing cell is indicated by the red arrows. Nuclei were visualized by DAPI staining. (d) HeLa cells were transfected with EGFP-NPMc+ (green) and immunostained with anti-HEXIM1 antibody (red). Co-localization of EGFP-NPMc+ and endogenous HEXIM1 in cytoplasm appears yellow in the overlay image.

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Fig. 9. Up-regulation of P-TEFb-dependent genes in AML3 cells. Quantitative RT-PCR was performed using RNAs isolated from AML2 and AML3 cells. Expression levels of P-TEFb-dependent (i.e., Mcl-1, cyclin D1, and Pbx1) and -independent genes (i.e., histone H2b) were determined. Fold changes were determined by comparing with the readings of AML2 cells.

tion inhibition was shown when NPM was knocked down (Fig. 6). Furthermore, when high-level overexpression of NPM was reached, a significant decrease in the protein level of HEXIM1 was observed (Fig. 5b). Our results showed that NPM negatively regulates HEXIM1 expression through the proteasome-dependent pathway (Fig. 5c), but not through transcription control (Supplementary Data Fig. 2). Over-expression of NPM has been detected in tumors of various histological origins and shown to increase cell growth and proliferation.34 Therefore, the activating effect of NPM over-expression on HEXIM1mediated RNA Pol II transcription may provide a possible molecular mechanism for the proto-oncogenic function of NPM. In a recent study, HEXIM1 has been demonstrated to bind to double-stranded RNAs (dsRNAs) including at least one microRNA.50 Our results suggest strongly that the effects of NPM are on P-TEFb-mediated transcription through the association with HEXIM1. However, we cannot rule out the possibility that NPM affects the ability of HEXIM1 to bind to dsRNA and microRNA, which in turn may influence RNA Pol II transcription directly or indirectly. More than half of P-TEFb in cells is associated with HEXIM1,14,22 suggesting an important role of HEXIM1 in regulating the balance between large and small PTEFb complexes, as well as the activity of RNA Pol II. Our data showed that NPMc+ sequestered a significant portion of HEXIM1 in the cytoplasm (Fig. 7d). Furthermore, the increased cytoplasmic localization of endogenous HEXIM1 was detected in the NPMc+ AML cell line, AML3 (Fig. 8). Because of the mislocalization of HEXIM1, it is possible that the equilibrium between large and small P-TEFb complexes is disturbed in AML3 cells. This could cause a significant difference in RNA Pol II transcription between AMLs with wild-type and mutant NPM.

Indeed, quantitative RT-PCR demonstrates the increased levels of P-TEFb-mediated gene expression in AML3 cells (Fig. 9). Collectively, the interaction between NPMc+ and HEXIM1 might have a broad effect on the transcription by RNA Pol II. Besides the anti-viral activity through the inhibition of P-TEFb,11,13,43 the pharmacological P-TEFb inhibitors, flavopiridol and CYC202, are currently being tested as potential cancer therapeutics in clinical trials.51,52 The mislocalization of HEXIM1 and the increased P-TEFb-dependent transcription caused by NPMc+ suggests that the misregulated activity of PTEFb may contribute to the tumorigenesis of NPMc+ AML. It is important to investigate the correlation between P-TEFb activity and NPMc+ AML, and evaluate the therapeutic potential of P-TEFb inhibitors, such as flavopiridol and CYC202, in NPMc+ AML patients.

Materials and Methods Cells, short interfering RNAs (siRNAs), and plasmids HeLa, HEK293, and 293T cells were obtained from the American Type Culture Collection. AML cell lines, OCIAML2 and OCI-AML3, were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. Non-specific siRNA, GL2 luciferase (Luc) siRNA, HEXIM1 siRNA, NPM-1 and NPM-7 siRNAs were purchased from Qiagen. The mammalian expression vector pcDNA6, which contains the human CMV MIE promoter, was purchased from Invitrogen. To generate Myc-tagged NPM plasmids, including wild-type NPM, NPM(1–117), NPM(1–191), NPM (120–294), and NPM(191–294), the NPM cDNA (obtained from Open Biosystems) was used as the template for PCR amplification, and the amplified DNA fragments were subcloned into pcDNA6. The CMV-EGFP plasmid, in which the expression of EGFP was driven by CMV MIE promoter,

314 was kindly provided by Dr Zhiwei Song (Bioprocessing Technology Institute, Singapore). The PCR-amplified NPM coding region was subcloned into CMV-EGFP to generate the CMV-EGFP-NPM vector. Plasmid containing the NPMc+ cDNA was used to generate the CMV-NPMc+ and CMVEGFP-NPMc+ expression plasmids. Plasmids coding for HA-NPM, HEXIM1-YFP, BR-YFP, FLAG-HEXIM1(wildtype), FLAG-HEXIM1(120–359), FLAG-HEXIM1(180– 359), FLAG-HEXIM1(1–180), FLAG-HEXIM1(1–150), FLAG-HEXIM1(ΔBR), and FLAG-HEXIM1 ILAA mutant were kindly provided by Dr B. Matija Peterlin,27 Dr Olivier Bensaude,15,26 and Dr Sergio Menendez (Institute of Molecular and Cell Biology, Singapore). The coding region of YFP was amplified by PCR using the HEXIM1-YFP plasmid as the template and subcloned into pcDNA6 to generate CMV-YFP. GL3 firefly luciferase reporter vector driven by HTLV LTR, HTLV-LTR-Luc, and HTLV Tax expression vector were kindly provided by Dr Arnold Rabson.53 GL2 firefly luciferase reporters, including CMV-Luc (driven by CMV MIE promoter), MLV-LTR-Luc (driven by Moloney MLV LTR), and HIV-LTR-Luc (controlled by HIV LTR) have been described.43,54 Purification of HEXIM1 protein A histidine-tagged HEXIM1 construct, pET21a-HEXIM1His, was generated by subcloning the human HEXIM1 coding sequence (Open Biosystems, IMAGE clone 3535529) into pET21a (Novagen). Expression and purification of HEXIM1 were performed essentially as described22 but with minor modifications. Briefly, the bacterial lysates were incubated with Ni-NTA agarose beads (Qiagen) and washed with ten column volumes of buffer 1 (10 mM Tris (pH 7.8), 500 mM NaCl, 1% (v/v) TritonX-100, 40 mM imidazole, complete EDTA-free protease inhibitors(Roche)) and ten column volumes of buffer 2 (10 mM Tris (pH 7.8), 100 mM NaCl, 1% TritonX-100, 40 mM imidazole, complete EDTA-free protease inhibitors cocktail). The column was eluted with buffer 2 containing 250 mM imidazole. Analysis of HEXIM1-associated proteins in HeLa cells Cell lysates were prepared from HeLa cells using the lysis buffer (10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.5% Triton X-100, 10 mM imidazole, Protease Inhibitor tablet (Roche)). The lysates were pre-incubated with NiNTA agarose beads to remove any non-specific binding. The pre-cleared cell lysates were then incubated with NiNTA beads only or the HEXIM1-His-immobilized NiNTA beads. After washing four times with the wash buffer (10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.5% Triton X-100, 40 mM imidazole, Protease Inhibitor tablet (Roche)), the HEXIM1–protein complexes containing the purified HEXIM1-His and unidentified HEXIM1-binding proteins were eluted with 250 mM imidazole and analyzed by SDS-PAGE with silver staining. The specific protein bands, which are not present in the control experiment (i.e., HEXIM1-His free Ni-NTA resin), were excised from the gel and soaked overnight at 4 °C in washing solution (25 mM ammonium bicarbonate in 50% (v/v) aqueous acetonitrile). The gel pieces were then dried in a SpeedVac (Savant) and 20 μl of 10 mM dithiothreitol in 100 mM ammonium bicarbonate was added to each gel piece and incubated at 56 °C for 1 h. After the incubation, 20 μl of 55 mM iodoacetamide in 100 mM ammonium bicarbonate was added and incubated at room temperature for 45 min in the dark. The gel pieces were then washed and dehydrated twice in

Nucleophosmin is a HEXIM1-binding Protein

100 mM ammonium bicarbonate and 100% acetonitrile, respectively. For proteolysis, the protein in each gel piece was digested with 10 μl of trypsin (Promega) at 0.02 μg/μl in 25 mM ammonium bicarbonate at 37 °C overnight with shaking. Following incubation, the supernatant was removed and dried in a SpeedVac and finally resolubilized in 9 μl of 1% (v/v) formic acid in 2% (v/v) methanol for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Each sample was injected onto a C-18 reversed-phase micro pre-column (LC-Packings) in 0.1% formic acid (Sigma). Peptides were then eluted from the cartridge by the application of a gradient from 0%–90% acetonitrile in 0.1% formic acid. The eluting peptides were further separated by passage through a fused silica capillary column that was packed in-house (75 μm × 100 mm volume of 5 μm C-18 reversed-phase packing material (Column Engineering)). Eluted peptides were ionized by the application of 2200 V distally to the column using a liquid junction and sprayed directly into a QSTAR-XL quadrupole-time-of-flight mass spectrometer (ABI/MDSSciex). Data were acquired in Information Dependent Acquisition mode over a parent ion mass range of 800– 2000 amu for 2+ and 3+ ions only with a threshold of 8 counts/s. The acquired MS/MS spectra were searched against the UniProt (EBI) protein database using the MASCOT (Matrix Science) search engine. The parameter settings included a parent ion mass tolerance of 100 ppm, a fragment mass tolerance of 0.2 Da, and variable modifications for the oxidation of methionine residues as well carbamidomethylation of cysteine residues. Co-immunoprecipitation (Co-IP) and Western blotting HeLa and HEK293 cell lysates were utilized for Co-IP, which was performed using an immunoprecipitation kit according to the manufacturers' instructions (Roche). For RNase assays, cell lysates were incubated with 200 μg/ml of RNase A (Qiagen) at 4 °C for 1 h, followed by Co-IP. Cell lysates were prepared using the buffer provided in the Roche immunoprecipitation kit. HEXIM1 antiserum was kindly provided by Dr Olivier Bensaude.15 Another HEXIM1 antiserum was generated by immunizing rabbits with the HEXIM1 C-terminal peptide, LHRQQERAPLSKFGD (i-DNA Biotechnology, Singapore). Other antibodies used in this study included anti-NPM (Santa Cruz Biotechnology), anti-Cdk9 (Santa Cruz Biotechnology), anti-actin (Abcam), anti-GFP (Abcam and Invitrogen), anti-FLAG (Sigma) antibodies. Both EGFP and YFP can be recognized by the anti-GFP antibodies. SDS-PAGE and Western blotting followed standard protocols. For MG132 experiments, HEK293 cells were transfected with pcDNA6 (i.e., empty vector) or HA-B23 plasmids for 48 h. HEK293 cells transfected with HA-B23 plasmids were treated with or without 10 μM proteasome inhibitor MG132 (Sigma) for 15 h before harvest. Lysates prepared from the HEK293 cells were examined by standard SDSPAGE and Western blotting protocols. Immunofluorescence and fluorescent microscopy HeLa cells were seeded on circular coverslips into six-well plates one day before transfection with the indicated expression vectors: YFP-HEXIM1, YFP-BR, EGFP-NPMc+, FLAG-HEXIM1, and HA-NPM. The cells were fixed with 4% (v/v) paraformaldehyde and permeabilized using 0.1% Triton X-100 in PBS 48 h post-transfection. The samples were then incubated with anti-NPM monoclonal antibody (Santa

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Cruz Biotechnology), anti-FLAG monoclonal antibody (Sigma), anti-HA monoclonal antibody (Sigma), or affinity-purified anti-HEXIM1 antibody.50 After washing with PBT (PBS and 0.1% (v/v) Tween 20), the cells were incubated with R-phycoerythrin-labeled anti-mouse antibody (DAKO), anti-sheep Alexa Fluor 488 or 594 antibody (Invirogen) for 1 h. The samples were finally mounted with 4′,6-diamidino-2-phenylindole (DAPI) mixed ProLong® Gold Antifade Reagent (Invitrogen). Fluorescent microscopy was carried out using Zeiss Axio Imager.Z1 microscope or Zeiss LSM 510 Meta confocal laser scanning microscope.

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5. Transient transfection and luciferase assays Transfections were performed using FuGENE 6 (Roche) or Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturers' instructions. To perform luciferasebased assays, HEK293 or HeLa cells were grown to 50–80% confluence in 96-well plates. Cells were transiently transfected with a firefly luciferase reporter and a Renilla luciferase plasmid, pRL-RSV (Promega), to normalize transfection efficiency. Firefly and Renilla luciferase activities were measured 48 h post-transfection using the DualGlo assay system (Promega) and the activities were determined using an Infinite 200 multiplate reader (Tecan). Quantitative RT PCR RNAs of the transfected cells were isolated using an RNeasy kit (Qiagen). The Improm II Reverse Transcription System (Promega) and SYBR Green PCR Master Mix (Applied Biosystems) were used to carry out reverse transcription and real time PCR, respectively. The mRNA of glyceraldehyde-3-phosphate dehydrogenase, a P-TEFbindependent gene,45 was used to normalize the readings of quantitative RT-PCR. Amplification and detection of specific mRNAs were performed using ABI Prism 7000 Thermal-Cycler (Applied Biosystems).

Acknowledgements We thank Dr Olivier Bensaude, Dr B. Matija Peterlin, Dr Arnold Rabson, Dr Zhiwei Song, and Dr Sergio Menendez for providing plasmids and antibodies; Dr May May Lee, Dr Sai Mun Leong, Lu Zhang and Ally Lau for expert technical assistance.

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Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2008.02.055

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