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Blood, 1 May 2004, Vol. 103, No. 9, pp. 3326-3335. Prepublished online as a Blood First Edition Paper on January 8, 2004; DOI 10.1182/blood-2003-05-1689.
HEMATOPOIESIS Characterization of DNA-binding-dependent and -independent functions of SCL/TAL1 during human erythropoiesisFrom the Department of Hematology, Institut Cochin, U567 INSERM, Paris, France.
The transcription factor TAL1 has major functions during embryonic hematopoiesis and in adult erythropoiesis and megakaryocytopoiesis. These functions rely on different TAL1 structural domains that are responsible for dimerization, transactivation, and DNA binding. Previous work, most often done in mice, has shown that some TAL1 functions do not require DNA binding. To study the role of TAL1 and the relevance of the TAL1 DNA-binding domain in human erythropoiesis, we developed an approach that allows an efficient enforced wild-type or mutant TAL1 protein expression in human hematopoietic CD34+ cells using a lentiviral vector. Differentiation capacities of the transduced cells were studied in a culture system that distinguishes early and late erythroid development. Results indicate that enforced TAL1 expression enhances long-term culture initiating cell (LTC-IC) potential and erythroid differentiation of human CD34+ cells as shown by increased  globin and porphobilinogen deaminase (PBGD) gene expressions and erythroid colony-forming units (CFU-Es), erythroid burst-forming units (BFU-Es), and glycophorin A-positive (GPA+) cell productions. Enforced expression of a TAL1 protein deleted of its DNA-binding domain (named  bTAL1) mimicked most TAL1 effects except for the LTC-IC enhancement, the down-regulation of the CD34 surface marker, and the GPA+ cell production. These results provide the first functional indications of DNA-binding-dependent and -independent roles of TAL1 in human erythropoiesis. (Blood. 2004;103:3326-3335)
Hematopoiesis is the process that leads to the generation of all mature blood cells. The cells that initiate this process lay in a small population, named hematopoietic stem cells (HSCs), that is subjected to various steps of differentiation, leading to the overall hematopoietic lineages. Several proteins are known to play a major role in the control of blood cell production, in particular the transcription factors. The TAL1 (named also TCL-5 or SCL) protein belongs to the class II basic helix-loop-helix (bHLH) transcription factor family. These factors display a tissue-restricted expression and are known to play critical roles in regulating differentiation of many cell types,1 as shown for MYF5 or MYOD in muscles or neurogenin in the brain. TAL1 is expressed in the vascular and in the hematopoietic systems, especially in hematopoietic progenitors and in erythroid, megakaryocytic, and mast cell precursors,2,3 where it has been implicated at different steps of development and differentiation.1 TAL1 participates to the onset of primitive hematopoietic development. Tal1 null embryos fail to develop any hematopoietic cells4-6 and this failure can be rescued by expressing TAL1 using retroviral transduction before the onset of hematopoiesis.6 TAL1 is also important for the establishment of definitive hematopoiesis since Tal1 null mouse embryonic stem (ES) cells injected into blastocysts do not participate in any adult hematopoietic lineage, whereas their contribution to other tissues of the mouse body is normal.5,6 The role of TAL1 in the homeostasia of adult hematopoiesis has been recently investigated using a conditional TAL1 deletion model.7,8 Interestingly, deletion of Tal1 in adult bone marrow (BM) hematopoietic cells did not interfere with the reconstitution properties of hematopoietic stem cells and early progenitors such as colony-forming unit-spleen (CFU-S) in transplantation but impaired their erythrocytic and megakaryocytic differentiation capacities. These results strengthened previous findings on the major role that TAL1 plays during erythroid and megakaryocytic cell differentiation since both lineages were strongly impaired in the absence of TAL1 protein.7,8
The biologic activity of TAL1 relies on 2 important domains of the protein. The helix-loop-helix (HLH) domain is common to the HLH transcription factor family and allows its members to homo- or heterodimerize. Hence, this domain is crucial for the heterodimerization of TAL1 with the members of the ubiquitously expressed E2A proteins (ie, E47, E12, and HEB), which are main partners of TAL1.9 The basic domain is present in many HLH proteins where it confers binding to DNA on the E-box consensus sequence CANNTG and further transactivation of target genes.10 Mutations and deletions of these 2 domains and studies of the mutated TAL1 proteins in functional assays have indicated that the DNA-binding domain, but not the HLH dimerization domain, is dispensable for some TAL1 properties. The TAL1 protein lacking its capacity to bind to DNA can rescue some of the primitive and definitive hematopoietic potentials of Tal1-null mouse ES cells and of the zebrafish mutant cloche.11 Moreover, the same The molecular mechanisms of TAL1 functions have long been thought to be similar to the myogenic or neurogenic bHLH protein ones; that is, a direct positive (or negative) transcriptional regulation of target genes through DNA binding to consensus E-box sequences present in the regulating sequences of several genes.13 In vitro transcription assays have demonstrated that TAL1 can participate in large protein complexes that comprise E12, the LIM protein LMO-2, Ldb1, and GATA-1 or SP-1 in the erythroid differentiation or LMO-2 and E12/E47 or GATA-3 in T-cell acute lymphoblastic leukemia (T-ALL) cells. These complexes display transcriptional activity on promoters containing an E-box, an SP-1, or a GATA binding site.14-16 Finally, results showing that mutated TAL1 proteins have some functional activities have led to the emergence of a new concept of the molecular mechanisms by which TAL1 may act, such as the sequestration of a repressor or the titration of other proteins.11,12 The role of TAL1 during erythropoiesis is documented in the mouse,1 and recent experiments performed on human CD34+ cells have shown the major role of this transcription factor for human erythroid differentiation.16,17 However, these experiments used enforced TAL1 expression by oncoretroviral transduction and are limited by the levels of gene transfer obtained in the human primary hematopoietic cells using such vectors.
We have recently developed an approach to transduce efficiently the overall hierarchy of human hematopoietic cells using lentiviral TRIP vectors18,19 that avoid the selection of transduced cells. Using a 2-step culture system that reproduced the early and late erythroid differentiation,20 we found that enforced expression of TAL1 into CD34+ cord blood cells enhances erythroid differentiation, leading to 2 times more erythroid burst-forming units (BFU-Es), 10 to 20 times more erythroid colony-forming units (CFU-Es), and 2 times more glycophorin A-positive (GPA+) cells than in control cells. The long-term culture initiating cell (LTC-IC) compartment is also increased, with up to 3 times more clonogenic progenitors produced in the presence of high levels of TAL1. Part of this activity is independent of DNA binding since enforced expression of TAL1 deleted of its DNA-binding domain enhances CFU-E production and
Lentiviral vector
The lentiviral vector TRIP
Lentiviral vector supernatants
Vector particles were produced by transient calcium phosphate cotransfection of 293T cells.21 The viral titers measured on 293T cells were 2.6 x 1010/mL to 10 x 1010/mL using the TRIP Collection and fractionation of hematopoietic CD34+ cells
Umbilical cord blood (UCB) samples were collected with the informed consent of the mothers, according to approved institutional guidelines. CD34+ cells were purified by immunomagnetic selection (Miltenyi Biotec, Paris, France).22 CD34+ cells (purity Transduction protocols Human CD34+ cells were plated at 1 x 106 cells/mL in serum-free medium (RMB00; Mabio, Tourcoing, France) in the presence of recombinant human (rhu) stem cell factor (SCF; 100 ng/mL; Amgen, Neuilly sur Seine, France), Flt3-ligand (FL; 100 ng/mL; Immunex, Seattle, WA), interleukin 3 (IL-3; 60 ng/mL; Novartis France, Rueil-Malmaison, France), and thrombopoietinmimetic peptide23 (TPOmp; 25 nM; Genosys Biotechnologies, St Quentin en Yvelines, France). Concentrated lentiviral vector particles were added at a concentration of 2500 ng/mL viral P24 twice at 24-hour intervals for a total of 72 hours as we recently described.19 Cells were then washed and cultured in conditions that support lymphoid and myeloid differentiation22 during 72 hours. EGFP expression in the CD34+ cell population was then analyzed by flow cytometry (FACScalibur; Becton Dickinson, Pont de Claix, France). Jurkat-L4, a Jurkat subclone that expresses a mutant TAL1 protein deleted in its carboxy terminal domain24, was transduced with the different vectors during 48 hours, in presence of a single dose of 350 ng/mL viral P24 in RPMI containing 10% fetal calf serum (FCS). Cells were then washed and established as cell lines in the same medium. Western blot analysis
Western blot analysis was performed as previously described.24 Briefly, 5 x 105 transduced CD34+-derived cells, Jurkat, Jurkat-L4, or Jurkat-L4-transduced cells were pelleted and lysed into Laemmli buffer (60 mM Tris-HCL, pH 6.8; 5% 2 beta-mercaptoethanol; 2% sodium dodecyl sulfate [SDS]; 15% glycerol) and total protein extracts were run onto a 10% SDS-polyacrylamide gel electrophoresis (PAGE) gel. After protein transfer, the TAL1 and Hematopoietic cell cultures Colony-forming cells (CFCs) and LTC-ICs were assayed as described.25 Erythrocytic potential was assessed in specific culture conditions. Cells were cultured for 6 days in serum-free medium in the presence of IL-3 (10 ng/mL), IL-6 (10 ng/mL) and stem cell factor (SCF) (25 ng/mL) then for 4 additional days in the presence of the same cytokines supplemented by 2000 IU/L erythropoietin (Epo).20 During this culture, cells were phenotyped at different time points by FACS analysis for expression of differentiation markers using monoclonal antibodies (MoAbs), CD36-phycoerythrin (PE) (clone CB38; Pharmingen, Pont de Claix, France), GPA-PE (clone 11E4B-7-6), CD34-PECy5 (clone QBEnd10), and CD34-allophycocyanin (APC) (clone 581) (all from Immunotech, Villepinte-Roissy CDG, France). In all the FACS analyses, nonspecific staining was measured using irrelevant labeled mouse immunoglobulin G1 (IgG1)-PE/PC5/APC (all from Immunotech) and IgM (Pharmingen) MoAbs. PCR analysis
Integration of the TRIP RNA extraction and cDNA synthesis Sorted cells (105 cells) were lysed in 200 µL TRIzol (Invitrogen, Groningen, The Netherlands) and total RNA was purified as recommended by the manufacturer. Total RNA was then reverse transcribed using random hexamers and the Superscript RT kit (Invitrogen) according to manufacturer's instructions. The cDNA product was diluted 10-fold prior to PCR amplification. Real-time quantification PCR
Real-time PCR was performed using a LightCycler rapid thermal cycler system (Roche Diagnostics, Lewes, United Kingdom) according to the manufacturer's instructions. Reactions were performed in a 10 µL volume with 0.5 µM primers, 3.5 mM MgCl2, and LightCycler-DNA Master SYBR Green I mix (Roche Diagnostics) including nucleotides Taq DNA polymerase and buffer. Typical PCR protocol consists of a TAQ polymerase activation step at 95°C for 10 minutes followed by 40 cycles with 60°C to 64°C annealing for 5 seconds, and 72°C elongation for 15 seconds. Primer sets were designed to span introns and were tested on dilution of cDNA from the UT7 cell line to ensure PCR efficiency and specificity. Primers used are as follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sense 5'-GGGAAACTGTGGCGTGAT-3', antisense 5'-GGAGGAGTGGGTGTCGCTGTT-3'; c-kit, sense 5'-TTCTTACCAGGTGGCAAAGG-3', antisense 5'-AAATGCTTTCAGGTGCCATC-3'; Statistical analyses Statistical analyses were done using a Student t test (paired, 2-sided). Data were considered statistically significant when P was less than .05.
Lentiviral transduction and expression of TAL1 and bTAL1 in human hematopoietic cells
The human TAL1 and
We have recently defined conditions that reproducibly allow high-efficiency transduction and expression of the lentiviral vector TRIP-EGFP in human CD34+ cells.19 To determine the gene transfer efficiency of the TRIP-TAL1 and TRIP-
The amount of transgenic TAL1 and
Altogether these results indicate that TAL1 and Function of TAL1 in early differentiation of human CD34+ cells into erythroid cells
To study the effect of TAL1 and
After transduction, more than 90% of the cells were CD34+, or whatever TRIP vector was used (Figure 3A). From that time, cells that overexpressed TAL1 displayed a similar differentiation profile to EGFP+ control cells (Figure 3A-B) with a gradual but rapid decrease of the proportion of CD34+ cells; at day 5, less than 20% of cells were CD34+ (Figure 3A). On the contrary, cells that expressed
These results show that overexpression of TAL1 does not affect the first steps of erythroid differentiation in liquid culture. However, TAL1 without its DNA-binding domain maintains cells that express the CD34 antigen. This suggests that TAL1 DNA binding plays an important role for efficient progression through erythroid differentiation or that the CD34 gene expression is dependent on bHLH proteins. Enforced TAL1 expression enhances CFU-E production independently of DNA binding
We next studied the progenitor compartments after transduction of CD34+ cells with the TRIP-TAL1 and TRIP-
To discriminate between a functional impact of
The transduced CD34+CD36+ cell population that mainly contained erythroid progenitors had similar cloning efficiencies with whatever TRIP vector had been used. However, the enforced expression of TAL1 and
In both experiments, TAL1 and Altogether, these results indicate that enforced expression of TAL1, with or without its DNA-binding domain, enhances erythroid differentiation through the production of erythroid progenitors, mainly CFU-Es.
Enforced expression of TAL1 but not
TAL1 is known to play a role during erythroid differentiation and in the development of hematopoiesis in the mouse.1 This effect has been described to be independent of the DNA-binding capacity of TAL1.11 As cells transduced with the TRIP-
Cells recovered after transduction or CD34+CD36- cells, sorted after 2 to 3 days in the presence of SCF, IL-3, and IL-6, were tested for their LTC-IC potentials. Results indicate that enforced TAL1 expression increases the number of colonies generated after 5 weeks in LTC up to 3-fold (x2.2 ± 0.8; n = 4) in cells immediately after transduction, and 2.4 and 3 times (n = 2) in the transduced CD34+CD36- sorted cells (Table 2). This increase is not related to the effect of TAL1 on BFU-Es, since more than 90% of the colonies were CFU-GMs.
These results indicate that expression of TAL1 without its DNA-binding domain in CD34+ cells has little impact on the human immature LTC-IC compartment and are in agreement with the results obtained with the conditional knockout of TAL1 in adult murine HSCs.7,8 In addition, we show that enforced expression of the wild-type TAL1 protein has a positive effect on the development of LTC-ICs. Altogether, these results suggest that this effect requires binding to DNA. The effect of enforced expression of TAL1 on progenitor cells is not mediated by an increase of c-KIT expression
As TAL1 and
Finally, we performed a Western blot analysis on transduced CD34+ cells to measure c-KIT protein levels at day 0 and day 5 of the erythroid culture. The results of 2 experiments indicated that enforced expression of TAL1 and
These results highlight modulations of the c-kit mRNA levels when TAL1 and Positive role of TAL1 on erythroid differentiation depends on DNA binding
To study the effect of enforced TAL1 expression during the erythroid differentiation, the onset of the GPA marker was followed by flow cytometry during the erythroid liquid culture. When cells transduced with TRIP-TAL1 were cultured in the presence of SCF, IL-3, and IL-6, but without Epo, a major effect was seen in the GPA+ population, which was raised to 19.8% ± 5% (n = 4) at day 3 and 45% ± 11% (n = 2) at day 5 compared with 7.3% ± 3% and 17.6% ± 1.9% in the EGFP+ cells (Figure 5A; P < .05). This result correlated with a 3- to 4-fold increase in the absolute number of GPA+ cells in the TRIP-TAL1-transduced cells compared with the EGFP+ cells at day 3 to day 5. Moreover, analysis of the
The increase in the percent and numbers of GPA+ cells with TAL1 overexpression progressively disappeared upon addition of Epo to the culture. At day 7, the difference in proportion and absolute cell numbers between TRIP-TAL1 and TRIP-EGFP cells was still significantly different (Figure 5A,C; P < .05) whereas at day 10 both populations were identical (Figure 5A). The effects of enforced TAL1 expression on the production of GPA+ cells are not mediated through an increased sensitivity to Epo since a low dose (50 IU/L) of Epo leads to a low production of cells, including GPA+ cells, with whatever TRIP (EGFP/TAL1/
Results were different with cells transduced with TRIP- These results show that enforced TAL1 expression allows an early wave of differentiation of CD34+ cells into mature GPA+ erythroid cells independently of the presence of Epo in the culture system. This effect is dependent on the presence of the DNA-binding domain. Moreover, overexpression of wild-type or mutant TAL1 protein transiently pertubates GPA+ cell production in the presence of Epo but independently of its dose.
In this study, we show that enforced TAL1 expression enhances human erythroid differentiation and that some of these effects need DNA binding.
We previously developed and described TRIP lentiviral vectors to transduce the whole hierarchy of human hematopoietic cells with a high efficiency.18,19 To our knowledge, this is the first description of the use of a lentiviral vector to study the role of enforced protein expression in primary human hematopoietic progenitor cells. Interpretation of previous works of enforced TAL1 expression in human CD34+ cells is limited by the low levels of gene transfer obtained with oncoretroviral vectors and by the type of cells targeted by this gene transfer, mainly mature clonogenic progenitors. Consequently, the effect of such enforced expression was studied on transduced cells selected on the resistance to antibiotics.16,17 Our approach offers a new way of studying the effect of overexpressing transgenic proteins in a high proportion of human hematopoietic progenitors, mature and immature, avoiding bias of selection and allowing expression of transgenes to be measured at the protein level. In these conditions, levels of TAL1 and
A major role for TAL1 during erythropoiesis has been suggested by previous reports performed in cell lines and primary hematopoietic cells.1 Our results definitively show the function of TAL1 as a positive regulator of human erythroid differentiation. TAL1 overexpression increases erythroid differentiation based on (1) an increased The absence of a negative effect of enforced TAL1 expression on CFU-GM production (Table 1) and on the production of macrophages or granulocytes in liquid cultures (E.R., D.R., M.T., et al, unpublished observations, December 2002) was unexpected. Down-regulation of TAL1 expression has been reported to occur in early myeloid differentiation and to be necessary for proper monocytic and granulocytic differentiation.29,30 Constitutive expression of TAL1 in cell lines such has TF-1, M1, HL-60, and 32D, further cultured in conditions that allow myeloid differentiation, interfered with the expected monocytic and granulocytic differentiation.28-30 When enforced TAL1 expression was obtained in primary human hematopoietic cells, contradictory results were reported that seemed to be dependent on the culture conditions.16,17 Our results on CFU-GMs suggest that the positive effect we observed on progenitors generated from LTC is related to an increase of the LTC-IC compartment rather than a direct effect on the CFU-GM progenitors. Enforced expression of TAL1 increased the number of progenitors detected after 5 weeks, progenitors that were mainly CFU-GMs as in controls. Of importance, BFU-Es were detected in these cultures only when TAL1 was overexpressed (not shown), suggesting that TAL1 can interfere with the potential of LTC-ICs. Whether these BFU-Es are derived from LTC-ICs or are clonogenic progenitors that survive during the LTC is presently not known. The study of the effect of enforced TAL1 expression, especially on primitive progenitors that require long-term experiments, is limited by the fact that TAL1 is constitutively expressed during the overall culture. As a first approach, we developed a system of conditional TAL1 function using a fusion between the cDNA of TAL1 and that of the mutated estrogen receptor ERt2. However, as recently described,31 adverse effects of ERt2 alone or of tamoxifen, the ERt2 ligand, on human hematopoiesis were detected (E.R., D.R., M.T., et al, unpublished results, November 2001).
Several studies have shown that some TAL1 functions do not need DNA binding, implying a different molecular mechanism for some activities of TAL1 than direct binding to an E-box present in its target genes.11,12,15,32 In this study, we distinguish DNA-binding-dependent and -independent functions of TAL1 during human erythropoiesis. The increase of
The molecular mechanisms that underlie TAL1 functions are still a matter of debate, as very few target genes are known. TAL1 has been reproducibly shown to interfere with apoptosis,24,28 and this effect could be mediated by cytokines and/or by their receptors. The main candidate that was described to regulate or be regulated by TAL1 is c-KIT, the SCF receptor,32,34-36 although contradictory data exist.37 Correlation between c-KIT and TAL1 expressions has been documented, as has interference of c-KIT expression, and thus survival of cells in response to SCF, when antisense TAL1 or In conclusion, this study gives new insights into the functions of TAL1 in human erythropoiesis. Specifically, we show that specific steps of erythroid differentiation that are regulated by TAL1 do not depend on DNA binding whereas others do. Whether these results suggest a dynamic turnover of the protein complexes | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
-encoding enhanced green fluorescence protein (EGFP) has been described.
80%) were used either immediately or after storage in liquid nitrogen. 
) and CD34+CD36+ (
) cells. Results (mean ± SD; n = 4) are compared with those of EGFP+ control cells, referred to as 100%. Inserted in the figure are results of CD34 mRNA expression in both CD34+CD36- and CD34+CD36+ populations obtained using quantitative RT-PCR analysis (representative of 2 experiments). Statistical analyses were performed using the Student t test (paired, 2-sided). * P < .05.
), TAL1- (
), and