Oncogene (2001) 20, 3897 ± 3905 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc 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 trans- genic 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).