Letters to the Editor 1067 previously described BCL6/IG translocations with a breakpoint P Bertrand1, C Maingonnat1, JM Picquenot1, N Dastugue2, 1 2 1 1 in the 50 UTR of BCL6, or in the distant ABR.2,6 Thus, in our D Penther , L Ysebaert , C Maisonneuve , H Tilly and C Bastard1 cases BCL6 deregulation can not be explained by promoter 1 substitution. In case no. 2 the inactivation of BCL6 negative Groupe d’e´tude des prolife´rations lymphoı¨des, INSERM U918, IFRMP23, Centre Henri Becquerel, Rouen, France and autoregulation sites by mutations can be hypothesized, but in 2INSERM U563, Centre de Physiopathologie, case no.1, for which no mutation was found inside these Toulouse, France regions, the most reliable hypothesis involves cis-acting E-mail: [email protected] elements provided by the partner sequences. The expression of TMEM75, the closest locus brought by the translocation was References investigated. The transcript was detected, at a level which did not differ significantly from the mean level of the 11 previous 1 Bertrand P, Bastard C, Maingonnat C, Jardin F, Maisonneuve C, controls. Thus, the activation of BCL6 by TMEM75 regulating Courel MN et al. Mapping of MYC breakpoints in 8q24 rearrange- sequences remains possible, but the effects of more distant ments involving non-immunoglobulin partners in B-cell lympho- regions can not be precluded. Indeed, it was demonstrated that mas. Leukemia 2007; 21: 515–523. enhancers, defined as position and orientation independent 2 Butler MP, Iida S, Capello D, Rossi D, Rao PH, Nallasivam P et al. 0 activators of transcription, can exert regulating effects several Alternative translocation breakpoint cluster region 5 to BCL-6 in B-cell non-Hodgkin’s lymphoma. Cancer Res 2002; 62: hundred kilobases from targets by DNA folding, thus bringing 7 4089–4094. together enhancer and the target. 3 Lossos IS, Levy R. Mutation analysis of the 50 noncoding regulatory Regarding the pathophysiological role of the t(3;8), several region of the BCL-6 in non-Hodgkin lymphoma: evidence for hypotheses can be raised as: (i) BCL6 expression was recurrent mutations and intraclonal heterogeneity. Blood 2000; 95: significantly increased as compared to controls although the 1400–1405. effects of mutations located in negatively regulating region 4 Jardin F, Buchonnet G, Parmentier F, Contentin N, Lepretre S, Lenain P et al. Follicle center lymphoma is associated with could also explain the gene overexpression; (ii) we have shown significantly elevated levels of BCL-6 expression among lymphoma previously that, in these cases MYC transcript was also subtypes, independent of 3q27 rearrangements. overexpressed; (iii) the formation of MYC-BCL6 complexes at Leukemia 2002; 16: 2318–2325. the level has been reported,8 with an effect on MYC half- 5 Kikuchi M, Miki T, Kumagai T, Fukuda T, Kamiyama R, Miyasaka N life, suppression of the synthesis of the p21CIP cell cycle arrest et al. Identification of negative regulatory regions within the gene, and inhibition of BCL6 acetylation. Therefore, the first exon and of the BCL6 gene. Oncogene 2000; 19: 4941–4945. synergistic effect of MYC and BCL6 could explain the survival 6 Akasaka H, Akasaka T, Kurata M, Ueda C, Shimizu A, Uchiyama T and clonal selection of a t(3;8) carrying cell in lymphoma et al. Molecular anatomy of BCL6 translocations revealed by long- progression. distance polymerase chain reaction-based assays. Cancer Res 2000; 60: 2335–2341. 7 Ratsch A, Joos S, Kioschis P, Lichter P. Topological organization of Acknowledgements the MYC/IGK locus in Burkitt’s lymphoma cells assessed by nuclear halo preparations. Exp Cell Res 2002; 273: 12–20. 8 Saito M, Phan RT, Morse HC, Pasqualucci L, Dalla-Favera R. This work was supported by the Ligue Contre le Cancer (Comite´ Pathologic co-expression and physical interaction of c-MYC and de Seine Maritime) and the Fe´de´ration des Centres de Lutte contre BCL6 in B-cell lymphomas. Blood (Am Soc Hematol Annu Meet le Cancer. Abstr) 2005; 106, (abstract [2]).

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Downregulation of microRNA-142 by proto-oncogene LMO2 and its co-factors

Leukemia (2008) 22, 1067–1071; doi:10.1038/sj.leu.2405001; LMO2 functions as a bridging molecule with the two arms of published online 1 November 2007 GATA1 and TAL1/E47 bound to relevant DNA motifs.4 MicroRNAs (miRNAs) are a recently discovered class of small Hematopoiesis is a tightly regulated multistage process. In the (E22-nt), non-coding RNAs that regulate via past decade, a number of transcription factors have been the RNA interference pathway either to promote degradation of identified as crucial regulators in cell proliferation, differentia- the target mRNA or to repress its translation. There was tion and apoptosis in hematopoiesis. The proto-oncogene, increasing evidence showing that miRNAs had crucial functions LMO2 (also named rbtn2 or ttg2), is such a factor. LMO2 was in hematopoiesis, as well as leukemia (reviewed in Ref. 5). first cloned from an acute T-lymphocyte leukemia (T-ALL) However, there were only a few reports about the expression patient with a (11;14;p13;q11) translocation,1 and subsequently profiles of miRNAs, and studies on miRNA expression regula- showed crucial functions in hematopoiesis, as well as in tions in hematopoiesis are limited. Until now, the best studied is angiogenesis.2,3 LMO2 encodes a LIM-only protein comprised miR-223, which has been shown to be regulated by C/EBPa6, of two LIM domains characterized as a cysteine-rich motif PU.1 and GATA1.7 consisting of two tandemly repeated zinc fingers and is MiR-142, which was confirmed to have similar functions with considered to be a transcriptional regulator through the miR-223 in promoting T-cell development, has rarely been interactions with its partners, including LDB1 (CLIM2/NLI1), studied before. In this study, we analyzed miR-142 expression GATA1, TAL1(SCL) and E47, to form a multi-complex in which profiles in the human erythroid/myeloid leukemia cell line,

Leukemia Letters to the Editor 1068

Figure 1 MiR-142 expression was correlated with LMO2. (a) Expression levels of miR-142 and LMO2 were shown by reverse transcriptase (RT)–PCR analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The agarose gel electrophoresis data was quantified by UV Band scanning. Data represent three independent experiments. (b) LMO2 protein expression was detected by western blot analysis using monoclonal anti-LMO2 antibody.

K562, the human T-cell lymphoblast-like cell line, Jurkat, and specific found to be directly regulated by the LMO2 the Burkitt lymphoma cell line, Raji, by semi-quantitative RT– complex.8 In addition, there were also two predicted transcrip- PCR. MiR-142 was detected in all these cell lines. We then tional start sites in this region, as shown in Figure 2a (committed analyzed LMO2 mRNA levels by semi-quantitative RT–PCR and by NNPP Neural Network Promoter Prediction; http:// LMO2 protein expression by immunoblot analysis in these cell www.fruitfly.org/seq_tools/promoter.html), but they seemed to lines. We found that LMO2 expression (both mRNA and protein) contribute with no prominent difference to the promoter activity was absent in Jurkat cells, which expressed high levels of miR- (Figure 2b N2 and N3). 142. In contrast, the other two cell lines that expressed high To investigate whether LMO2 physically interacted with the levels of LMO2 showed reduced miR-142 expression (Figure 1). miR-142 promoter region in vivo, we performed ChIP in K562 These data suggested that there might be some relationship cells. Anti-LMO2 antibody, as well as anti-FLAG antibody used between miR-142 and LMO2. as a control, was used to immunoprecipitate the fragmented To identify possible regulators of miR-142 expression, we chromatin. After reversal of the crosslink, a segment of the miR- cloned a 2320 bp region 50 flanking of pre-miR-142 from the 142 promoter (À2000 to À1703 bp, 298 bp) containing the and then confirmed by sequencing, named N1 E-box/GATA site was amplified by PCR using specific spanning (À2320 to À1 bp). Then a set of 50 deletions were performed primers. Primers recognizing the 30-distal region were used as a according to the restriction sites that were suitable for insertions negative control in the PCR reactions, as shown in Figure 3a. to pGL4 vector and another two reporter constructs, named N2 The result suggested that LMO2 could specifically bind to the (À1274 to À1 bp) and N3 (À1144 to À1 bp), were generated. À2000 to À1703 bp (298 bp) region of the miR-142 promoter These constructs were assayed in transient transfection experi- (Figure 3b), providing strong evidence that LMO2 was involved ments in K562 and control (HEK293) cell lines. All these in the regulation of the miR-142 gene. reporters showed potent promoter activity in K562 cells, in To further study, the regulatory effect of LMO2 on the which the miR-142 gene was endogenously expressed. How- miR142-promoter, we performed a LMO2 overexpression assay ever, the reporters were virtually silent in the control cell line. and knockdown assay using RNAi. The LMO2 siRNA construct One notable thing was that the promoter activity was (pSilencer4.1-siLMO2), or an unrelated control siRNA construct significantly increased when the À2320 to À1274 region was (pSilencer4.1-control), was transfected into K562 cells. LMO2 deleted. This indicated that this region might contain some silencing efficiency was confirmed using western blot analysis repressor elements contributing to the regulating miR-142 and the results showed that the protein level was reduced to less expression in K562 cells (Figure 2b). The putative binding sites than 20% (Figure 3b). In the same cell lysates, we found a for transcription factors were explored in this region using the dramatic increase of N1 reporter activity compared to the cells Transcription Element Search System (URL: http://www.cbil.u- transfected with control siRNA. Meanwhile, we also found that penn.edu/tess). Notably, one putative GATA-binding site and an when overexpressing LMO2 in K562 cells, the reporter activity E-box (CACGTG) on the transcriptional minus strand were decreased by about 50% (Figure 3a). found. The distance between the E-box and the GATA site in this According to our data, LMO2 appeared to play a pivotal bipartite sequence was 9 bp, similar to the bipartite DNA- function in negatively regulating the expression of miR-142. binding motif present in the promoter of several hematopoietic- Some reports pointed out that LMO2 functioned as a bridging

Leukemia Letters to the Editor 1069

Figure 2 Transcriptional activity of promoter reporter constructs in K562 and control cells. (a) The diagram shows the upstream structure of the human miR-142 promoter. The putative LMO2 complex binding site and transcription start sites are indicated. (b) The activity of the indicated constructs following transfection into K562 cells and HEK293 cells. Cell lysates were collected 24 h after transfection and the luciferase assay was performed. The graph displays the mean and standard error from at least three separate experiments. *Po0.05, unpaired Student’s t-test, compared with vector alone.

Figure 3 LMO2 associated with the endogenous human miR-142 promoter. (a) The diagram of the ChIP assay, the binding sites of the spanning primers, and the control primers are indicated. (b) Association of LMO2 with the hmiR-142 promoter was analyzed in vivo by CHIP analyses in K562 cells, using monoclonal antibody against LMO2 or unrelated Flag. The PCR was performed using a spanning primer to detect LMO2-binding and using crosslinked IP products as templates (lanes 4 and 6). Control primer was used as a negative control for PCR (lanes 3 and 5). PCR reactions with a spanning primer and a control primer using input K562 genomic DNA as the template were performed as the positive control of the PCR reaction (lanes 1 and 2). molecule to assemble TAL1, LDB1, E47 and GATA1 to form a GATA1, TAL1 and E47, were transfected separately or in DNA-binding complex; and in the complex, GATA1 and TAL1 combination to examine the function of these transcription could bind to their relevant DNA motifs, while LMO2 itself was factors on the promoter. The relative luciferase activity of single considered having no DNA-binding ability.8 This reminded us transfection with N1 reporter was marked as the control (100%). that it probably also required the presence of GATA1, LDB1 and As shown in Figure 4c, co-transfection of the N1 reporter with TAL1/E47, besides LMO2 to efficiently regulate the expression the LDB1, TAL1 or E47 expression vector did not prominently of miR-142. To investigate this question, we performed affect the reporter activity, whereas overexpression of GATA1 transactivation assays using K562 cells and the longest promoter conferred a slight inhibitory effect and overexpression of LMO2 reporter, N1, which contained the LMO2 complex-binding inhibited the N1 reporter activity to a modest degree, similar to motif. Expression vectors for LMO2 and its partners, LDB1, the result shown in Figure 4a. Notably, when we co-expressed

Leukemia Letters to the Editor 1070

Figure 4 LMO2 together with its co-factors negatively regulated miR-142 expression and the efficacy was enhanced by the existence of its co- factors. (a) K562 cells were co-transfected with pGL4-N1 reporter together with LMO2 expression vector pcDNA6B-LMO2 or LMO2-specific siRNA construct pSilencer4.1-siLMO2 or si-negative control (si-NC) construct pSilencer4.1-control, respectively. Cell lysates were collected 24 h after transfection and the luciferase assay was performed. *Po0.05, unpaired Student’s t-test, compared with vector alone. (b) The efficiency of LMO2 protein knockdown was determined by western blot using monoclonal anti-LMO2 antibody. (c) K562 cells were co-transfected with pGL4- N1 reporter together with pcDNA6B as a negative control (NC) or TAL1 or GATA1 or LMO2 or LDB1 or E47 expression vectors or the complex containing LMO2, LDB1, GATA1, TAL1 and E47, respectively. Cell lysates were collected 24 h after transfection and the luciferase assay was performed. (d) K562 cells were transfected with LMO2 or the complex containing LMO2, LDB1, GATA1, TAL1 and E47 expression vectors or LMO2-specific siRNA construct pSilencer4.1-siLMO2 or EGFP expression vectors as a negative control (NC). The endogenous miR-142 precursor expression levels were measured by RT–PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control.

LMO2 with GATA1, TAL1, LDB1 and E47, the N1 reporter Thus, LMO2 and its co-factors may also play crucial roles in activity was repressed remarkably to 20% compared to the hematopoiesis by regulating the expression levels of miRNAs. control, showing a synergistic pattern of these members. In addition, to detect the affect of LMO2 on endogenous miR- 142 expression, we transiently transfected K562 cells with Acknowledgements LMO2 expression vector alone, or the LMO2 complex including LMO2, GATA1, LDB1, TAL1 and E47 expression vectors, or the This work was supported by the National Nature Science LMO2 siRNA construct, or the EGFP expression vector as a Foundation of China (No.30771054). negative control. In cells overexpressing LMO2 or the LMO2 W Yuan1, W Sun1, S Yang, J Du, C-L Zhai, Z-Q Wang, J Zhang complex, the endogenous miR142 precursor expression levels and T-H Zhu were decreased significantly, whereas in the cells transfected Laboratory of Molecular Genetics, Medical College, Nankai with LMO2 siRNA construct, the miR142 precursor expression University, Tianjin, China level showed a strong increase compared with the negative E-mail: [email protected] 1 control (Figure 4d). These data confirmed that LMO2, together These authors contributed equally to this work. with its co-factors, could negatively regulate the expression of miR-142 in vivo. Previously, it was considered that the LMO2 complex References customarily functioned by promoting the transcription of its target genes, and consequently upregulated a set of its down- 1 Royer-Pokora B, Loos U, Ludwig WD. TTG-2 a new gene encoding 4,8 stream genes. We reported here that miRNA-142 was a cysteine-rich protein with LIM motif, is overexpressed in acute regulated by LMO2 together with its co-factors in a negative T-cell leukaemia with the t(11;14)(p13;q11). Oncogene 1991; 6: manner. This result indicated that the LMO2 complex could 1887–1893. inhibit the expression of some genes directly. Considering the 2 Yamada Y, Warren AJ, Dobson C, Forster A, Pannell R, Rabbitts TH. ability of miR-142 on promoting T-cell development and the The T cell leukemia LIM protein Lmo2 is necessary for adult mouse hematopoiesis. Proc Natl Acad Sci USA 1998; 95: differentiation arrest caused by abnormal expression of LMO2 in 3890–3895. T-cell leukemia, our findings might partially explain the 3 Yamada Y, Pannell R, Forster A, Rabbitts TH. The oncogenic LIM- pathologic mechanism of LMO2 in T-cell leukemia, that is, only transcription factor Lmo2 regulates angiogenesis but not LMO2 might exhibit its oncogenic property through inhibiting vasculogenesis in mice. Proc Natl Acad Sci USA 2000; 97: the expression of miR-142. 320–324. Recent studies described several miRNAs regulated by classic 4 Wadman IA, Osada H, Grutz GG, Agulnick AD, Westphal H, Forster A et al. The LIM-only protein Lmo2 is a bridging molecule transcription factors. Our results showed that in hematopoiesis, assembling an erythroid, DNA-binding complex which includes the miR-142 was also regulated by the transcriptional regulators, TAL1, E47, GATA-1 and Ldb1/NLI . EMBO J 1997; 16: LMO2, LDB1, GATA1 and TAL1/E47, but in a negative manner. 3145–3157.

Leukemia Letters to the Editor 1071 5 Shivdasani RA. MicroRNAs: regulators of gene expression and cell 7 Fukao T, Fukuda Y, Kiga K, Sharif J, Hino K, Enomoto Y et al. An differentiation. Blood 2006; 108: 3646–3653. evolutionarily conserved mechanism for microRNA-223 expression 6 Fazi F, Rosa A, Fatica A, Gelmetti V, Marchis MLD, Nervi C et al. revealed by microRNA gene profiling. Cell 2007; 129: 617–631. A minicircuitry comprised of microRNA-223 and transcription 8 Nam CH, Rabbitts TH. The role of LMO2 in development and in factors NFI-A and C/EBPalpha regulates human granulopoiesis. Cell T cell leukemia after chromosomal translocation or retroviral 2005; 123: 819–831. insertion. Mol Ther 2006; 13: 15–25.

Spectrum of p53 mutations in low-grade B-cell malignancies

Leukemia (2008) 22, 1071–1073; doi:10.1038/sj.leu.2405002; ment. Our p53 mutation rate of 4% in CLL, although lower than published online 8 November 2007 many early studies, is in line with the only other study that we are aware of on a similar population of patients by Oscier et al. There has been considerable interest in p53 mutation in low- (when patients specifically selected for inclusion due to known grade B-cell malignancies over the years, particularly in chronic p53 mutation are excluded, an overall p53 mutation rate of 4% lymphocytic leukaemia (CLL). CLL exhibits a highly variable was found).4 Many early studies did not benefit from the clinical course; p53 mutation confers a worse prognosis and improved classification of patients that is now possible due to adversely affects response to purine analogue therapy. Reported flow cytometric, cytogenetic and molecular biological techni- rates of mutation of the p53 gene in B-cell malignancies vary ques, resulting in inclusion of pathologies such as mantle cell between studies and subtypes, but are not common.1 The lymphoma in leukaemic phase with CLL. When strict classifica- majority of studies have restricted mutation screening to exons tion is applied the rate of cytogenetic abnormalities in CLL 5–8, so the true rate of mutation outside of this region is not appears reduced.3 Most studies report the rate of p53 known. abnormalities as a combination of deletion and mutation, the The non-isotopic RNase cleavage assay (NIRCA) works on the mutation rate alone is therefore lower than the 10–15% often principle that RNase cleaves both strands of duplex RNA targets quoted. at mismatched bases and has been previously developed and A total of 24 patients were found to have a p53 mutation and validated as a method for screening for the presence of mutated 23 of these mutations were in the DNA binding domain of the p53 mRNA.2 Briefly single stranded RNA is synthesized from p53 gene (exons 5–8), suggesting that restricting screening to opposing strands of patient PCR product and wild type control exons 5–8 in low-grade B-cell malignancies will not exclude product, of the entire p53 coding region (from viral promoter detection of a large number of mutations. The aberrant mRNA sequences incorporated during PCR) and opposing RNA strands present in the remaining patient (Patient 1) was missing exon 4 are hybridized. Subsequent digestion with RNase and analysis entirelyFthis could have arisen due to a splice site mutation, by agarose gel electrophoresis and ethidium bromide staining such mutations have been previously reported in CLL.5 Studies results in the observation of digested product for those samples over the years have reached different conclusions as to the containing a mismatch between patient and wild type sequence. distribution of p53 mutations in B-cell malignancies, few We have used NIRCA to screen the entire coding region of the have screened the complete coding region of the gene. p53 gene from 480 patients diagnosed with a low-grade B-cell However Cordone et al.6 reported 4/14 mutations to be outside malignancy for p53 mutations (see Table 1 for disease of the evolutionary conserved region in direct contrast to our classification). findings. The population of patients seen at our centre are very different Trbusek et al.7 recently published a study using a functional from those seen at referral centres (where the majority of studies yeast assay in which they found 15/168 CLL patients to be p53 are carried out) and could be considered a more genuine mutated; surprisingly, all of the mutations were different and representation of an unselected CLL population. In our patient only one affected a mutation hotspot codon for p53 mutation population, 62.5% have never required treatment, 83% have (codon 248). However, only 3/15 of their reported mutations did stage A disease and many never require a follow up appoint- not affect codons with over 100 reported mutations on the UMD p53 mutation database.8 In our present cohort of patients (Table 2), 7/24 mutations affected the classic ‘hotspot’ codons Table 1 Disease classification and p53 mutation rate 175, 248 and 273 with only 6 mutations that did not affect a commonly mutated codon.8 Most studies report a similar Disease classification No. of patients No. of p53 distribution of p53 mutation in haematological malignancy. tested mutation (%) Two patients exhibited identical mutations at codon 248 which CLL 351 13 (4) resulted in substitution of arginine by glycine rather than the NHL leukaemic phase 34 3 (9) commonly reported substitution by tryptophan or glutamine. PLL 3 0 (0) There are reports of this mutation in the UMD p53 database in CLL/PL 29 2 (7) solid tumours (12 cases), Burkitts lymphoma (1 case), Diffuse UNa 32 2 (6) Mantle cell lymphoma 22 3 (14) Large B-cell lymphoma (1 case) and CLL (1 case) who is Waldenstro¨ ms 2 1 (50) included in this study and in a previous report by this Hairy cell leukaemia 7 0 (0) laboratory.8 Although rare, this particular substitution results in almost complete abolition of p53 activity in common with the Abbreviations: CLL, chronic lymphocytic leukaemia; NHL, non- 8 Hodgkin’s lymphoma; PL, prolymphocytes; PLL, prolymphocytic other recurrent mutations of this codon. The mutation at codon leukaemia. 273 seen in two patients resulted in the frequently found aUnclassifiable group according to Howe et al.3 arginine to histidine substitution.

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