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Inhibition of the Htert Promoter by the Proto-Oncogenic Protein TAL1

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Inhibition of the Htert Promoter by the Proto-Oncogenic Protein TAL1 Leukemia (2009) 23, 2081–2089 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu ORIGINAL ARTICLE Inhibition of the hTERT promoter by the proto-oncogenic protein TAL1 J-M Terme1, V Mocquet1, A-S Kuhlmann2, L Zane3, F Mortreux3, E Wattel3, M Duc Dodon2 and P Jalinot1 1Laboratoire de Biologie Mole´culaire de la Cellule, Unite´ Mixte de Recherche 5239, Centre National de la Recherche Scientifique, Ecole Normale Supe´rieure de Lyon, Institut Fe´de´ratif de Recherche 128 Biosciences Lyon Gerland, Lyon cedex 07, France; 2Virologie Humaine, Unite´ 758, Institut National de la Sante´ et de la Recherche Me´dicale, Ecole Normale Supe´rieure de Lyon, Institut Fe´de´ratif de Recherche 128 Biosciences Lyon Gerland, Lyon cedex 07, France and 3Unite´ d’Oncovirologie et de Biothe´rapies, Formation de Recherche en Evolution 3011, Centre National de la Recherche Scientifique, Centre Le´on Be´rard, Lyon cedex 03, France Telomerase activity, which has fundamental roles in develop- expression is regulated by many transcription factors (for a ment and carcinogenesis, strongly depends on the expression review, see Kyo et al.8). Its proximal transcription promoter of human telomerase reverse transcriptase (hTERT), its cataly- tic subunit. In this report, we show that the basic helix-loop- sequences include two E-boxes and five Sp1-binding sites. These helix factor, TAL1 (T-cell acute lymphoblastic leukemia 1), is a E-boxes have been shown to be regulated positively by the Myc– 9–12 negative regulator of the hTERT promoter. Indeed, TAL1 Max complex and negatively by Mad–Max. overexpression leads to a decrease in hTERT mRNA abundance An interesting model of hTERT regulation focused on the and hence to reduced telomerase activity. Conversely, suppres- opposite effects exerted by the Tax and HBZ (HTLV-1 basic sion of TAL1 by RNA interference in Jurkat cells increases leucine zipper) proteins expressed by the human T-cell hTERT expression. Analysis by chromatin immunoprecipitation assays showed that TAL1 binds to the hTERT proximal leukemia virus type 1 (HTLV-1), the etiological agent of adult promoter and recruits HDAC1. Considering the relationship T-cell leukemia (ATL). Indeed, it has been shown that the Tax recently established between TAL1 and the human T-cell transactivator decreases the activity of the hTERT promoter leukemia virus type 1 (HTLV-1) Tax protein, which was through the first E-box.13 Conversely, the HBZ protein, a factor confirmed in T lymphocyte clones derived from adult T-cell expressed by the antisense strand of the provirus, activates in leukemia patients, we analyzed the effect of TAL1 with respect cooperation with JunD, the hTERT promoter activity.14 This led to the earlier characterized effects of Tax and HBZ (HTLV-1 basic leucine zipper) on hTERT expression. TAL1 was observed to a model suggesting that early after infection Tax decreases to reinforce the negative effect of Tax, whereas hTERT telomerase activity, thereby favoring genomic instability. At a transactivation by the HBZ–JunD complex was repressed by later stage, Tax expression is repressed and the expression of TAL1 overexpression. Moreover, HBZ was found to induce HBZ reactivates telomerase expression allowing evolution proteasome-mediated degradation of TAL1. These observa- toward fully transformed leukemic cells.14 tions support a model in which Tax and TAL1 by repressing Recently, we have shown that TAL1 (T-cell acute lympho- hTERT would initially favor genomic instability, whereas expression of factors such as HBZ allows at a later stage an blastic leukemia 1), which is known to have a key function in increase in hTERT production and consequently in telomerase T-cell acute lymphoblastic leukemia (T-ALL) by impairing 15 16 activity. differentiation, is a target of Tax. Indeed, through activation Leukemia (2009) 23, 2081–2089; doi:10.1038/leu.2009.131; of its transcription promoter 1b, Tax increases TAL1 expression. published online 9 July 2009 By considering these data, we asked whether TAL1 is involved Keywords: T-ALL; hTERT; TAL1; HTLV-1; Tax; HBZ in hTERT regulation both in T-ALL and ATL cells. This factor is known to activate or repress many cellular genes.17 This class II basic helix-loop-helix factor, which has a key function in the differentiation of both hematopoietic and endothelial cells, has Introduction been shown to associate with E-boxes as a heterodimer with E protein as E47.18 TAL1 has also been shown to associate with Telomerase enzyme, which has the unique property of increas- many different transcriptional regulators, including the LIM-only ing the length of the telomeres, has a key function in cancer 1,2 proteins LMO1 and LMO2, GATA factors, Sp1, mSIN3A and cells. It has been shown that telomerase is active in many 19–23 3,4 p300. Depending on the combination of factors in which leukemic cells. Telomerase consists of an RNA molecule, TAL1 is engaged, its effect on a target transcriptional promoter is hTR, and of a catalytic protein subunit, human telomerase 5 either positive or negative. reverse transcriptase (hTERT). The high telomerase activity in We show in this report that TAL1, similar to Tax, is a repressor leukemic cells is often linked to an elevated expression of 6 of hTERT promoter activity. This effect relies on the Sp1 sites hTERT, and in various types of leukemia a decrease in hTERT present in the hTERT promoter. Interestingly, Tax and TAL1 can expression has been linked to reduced proliferation and to 7 cooperate for hTERT repression, but their effect is counteracted by higher sensitivity to drug treatment. The hTERT subunit JunD and HBZ. Our results suggest that TAL1 by inhibiting hTERT might favor genome instability both in T-ALL and ATL cells. Correspondence: Dr P Jalinot, Laboratoire de Biologie Mole´culaire de la Cellule, Unite´ Mixte de Recherche 5239, Centre National de la Recherche Scientifique, Ecole Normale Supe´rieure de Lyon, Institut Materials and methods Fe´de´ratif de Recherche 128 Biosciences Lyon Gerland, 46 Alle´e d’Italie, 69364 Lyon cedex 07, France. E-mail: [email protected] Constructs Received 29 October 2008; revised 27 May 2009; accepted 2 June pSGF-TAL1, pSUPER-TAL1 and pSG5-Tax have been described 2009; published online 9 July 2009 earlier.16 pSG5-MYC-TAL1 was generated by amplifying it with hTERT inhibition by TAL1 J-M Terme et al 2082 the appropriate primers, the TAL1-coding sequences from diluted 1:1000 or as indicated by the manufacturer, and detection a plasmid including the complete human TAL1 open-reading was performed by chemiluminescence using enhanced chemilu- frame, provided by D Mathieu. The PCR product was inserted minescence (ECL) or ECL þ kits (GE Healthcare). between the BglII and XhoI restriction sites in the pSG5-MYC Antibody against HTLV-1 Tax was obtained from the AIDS vector. pCDNA3-HBZ-myc and pCMV-JunD-Flag have been Research and Reference Reagent program catalog. The follow- described earlier.14 pCMV-Sp1 was kindly provided by Katie ing antibodies were purchased: FLAG (clone M2; Sigma- Flaig.24 pGL3-hTERTÀ3300, pGL3-hTert-378 and pGL3-hTert- Aldrich), b-actin (Sigma-Aldrich) and MYC (clone 9E10; 181 wild-type (WT), mt1, mt2, mt and mutSp1 were kindly Sigma-Aldrich). provided by Satoru Kyo.10 pCMV-p300 was kindly provided by Louis Gazzolo.25 Real-time quantitative telomeric repeat amplification protocol Cell culture, transfection and T-cell limiting dilution Stably transfected HeLa cells were trypsinized, washed twice in cloning phosphate-buffered saline and resuspended in 3-((3-cholamido- Jurkat cells were cultured in RPMI 1640 medium with 10% fetal propyl)-dimethylammonio)-1-propanesulfonate (CHAPS) lysis calf serum (FCS) (Sigma-Aldrich, St Louis, MO, USA), 100 U/ml buffer (10 mM Tris-HCl (pH 7.5), 1 mM MgCl2,1mM EGTA, penicillin and 100 mg/ml streptomycin (Gibco Invitrogen, San 0.5% CHAPS (w/v), 10% (w/v) glycerol, 0.1 mM phenylmethyl- 7 Diego, CA, USA) at 37 1C in a 5% CO2-humidified atmosphere. sulfonyl fluoride, 5 mM b-mercaptoethanol) at 10 cells per ml. HeLa cells were cultured in Dulbecco’s modified Eagle’s After 30 min lysis at 4 1C, lysates were centrifuged at 14 000 g for medium with 10% FCS (Gibco), 100 U/ml penicillin and 30 min at þ 4 1C and the supernatants were stored at À80 1C. 100 mg/ml streptomycin at 37 1C in a 5% CO2-humidified Samples were diluted at a final concentration of 300 ng/ml. The atmosphere. For transfection, FCS concentration was reduced sample dilution (1 ml) was used to perform the real-time to 5%. Jurkat cells were transfected using the Nucleofector kit quantitative telomeric repeat amplification protocol (RQ-TRAP) (Amaxa, Cologne, Nordrhein-Westfalen, Germany). HeLa cells assay with 5 ng/ml of TS primer (50-AATCCGTCGAGCAGAGT were stably transfected with the pCEP-FLAG-TAL or the control T-30), 2.5 ng/ml of ACX primer (50-GCGCGGCTTACCCT 16 0 pCEP-FLAG-GFP constructs, as described earlier. Expression TACCCTTACCCTAACC-3 ), 0.5 mM MgCl2 and the reactions vectors were transfected using the calcium phosphate precipita- were carried out in 20 ml reactions containing the LC-FastStart tion method as described earlier.16 To achieve inhibition of the DNA Master SYBR Green I mix (Roche, Basel, Basel-Stadt, proteasome, we added MG132 (Sigma-Aldrich) at 10 mM for 6 h. Switzerland). Using the LightCycler (Roche), we incubated the Cellular extracts were normalized with respect to protein samples for 30 min at 30 1C, for 10 min at 95 1C and 40 cycles concentrations that were quantified with the DC protein assay were performed: 30 s at 95 1C and 90 s at 60 1C. The values kit (Bio-Rad, Hercules, CA, USA).
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