A DNA-binding mutant of TAL1 cooperates with LMO2 to cause T cell leukemia in mice

Abstract

The most common translocation in childhood T-cell acute lymphoblastic leukemia (T-ALL) involves the LMO2 locus, resulting in ectopic expression of the LMO2 gene in human thymocytes. The LMO2 gene was also activated in patients with X-linked Severe Combined Immune Deficiency treated with gene therapy because of retroviral insertion in the LMO2 locus. The LMO2 insertions predisposed these children to T-ALL, yet how LMO2 contributes to T cell transformation remains unclear. The LIM (Lin 11, Isl-1, Mec-3) domain containing LMO2 protein regulates erythropoiesis as part of a large transcriptional complex consisting of LMO2, TAL1, E47, GATA1 and LDB1 that recognizes bipartite E-box-GATA1 sites on target genes. Similarly, a TAL1/E47/LMO2/LDB1 complex is observed in human T-ALL and Tal1 and Lmo2 expression in mice results in disease acceleration. To address the mechanism(s) of Tal1/Lmo2 synergy in leukemia, we generated Lmo2 transgenic mice and mated them with mice that express wild-type Tal1 or a DNA-binding mutant of TAL1. Tal1/Lmo2 and MutTAL1/Lmo2 bitransgenic mice exhibit perturbations in thymocyte development due to reduced E47/HEB transcriptional activity and develop leukemia with identical kinetics. These data demonstrate that the DNA-binding activity of Tal1 is not required to cooperate with Lmo2 to cause leukemia in mice and suggest that Lmo2 may cooperate with Tal1 to interfere with E47/HEB function(s).

Figure 1

The DNA-binding properties of TAL1 are not required to cooperate with Lmo2 to cause leukemia in mice. (a) Schematic representation of the prox lck/Lmo2 transgene. The murine Lmo2 cDNA is expressed under control of the proximal lck promoter and human growth hormone (hGH) splice and poly(A)+ addition sequences. (b) Expression of the Lmo2 transgene. RNA was isolated from a murine erythroleukemic cell line (MEL) and from thymocytes isolated from wild-type (wt) or Lmo2 founder mice designated F5, F19, F41 and F42. Lmo2 mRNA levels were determine using quantitative PCR with β-actin serving as an internal control. (c) Survival curves for the Tal1, mutTAL1, Lmo2, Tal1/Lmo2 and mutTAL1/Lmo2 mice. Mice were monitored daily for evidence of disease, upon which the mice were sacrificed and a post-mortem examination performed. The cohort of Tal1/Lmo2 mice consisted of 15 animals and the cohort of mutTAL1/Lmo2 mice consisted of 11 animals. The Lck-Lmo2 only cohort consisted of 26 mice. Tal1 and mutTAL1 survival curves have been published previously (Kelliher et al., 1996; O’Neil et al., 2001).

Figure 2

A DNA-binding mutant of TAL1 cooperates with Lmo2 to perturb thymocyte development. (a) Significant decreases in thymic cellularity are observed in Tal1/Lmo2 and mutTAL1/Lmo2 bitransgenic mice. Total thymocyte numbers from wild-type, Tal1, mutTAL1, Lmo2, Tal1/Lmo2 and mutTAL1/Lmo2 transgenic mice. When compared with wild type, Lmo2 expression causes a decrease in thymic cellularity (P = 8.0 × 10⁻⁶) that is exacerbated when either Tal1 or mut-TAL1 is co-expressed (P = 7.8×10⁻¹⁵ and P = 3.1×10⁻⁶, respectively). (b) Co-expression of Lmo2 with either Tal1 or mutTAL1 perturbs thymocyte development. Thymocytes from 4-week old, preleukemic mice were stained with CD4-PE and CD8-FITC and analyzed by flow cytometry. A total of 14 Lmo2, 19 Tal1/Lmo2 and 13 mutTAL1/Lmo2 preleukemic mice were analyzed. Representative profiles are shown. (c) DN thymocyte development is altered when Lmo2 is co-expressed with either Tal1 or mutTAL1. Thymocytes from 4-week old mice were stained with antibodies for the lineage markers (CD4, CD8, CD3, B220, Mac1, Gr1 and Terr119) and the lineage-negative cells were isolated and stained with CD44-APC and CD25-FITC. A total of 14 Lmo2, 19 Tal1/Lmo2 and 13 mutTAL1/Lmo2 preleukemic mice were examined. Representative profiles are shown. (d) Increase in the absolute numbers of DN3 and DN4 thymic progenitors in preleukemic Tal1 and mutTAL1 transgenic mice. Thymocytes from wild-type (n = 12), Tal1 (n = 7), MutTAL1 (n = 8), Lmo2 (n = 15), Tal1/Lmo2 (n = 11) and mutTAL1/Lmo2 (n = 12) mice were stained with CD4 and CD8 and lineage negative cells were stained with CD44 and CD25. Absolute numbers of DN1, DN2, DN3 and DN4 cells are indicated for each strain. A representative experiment is shown.

Figure 2

A DNA-binding mutant of TAL1 cooperates with Lmo2 to perturb thymocyte development. (a) Significant decreases in thymic cellularity are observed in Tal1/Lmo2 and mutTAL1/Lmo2 bitransgenic mice. Total thymocyte numbers from wild-type, Tal1, mutTAL1, Lmo2, Tal1/Lmo2 and mutTAL1/Lmo2 transgenic mice. When compared with wild type, Lmo2 expression causes a decrease in thymic cellularity (P = 8.0 × 10⁻⁶) that is exacerbated when either Tal1 or mut-TAL1 is co-expressed (P = 7.8×10⁻¹⁵ and P = 3.1×10⁻⁶, respectively). (b) Co-expression of Lmo2 with either Tal1 or mutTAL1 perturbs thymocyte development. Thymocytes from 4-week old, preleukemic mice were stained with CD4-PE and CD8-FITC and analyzed by flow cytometry. A total of 14 Lmo2, 19 Tal1/Lmo2 and 13 mutTAL1/Lmo2 preleukemic mice were analyzed. Representative profiles are shown. (c) DN thymocyte development is altered when Lmo2 is co-expressed with either Tal1 or mutTAL1. Thymocytes from 4-week old mice were stained with antibodies for the lineage markers (CD4, CD8, CD3, B220, Mac1, Gr1 and Terr119) and the lineage-negative cells were isolated and stained with CD44-APC and CD25-FITC. A total of 14 Lmo2, 19 Tal1/Lmo2 and 13 mutTAL1/Lmo2 preleukemic mice were examined. Representative profiles are shown. (d) Increase in the absolute numbers of DN3 and DN4 thymic progenitors in preleukemic Tal1 and mutTAL1 transgenic mice. Thymocytes from wild-type (n = 12), Tal1 (n = 7), MutTAL1 (n = 8), Lmo2 (n = 15), Tal1/Lmo2 (n = 11) and mutTAL1/Lmo2 (n = 12) mice were stained with CD4 and CD8 and lineage negative cells were stained with CD44 and CD25. Absolute numbers of DN1, DN2, DN3 and DN4 cells are indicated for each strain. A representative experiment is shown.

Figure 3

Rag1 and Rag2 expression is further repressed in Tal1 and mut-TAL1 transgenic thymocytes when Lmo2 is co-expressed. Thymocytes were isolated from wild-type or preleukemic Tal1, mutTAL1, Lmo2, mutTAL1/Lmo2 or Tal/Lmo2 transgenic mice at 4–6 weeks of age. RNA was isolated and Rag1 and Rag2 expression levels quantified by qPCR. The copy number for the gene of interest was normalized to the copy number for β-actin. Data is represented as percent of wild-type control.

Figure 4

The DNA-binding activity of TAL1 is required to stably bind and repress CD4. (a) The DNA-binding properties of TAL1 contribute to CD4 repression. RNA was isolated from Tal1/Lmo2 and mutTAL1/Lmo2 leukemic cell lines and CD4 expression levels quantified by qPCR. The copy number for CD4 was normalized to the copy number for β-actin. (b) The TAL1 DNA-binding mutant is impaired in its ability to bind the CD4 enhancer. Chromatin immunoprecipitation was performed on mutTAL1/Lmo2 and Tal1/Lmo2 leukemic cell lines using a rabbit IgG antibody or antibodies to HEB, mSin3a, mouse Tal1 (m258c), human TAL1 (Millipore, BTL73) or a TAL1 antibody that recognizes mouse and human TAL1 (Santa Cruz, C-21). Input-sheared DNA served as a positive control. PCR amplification was performed on the immunoprecipitated DNA with primers specific for the mouse CD4 enhancer region.