TAL1 and LIM-only proteins synergistically induce retinaldehyde dehydrogenase 2 expression in T-cell acute lymphoblastic leukemia by acting as cofactors for GATA3

Abstract

Previously, we have shown that TAL1 and the LIM-only protein gene (LMO) are regularly coactivated in T-cell acute lymphoblastic leukemia (T-ALL). This observation is likely to relate to the findings that TAL1 and LMO are highly synergistic in T-cell tumorigenesis in double-transgenic mice. To understand the molecular mechanisms of functional synergy between TAL1 and LMO in tumorigenesis and transcriptional regulation, we tried to identify downstream target genes regulated by TAL1 and LMO by a subtractive PCR method. One of the isolated genes, that for retinaldehyde dehydrogenase 2 (RALDH2), was regularly expressed in most of the T-ALL cell lines that coexpressed TAL1 and LMO. Exogenously transfected TAL1 and LMO, but not either alone, induced RALDH2 expression in a T-ALL cell line, HPB-ALL, not expressing endogeneous TAL1 or LMO. The RALDH2 transcripts in T-ALL were, however, mostly initiated within the second intron. Promoter analysis revealed that a GATA site in a cryptic promoter in the second intron was essential and sufficient for the TAL1- and LMO-dependent transcriptional activation, and GATA3 binds to this site. In addition, forced expression of GATA3 potentiated the induction of RALDH2 by TAL1 and LMO, and these three factors formed a complex in vivo. Furthermore, a TAL1 mutant not binding to DNA also activated the transcription of RALDH2 in the presence of LMO and GATA3. Collectively, we have identified the RALDH2 gene as a first example of direct transcriptional target genes regulated by TAL1 and LMO in T-ALL. In this case, TAL1 and LMO act as cofactors for GATA3 to activate the transcription of RALDH2.

FIG. 1

Induction of RALDH2 by TAL1 and LMO in T-ALL cell lines. (A) Northern blot analysis for RALDH2 in HPB-ALL clones. HPB-ALL cells were transfected with the indicated combinations of expression vectors, and stable transformants were isolated. Two clones were analyzed for each combination. Poly(A)⁺ RNA (2 μg each) was electrophoresed, blotted onto a filter, and hybridized with the ³²P-labeled 3′ untranslated region of RALDH2 cDNA obtained by subtractive PCR. The same filter was reprobed for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as an internal control. (B) RT-PCR analysis for RALDH2 in HPB-ALL cells transiently expressing the indicated combinations of TAL1 and LMO. HPB-ALL cells were cotransfected with 15 μg each of the indicated expression vectors and 1 μg of pRC/CMV-luc by electroporation. Total RNA was prepared 20 h after transfection and subjected to RT-PCR analysis for RALDH2 and E2A (control). The amounts of total RNA used were 10, 2, and 0.4 ng from left to right. Amplification products were electrophoresed on 2% agarose and stained with ethidium bromide. Transfection efficiency was tested by luciferase assay and varied within threefold. (C and D) Northern blot analysis for RALDH2 in T-ALL-derived cell lines (C) and other hematopoietic cell lines (D). Poly(A)⁺ RNA (2 μg each) was examined as described for panel A. BALL-1 and Raji, B-cell lines, HL60, promyelocytic cell line; U937, monocytoid cell line; K562, erythroleukemia cell line.

FIG. 2

Structures of the RALDH2 gene and mRNA. (A) Schematic illustration of the RALDH2 gene. Boxes and lines correspond to exons and introns, respectively. The transcription start sites of the full-length RALDH2 and the T-ALL-type RALDH2 (RALDH2-T) are marked by arrows. (B) 5′ structure of RALDH2 mRNA in T-ALL cell lines. Two types of RALDH2 cDNA and the positions of primers used for RT-PCR to distinguish each type are shown schematically at the top. Total RNA samples prepared from the indicated cell lines were subjected to RT-PCR. Amplification products were electrophoresed on 2% agarose and stained with ethidium bromide. Two clones were analyzed for HPB-ALL cells stably transfected with TAL1α and LMO1. (C) In vitro translation from two types of RALDH2 mRNA. Both types of RALDH2 cDNA were transcribed in vitro and translated in rabbit reticulocyte lysate in the presence of [³⁵S]methionine. Translation products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography.

FIG. 3

Induction of the RALDH2-T promoter by TAL1 and LMO. (A) Nucleotide sequence around the promoter region of RALDH2-T. Exon sequences are boxed. A consensus GATA site, an optimal E-box, and the putative initiation codon of RALDH2-T are underlined. Major transcriptional initiation sites are indicated by arrows. (B) HPB-ALL cells were cotransfected with 20 μg of a luciferase reporter plasmid containing the 1.7-kb genomic fragment of the RALDH2-T promoter (−1.7-luc), 0.1 μg of pRL-CMV for normalization of transfection efficiency, and 5 μg each of expression plasmids without inserts (−) or with the indicated inserts. Relative luciferase activity compared to that of the reporter plasmid alone was determined. Means and standard deviations for three independent experiments are shown.

FIG. 4

Requirement of a GATA site but not an E-box in the RALDH2-T promoter for induction by TAL1 and LMO. (A) HPB-ALL cells were cotransfected with one of the indicated RALDH2-T reporter plasmids and expression vectors for TAL1α and LMO1. The results show fold activation in luciferase activity from the reporter alone. Means and standard deviations for three independent experiments are shown. (B) Mutations introduced in −1.7GM-luc.

FIG. 5

Involvement of GATA3 in induction of RALDH2-T by TAL1 and LMO. (A) GATA3 binds to the GATA site in the RALDH2-T promoter. EMSA was carried out by using nuclear extracts prepared from HPB-ALL cells and a labeled oligonucleotide spanning the GATA site in the RALDH2-T promoter. Cold competitors in 200-fold excess or anti-GATA3 MAb was added as indicated. (B) The GATA3-CFM mutant does not bind to DNA. COS cells were transfected with wild-type (WT) or mutant GATA3 expression vectors. EMSA was carried out by using cell lysates from transfected COS cells and a labeled oligonucleotide spanning the GATA site in the TCRα enhancer. Cold competitors in 200-fold excess or anti-FLAG MAb was added as indicated. Expression of GATA3 was confirmed by Western blotting (W) with anti-FLAG MAb (αFLAG). The asterisk indicates nonspecific binding. (C) Interaction between GATA3 and LMO1 or LMO2 in vivo. Cell lysates from 293E cells transiently transfected with expression vectors for the indicated tagged proteins were precipitated with anti-FLAG MAb. Immunoprecipitates (IP) and cell lysates were immunoblotted with anti-FLAG or anti-HA MAb as indicated. The asterisk indicates immunoglobulin H. (D) Complex formation between TAL1, LMO2, and GATA3. 293E cells were transiently transfected with expression vectors for FLAG-GATA3 and HA-TAL1 with or without that for LMO2, and cell lysates were immunoprecipitated with anti-FLAG MAb. Immunoprecipitates and cell lysates were immunoblotted with anti-FLAG or anti-HA MAb as indicated. The asterisk indicates immunoglobulin H or L. (E and F) Effect of GATA3 on induction of the RALDH2-T promoter by TAL1 and LMO. HPB-ALL (E) and BALL-1 (F) cells were cotransfected with 15 μg of a luciferase reporter containing the 1.7-kb genomic fragment of the RALDH2-T promoter (−1.7-luc), 0.1 μg of pRL-CMV for normalization of transfection efficiency, and 5 μg each of expression plasmids without inserts (−) or with the indicated inserts. Relative luciferase activity compared to that of the reporter alone was determined. Means and standard deviations for three independent experiments are shown.

FIG. 6

No requirement of DNA binding activity of TAL1 for induction of RALDH2-T. (A and B) TAL1β-BM can interact with E47 and LMO2. Cell lysates from 293E cells transiently transfected with expression vectors for each tagged proteins were immunoprecipitated (IP) with anti-FLAG MAb (αFLAG). Immunoprecipitates and cell lysates were immunoblotted (W) with anti-FLAG or anti-HA MAb as indicated. The asterisk indicates immunoglobulin L. WT, wild type. (C) TAL1β-BM does not bind to DNA. 293E cells were transfected with expression vectors for wild-type or mutant TAL1β and E47S. EMSA was carried out by using cell lysates from transfected 293E cells and the labeled consensus E-box oligonucleotide. Cold competitors in 200-fold excess or anti-FLAG MAb was added as indicated. Expression of TAL1β and E47S was confirmed by Western blotting with anti-FLAG MAb. Asterisks indicate nonspecific binding. (D and E) TAL1β-BM induces transcription from the RALDH2-T reporter in collaboration with LMO and GATA3. HPB-ALL (D) and BALL-1 (E) cells were cotransfected with 20 μg (D) or 15 μg (E) of a luciferase reporter containing the 1.7-kb genomic fragment of the RALDH2-T promoter (−1.7-luc), 0.1 μg of pRL-CMV for normalization of transfection efficiency, and 5 μg each of expression plasmids without inserts (−) or with the indicated inserts. Relative luciferase activity compared to that of the reporter alone was determined. Means and standard deviations for three independent experiments are shown. (F) TAL1β-BM induces endogeneous RALDH2-T expression with LMO in HPB-ALL cells. HPB-ALL cells were cotransfected with 15 μg each of indicated expression vectors and 1 μg of pRC/CMV-luc by electroporation. Total RNA was prepared 20 h after transfection and subjected to RT-PCR analysis for RALDH2 and E2A (control). The amounts of total RNA used were 10, 2, and 0.4 ng from left to right. Amplification products were electrophoresed on 2% agarose and stained with ethidium bromide. Transfection efficiency was tested by luciferase assay and varied within threefold.

FIG. 7

Transactivation of an artificial reporter consisting of three tandem repeats of the GATA site by TAL1, LMO, and GATA3. BALL-1 cells were cotransfected with 15 μg of an artificial luciferase reporter (GATA×3-tk-luc or tk-luc), 0.1 μg of pRL-CMV for normalization of transfection efficiency, and 5 μg each of expression plasmids without inserts (−) or with the indicated inserts. tk-luc contains only the HSV tk minimal promoter. GATA×3-tk-luc contains three copies of the GATA site from the RALDH2-T promoter linked to the minimal HSV tk promoter. Relative luciferase activity compared to that of the reporter alone was determined. Means and standard deviations for three independent experiments are shown. WT, wild type.

FIG. 8

Model of transcriptional activation of the RALDH2-T promoter by TAL1, LMO, and GATA3. In normal T cells, GATA3 binds to the GATA site in the RALDH2-T promoter but does not activate transcription. When TAL1 and LMO are ectopically expressed in T-ALL, a large complex containing TAL1, LMO, and GATA3 is formed on the GATA site in the RALDH2-T promoter to activate transcription from a downstream initiator (Inr) (see Discussion).