The DNA Binding Activity of TAL-1 Is Not Required to Induce Leukemia/ Lymphoma in Mice

The DNA binding activity of TAL-1 is not required to induce leukemia/ lymphoma in mice

Jennifer O'Neil1, Marilisa Billa1, Sarah Oikemus1 and Michelle Kelliher*,1

1University of Massachusetts Medical School, Department of Molecular Genetics and Microbiology and the Cancer Center, 373 Plantation Street, Worcester, Massachusetts, MA 01605, USA

Activation of the basic helix ± loop ± helix (bHLH) gene (Kelliher et al., 1996; Condorelli et al., 1996), further TAL-1 (or SCL) is the most frequent gain-of-function demonstrating the oncogenicity of tal-1. Yet, the mutation in pediatric T cell acute lymphoblastic mechanism(s) of tal-1-induced leukemogenesis remains leukemia (T-ALL). Similarly, mis-expression of tal-1 in unclear. the thymus of transgenic mice results in the development In human leukemic Jurkat cells, tal-1 does not of clonal T cell lymphoblastic leukemia. To determine homodimerize, but forms stable heterodimers with the the mechanism(s) of tal-1-induced leukemogenesis, we ubiquitously expressed bHLH E2A proteins, E12 and created transgenic mice expressing a DNA binding E47 (Hsu et al., 1994b). Members of the E2A family mutant of tal-1. Surprisingly, these mice develop disease, include E2-2, HEB and the products of the E2A gene demonstrating that the DNA binding properties of tal-1 E47 and E12 (Murre et al., 1989; Henthorn et al., 1990; are not required to induce leukemia/lymphoma in mice. Hu et al., 1992). Tal-1/E2A heterodimers preferentially However, wild type tal-1 and the DNA binding mutant recognize the E-box consensus sequence CAGATG both form stable complexes with E2A proteins. In (Hsu et al., 1994a) and exhibit transcriptional addition, tal-1 stimulates dierentiation of CD8-single transactivation activity (Hsu et al., 1994c). Conse- positive thymocytes but inhibits the development of CD4- quently, it was proposed that tal-1 functions as a direct single positive cells: eects also observed in E2A- transcriptional activator in leukemia. de®cient mice. Our study suggests that the bHLH In erythroid cells, tal-1 associates with E12 and E47 protein tal-1 contributes to leukemia by interfering with (Hsu et al., 1991, 1994, Condorelli et al., 1995), the E2A protein function(s). Oncogene (2001) 20, 3897 ± cysteine-rich LIM-only proteins LMO2 and Ldb-1 3905. (Valge-Archer et al., 1994; Visvader et al., 1997; Wadman et al., 1997) and the erythroid-speci®c zinc Keywords: Tal-1; E2A; leukemia ®nger protein GATA-1 (Wadman et al., 1997). The tal- 1/E2A/LMO2/GATA-1 complex binds a composite E- box GATA site (Wadman et al., 1997) and presumably Introduction regulates genes involved in erythroid dierentiation. E- box-GATA sites have been identi®ed in several The basic helix ± loop ± helix protein TAL-1 is normally erythroid genes, including enhancers of the erythroid expressed in hematopoietic progenitors and erythroid, speci®c EKLF and GATA-1 transcription factors megakaryocytic and mast cell precursors, as well as (Anderson et al., 1998; Cohen-Kaminsky et al., 1998; endothelial cells and the central nervous system Vyas et al., 1999). Hence, tal-1/E2A heterodimers may (reviewed in Begley and Green, 1999). Gene targeting function as direct transcriptional regulators in both experiments in mice have established tal-1 as an hematopoietic development and leukemia. essential regulator of blood cell and vascular develop- Tal-1/E2A heterodimers are reported to be relatively ment (Shivdasani et al., 1995; Porcher et al., 1996; weak transcriptional activators compared to E2A Visvader et al., 1998). Deregulated expression of TAL- homodimers (Hsu et al., 1994c; Doyle et al., 1994; Park 1 in humans by either chromosomal translocation, and Sun, 1998). However, under physiologic conditions interstitial deletion or mutation occurs in greater than where inhibitory HLH Id proteins are expressed, tal-1/ 60% of pediatric patients with T cell acute lympho- E2A heterodimer is a signi®cantly better transcriptional blastic leukemia (T-ALL) (Bash et al., 1995). Ectopic activator than the E2A homodimer (Voronova and Lee, expression of tal-1 in the thymus of mice results in the 1994). In addition, tal-1 has been shown to interact with development of clonal T cell leukemia/lymphomas both transcriptional co-repressors and co-activators (Wadman et al., 1994; Huang et al., 1999; Huang and Brandt, 2000; Huang et al., 2000). Thus, tal-1-induced leukemogenesis may re¯ect the aberrant activation of *Correspondence: M Kelliher, Two BioTech, 373 Plantation Street, novel target genes or alternatively, sequestration of E2A Worcester, MA 01605, USA proteins and alteration of E2A-target genes. Support for E-mail: [email protected] Received 17 January 2001; revised 3 April 2001; accepted 9 April the sequestration model comes largely from studies on 2001 E2A-de®cient mice, where approximately 10% of DNA binding mutant of tal-1 induces leukemia in mice J O'Neil et al 3898 E2A7/7 mice develop spontaneous T cell lymphomas/ leukemias (Bain et al., 1997). To determine whether tal-1 transforms thymocytes by acting as a direct transcriptional activator, we created transgenic mice expressing a known DNA binding mutant of tal-1 (Hsu et al., 1994c). Mutagen- esis of the myogenic bHLH proteins, myogenin and MyoD1, identi®ed amino acid residues within the basic domain critical for DNA binding (Davis et al., 1990; Brennan et al., 1991). Replacement of two of the conserved, contact arginines with glycines within the basic domain of tal-1, (designated tal-1R188G;R189G), obliterated binding to the tal-1/E47 consensus sequence and destroyed E-box reporter activity (Hsu et al., 1994c). To elucidate the mechanism(s) of tal-1-induced leukemia, we tested the transforming potential of the tal-1R188G;R189G DNA binding mutant. Three transgenic lines of mice expressing tal-1R188G;R189G in the thymus were generated and characterized. Approxi- mately half of the mice expressing a DNA binding mutant of tal-1 developed disease. This study provides direct evidence that DNA binding activity of tal-1 is Figure 1 Structure and expression of the tal-1R188G;R189G transgene. (A) Diagrammatic representation of the lck-tal- not required to induce leukemia/lymphoma in mice and 1R188G;R189G fusion construct used to create transgenic mice. demonstrates that tal-1 contributes to leukemia by The human tal-1R188G;R189G cDNA subcloned into a vector interfering with E2A protein function(s). with the proximal lck promoter and human growth hormone (hGH) splice and poly(A)+ addition sequences was used to establish four lines of transgenic mice designated 2, 5, 6, and 23. (B) Expression of the tal-1R188G;R189 transgene. RNA prepared Results from thymus of wild type (+/+) and transgenic (mut tal-1 founder lines 2, 5, 6 and 23) was subjected to RNase protection analysis with an antisense riboprobe speci®c for human TAL-1. Tal-1R188G;R189G transgenic mice The human tal-1R188G;R189G mRNA protects a band of 500 bases. RNA levels were compared to the human TAL-1 A transgenic construct was generated by placing the expressing T-ALL cell line, Jurkat. RNA from the U937 and human TAL-1 cDNA containing the R188G;R189G yeast tRNA served as negative controls. (C) Expression of the mutations (gift of Dr Richard Baer, Columbia TAL-1 protein. The 42 kDa TAL-1 polypeptide was detected in University) under control of the lck proximal promoter the thymocytes of 4-week-old tal-1R188G;R189G transgenic mice from lines 2, 5, and 23 (mut tal-1) by immunoblotting with an (Figure 1A). The 3' untranslated region of this anti-human TAL-1 antibody (gift of Dr Richard Baer). Similar construct contains introns, exons and the poly A levels of TAL-1 protein expression were detected in the addition site of the human growth hormone gene thymocytes of age-matched tal-1 transgenic mice (tal-1), using (Abraham et al., 1991). The lck-tal-1R188G;R189G an anti-mouse tal-1 antibody (gift of Dr Richard Baer, Columbia construct was microinjected into the pronuclei of University). A murine erythroleukemic cell line (M) and Jurkat cells (J) were used as a positive controls and wild type thymus fertilized FVB/N oocytes (Taketo et al., 1991). Four (lane 6) as a negative control for pp42-tal-1 protein expression transgenic founders were identi®ed initially and three were studied in detail. The three tal-1R188G;R189G lines expanded for study expressed high levels of tal- 1R188G;R189G mRNA in thymocytes as shown by (Figure 2). Twenty-nine of 62 (48%) tal-1R188G; ribonuclease protection assay; the fourth line expressed R189G mice from three lines developed disease less tal-1R188G;R189G mRNA and was not studied compared to 21 of 75 (28%) of wild type tal-1 further (Figure 1B, lanes muttal-1/+). As expected, no transgenic mice (Figure 2 and Kelliher et al., 1996). tal-1 message was detected in wild type thymus. Both the wild type tal-1 and the tal-1R188G;R189G Thymocytes from the three tal-1R188G;R189G lines transgenic animals exhibit respiratory distress, rued expressed similar levels of tal-1R188G;R189G protein coat and weight loss. Necropsy revealed the presence of (Figure 1C lanes muttal/+). Furthermore, the protein a thymic mass, often accompanied by hepatospleno- expression levels of the tal-1R188G;R189G mutant megaly. Histological examination of the thymus were similar to that observed in the human leukemic revealed eacement of the normal thymic architecture cell line, Jurkat (J) (Figure 1C). by a monomorphic in®ltrate of lymphoblastic cells with prominent nucleoli and scant cytoplasm (Figure 3A,D). Similar cells invade the surrounding para-sternal T cell acute lymphoblastic leukemia/lymphoma in muscle, pericardium and other organs such as spleen, tal-1R188G;R189G transgenic mice liver and kidney (Figure 3B,C,E and F). Lymphoblasts The three tal-1R188G;R189G transgenic lines devel- were detected in the peripheral blood of diseased oped leukemia with a median survival of 215 days animals at the time of sacri®ce.

Oncogene DNA binding mutant of tal-1 induces leukemia in mice J O'Neil et al 3899 The histologic appearance of the thymic tumors as well as the leukemic blood pro®les of the tal- 1R188G;R189G mice were indistinguishable from that previously observed for wild type tal-1 transgenic mice (Kelliher et al., 1996). This study demonstrates that the DNA binding properties of tal-1 are not required to induce leukemia/lymphoma in mice and suggests that tal-1 transforms via an Id-like mechanism, potentially by sequestering E proteins.

Casein kinase II accelerates leukemia/lymphoma induced by tal-1 R188G;R189G CKII has been shown to modulate the activity of several transcription factors in vitro and to synergize dramatically with myc and with wild type tal-1 in inducing lymphocytic leukemia in bitransgenic mice (Seldin and Leder, 1995; Kelliher et al., 1996). The presence of CKII consensus phosphorylation sites in tal-1 and E47 and the fact that CKII phosphorylation has been shown to inactivate E47 DNA binding activity (Johnson et al., 1996) prompted us to test Figure 2 Kaplan ± Meier survival plot of tal-1, tal- whether CKII might collaborate with a DNA binding 1R188G;R189G, tal-1/CKIIa and tal-1R188G;R189G/CKIIa trans- mutant of tal-1 to induce leukemia in mice. To test genic mice. Survival plot for the tal-1 (tal); tal-1 R188G;R189G this, one tal-1R188G;R189G transgenic line (F 23) was (mut tal), tal-1/CKIIa (tal/CKII) and tal-1R188G;R189G/CKIIa 0 (mut tal/CKII) transgenic and bi-transgenic lines. The cohort of mated with mice which expressed the catalytic subunit tal-1 mice consisted of n=75 animals, the tal-1R188G;R189G of CKII in lymphocytes via the immunoglobulin heavy mice consisted of n=62 animals, the tal-1/CKIIa bi-transgenic chain promoter-enhancer (Seldin and Leder, 1995). The cohort consisted of n=14 animals, and the tal-1R188G;R189G/ CKII transgenic mice develop clonal T cell lymphomas CKII bi-transgenic cohort consisted of n=30. All animals were after a long latency (median survival of 400 days) monitored daily for any evidence of disease. Upon onset of disease, the mice were sacri®ced and a post-mortem examination (Seldin and Leder, 1995). Previously, we had shown was performed that wild type tal-1 and CKII cooperate to induce

Figure 3 Histology of the lymphoproliferative disease in tal-1R188G;R189G transgenic mice. A thymus from an adult tal- 1R188G;R189G transgenic mouse that developed a thymoma shows the eacement of the normal thymic architecture (A; 1006) and the proliferation of large lymphoblasts with prominent nucleoli (D; 4006). Similar cells invaded the visceral organs such as the kidney (B; 1006and E; 4006) and the liver (C; 1006and F; 4006)

Oncogene DNA binding mutant of tal-1 induces leukemia in mice J O'Neil et al 3900 disease in mice. All bitransgenic tal-1/CKII animals bands were evident in all tissues. As expected, the developed leukemia with a median survival of 72 days immunoglobulin heavy chain locus was retained in its (Figure 2 and Kelliher et al., 1996). When mated to the germline con®guration (data not shown). tal-1R188G;R189G transgenic mice, a strikingly similar acceleration of disease onset and increase in disease Tal-1R188G;R189G thymomas do not express CD4 penetrance was observed. All bitransgenic tal- 1R188G;R189G/CKII animals developed aggressive Tumors from the tal-1R188G;R189G and tal- disease with a median survival of 69 days (Figure 2). 1R188G;R189G/CKII mice were examined by ¯ow In the tal-1R188G;R189G/CKII animals, the disease cytometry to determine the phenotype(s) of the was characterized by thymic enlargement, often primary tumor. All of the tumors appeared to be of accompanied by splenomegaly and lymphadenopathy. T cell origin, although at varying stages of thymocyte As observed in wild type tal-1 transgenic mice, the development (Table 1). None of the tal-1R188G;R189G thymic architecture was obliterated by neoplastic cells or tal-1R188G;R189G/CKII tumors expressed the B cell and numerous clusters of apoptotic cells were observed. speci®c antigen B220 or surface immunoglobin heavy The nearly identical survival curves observed for tal- chain (data not shown). 1/CKII and tal-1R188G;R189G/CKII suggests that Two predominant immunophenotypes were ob- CKII does not synergize by potentiating the transcrip- served: four of seven tumors consisted of predomi- tional activity of tal-1. Furthermore, this experiment nantly CD3-positive, CD4-negative and CD8-positive supports the idea that wild type tal-1 and its DNA cells, presumably arising from the mature, single binding mutant (tal-1R188G;R189G) transform thymo- positive thymocyte population. The remaining three cytes by similar mechanisms. tal-1R188G;R189G tumors expressed CD3 but failed to express either CD4 or CD8. The bitransgenic tal- 1R188G;R189G/CKII tumor cells exhibited similar Tal-1 R188G;R189G induces clonal or oligoclonal disease immunophenotypes. Half (2/4) of the tumors analysed The dramatic acceleration of disease onset in the tal- were CD4-negative and CD8-positive and the remain- 1R188G;R189G/CKII bitransgenic mice prompted us to ing two tumors examined were both CD4-negative and determine the clonal nature of the disease. DNA was CD8-negative. isolated from cell lines derived from the tumors Interestingly, no CD3-positive, CD4-positive, CD8- restricted with HindIII and examined by ®lter negative tumors were observed in either the tal- hybirdization with the TCR Jb2 probe. Clonal or 1R188G;R189G or tal-1R188G;R189G/CKII mice, sug- oligoclonal rearrangements were detected in both the gesting that tal-1R188G;R189G expression stimulates tal-1R188G;R189G and in the bitransgenic tal- CD4-negative, CD8-positive thymocyte dierentiation 1R188G;R189G/CKII tumor cells analysed and in most and inhibits development (or survival) of CD4-positive, cases, both TCR b alleles were rearranged (Figure 4). CD8-negative thymocytes. In animals where disease involved multiple organs such as thymus, spleen, liver and kidney, the speci®c bTCR Thymic expression of wild type tal-1 and tal-1R188G;R189G perturbs thymocyte development The absence of CD4-positive tumor target cells derived from tal-1R188G;R189G mice prompted us to further examine the eects of tal-1 expression on thymocyte development. Thymus from disease-free, 4-week-old, tal-1, tal-1R188G;R189G transgenics and control littermates was stained with antibodies to CD4 and CD8 and analysed by ¯ow cytometry. The wild type

Table 1 Immunophenotypes of tal-1 R188G;R189G and tal-1 R188G;R189G/CKIIa tumors Age Per cent of cells stained with antibody Animal Genotype (days) CD3 CD4 CD8

5721 Mut tal-1/+ 139 15 1 98 Figure 4 Tal-1R188G;R189G and tal-1R188G;R189G/CKIIa tu- 5578 Mut tal-1/+ 183 86 1 96 mors are clonal or oligoclonal. DNA prepared from tumor cell 5591 Mut tal-1/+ 115 33 3 86 lines and wild type genomic tail DNA was digested with HindIII 5577 Mut tal-1/+ 146 26 1 77 and analysed by Southern blot analysis. T cell receptor Jb chain 1326 Mut tal-1/+ 147 59 2 3 rearrangements were detected with a probe that identi®ed a 5 kb 5378 Mut tal-1/+ 140 14 51 51 DNA fragment in the germline position of genomic tail DNA 1320 Mut tal-1/+ 185 72 4 51 (lane T). Mut tal-1 lanes contain DNA isolated from tumor cell 5495 Mut tal-1/CKII 51 34 3 30 lines derived from tal-1R188G;R189G mice, whereas, mut tal-1/ 5522 Mut tal-1/CKII 85 99 4 5 CKII lanes contain DNA from tal-1R188G;R189G/CKIIa bi- 5535 Mut tal-1/CKII 83 5 1 20 transgenic tumor cell lines 5538 Mut tal-1/CKII 55 96 7 51

Oncogene DNA binding mutant of tal-1 induces leukemia in mice J O'Neil et al 3901 tal-1 transgenic mice had 2 ± 3-fold fewer thymocytes bHLH transcription factors. Consistent with this idea, compared to tal-1R188G;R189G transgenic or control similar thymocyte developmental abnormalities have littermates (not shown). Yet, analysis of the CD4/CD8 been observed in both HEB-de®cient mice (Zhuang et thymic pro®les revealed the presence of all thymocyte al., 1996) and E2A-de®cient mice (Bain et al., 1997). subpopulations. However, mice expressing either wild type tal-1 or the tal-1 DNA binding mutant Wild type tal-1 and the DNA-binding mutant (R188G;R189G) consistently showed signi®cant de- tal-1R188G;R189G form stable heterodimers with E47 creases in the percentage of the CD4-positive, CD8- negative population (Table 2). Tal-1-expressing thymo- The tissue-speci®c bHLH proteins, like tal-1, do not cytes exhibited concomitant increases in the percentage bind DNA because they do not form homodimers of CD4-negative, CD8-positive population, resulting in (Littlewood and Evan, 1998). Thus, heterodimer markedly dierent CD4/CD8 ratios (0.2 for tal/+; 0.8 formation is essential for the DNA-binding activity for tal-1R188G;R189G/+ compared to 3.45 for wild and functional properties of these proteins. A stable type littermates (Table 2). tal-1/E2A complex has been detected in Jurkat cells Interestingly, similar percentages of CD4-positive, (Hsu et al., 1994b). However, it remains unclear CD8-positive `double positive' thymocytes were ob- whether this complex is a consistent feature associated served in wild type tal-1 and tal-1R188G;R189G with tal-1-induced leukemia. To test whether a tal-1/ thymocytes, indicating that tal-1 does not inhibit the E2A complex contributes to leukemia development in initial expression of the CD4 coreceptor. These data the mouse, tal-1 tumor cell lysates were immunopreci- suggest that tal-1 may interfere with CD4/CD8 pitated with either a preimmune (P) or anti-tal-1 coreceptor silencing, potentially by sequestering other polyclonal antiserum (I). Co-precipitating proteins were tested for the presence of E47 by immunoblotting with an anti-E47 monoclonal antibody. E47 coprecipi- tated with tal-1 in murine erythroleukemia cells (MEL) Table 2 Expression of wild type tal-1 and tal-1 R188G;R189G(Mut and in the tal-1-induced thymomas tested (Figure 6A, tal-1) perturbs thymocyte development lanes 2, 4 and 6). Thus, a stable tal-1/E47 heterodimer %CD47/ %CD4+/ is present in the leukemic cells of the tal-1 transgenic CD87 CD8+ %CD4+ %CD8+ CD4:CD8 mice. +/+ 4 83 11 3 3.6 Although cells expressing the tal-1R188G;R189G +/+ 3 82 11 4 2.8 DNA binding mutant have been shown to lack DNA +/+ 3 76 16 4 4.0 binding activity (Hsu et al., 1994c), it was unclear tal/+ 9 66 4 21 0.2 whether mutation of the tal-1 basic domain might tal/+ 4 72 4 19 0.2 tal/+ 8 68 5 19 0.3 interfere with its ability to interact with E47. To test Mut tal/+ 7 73 10 11 0.9 whether the mutant tal-1R188G;R189G protein is Mut tal/+ 6 79 8 8 1.0 capable of forming stable heterodimers in vivo, lysates Mut tal/+ 6 79 6 10 0.6 were prepared from thymomas derived from the tal- Thymocytes from disease-free, aged-matched wild type, tal-1 1R188G;R189G mice. Tal-1R188G;R189G/E47 com- transgenic and tal-1 R188G; R189G transgenic mice were stained plexes were readily detected in the two thymomas with CD4-FITC and CD8-PE and analysed by ¯ow cytometry tested, demonstrating that these basic domain muta-

Figure 5 Thymic expression of wild type tal-1 and tal-1R188G;R189G (mut tal-1/+) perturbs thymocyte development. Thymocytes from four week-old, disease-free, wild type, tal-1 transgenic and tal-1R188G;R189G (mut tal-1) transgenic mice were stained with CD4-FITC and CD8-PE and analysed by ¯ow cytometry

Oncogene DNA binding mutant of tal-1 induces leukemia in mice J O'Neil et al 3902 of E47. Similar amounts of E47 were detected in nuclear extracts prepared from MEL cells and tal-1 tumor cells (data not shown). Hence, LMO2 activation does not contribute to tal-1-induced leukemogenesis in mice.

Tal-1R188G;R189G/E2A complexes isolated from leukemic cells fail to bind DNA Although previously shown to obliterate DNA binding in vitro, it remained possible that tal- 1R188G;R189G/E2A protein complex retained some ability to bind DNA in vivo. To test this possibility, nuclear extracts were prepared from Figure 6 Wild type tal-1 and mutant tal-1 form stable thymomas and subjected to gel mobility shift heterodimers with E2A proteins (A). Wild type tal-1 and mutant analysis using the tal-1/E47 consensus E-box motif tal-1 leukemic cell lines were lysed under low stringency (CAGATG) as a probe (Hsu et al., 1994a). conditions and the lysates were immunoprecipitated with either Incubation of this probe with nuclear extracts anti-tal antiserum or the corresponding pre-immune serum. The samples were fractionated by SDS ± PAGE and co-precipitating from Jurkat cells generated three distinct protein- proteins detected by immunoblotting with an anti-E47 or an anti- DNA complexes (Figure 7). All three complexes E12 monoclonal antibody. A murine erythroleukemic cell line was were eliminated by incubating the extract with an used as a positive control. (B) LMO2 expression is not required E2A polyclonal antisera. The middle two complexes for tal-1-induced leukemia. 293T cells were transfected with the EF1-a puro mammalian expression vector or with the vector were also abrogated by incubating the extract with expressing LMO2. Nuclear extracts containing an equal amount an antiserum raised against human TAL-1 but not of protein from the LMO2-transfected 293T cells, MEL cells, and with the corresponding preimmune serum. Tal-1 tal-1 tumor cells were resolved by SDS ± PAGE and LMO2 encodes two phosphoproteins; the full length pp42 protein detected by immunoblotting with an anti-LMO2 rabbit and a truncated polypeptide pp22. The upper tal-1/ polyclonal antisera E2A complex corresponds to a pp42TAL/E2A heterodimer whereas the pp22TAL/E2A forms the lower complex (Hsu et al., 1994a). Nonspeci®c complexes were also detected (labeled n.s.). tions do not interfere with formation/stability of the Similar complexes were detected when the tal-1/ tal-1/E47 complex in tal-1R188G;R189G thymomas. E47 probe was incubated with nuclear extracts from tal-1-induced mouse thymomas (Figure 7, tal- 1 tumor lanes). All three complexes were depleted LMO2 expression is not required for tal-1-induced disease by incubating the extract with the anti-E2A Stable complexes between tal-1/E47 and the cysteine- antisera but not with the corresponding preimmune rich LIM-only protein LMO2 have been detected in antisera. The two lower complexes were depleted the leukemic cells of some T-ALL patients (Wadman when the mouse tumor extract was preincubated et al., 1994) and transgenic coexpression of tal-1 and with an anti-tal-1 antisera, demonstrating the LMO2 results in accelerated tumor development presence of a pp42tal/E2A and pp22tal/E2A com- (Larson et al., 1996). Moreover, gene targeting plexes in mouse leukemic cells. experiments support a cooperative relationship between tal-1 and LMO2, as mice de®cient for either gene exhibit defects in erythropoiesis (Warren et al., 1994; Shivdasani et al., 1995). Together, these studies suggest that tal-1 may induce LMO2 expression in leukemia. To test whether LMO2 expression is required for tal- 1-induced leukemogenesis in mice, nuclear extracts were prepared from tal-1 and tal-1R188G;R189G- induced thymomas and analysed for LMO2 expression by immunoblotting with an anti-LMO2 antisera (gift of Dr Stuart Orkin, Harvard Medical School and Figure 7 Mutant tal-1/E2A complexes fail to bind DNA. Children's Hospital, Boston, MA, USA). The 22 kD Nuclear extracts from Jurkat cells, a wild type tal-1 leukemic LMO2 protein was detected in LMO2-transfected 293T cell line and mutant tal-1R188G;R189G leukemic cell lines were cells and in the MEL cells, however, no LMO2 incubated with a radiolabeled oligonucleotide probe correspond- expression was detected in any of the tal-1 or tal- ing to the tal-1/E47 consensus binding sequence (Hsu et al., 1994). 1R188G;R189G tumors examined (Figure 6B). To In some cases, the reaction was supplemented with the indicated antiserum. The binding reactions were fractionated on a 5% ensure that samples contained equivalent amounts of nondenaturing, polyacrylamide gel and the DNA-protein com- nuclear protein, extracts were examined for expression plexes were detected by autoradiography

Oncogene DNA binding mutant of tal-1 induces leukemia in mice J O'Neil et al 3903 To test whether the tal-1R188G;R189G DNA analyses of genes activated/repressed upon tal-1 expres- binding mutant protein exhibited DNA binding activity sion or E47 deletion should identify the cooperating in vivo, nuclear extracts were prepared from thymomas oncogene(s). isolated from the tal-1R188G;R189G transgenic mice. As expected, no tal-1R188G;R189G/E2A heterodimers bound the tal-1/E47 consensus sequence. Taken Materials and methods together, this study argues that tal-1 contributes to Generation of transgenic mice leukemia by interfering with E2A protein function(s) in thymocytes. The human TAL-1 cDNA containing the R188G;R189G mutations (generously provided by Dr Richard Baer) was isolated as a BamHI ± BglII fragment and cloned into the BamHI cloning site of p1017, a plasmid cassette containing Discussion the proximal lck promoter and the human growth hormone splice and poly A addition sites (Abraham et al., 1991). After We have demonstrated that the DNA binding proper- being checked for proper orientation and sequenced to ties of tal-1 are not required to induce leukemia in con®rm presence of the R188G;R189G mutations, the mice. Forty-eight per cent of tal-1R188G;R189G mice plasmid was linearized by digestion with SpeI and micro- from three lines died of clonal T lymphoblastic injected into the FVB/N pronuclei. Transgenic mice were leukemia. Furthermore, we show that in all the tal-1- identi®ed by probing Southern blots of EcoRI- digested tail induced thymomas tested, a stable tal/E47 complex was DNA with a 32-P-labeled random-primed 585 bp EcoRI ± detected. This provides direct evidence that transforma- EcoRI tal-1 partial cDNA fragment. Southern blots were tion by tal-1 does not require DNA binding and hybridized and washed as previously described (Kelliher et al., 1996). Transgenic lines were propagated by crossing demonstrates that tal-1 transforms by an Id-like founder animals with FVB/N animals. mechanism, interfering with the formation of E protein homodimers. Tal-1 encodes a basic helix ± loop ± helix protein that Histology is required for embryonic hematopoietic and vascular Upon necropsy, all tissue samples were preserved in Optimal development (Shivdasani et al., 1995; Porcher et al., Fix (American Histology Reagent Company, Inc.). Four mm 1996; Visvader et al., 1998). A recent structure-function sections were cut and stained with hematoxylin and eosin for analysis of the regions of tal-1 required for hematopoi- histologic evaluation in the Transgenic Core Pathology esis revealed that DNA binding by tal-1 is not required Laboratory at the University of California at Davis. for primitive erythropoiesis in embryonic stem cells (Porcher et al., 1999). Thus, the DNA binding activity Antibodies and fluorescence-activated flow cytometry analysis of tal-1 is dispensable in both embryonic hematopoiesis Mouse thymomas were gently teased with frosted glass slides and in leukemia. in order to produce single cell suspensions. The cells were When expressed in the thymus, wild type tal-1 and washed with PBS and stained with ¯uorescent-labeled the DNA binding mutant (tal-1R188G;R189G) perturb antibodies and subjected to ¯ow cytometry at the FACS thymocyte dierentiation, stimulating CD8-single posi- facility at the University of Massachusetts Medical Center. tive thymocytes and inhibiting the development of Antibodies used in ¯ow cytometry included FITC-conjugated CD4-single positive thymocytes. The E proteins anti-mouse CD3, FITC-anti-mouse L3T4 (CD4), FITC-anti- participate in lymphocyte development (Bain et al., mouse Ly-2 (CD8), FITC-anti-mouse IL-2 receptor (CD25), 1997) and are involved in transcriptional activation of FITC-anti-B220, FITC-polyclonal goat anti-rat immunoglo- the immunoglobulin and CD4 coreceptor genes (Murre bulin antibody and PE-conjugated anti-mouse L3T4 (Phar- et al., 1989; Sawada and Littman, 1993). The ratio of Mingen, San Diego, CA, USA). Dead cells were eliminated by gating for cells which stained with LDS-751 (Exciton). CD4 to CD8 single positive thymoctyes is similarly Data were analysed using FlowJo software (Treestar, Inc.). aected in E2A-de®cient mice (Bain et al., 1997), further implicating E2A protein sequestration in tal-1- induced disease. Tumor DNA analysis These studies have major implications for current Southern blots of HindIII-digested DNA (10 mg) obtained work focused on identifying tal-1 target genes. Our work from primary tumors and from tumor cell lines were argues that tal-1 transforms by interfering with genes hybridized with a 32P-labeled 2 kb EcoRI fragment containing activated or repressed by the E2A proteins, E12 or E47. the murine TCR Jb2B exon (Malissen et al., 1984). Genomic One potential E47 target gene is the cyclin-dependent DNA from tail samples was also digested with EcoRI, kinase inhibitor p21CIP1/WAF1/Sdi1 (Prabhu et al., 1997). Tal- transferred to GeneScreen Plus (New England Nuclear) and 32 1 has been shown to inhibit E47-mediated activation of a hybridized to a P-lableled 1.5 kb PstI m fragment (Early et al., 1980). Blots were washed in 16SSC, 1% SDS, followed p21 reporter construct in HeLa cells (Park and Sun, by a higher stringency wash containing 0.16SSC, 0.1% SDS. 1998), implicating p21CIP1/WAF1/Sdi1 as a potential target gene in human T-ALL. However, no dierences in p21CIP1/WAF1/Sdi1 expression levels were observed in tumors RNase protection analysis isolated from tal-1 (or tal-1R188G;R189G) transgenic Total RNA was isolated from thymus from age-matched, mice (data not shown), indicating that other E47 target disease-free tal-1R188G;R189G transgenic mice and control genes are likely involved. Future cDNA microarray littermates. T3 and T7 antisense probes were synthesized and

Oncogene DNA binding mutant of tal-1 induces leukemia in mice J O'Neil et al 3904 hybridized to RNA samples as described in (Krieg and transfected with the pEFpGKpuro expression vector contain- Melton, 1987). The probe for human tal-1R188G;R189G was ing the LMO2 cDNA (provided by Dr Stuart Orkin, Harvard derived by linearization of a 625 bp partial cDNA clone with Medical School and Children's Hospital, Boston, MA, USA) SacI, resulting in a probe that protects 500 nucleotides. or with vector alone. Equal amounts of total protein were examined by immunoblotting with an anti-LMO2 speci®c antisera (provided by Dr Stuart Orkin, Harvard Medical Immunoprecipitation and Western blotting School). To analyse transgene expression levels, lysates were prepared from the thymus of 4-week-old tal-1R188G;R189G transgenic Gel mobility shift assay mice and control littermates and tal-1 protein detected by immunoblotting with anti-tal-1 polyclonal antisera (gift of Dr Nuclear extracts were prepared from Jurkat cells and from Richard Baer, Columbia University). Tal-1 protein levels tumors isolated from tal-1/+ and tal-1R188G;R189G trans- were compared to levels expressed in Jurkat and mouse genic mice (mut tal-1) as described in Grimm et al. (1988). erythroleukemia cells. For the co-immunoprecipitation ex- EMSAs were performed with equivalent amounts of nuclear periments, tal-1 tumor cell lines and murine erythroleukemia extract (20 mg), incubated with a 32P-labeled double-stranded cells were lysed in a lo-stringency lysis buer (10 mM HEPES oligonucleotide probe containing the preferred sequence for pH 7.6, 250 mM NaCl, 0.1% NP-40, 5 mM EDTA) (Lassar et tal-1/E47 heterodimers (sense strand ACCTGAACA- al., 1991), pre-cleared with protein A-agarose and immuno- GATGGTCGGCT, Hsu et al., 1994a). Some reactions were precipitated with either pre-immune or anti-tal-1 polyclonal supplemented with 1 ml of a rabbit anti-tal-1 or anti-E2A antiserum (provided by Dr Richard Baer, Columbia Uni- antisera (gift of Dr Richard Baer). versity). The immune complexes were washed twice in lysis buer and resolved by SDS ± PAGE. The tal- 1R188G;R189G-associated proteins were detected by immunoblotting with either an anti-E47 or anti-E12/HEB monoclonal antibody (PharMingen). To determine whether LMO2 Acknowledgments expression contributed to tal-1-induced disease, cell lysates The authors would like to acknowledge the expert technical were prepared from murine erythroleukemic cells (M), ®ve assistance of the University of Massachusetts Medical tal-1/+ tumors and one tal-1R188G;R189G tumor. As a School Transgenic Mouse Core Facility. M Kelliher is a positive control for LMO2 expression, 293T cells were recipient of a Sidney Kimmel Cancer Scholar Award.

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