Leukemia-related transcription factor TEL/ETV6 expands erythroid precursors and stimulates hemoglobin synthesis

Abstract

TEL/ETV6 located at chromosome 12p13 encodes a member of the E26 transformation-specific family of transcription factors. TEL is known to be rearranged in a variety of leukemias and solid tumors resulting in the formation of oncogenic chimeric protein. Tel is essential for maintaining hematopoietic stem cells in the bone marrow. To understand the role of TEL in erythropoiesis, we generated transgenic mice expressing human TEL under the control of Gata1 promoter that is activated during the course of the erythroid-lineage differentiation (GATA1-TEL transgenic mice). Although GATA1-TEL transgenic mice appeared healthy up to 18 months of age, the level of hemoglobin was higher in transgenic mice compared to non-transgenic littermates. In addition, CD71+/TER119+ and c-kit+/CD41+ populations proliferated with a higher frequency in transgenic mice when bone marrow cells were cultured in the presence of erythropoietin and thrombopoietin, respectively. In transgenic mice, enhanced expression of Alas-e and beta-major globin genes was observed in erythroid-committed cells. When embryonic stem cells expressing human TEL under the same Gata1 promoter were differentiated into hematopoietic cells, immature erythroid precursor increased better compared to controls as judged from the numbers of burst-forming unit of erythrocytes. Our findings suggest some roles of TEL in expanding erythroid precursors and accumulating hemoglobin.

Figure 1

Expression of TEL transgene in GATA1‐TEL transgenic mice. (a) Schematic representation of the Gata1 promotor region and the pIE3.9int‐TEL construct used for the generation of GATA1‐TEL transgenic mice. The pIE3.9int vector contains 3.9 kb Gata1 promoter region upstream of exon IE.^{( 30 )} The coding sequence of wild‐type human TEL cDNA was connected to the first ATG codon in exon II of mouse Gata1 gene. Abbreviations for the restriction enzyme sites are E, EcoRI; B, BamHI; S, SacI. (b) Expression of Gata1‐driven TEL transgene was confirmed by reverse transcription polymerase chain reaction (RT‐PCR) using bone marrow cells extracted from GATA1‐TEL transgenic mice of lines 460, 462 and 464. Forward primers for the first and second PCR were both located on exon IE of mouse Gata1 gene and reverse primers on exon II of human TEL gene. Tg, Gata1‐TEL transgenic mouse; nTg, non‐transgenic littermate.

Figure 2

Differentiation of bone marrow cells into erythroid and megakaryocytic precursors. Bone marrow cells were extracted and cultured in the presence of recombinant murine (a) erythropoietin (EPO) (3 U/mL) and Stem cell factor (SCF) (50 ng/mL), or (b) thrombopoietin (TPO) (20 ng/mL), interleukin (IL)‐3 (10 ng/mL) and IL‐6 (10 ng/mL). Cells were examined after 8 days of culture by fluorescence‐activated cell sorter, which revealed that bone marrow cells obtained from GATA1‐TEL transgenic mice showed higher populations of (a) CD71^high/TER119⁺ cells, or (b) c‐kit⁺/CD41⁺ cells compared to those from littermate controls. In the left panels, the representative data from non‐transgenic (nTg) and transgenic (Tg) mice of line 464 are shown. In the right panel, indicated are average and standard deviation of five (a) or four (b) independent experiments using lines 460, 462 and 464. Numbers in parenthesis indicate numbers of mice analyzed in each group. Tg, GATA1‐TEL transgenic mouse; nTg, non‐transgenic littermate.

Figure 3

Quantitative PCR of the genes involved in erythropoiesis. (A) To compare the expression of erythroid‐related genes between GATA1‐TEL transgenic mice and control littermates, bone marrow cells were sorted for CD71^int/TER119⁻ (a), CD71^high/TER119⁺ (b) and CD71⁻/TER119⁺ (c), representing different stages of erythroid differentiation, and then subjected to quantitative PCR analysis. The result of FACS analysis shown in Fig. 3 A came from a non‐transgenic litter mouse. There was no difference in the expression pattern of each population between non‐transgenic and transgenic mice. (B) Representative results of quantitative PCR for endogenous Gata1 and endogenous + exogenous TEL in each stage of erythroid differentiation from animals of line 460. The highest expression of TEL gene was obtained in CD71^high/TER119⁺ population in the GATA1‐TEL transgenic mice, in concordance with the highest expression of endogenous Gata1 among the three populations. (C) Representative results of quantitative PCR for Alas‐e and β‐major globin genes from animals of line 460. Tg, GATA1‐TEL transgenic mouse; nTg, non‐transgenic littermate.

Figure 4

Expression of Gata1 and TEL during differentiation of embryonic stem (ES) cells. (a) Undifferentiated original J1, mock‐transfected (Mock2) and Gata1‐TEL‐overexpressing (GATA1‐TEL3, 29 and 30) ES cells were deprived of leukemia inhibitory factor to initiate differentiation and analyzed for the expression of endogenous Gata1 gene and TEL transgene under the control of Gata1 IE3.9int promoter. Expression of TEL transgene was observed from day 5 of differentiation in concordance with the expression of endogenous Gata1 gene. (b) Total amount of TEL transcript (endogenous + exogenous) in differentiating embryoid body (EB) cells. Mouse and human TEL transcripts were simultaneously amplified as described in Materials and methods using primers TEL‐829f and TEL‐921r located on exons V and VI of mouse and human TEL gene. Average and standard deviation of two independent experiments are shown. After day 6 of differentiation, total amount of TEL was higher in GATA1‐TEL EBs than in mock EBs.

Figure 5

Enhanced erythroid colony formation in GATA1‐TEL embryoid body (EB) cells. Undifferentiated J1, mock‐transfected (Mock1) and Gata1‐TEL‐overexpressing (GATA1‐TEL25, 29 and 30) embryonic stem cells were deprived of leukemia inhibitory factor to form differentiated EBs. EBs at day 7 of differentiation were collected and subjected to BFU‐E (supplemented with SCF, thrombopoietin [TPO] and erythropoietin [EPO]) and CFU‐GEMM (supplemented with SCF, TPO, EPO, interleukin [IL]‐11, IL‐3, GM‐CSF, G‐CSF, M‐CSF and IL‐6) assays. Average and standard deviation of at least two independent experiments are shown. Gata1‐TEL‐expressing EB cells showed higher BFU‐E activity than controls, while no difference was observed in CFU‐GEMM activity. GEMM, mixed colony; E, erythroid colony; M, myeloid colony.

Figure 6

In vitro erythroid differentiation of embryoid body (EB)‐derived c‐kit⁺/CD71⁺ cells.
Undifferentiated embryonic stem cells were deprived of leukemia inhibitory factor to form differentiated EBs. After 6 days of differentiation, c‐kit⁺/CD71⁺ cells (shown in panel a) were separated by fluorescence‐activated cell sorter (FACS) and subjected to erythroid differentiation assay on OP9 stromal cell layer. The result of FACS analysis shown in (a) came from non‐transgenic cells. There was no difference in the population of EB‐derived c‐kit⁺/CD71⁺ cells between non‐transgenic and transgenic cells. (b) The CD71^high/TER119⁺ fraction after 8 days of culture with erythropoietin and SCF on OP9 layer. In the left panel, the representative data of mock‐transfected (Mock1) and GATA1‐TEL30 are shown. In the right panel, average and standard deviation of control and transgenic (Tg) cells are shown. The results of control and Tg are derived from the combined data in at least two independent experiments of J1, Mock1 and 2, and GATA1‐TEL3, 25 and 30, respectively. Day 6 transgenic EB‐derived c‐kit⁺/CD71⁺ cells produced higher numbers of CD71^high/TER119⁺ cells compared to the controls.