Formation of in Vivo Complexes Between the Tall and E2A

Proc. Nati. Acad. Sci. USA Vol. 91, pp. 3181-3185, April 1994 Medical Sciences Formation of in vivo complexes between the TALl and E2A polypeptides of leukemic T cells (leukeinia/transription factor/hellx4oop-heix protein) HAI-LING Hsu, ISOBEL WADMAN, AND RICHARD BAER Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75235 Communicated by Jonathan W. Uhr, December 21, 1993

ABSTRACT Tumor-specific activation of the TALI gene and Max bHLH proteins have also been demonstrated (20- occurs in "r.25% of patients with T-cell acute lymphoblastic 22). leukemia (T-ALL). The TALI gene products possess a basic The E2A gene products (E12 and E47) are expressed in a helix-oop-belix (bHLH) domain that interacts in vitro with the broad spectrum of cell types, where they may exist in bHLH proteins (E12 and E47) encoded by the E2A locus. We heterologous complexes, presumably heterodimers, with have now applied two independent methods, the two-hybrid certain tissue-specific bHLH proteins (15). For example, the procedure and co-immunoprecipitation analysis, to demon- E2A gene products form heterodimers with muscle-specific strate that TALl and E2A polypeptides also associate in vivo. bHLH proteins (e.g., MyoD1) that govern the myogenic These studies show that the bHLH domain of TALl selectively conversion ofmultipotential mesodermal cells (23-26). These interacts with the bHLH domains ofE12 and E47, but not with heterodimers apparently function as transcription factors the Idl helix-oop-helix protein. TALl does not self-asiate that promote myogenesis by inducing the expression of to form homodimeric complexes, implying that the in vivo muscle-specific genes. functions of TALl depend on heterologous interaction with The tissue-specific bHLH proteins involved in myogenesis other bHLH proteins such as E12 and E47. Co-immunopre- (e.g., MyoD1) do not bind DNA by themselves, in large part cipitation analysis revealed the presence of endogenous because they do not form homodimers under physiologic TAL1/E2A complexes in Jurkat cells, a leukemic line derived conditions (23-26). Thus, heterodimer formation is essential from a T-ALL patient. Thus, the malignant properties ofTALl for the DNA-binding activity and functional properties of may be due to obligate interaction with the E2A polypeptides. these proteins (15). To determine whether TAL1 also interacts in an obligate manner with ubiquitous bHLH proteins, we initially examined the protein-dimerization and DNA- Activation of the TALI gene is the most common genetic recognition properties of TAL1 in vitro (27). These studies lesion associated with T-cell acute lymphoblastic leukemia demonstrated that the bHLH domain of TAL1 mediates (T-ALL). Thus, approximately 25% of T-ALL patients har- complex formation with both E12 and E47 and that the bor tumor-specific rearrangements of the TALI locus (also resultant heterodimers (TAL1/E12 and TAL1/E47) bind called TCL5 or SCL) (1-5). TALI encodes at least two DNA in a sequence-specific manner (27). Our previous phosphoproteins, the full-length gene product, pp42TAL results were based on in vitro assays; we now provide (amino acid residues 1-331), and a truncated species, evidence that TALl and E2A polypeptides also associate in pp22TAIl (residues 176-331) (6). Both polypeptides contain vivo and that TAL1/E2A complexes exist in leukemic T cells. the basic helix-loop-helix (bHLH) motif (1, 3, 7), a DNA- Thus, the leukemic properties of TAL1 are likely to require binding and protein-dimerization domain common to several in vivo interaction with ubiquitous bHLH proteins such as known transcriptional regulatory factors (8). Although more E12 and E47. than 60 different bHLH proteins have been identified to date, the bHLH domain of TAL1 is most closely related to the bHLH domains encoded by LYLI and TAI2-distinct genes MATERIALS AND METHODS that are also activated by chromosomal rearrangement in Expression Plasmids and Rabbit Antisera. Plasmids encod- T-ALL (9, 10). Hence, TAL1, TAL2, and LYL1 constitute a ing the GAL4-TAL1 and VP16-TAL1 hybrid polypeptides discrete subgroup of bHLH proteins implicated in human were constructed by inserting TAL1 cDNA sequences T-cell leukemia (10). (codons 176-331) into the pSG424 and pNLVP16 expression The bHLH domain forms two amphipathic a-helices sep- vectors, respectively (28, 29). To produce a plasmid encoding arated by an intervening loop (8, 11). Most helix-loop-helix GAL4-E47, a short oligonucleotide (containing a Cla I site) proteins, including those implicated in T-ALL, harbor a was inserted into the EcoRI site ofpE47S, a cDNA clone that cluster of basic residues at the amino terminus of the first specifies 85 amino acid residues of E47, including the entire helix. Mutational analyses and structural studies show that bHLH domain (8); a Cla I fragment encompassing the E47 the amphipathic helices mediate protein dimerization, while sequence was then transferred into the pGAL0 expression the basic residues participate in DNA recognition (11-14). vector (29). A plasmid encoding VP16-E47 was generated by Many bHLH proteins interact in vitro to form stable ho- excising a Sal I-Sac I E47 cDNA fragment from the GAL4- modimers and heterodimers with DNA-binding activity (15). E47 expression plasmid and inserting it into pNLVP16. However, in few cases have these bHLH complexes been Expression vectors encoding GAL4-E12, GAL4-Idl, and shown to exist in vivo. Hence, the E2A gene products can VP16-Idl were kindly provided by Toren Finkel and Chi associate intracellularly with the myogenic bHLH factors, Dang (18). The TAL1m176/pCMV4 expression plasmid has MyoD1 and myogenin, as well as with the regulatory Idl been described (30). The AE12/pCMV4 plasmid encodes a polypeptide (16-19). In vivo interactions between the c-Myc truncated form of E12 (residues 217-654); it was constructed

The publication costs ofthis article were defrayed in part by page charge Abbreviations: bHLH, basic helix-loop-helix; T-ALL, T-cell acute payment. This article must therefore be hereby marked "advertisement" lymphoblastic leukemia; GST, glutathione S-transferase; CAT, in accordance with 18 U.S.C. §1734 solely to indicate this fact. chloramphenicol acetyltransferase.

3181 Downloaded by guest on September 24, 2021 3182 Medical Sciences: Hsu et al. Proc. Natl. Acad. Sci. USA 91 (1994) by transferring a 1.3-kb EcoRI-HindIII fragment from the of extract was added to 250 pI of luciferase assay buffer (7 pE12R cDNA plasmid (23) into the Sma I site ofpCMV4 (31). mM ATP, pH 7.8/1 mM dithiothreitol/24 mM MgSO4/2.5 Antibodies specific for the E2A polypeptides (E12 and E47) mM EDTA/0.4 mg of bovine serum albumin per ml/45 mM were generated by immunizing rabbits with GST-E2A (217- glycylglycine, pH 7.8). The light emitted during the 10 sec 371), a glutathione S-transferase fusion protein that includes immediately following injection of 100 pI1 of luciferin (150 amino acid residues 217-371 of E2A [numbering system of pug/ml) was measured by using an Optocomp II luminometer. Kamps et al. (32)] as described previously (33). Antibodies The luciferase activity of each sample was normalized with specific for the carboxyl-terminal 19 residues of TALl were respect to protein concentration, as measured by the Brad- generated by immunizing rabbits with a synthetic peptide ford assay. conjugated to keyhole-limpet hemocyanin (33). Two-Step Co-immunoprecipitation. Methods for DNA The Two-Hybrid Assay in Fibroblasts. Approximately 3 x transfection of COS1 cells and [35S]methionine labeling of 1i0 C3H/10T½ fibroblasts were seeded onto each 100-mm cultured cells have been described (6). The two-step ci- plate and cultured in 10 ml of growth medium (Dulbecco's immunoprecipitation procedure was modified from Lassar et modified Eagle's medium plus 10o fetal calf serum). After 2 al. (19). Thus, 1-ml lysates were prepared from 107 radiola- days the adherent cells were treated with the calcium phos- beled cells as described previously (6), except that a low phate transfection system (GIBCO/BRL) according to man- stringency Nonidet P40 buffer was used for cell lysis (10mM ufacturer's instructions. Each 100-mm culture was trans- Hepes, pH 7.6/250 mM NaCl/0.1% Nonidet P40/5 mM fected with 5 ug of the G5E1bCAT reporter plasmid (34), 10 EDTA). Ten microliters of immune or preimmune rabbit pg of a GAL4-hybrid expression plasmid, and/or 10 pg of a serum (the "first antibody") was added to each lysate, and VP16-hybrid expression plasmid; where necessary 10 pg of after gentle rocking at 4°C for 1 hr, 50 ,d of staphylococcal salmon sperm DNA was added to provide a constant DNA protein A-Sepharose (Pharmacia) was also added. After mass (25 pg) for transfection of each 100-mm culture. After rocking at 4°C for an additional hour, each lysate was washed 24 hr the adherent cells were washed twice in TBS buffer (6) four times in low-stringency buffer. Five hundred microliters and cultured in fresh growth medium for an additional 24 hr. of high-stringency RIPA buffer (6) was added to each pellet, Cell lysates (150 yd) were then prepared from each 100-mm and the slurry was then heated to 100°C for 10 min. After a culture by three cycles of freezing and thawing in 0.25 M brief centrifugation, each supernatant was removed, chilled Tris HC1, pH 8.0. The protein content of each lysate was on ice, and provided with 10 01 of immune or preimmune determined by the Bradford assay (Bio-Rad), and equivalent serum (the "second antibody"). After rocking at 4°C for 1 hr, amounts (150 pg) were examined for chloramphenicol ace- 50pI ofprotein A-Sepharose was added to each sample. After tyltransferase (CAT) activity by using [14C]chloramphenicol an additional hour of rocking at 4°C, each pellet was washed and n-butyryl-coenzyme A. Each reaction mixture was then and analyzed by SDS/PAGE as described previously (6). fractionated by thin-layer chromatography, and the conversion of [14C]chloramphenicol into its butyrylated forms was measured quantitatively with a Betascope 603 (Betagen, RESULTS Waltham, MA). Two-Hybrid Analysis of TALl Interactions in Fibroblasts. Construction of the G5E1bLUC Reporter. Five GAL4- We previously showed that TAL1 polypeptides form heter- binding sites together with the Elb TATA sequence were ologous complexes in vitro with bHLH proteins encoded by inserted upstream of the firefly luciferase gene to construct the E2A gene; these results were based on binding assays the reporter plasmid G5E1bLUC. The GAL4-binding sites involving in vitro translated polypeptides and GST fusion and Elb TATA sequence were PCR amplified from the CAT proteins produced in bacteria (27). To determine whether reporter plasmid G5E1bCAT (34), using forward (5'- TAL1 and E2A also associate in vivo we applied the two- TTQAICICAGCTTGCATGCCTGCAG-3') and reverse hybrid system, a method to detect in vivo protein-protein (5'-AAAAQCICGGTACCCGGGGATC-3') oligonucleo- interactions based on functional reconstitution of the artifi- tide prmers. The 160-bp PCRfragment was purified, digested cial transcription factor GAL4-VP16 (29, 35). This factor, with Sac I and HindIII, and ligated into the vector pGL2- which contains the DNA-binding domain of the yeast GAL4 Basic (Promega). protein fused to the transactivation domain ofthe herpesvirus The Two-Hybrid Assay in Lymphocytes. Logarithmic-phase VP16 protein, efficiently induces transcription of GAL4- Jurkat cells were harvested at a density between 5 x 105 and regulated reporter genes (36). Expression vectors were con- 1.2 x 106 cells per ml. Cells were centrifuged and washed with structed that encode segments ofTAL1 (residues 176-331) or 50 ml of "complete RPMI" (RPMI-1840 growth medium E47 (residues 524-651) fused to either the GAL4 DNA- supplemented with penicillin at 100 units/ml, streptomycin at binding domain or the VP16 transactivation domain (Fig. 1). 100 pg/ml,2 mM glutamine, 1 mM sodium pyruvate, and 10%6 These hybrid polypeptides should not activate the reporter fetal bovine serum). Aliquots containing 107 cells (in 0.5 ml gene on their own, since they lack either a transactivation ofcomplete RPMI) were gently mixed with a 50-pg sample of domain (GAL4-bHLHx) or the appropriate DNA-binding plasmid DNA (in 100 1.d ofwater). The plasmid DNA sample domain (VP16-bHLHy). If, however, the bHLH moieties of included 15 pg of the G5E1bLUC reporter, 15 pg of a these hybrids interact stably in vivo, then a functional tran- GALA-hybrid expression vector, and/or 15 pg of a VP16- scription factor is reconstituted by protein dimerization (i.e., hybrid expression; this was supplemented with carrier DNA GAL4-bHLHx/VPI6-bHLHy) and expression of the re- (pUC18X) to make a total of 50 pg of DNA per sample. porter gene is induced (Fig. 1) (17, 18). Transfections were performed by electroporation using a The expression vectors were transfected singly or pairwise GIBCO/BRL Cell-Porator at 1180 1LF and 250 V (a field into C3H/10TY2 fibroblasts along with G5E1bCAT, a GAL4- strength of 625 V/cm). The transfected cells were then responsive reporter plasmid that contains five GAL4-binding diluted into 9 ml ofcomplete RPMI. After 24 hr ofculture the sites upstream of the CAT gene (34). Transcription of the cells were harvested by centrifugation and the cell pellets reporter plasmid was then evaluated by measuring the CAT were washed twice with 5 ml of phosphate-buffered saline. activity of lysates prepared from transfected cells. As illus- The cells were then lysed in 150 ,ul of lysis buffer (0.1% trated in Fig. 2A, expression of either the GAL4-E47 or the Nonidet P40/2 mM MgCl2/0.7 mM dithiothreitol/5 pg of VP16-TALl polypeptide failed to induce significant CAT aprotimin per ml/250 pug of soybean trypsin inhibitor per activity in transfected C3H/1OTY2 fibroblasts. However, ml/70 mM K2HPO4/55 mM Tris, pH 7.8), and the cell debris co-expression of these polypeptides generated a large in- was removed by centrifugation ofthe lysate. Fifty microliters crease in CAT activity to levels 40-fold or 200-fold higher, Downloaded by guest on September 24, 2021 Medical Sciences: Hsu et al. Proc. Natl. Acad. Sci. USA 91 (1994) 3183

A sbHLHxie El A GAL4-E47 GAL4-E47 VP1 6 -TALl - _ \ ,E VP1 6-TALl

VP1 6-TALl GAL4 -TALl B bHLH GAL4 -TAL1 FRMMWNFFF -~~~ ---I GAL4 -TALl bHLH 0 5 10 15 20 GAL4-E47 bHLH B GAL4 -TALl _~~~~ --- VP1 6-TALl GAL4 -TALl VP16-E47 bHLH VP16 -E47E _ VP16-E47 VP16-Idl FIG. 1. The two-hybrid assay for detection of in vivo protein- GAL4 -TALl protein interaction. (A) Schematic illustration of a reconstituted transcription factor inducing expression of a GAL4-responsive re- VP1 6- Idl porter gene. The transcription factor is reconstituted by dimerization oftwo hybrid polypeptides (GAL4-bHLHx and VP16-bHLHy) upon 0 2 4 6 83 in vivo interaction of their bHLH moieties. (B) Hybrid polypeptides used in this study. Shaded bars represent either GAL4 or VP16 sequences, while open bars denote TAL1 or E47 sequences. The C VP16-TALl i bHLH domains of TALl or E47 are also indicated. Other hybrid VP16-TAL1 polypeptides used in the study (GAL4-E12, GAL4-Idl, and VP16- WL4G.AI A-F1- C I Z5 Idl) have been described by Finkel et al. (18). GAL4- El2

VP1 6 -TALl respectively, than those found with GAL4-E47 or VP16- GAL4-Idl TAL1 alone (Fig. 2A). This degree of induction was similar ~ ~ ~ to that observed upon co-expression ofGAL4-fos and VP16- GAL4-Idl jun hybrid proteins (data not shown). These data suggest that 0 heterodimers are formed in vivo through a stable interaction 1 2 3 between the bHLH domains ofTALl and E47. Experiments CAT activity (cpm x 100) with the reciprocal hybrids, GAL4-TAL1 and VP16-E47, also revealed in vivo association between TALl and E47; as FIG. 2. In vivo interactions ofTALl evaluated by the two-hybrid assay. C3H/1OTY2 cells were transiently transfected with 5 Pg ofthe shown in Fig. 2B, co-expression of these polypeptides in- pG5E1bCAT reporter plasmid and 10 pg ofeach expression plasmid. duced 150-fold higher CAT activity than either GAL4-TAL1 The hybrid polypeptides encoded by the expression plasmids are or VP16-E47 alone. In contrast, pairwise expression of either illustrated in Fig. 1 or described by Finkel et al. (18). Duplicate GAL4-TAL1 and VP16-TALl did not produce a detectable transfections were performed for each combination of expression increase in CAT activity (Fig. 2A). Thus, the TALl bHLH plasmids, and the CAT activities generated by each transfection are domain, like those of other tissue-specific bHLH proteins illustrated. (e.g., MyoDl), does not mediate homodimer formation in vivo, at least as measured by this assay. Two-hybrid analysis Co-immunoprecipitation of TALl and E2A. To confirm that also revealed TALl association with E12 (Fig. 2C) but not TAL1/E2A complexes exist in vivo, we attempted to co- with Idl (Fig. 2 B and C), a negative regulator of bHLH immunoprecipitate these complexes from mammalian cells function (37). with rabbit antisera specific for TALl or E2A. Therefore, TALl Interactions in Lymphocytes. The two-hybrid exper- COS-1 cells were cotransfected with expression plasmids iments demonstrate in vivo association between TALl and that encode pp22TAL1 (TAL1m176/pCMV4) and a truncated E2A in fibroblasts. To extend this observation to a setting form of E12 (AE12/pCMV4); the expressed sequences in- more relevant to human leukemia, it was necessary to adapt clude the intact bHLH domains of TAL1 and E12, respec- the two-hybrid assay for use in lymphocytes. For this pur- tively. After labeling with [35S]methionine, the cells were pose, a GAL4-responsive reporter plasmid (G5E1bLUC) lysed under mild conditions (Nonidet P-40 buffer) that are encoding firefly luciferase was constructed. Jurkat cells, a compatible with the maintenance of bHLH dimerization leukemic line derived from a T-ALL patient (38), were then complexes (19). Aliquots of the lysate were immunoprecip- cotransfected with the G5E1bLUC reporter and various itated under low-stringency conditions with an anti-TAL1 combinations ofexpression plasmids. As illustrated in Fig. 3, serum as the first antibody. To determine whether E2A co-expression of the GAL4-E47 and VP16-TALl polypep- polypeptides were co-immunoprecipitated with the TALl tides generated an enormous increase in luciferase activity, to immune complex, the precipitate was resuspended in a levels 1000-fold higher than those observed with either poly- high-stringency buffer that disrupts bHLH dimerization peptide alone. A 20-fold induction of luciferase activity was (RIPA buffer), and the resultant supernatant was reprecipi- produced in the reciprocal experiment upon co-expression of tated under high-stringency conditions with an anti-E2A GAL4-TAL1 and VP16-E47. Thus, protein-protein interac- serum as the second antibody. The final precipitates were tions between TALl and E47 are readily detected by two- then fractionated by SDS/PAGE. As illustrated in Fig. 4, a hybrid analysis in lymphocytes. Despite the sensitivity ofthis single radiolabeled species was present in the final precipitate assay, co-expression of GAL4-TAL1 and VP16-TALl did when anti-TALl and anti-E2A sera were used as the first and not produce a noticeable induction ofluciferase activity (Fig. second antibodies, respectively (lane 2). This molecule is 3). This result provides further evidence that TALl ho- likely to be the truncated E2A polypeptide encoded by modimerization is unlikely to occur in vivo. AE12/pCMV4 because it has the expected molecular mass of Downloaded by guest on September 24, 2021 3184 Medical Sciences: Hsu et al. Proc. Natl. Acad Sci. USA 91 (1994)

....frr'af: !9lMolt GAL4 AA.b D E .. .__...... - GAL4-E47 ~~~~~~~~~~~~~~~~~~~~. GAL4-E47 VP16-TALl

VP16-TAL1

VP16-TALl GAL4-TAL1 l GAL4-TALl I GAL4-TALl VP16-E47

VP16-E47 ___/_I FIG. 5. Co-immunoprecipitation of endogenous TALl and E2A

0 0.5 1.0 1.5 2.0 2.5 10.0 15.0 polypeptides from T-ALL cell lines. Leukemic cells that do (Jurkat) or do not (Molt-13) express TAL1 were labeled with [35S]methionine Luciferase activity (RLU x 100,000) and lysed under mild conditions. Aliquots of these lysates were subject to the two-step co-immunoprecipitation procedure using FIG. 3. Two-hybrid analysis of TALl interactions in lympho- anti-TAL1 serum (1), anti-E2A serum (E), or the corresponding cytes. Jurkat cells were transiently transfected with 15 pg of the preimmune sera (pre-T and pre-E, respectively) as either the first or G5E1bLUC reporter and 15 pg of each expression plasmid. The second antibody. Final precipitates were fractionated by SDS/ hybrid polypeptides encoded by the expression plasmids are illus- PAGE. Arrows denote the mobilities of the E2A polypeptides, as trated in Fig. 1B. Duplicate transfections were conducted for each well as the hyperphosphorylated (pp42TALl) and underphosphory- combination of expression plasmids, and the luciferase activities lated (p41TAL1) TAL1 polypeptides. Molecular mass standards (kDa) generated by each transfection are illustrated. RLU, relative light are indicated on the left of the autoradiogram. units. in the final precipitate when anti-E2A and anti-TALl sera was E2A =47 kDa and because it not observed when pre- were used as the first and second antibodies, respectively immune serum was used as the second antibody (lane 3). (Fig. 4, lane 5). Again, the co-immunoprecipitation was Moreover, co-immunoprecipitation of AE12 was clearly de- specific in the sense that pp22TAL1 was not recovered when pendent on the presence of pp22TAl, since it did not occur a preimmune serum was used as either the first or second when TAL1 preimmune serum was used as the first antibody antibody (lanes 4 and 6, respectively). These data suggest (lane 1). Evidence for complex formation between pp22TAL1 that TALl and E2A form in vivo complexes upon overex- and AE12 was also obtained by the reciprocal co- pression in COS-1 cells. immunoprecipitation experiment. Thus, pp22TAL' appeared The Presence of TAL1/E2A Complexes in Leukemic Cells. We then sought to determine whether TAL1/E2A complexes _ Ab| T T p-E tI are also formed from the endogenous polypeptides of leuke- 2nd Ab E_ pE | T T p | mic T-ALL cells. Therefore, the same co-immunoprecipitation assay was applied to radiolabeled cell lysates prepared from established T-ALL lines that either do (Jurkat) or do not (Molt-13) express endogenous TALI gene products (6). As illustrated in Fig. 5, a major E2A species of =65 kDa was recovered in the final precipitate from Jurkat cells when ---AEl2 anti-TALl and anti-E2A sera were used as the first and second antibody, respectively (lane 2). Since the anti-E2A serum does not distinguish between the comigrating E12 (654 amino acids) and E47 (651 amino acids) polypeptides, we cannot ascertain whether this species contains E12, E47, or both. Nevertheless, the appropriate control experiments (lanes 1 and 3) indicate that the E2A species is specifically associated with endogenous TALl polypeptides of Jurkat cells. Furthermore, the reciprocal experiment demonstrated that both hyperphosphorylated (pp42) and underphosphory- lated (p41) TAL1 polypeptides are specifically co- Lane 2 3 4 5 6 immunoprecipitated from Jurkat cells by the anti-E2A serum FIG. 4. Co-immunoprecipitation of exogenous TALl and E2A (lanes 4-6) (30). As expected, TAL1/E2A complexes were polypeptides from transfected COS1 cells. The cells were cotrans- not observed in Molt-13, a T-ALL cell line that does not fected with expression vectors encoding pp22TALl (TALlml76/ express TALI gene products (lanes 7 and 8). In sum, these pCMV4) and a truncated E12 polypeptide (AE12/pCMV4). After data suggest that TAL1/E2A complexes are formed from the labeling with [35S]methionine, the cells were lysed under mild endogenous polypeptides of T-ALL leukemic cells. conditions, and aliquots of the cell lysate were subjected to the two-step co-immunoprecipitation procedure, using anti-TAL1 serum (T), anti-E2A serum (E), or the corresponding preimmune sera (p-T DISCUSSION and p-E, respectively) as either the first or second antibody. Final immunoprecipitates were fractionated by SDS/PAGE. The immu- During normal hematopoiesis TALI transcripts can be found noprecipitated AE12 and p22TAL1 polypeptides are denoted with in a spectrum of cell types that includes megakaryocytes, arrows; mobilities of the molecular mass standards (kDa) are indi- erythroblasts, and basophilic granulocytes, as well as their cated on the left of the autoradiogram. committed progenitors (39). Despite this diverse pattern of Downloaded by guest on September 24, 2021 Medical Sciences: Hsu et al. Proc. NatL. Acad. Sci. USA 91 (1994) 3185 hematopoietic expression, TALI gene products have not as 3. Bernard, O., Lecointe, N., Jonveaux, P., Souyri, M., Mauchauffe, yet been observed in normal lymphoid cells. Nevertheless, M., Berger, R., Larsen, C. J. & Mathieu-Mahul, D. (1991) Onco- DNA gene 6, 1477-1488. nearly 25% of T-ALL patients harbor a tumor-specific 4. Aplan, P. D., Lombardi, D. P., Reaman, G. H., Sather, H. N., rearrangement that results in the production of TAL1 tran- Hammond, G. D. & Kirsch, I. R. (1992) Blood 79, 1327-1333. scripts and polypeptides in their leukemic cells (1-5). Ectopic 5. Bash, R. O., Crist, W. M., Shuster, J. J., Link, M. P., Amylon, M., TAL1 expression also occurs in the malignant cells of some Pullen, J., Carroll, A. J., Buchanan, G. R., Smith, R. G. & Baer, R. T-ALL patients that do not exhibit a detectable alteration of (1993) Blood 81, 2110-2117. the TALI locus (6). These observations imply that the un- 6. Cheng, J.-T., Hsu, H.-L., Hwang, L.-Y. & Baer, R. (1993) Onco- scheduled expression of TAL1 in lymphoid cells, whether gene 8, 677-683. achieved by tumor-specific DNA rearrangement or by other 7. Begley, C. G., Aplan, P. D., Denning, S. M., Haynes, B. F., Wald- mann, T. A. & Kirsch, I. R. (1989) Proc. Natl. Acad. Sci. USA 86, means, is a critical component of T-cell leukemogenesis in a 10128-10132. significant proportion of T-ALL patients. 8. Murre, C., McCaw, P. S. & Baltimore, D. (1989) Cell 56, 777-783. To understand how ectopic TAL1 expression promotes 9. Mellentin, J. D., Smith, S. D. & Cleary, M. L. (1989) Cell 58, T-ALL, it is important to identify the molecular complexes 77-83. formed by TAL1 polypeptides in leukemic T cells. Our 10. Xia, Y., Brown, L., Yang, C. Y.-C., Tsan, J. T., Siciliano, M. J., previous studies showed that the bHLH domain of TALl Espinosa, R., Im, Le Beau, M. M. & Baer, R. J. (1991) Proc. Natl. mediates in vitro interaction with the E2A proteins to form Acad. Sci. USA 88, 11416-11420. 11. Ferr6-D'Amar6, A. R., Prendergast, G. C., Ziff, E. B. & Burley, heterodimeric bHLH complexes (TAL1/E12 and TAL1/ S. K. (1993) Nature (London) 363, 38-45. E47) that bind DNA in a sequence-specific fashion (27, 33). 12. Voronova, A. & Baltimore, D. (1990) Proc. Nat!. Acad. Sci. USA We now report that the postulated TAL1/E2A complexes are 87, 4722-4726. stable in vivo and, more importantly, that these complexes 13. Davis, R. L., Cheng, P.-F., Lassar, A. B. & Weintraub, H. (1990) exist within leukemic cells derived from T-ALL patients. Our Cell 60, 733-746. results also indicate that the bHLH domain of TAL1 asso- 14. Brennan, T. J., Chakraborty, T. & Olson, E. N. (1991) Proc. Nat!. ciates with E2A For example, the Acad. Sci. USA 88, 5675-5679. selectively polypeptides. 15. Kadesch, T. (1993) Cell Growth Dffer. 4, 49-55. two-hybrid assay readily detects in vivo TAL1 interactions 16. Jen, Y., Weintraub, H. & Benezra, R. (1992) Genes Dev. 6, with either product of the E2A gene (E12 and E47) but not 1466-1479. with the Idl helix-loop-helix protein; the latter observation 17. Chakraborty, T., Martin, J. F. & Olson, E. N. (1992)J. Biol. Chem. is consistent with a previous study which showed that TAL1 267, 17498-17501. and Idl do not interact in vitro (40). Moreover, the bHLH 18. Finkel, T., Duc, J., Fearon, E. R., Dang, C. V. & Tomaselli, G. F. domain ofTAL1 does not self-associate to form homodimeric (1993) J. Biol. Chem. 268, 5-8. that be detected in the 19. Lassar, A. B., Davis, R. L., Wright, W. E., Kadesch, T., Murre, complexes can two-hybrid system. C., Voronova, A., Baltimore, D. & Weintraub, H. (1991) Cell 66, Thus, as postulated for the tissue-specific factors involved in 305-315. myogenesis (23-26), the functional properties of TAL1 may 20. Kato, G. J., Lee, W. M. F., Chen, L. & Dang, C. V. (1992) Genes depend on heterologous interactions with other bHLH pro- Dev. 6, 81-92. teins such as E12 and E47. 21. Blackwood, E. M., Luscher, B. & Eisenman, R. N. (1992) Genes It remains to be established whether the leukemic proper- Dev. 6, 71-80. ties ofTAL1 are due to the TAL1/E2A complexes present in 22. Littlewood, T. D., Amati, B., Land, H. & Evan, G. I. (1992) Oncogene 7, 1783-1792. T-ALL cells, and, if so, whether these complexes promote 23. Murre, C., McCaw, P. S., Vaessin, H., Caudy, M., Jan, L. Y., Jan, T-ALL by serving as transcriptional regulatory factors. Y. N., Cabrera, C. V., Buskin, J. N., Hauschka, S. D., Lassar, These complexes may influence gene expression in T-ALL A. B., Weintraub, H. & Baltimore, D. (1989) Cell 58, 537-544. cells by direct binding to the cis-acting regulatory elements of 24. Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., subordinate target genes. Ectopic TAL1 expression may also Benezra, R., Blackwell, T. K., Turner, D., Rupp, R., Hollenberg, affect RNA transcription in an indirect manner by disturbing S., Zhuang, Y. & Lassar, A. (1991) Science 251, 761-766. 25. Funk, W. D., Ouellette, M. & Wright, W. E. (1991) Mol. Biol. Med. the normal balance of interacting bHLH proteins in T cells; 8, 185-195. thus, through its ability to associate with E2A polypeptides, 26. Olson, E. N. (1990) Genes Dev. 4, 1454-1461. TAL1 might reduce the formation ofother homodimeric and 27. Hsu, H.-L., Cheng, J.-T., Chen, Q. & Baer, R. (1991) Mol. Cell. heterodimeric complexes involving E2A, and thereby influ- Biol. 11, 3037-3042. ence the transcription of subordinate genes normally regu- 28. Sadowski, I. & Ptashne, M. (1989) Nucleic Acids Res. 17, 7539. lated these complexes. Whatever the precise mechanisms 29. Dang, C. V., Barrett, J., Villa-Garcia, M., Resar, L. M. S., Kato, by G. J. & Fearon, E. R. (1991) Mol. Cell. Biol. 11, 954-962. involved, it seems likely that the inappropriate presence of 30. Cheng, J.-T., Cobb, M. H. & Baer, R. (1993) Mol. Cell. Biol. 13, TAL1 polypeptides alters the existing pattern of gene ex- 801-808. pression in T-lineage cells in a manner that promotes T-cell 31. Andersson, S., Davis, D. N., Dahlback, H., Jornvall, H. & Russell, leukemogenesis. D. W. (1989) J. Biol. Chem. 264, 8222-8229. 32. Kamps, M. P., Murre, C., Sun, X.-H. & Baltimore, D. (1990) Cell We thank Julia Tsou Tsan for technical assistance and Susan 60, 547-555. for the We are also to 33. Hsu, H.-L., Huang, L., Tsan, J. T., Funk, W., Wright, W. E., Hu, Campbell preparing manuscript. very grateful J.-S., Kingston, R. E. & Baer, R. (1994) Mol. Cell. Biol. 14, David Baltimore, Chi Dang, Toren Finkel, Michael Green, Kathy 1256-1265. Martin, Cornelius Murre, and Ivan Sadowski for generously provid- 34. Lillie, J. W. & Green, M. R. (1989) Nature (London) 338, 39-44. ing plasmid clones. 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