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Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, and Departments of Pharmacology, Biochemistry, and Molecular Biology and Institute of Immunovirology and Cancer, University of Montréal, Montréal, Québec H3C 3J7, Canada
Received 7 July 2003/ Returned for modification 22 August 2003/ Accepted 7 November 2003
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ABSTRACT |
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INTRODUCTION |
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Like other tissue-specific bHLH factors, SCL forms E-box (CANNTG) binding heterodimers with ubiquitous bHLH partners known as E-proteins, which include products of the E2A gene (E12 and E47), HEB, and E2-2 (27). In erythroid cells, SCL is found in a multifactorial complex (SCL complex) with E47, LMO2, Ldb1, and GATA-1 (68). Although potential binding sites for the SCL complex are found in erythroid genes such as GATA-1 and EKLF (1, 66), functional dissection of the mechanism of action of SCL on erythroid targets remains to be documented. For example, the importance of the N-terminal transactivation domain and the basic domain of SCL remains controversial, as they are both dispensable for the genetic rescue of specification of the hematopoietic cell fate (44) and for c-kit transcription activation (31) and yet DNA binding-defective mutants of SCL fail to rescue the maturation of definitive hematopoietic lineages in SCL-/- ES cells (44) and to induce erythroid differentiation in established cell lines (3). In more primitive hematopoietic progenitors, GATA-2 can function within the SCL complex (as has been observed in the context of the c-kit promoter) (31). This study also identified Sp1 as a novel component of the SCL complex (consistent with the importance of Sp1 in hematopoietic gene regulation) (57). In leukemic cells, SCL also associates with E2A gene products LMO2 and Ldb1 (22); in this cellular context, however, SCL and LMO1/2 may inhibit the normal functions of E-proteins, which are crucial regulators of lymphoid cell differentiation. Thus, the functions of SCL may differ depending on the cellular context and target genes. To clarify our understanding of the functions played by SCL in different hematopoietic lineages, it is crucial to define the mechanisms by which SCL and its partners regulate the expression of candidate target genes in these cellular compartments.
We previously demonstrated that ectopic expression of SCL in TF-1 cells, a bipotent cell line that can be induced to differentiate along the erythroid or monocyte/macrophage lineages, increases cell surface expression of the erythroid marker glycophorin A (GPA) and renders the induction of erythroid differentiation more efficient (26). GPA is one of the most abundant erythrocyte membrane proteins, and its highly glycosylated sialic acid-rich extracellular domain is predominantly responsible for the negative charge of the red cell membrane. Despite the recognition that SCL collaborates with its partners to activate transcription and determine the hematopoietic fate (31, 33, 68), there is little evidence for the formation of a functional high-molecular-weight SCL complex with regulatory sequences of physiological target genes. In the present report, we show that the SCL complex determines GPA gene expression and that the main function of SCL is to assemble this complex on target gene regulatory elements to activate transcription.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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G (13), and E47-bHLH (46) were generously provided by Stuart H. Orkin (Harvard Medical School, Boston, Mass.), John D. Crispino (Ben May Institute for Cancer Research, Chicago, Ill.), and Jacques Drouin (Institut de Recherches cliniques de Montréal, Montreal, Quebec, Canada), respectively. For retroviral infections, the FOG and E47-bHLH cDNAs were subcloned into the MSCV-neo vector. GPA promoter fragments -456, -116, -84, and -79 were PCR amplified (using forward primers [-456 to -440, -116 to -100, -84 to -68, and -75 to 60, respectively] and a reverse primer [+56 to +40]) from human genomic DNA. Amplified fragments were digested with BglII/KpnI and ligated upstream of the luciferase gene in the pXPIII plasmid (31). Nucleotide positions are numbered relative to the transcription initiation site as described by Rahuel and colleagues (48, 49). GPA promoter point mutations were generated by three-step PCR and resulted in the nucleotide substitutions indicated (see Fig. 3). Vectors encoding GST-GATA-1, GST-LMO2, and GST-SCL were generated by cloning PCR-amplified cDNAs into the pGex2T plasmid (Amersham Pharmacia Biotech, Piscataway, N.J.), while the origin of the GST-Sp1 vector was described previously (31). All vectors were verified by sequencing.
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bSCL, SCL-Nt, AS-Ldb1, AS-LMO2, FOG, and E47-bHLH and their MSCV control were produced by transfection of the 293 GPG retroviral packaging cell line (42). The viruses were concentrated by ultracentrifugation, and TF-1 cells were then grown for 24 h in the presence of concentrated viruses and 8 µg of Polybrene/ml. Following the infections, the cells were recovered and polyclonal populations were analyzed 1 week after selection in G418 at 1 mg/ml. For infections of primary hematopoietic cells, fetal livers from E14.5 embryos were dissected, disaggregated into single-cell suspensions, and washed in IMDM containing 10% FCS. The cells were then incubated overnight with control (MSCV-YFP) and AS-SCL-expressing (AS-SCL-YFP) retroviruses in the presence of 4 µg of Polybrene/ml. Following infection, cells were washed and cultured in IMDM containing 10% FCS, 1U of erythropoietin/ml, 100 ng of Steel factor/ml, and 50 ng of interleukin-3/ml. After 24 h, infected cells were analyzed by fluorescence-activated cell sorter (FACS) and reverse transcription-PCR (RT-PCR) as described below.
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RT-PCR analysis. Total RNA from TF-1 or fetal liver cells was prepared as detailed previously (25) and was reverse transcribed using a Superscript first-strand cDNA synthesis system (GIBCO Invitrogen Corporation). For PCR amplifications, 2 µl of a cDNA sample was added to mixtures containing 1 µM of forward and reverse primers, 20 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 5% dimethyl sulfoxide, 0.2 mM deoxynucleoside triphosphate, and 1.25 U of TaqDNA polymerase. Samples were amplified for 18, 20, and 22 cycles (94°C for 30 s, 55°C for 30 s, and 72°C for 30 s), and PCR products were migrated on a 1.5% agarose gel, transferred on nylon membranes, and hybridized with internal probes. The blots were exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, Calif.). Human GPA, murine GPA, SCL, and lysozyme mRNA levels were quantified using ImageQuant software (Molecular Dynamics) and are expressed as ratios over hS14 or mS16 signals. Data for oligonucleotides used for amplifications are presented in Table 1.
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-monothioglycerol. Colony formation was monitored at appropriate times, and day 7 mixed hematopoietic colonies were picked, washed in PBS, and subjected to RNA extraction and RT-PCR. Transfections and nuclear extracts. Transactivation assays were performed essentially as previously described (31). NIH 3T3 cells were transfected using calcium phosphate 24 h after plating 30,000 cells/well in 12-well culture plates. GPA reporter constructs were kept at 1.5 µg/well, while 100 ng of cytomegalovirus (CMV)-ß-galactosidase (CMV-ß-Gal)/well was included in each transfection mixture as an internal control for normalization. Expression vector doses are indicated in the figure legends. The total DNA was kept constant at 4.5 µg/well with pGem4 (Promega, Madison, Wis.). Luciferase and ß-Gal activities were measured 36 h posttransfection. All luciferase values were normalized using ß-Gal values. Results are shown as the means ± standard deviations (SD) of one experiment performed in triplicate and are representative of three or more independent experiments (depicted in the figures).
TF-1 cell nuclear extracts were prepared as previously described (30). For BOSC23 cell extracts, the cells (4.2 x 106) were first plated and then transfected 24 h later with the expression vectors for LMO2 and Ldb1 (11.25 µg) as well as for GATA-1, E47, and SCL or SCL (2.25 µg) mutants. At 36 h after transfection, the cells were harvested, washed twice in cold PBS, and subjected to nuclear extraction as indicated above.
Gel shift, pulldown, and chromatin immunoprecipitation (ChIP) assays. Binding reactions were performed at room temperature for 15 min in the presence of 0.5 µg of poly(dI-dC) in 20 mM HEPES (pH 7.5)-50 mM KCl-1 mM dithiothreitol-1 mM EDTA-5% glycerol-10 µg of bovine serum albumin-15,000 cpm of double-stranded probe-1 to 20 µg of TF-1 or BOSC23 cell nuclear extract in a total volume of 20 µl. The sequences of the GPA-84 probe and mutant promoter fragments used for competition experiments are indicated (see Fig. 3). For antibody supershift assays, 1 µg of the following affinity-purified antisera was used: goat anti-GATA-1 (M20), mouse anti-E2A (YAE), rabbit anti-E47 (N-649), goat anti-Ldb1/CLIM-2 (N-18), and rabbit anti-Sp1 (PEP-2) antibodies (all from Santa Cruz Biotechnology Inc., Santa Cruz, Calif.). The BTL73 mouse anti-SCL antibody was kindly provided by D. Mathieu (Institut de Génétique Moléculaire, Montpellier, France). As a control, equal amounts of species-matched serum Ig (Sigma, St. Louis, Mo.) were added to the binding reactions. Protein complexes were resolved by electrophoresis at 150 V on 4% polyacrylamide gels in 0.5x Tris-borate-EDTA at 4°C.
For pulldown assays, glutathione S-transferase (GST), GST-SCL, GST-LMO2, GST-GATA-1, and GST-Sp1 were purified from bacteria and coupled to glutathione Sepharose beads (Amersham Pharmacia Biotech). A TNT-coupled reticulocyte lysate system (Promega) was used to translate SCL, GATA-1, LMO2, Ldb1, and luciferase in vitro in the presence of [35S]methionine. Labeled proteins (15 µl) were incubated with 2 µg of immobilized GST fusion proteins in 400 µl of binding buffer (50 mM Tris-HCl [pH 8.0], 2 mM EDTA, 1% Nonidet P-40 [NP-40], 5 mM dithiothreitol, 10% glycerol, 200 µg of ethidium bromide/ml) for 2 h at 4°C with agitation and then centrifuged for 1 min at 3,000 x g. Samples were washed three times with binding buffer, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, and visualized and quantified using ImageQuant software (Molecular Dynamics).
ChIP assays were performed essentially as described previously (31, 59). Briefly, exponentially growing TF-1 cells were fixed by incubation with formaldehyde (1% final) for 10 min at room temperature. After the formaldehyde was quenched with glycine (0.125 M final concentration), cells were sequentially washed and sonicated to make chromatin extracts (ranging in size from 500 to 1,000 bp) as formerly detailed (31). Protein concentrations were determined by Bradford staining, and 500 µg of chromatin extract was incubated overnight at 4°C with specific antisera against SCL and its partners or control Ig (which are described above). An aliquot of chromatin extract was kept for isolation of input DNA. Samples were then precipitated by the addition of Pansorbin cells (Calbiochem, San Diego, Calif.) for 30 min at 4°C. Precipitated chromatin samples were sequentially washed and eluted as previously detailed (31) and incubated overnight at 65°C to reverse cross-linking. After RNA and proteins were degraded (using 30 µg of RNase A for 30 min at 37°C and 120 µg of proteinase K for 2 to 3 h at 37°C), DNA was phenol-chloroform extracted and precipitated with ethanol in the presence of 10 µg of tRNA as a carrier. PCRs were then performed on precipitated samples as described previously (31). PCR products were migrated on a 1.5% agarose gel, transferred to nylon membranes, and hybridized with internal oligonucleotide probes. Oligonucleotide data are indicated in Table 1.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether this correlation was also observed in primary hematopoietic cells, we next infected primary E14.5 fetal liver cells with control (MSCV-YFP) and AS-SCL (AS-SCL-YFP)-expressing retroviruses, which also encode the fluorogenic protein YFP, allowing for analysis of infected cells in the YFP+ fraction. It has recently been demonstrated that the Ter119 antibody, which specifically labels cells of the erythroid lineage, recognizes an epitope on mouse GPA (5). Flow cytometric analysis of viable fetal liver cells following gene transfer revealed a dramatic reduction in Ter119 reactivity in the YFP+ fraction of AS-SCL-infected cells compared to that seen with control samples, as the proportion of Ter119+ cells dropped from 16 to 3% (Fig. 1D). In contrast, the proportion of Ter119-reactive cells in the uninfected population (YFP-) was 32% for both AS-SCL and control MSCV cells. In AS-SCL-infected cells, this reduction in Ter119 labeling is concomitant with a threefold decrease in GPA mRNA expression as determined by RT-PCR analysis (Fig. 1E). Together, these results demonstrate a close correlation in SCL and GPA levels in an erythroid progenitor cell line (TF-1) and in primary fetal liver erythroid cells. In both cell types, the level of GPA per cell decreases when SCL levels are lowered, as flow cytometry analysis reveals fluorescence signals at the single-cell level.
To assess whether GPA expression in primary hematopoietic cells increases following SCL gain of function, we next analyzed SCL and GPA expression in multipotent colonies (CFU-GEMM) derived from bone marrows of wild-type or SIL-SCLtg mice, which express SCL ubiquitously (2). Total RNA was extracted from individually harvested multipotent colonies, and gene expression was assessed by RT-PCR analysis. Gene expression within single multipotent colonies was normalized on the basis of the control S16 mRNA level (Fig. 1F) and quantified as the ratio over S16 (Fig. 1G). This analysis revealed a linear relationship between the expression levels of SCL and GPA in individual colonies from both wild-type and SCLtg mouse bone marrows, with a correlation coefficient of 0.7. Furthermore, in colonies from SCLtg mice, which exhibit on average a twofold-increased level of SCL, GPA expression was increased fourfold whereas levels of the myeloid marker lysozyme remained constant (Fig. 1H). Together, data shown in Fig. 1 indicate a close correlation between SCL and GPA levels in the TF1 cell line and in primary hematopoietic cells.
The GPA promoter is activated by a complex containing SCL, E47, LMO2, Ldb1, and GATA-1. Increased GPA expression in SCLtg mouse colonies might be due to a direct effect of the SCL transgene on GPA expression or to an indirect increase in the erythroid content of each colony. To address the question of whether GPA is a direct target of SCL, we performed transactivation assays using heterologous NIH 3T3 cells and a reporter vector in which the GPA promoter region (from position -456 to position +56) was inserted in front of the luciferase gene (GPA-456). Using this assay, Lécuyer et al. previously demonstrated that the regulation of c-kit promoter sequences by SCL requires its integration within a multifactorial complex (SCL complex) containing E47, LMO2, Ldb1, and GATA-1/-2 (31). When transfected on its own, the GPA-456 reporter shows a low background level of luciferase expression comparable to the promoter-less reporter pXPIII (data not shown). GATA-1 could activate GPA-456 on its own by three- to fourfold (Fig. 2A) (consistent with a previous report on the glycophorin B gene [GPB] promoter, which is highly homologous to the GPA promoter) (49). In contrast, expression of SCL and E47 had no effect on promoter activity. When we coexpressed SCL with E47, LMO2, Ldb1, and GATA-1, strikingly, the GPA promoter was synergistically activated by 20- to 25-fold over its basal level (Fig. 2A and B). Omission of either one of the expression vectors from the transfection mixtures severely reduced promoter activation, demonstrating that each partner was required for synergistic transactivation (Fig. 2B). These results suggest that SCL and its partners regulate GPA expression through direct activation of the proximal GPA promoter.
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The SCL complex activates the GPA promoter through an E-box motif, two GATA binding sites, and a Sp1 binding site. We next sought to identify the cis elements that were required to recruit the SCL complex to the GPA promoter. We first generated a series of GPA promoter 5' deletion mutants to identify the minimal promoter sequence that remained maximally activated by the SCL complex. As shown in Fig. 3A, a promoter segment lacking the sequence up to position -84 (GPA-84) was still maximally activated and further deletion up to position -75 (GPA-75) resulted in a dramatic decrease in activation. Segments of the proximal GPA and GPB promoters were previously shown to contain sequences necessary for erythroid-specific expression (references 48 and 49 and data not shown). These studies had highlighted the presence of functionally important GATA motifs at positions -36 (G1) and -74 (G2) and an Sp1 binding site at -48 (Sp1) (Fig. 3B). An E-box sequence at position -70 (E) was also previously characterized, although its involvement in erythroid cell-specific expression of the glycophorin promoters remained unclear (10). The E and G2 elements are overlapping between positions -79 and -70 of the promoter, the G2 site being arranged in an opposite orientation relative to the GPA gene (Fig. 3B). In the GPA-75 construct, which shows a severe decrease in activation, both of these motifs were affected by the deletion, demonstrating that they are important for responsiveness to the SCL complex (Fig. 3A). To address whether the G1, G2, Sp1, and E elements were required for activation by the SCL complex, point mutations were introduced into these motifs as indicated in Fig. 3B. Promoter activation was greatly reduced when mutations were introduced into each of these elements (G1 M, G2 M, E M, and Sp1 M) and was completely abolished when all of the sites were simultaneously mutated (Fig. 3A). The requirement for an Sp1 binding site for promoter activation by the SCL complex is not unexpected, since Lécuyer et al. have previously demonstrated that Sp1 helps to recruit the SCL complex to the proximal c-kit promoter in hematopoietic progenitor cells (31). These results demonstrate that several cis elements collaborate to confer transcription activation by the SCL complex to the GPA promoter.
SCL and its partners form a low-mobility complex on the GPA promoter. We next addressed the question of whether the SCL complex assembles on the GPA proximal promoter sequence. For this we performed electrophoretic mobility shift assays (EMSA) using the GPA-84 probe, which spans position -84 to position -32 of the GPA promoter (Fig. 3B). We initially performed a titration experiment (in which various concentrations of TF-1 cell nuclear extracts were incubated with the GPA-84 probe) and found that a very-low-mobility complex was formed on this probe at higher concentrations of protein extract (Fig. 4A). This slowly migrating complex was not observed on probes containing the G1 and G2E motifs alone (data not shown). Addition of specific antibodies against SCL, E2A, Ldb1, GATA-1, and Sp1 supershifted or disrupted the migration of the low-mobility complex (Fig. 4B, lanes 2 to 6 and 10), whereas control Ig had no effect (lanes 7 to 8 and 11). This demonstrates that the SCL complex can indeed directly associate with the GPA promoter in vitro.
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EMSA with TF-1 cell nuclear extracts allowed us to demonstrate that the SCL complex associates with the GPA promoter. To assess whether SCL and its partners are necessary and sufficient to form a complex on the GPA promoter, we next sought to reconstitute the complex by ectopic expression in heterologous cells. When EMSA were performed with the GPA-84 probe and nuclear extracts of BOSC23 cells transfected with SCL and its partners, we observed the appearance of a low-mobility complex that was distinct from the background seen with untransfected BOSC23 extracts (Fig. 4D, lanes 1 and 2). To determine whether each partner was required for complex formation, we prepared extracts of BOSC23 cells in which the expression vector for each factor was sequentially omitted from the transfection mixtures. We found that subtracting either partner compromised the formation of the complex on the GPA-84 probe (lanes 3 to 7). Together, these results indicate that consistent with their crucial contribution in promoter transactivation, the presence of all SCL partners is required for the formation of a complex on the GPA promoter.
Partners of the SCL complex occupy the GPA promoter in hematopoietic cells. To address the question of whether SCL and its partners indeed associate with the GPA promoter in vivo in hematopoietic cells, we next performed ChIP assays with TF-1 cell chromatin extracts (31). Exponentially growing TF-1 cells were treated with formaldehyde, and fragmented chromatin was then subjected to immunoprecipitation with antibodies directed against SCL and its partners. Following immunoprecipitation, cross-linking was reversed and associated DNA fragments were purified, serially diluted, and subjected to PCR with primers specific for the GPA promoter region (as well as for the promoter segment of the ubiquitously expressed hypoxanthine phosphoribosyltransferase [HPRT] gene as a control). To confirm the specificity of amplification, fragments were hybridized with 32P-labeled internal oligonucleotide probes. We found that antibodies against SCL, E2A, and GATA-1 were able to precipitate the proximal GPA promoter 10- to 15-fold more efficiently than the HPRT promoter region, whereas control Ig did not bring down these sequences (Fig. 5). In contrast, immunoprecipitation with the anti-Ldb1 antibody showed a modest twofold enrichment of the GPA promoter compared to the levels seen with HPRT. Since Ldb1 is a ubiquitously expressed, it is possible that this factor regulates the expression of both tissue-specific and more widely expressed genes. Nevertheless, these results demonstrate that partners of the SCL complex directly and specifically associate with the GPA promoter in hematopoietic cells.
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bSCL) from the basic domain, which renders them unable to bind to DNA (31, 44), would still be functionally active. These DNA binding-defective mutants were less efficient than wild-type SCL at lower doses of expression vector, although they were active at higher doses. These results suggest that DNA binding by SCL is important for maximal GPA promoter activation, although it is not essential, which is similar to findings regarding the intermediate effect on promoter activation that were observed upon mutation of the GPA E-box motif (Fig. 3A).
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To assess whether the transactivation and DNA binding domains of SCL were required for the induction of endogenous gene GPA expression, we next infected TF-1 cells with viruses encoding SCL, bSCL, and SCL-Nt and monitored GPA mRNA expression by RT-PCR analysis. Immunoblotting was first performed to confirm that retrovirus-mediated gene delivery resulted in efficient overexpression of SCL, bSCL, and SCL-Nt (Fig. 7C, lanes 2 to 4) compared to that seen with mock-infected cells (lane 1). Interestingly, we found that both bSCL and SCL-Nt could induce endogenous gene GPA expression at levels that were similar to those of wild-type SCL (from 3- to 3.5-fold) (Fig. 7D). The high levels of expression attained with retroviral infection (Fig. 7C) may explain why the bSCL is as efficient as wild-type SCL, since transactivation assays revealed a difference between the two proteins at low doses but not at high doses (Fig. 7A). Finally, in consistency with the results of transient assays, the integrity of the putative transactivation domain of SCL is dispensable for the induction of endogenous gene GPA expression. We therefore conclude that SCL functions mainly as a nucleation factor for a multifactorial complex endowed with a capacity to drive erythroid gene expression.
LMO2, Ldb1, GATA factors, and E47 are required for SCL complex assembly on DNA and for gene GPA expression in hematopoietic cells.
Transient reporter assays with GPA regulatory sequences indicate that LMO2, Ldb1, E47, and GATA-1 comprise a transcriptionally active SCL complex. To determine whether the same partners are required for GPA expression in chromatin, we next sought to interfere with members of the complex through diverse strategies. For LMO2 and Ldb1, we generated stable TF-1 cells lines exhibiting reduced LMO2 or Ldb1 protein expression through retrovirus-mediated delivery of AS RNA molecules (AS-LMO2 or AS-Ldb1) (Fig. 8A, lanes 1 to 4). For GATA factors, our results indicate that both GATA-1 and GATA-2 can contribute to the activity of the SCL complex although GATA-1 is more active on the GPA promoter. TF-1 cells express GATA-1 and GATA-2, and both factors interact with FOG (62), a modulator of GATA activity (20, 62). In the context of the GPA promoter, our transactivation assay revealed that FOG coexpression drastically reduced GPA promoter activation by SCL and its partners (Fig. 8B). This inhibitory effect of FOG was due to its direct interaction with GATA-1, since the GATA-1V205G mutant, which is deficient with respect to FOG interaction (13), efficiently replaced normal GATA-1 in the SCL complex to activate the GPA promoter while conferring resistance to inhibition by FOG (Fig. 8D). We therefore generated a TF-1 cell line stably expressing FOG to repress both GATA-1 and GATA-2 activities (Fig. 8A, lanes 5 and 6). Finally, E47 is part of a family of widely expressed proteins that have overlapping functions comparable to GATA factors. We therefore assessed the activity of a truncated E47 protein, comprising the bHLH domain of E47 (E47-bHLH), within the SCL complex. In sharp contrast to the results seen with the SCL-bHLH construct, this truncated protein fails to collaborate with other members of the SCL complex in transient assays (Fig. 8C), indicating that the N-terminal domain of E47 is essential for transcription activation by the SCL complex. Furthermore, this truncated protein was found to be dominantly negative over wild-type E47 (Fig. 8C). We therefore stably expressed the truncated E47-bHLH mutant in TF-1 cells.
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Finally, these interventions significantly decreased GPA mRNA expression as assessed by RT-PCR analysis (Fig. 8E), resulting in a twofold reduction when Ldb1 and LMO2 protein levels are decreased or when E47 binding is displaced by E47-bHLH. Furthermore, there was a fivefold decrease in GPA mRNA levels when GATA factors were sequestered by FOG. Strikingly, these reductions in GPA mRNA levels closely correlate with the decrease in DNA binding observed by EMSA (compare Fig. 8D and E). Taken together, these results strengthen the view that LMO2, Ldb1, GATA factors, and E proteins (more specifically, E47) are important components of SCL-containing complexes and that they are indeed required for GPA gene expression in hematopoietic cells.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SCL in erythropoiesis. The catastrophic consequences of SCL gene ablation in mice, which results in early embryonic lethality due to a complete absence of hematopoietic cells (45, 52, 53, 55), has complicated the assessment of the roles that SCL might play in the maturation of particular blood cell lineages. Several lines of evidence point to SCL as an important regulator of erythropoiesis. First, during development SCL is expressed in both primitive and definitive erythroid cells of the yolk sac blood islands and fetal liver (15, 29, 47). Analysis of hematopoietic precursors has shown that SCL is highly expressed in committed erythroid progenitors (BFU-E and CFU-E/proerythroblasts), whereas it becomes down regulated in terminally differentiated red cells (7, 26, 29). Therefore, the pattern of SCL expression suggests that it might be involved in the initial stages of commitment or consolidation of the erythroid cell fate with respect to pluripotent progenitors. Second, enforced SCL expression in hematopoietic cell lines and primary bone marrow cells favors erythroid differentiation (3, 17, 26, 63). Third, the genetic rescue of SCL-deficient mice and recent conditional gene targeting experiments have demonstrated that SCL is required for proper erythroid differentiation in vivo (23, 36, 54). Indeed, Sanchez et al. (54) showed that a transgene driving SCL expression in stem cells was able to rescue early hematopoietic progenitors in SCL-/- embryos; however, these mice still exhibited a defect in erythroid differentiation that resulted in embryonic lethality, demonstrating that sustained SCL expression is required for erythropoiesis. In recent conditional knockout studies in which floxed SCL alleles were lacking in mice expressing an interferon-inducible Cre recombinase, two groups have demonstrated that SCL inactivation leads to a complete block of erythroid and megakaryocytic cell maturation, seemingly without affecting hematopoietic stem cells (23, 36). Interestingly, Mikkola et al. (36) observed that a population of Ter119+ CD71+ cells (representing normal erythroid precursors) disappears following SCL inactivation, leading to the accumulation of an abnormal Ter119lo/- CD71+ population. Since Ter119 recognizes an epitope on murine GPA (5), the observation made by Mikkola et al. provides additional genetic evidence for the importance of SCL in driving GPA expression during murine erythropoiesis (shown herein). It seems, therefore, that once SCL has specified the hematopoietic cell fate from uncommitted mesodermal precursors, its sustained expression is not required for stem cell function but becomes required anew for the generation of red blood cells and megakaryocytes. Together, these results clearly demonstrate the essential role played by SCL in activating the transcription of erythrocyte-specific genes and driving the erythroid lineage.
Glycophorin genes and GPA promoter regulation. The human glycophorins A, B, and E are part of a family of erythrocyte-specific membrane glycoproteins, which contribute to the expression of blood group antigens and determine the invasion and growth of parasites such as the malaria pathogen Plasmodium falciparum (11, 12). GPA is thought to form complexes with other erythroid membrane components (such as band 3, ankyrin, and protein 4.2), and their association appears to regulate the mechanical properties of the red cell membrane (8, 24). GPA-deficient human red blood cells show decreased sulfate anion transport due to the association between GPA and band 3, the human erythrocyte anion transporter (9). The GPA, GPB, and GPE genes are clustered on chromosome 4q28-q31 and seem to have evolved from successive duplications of the gene GPA (40). The cis-regulatory elements found here to be important for GPA promoter activation by the SCL complex are perfectly conserved in the GPB and GPE promoters (48), suggesting that these genes might also be direct targets of the SCL complex.
Previous studies of glycophorin promoter regulation, mainly focusing on the GPB promoter, demonstrated that these sites are protected from DNase I digestion in the presence of erythroid cell extracts and are required for promoter function (10, 48, 49). While the authors found that GATA-1 was the main factor binding to the G1 and G2 sites in EMSA and that the Sp1 motif was important for promoter activity, they did not identify other partners of the SCL complex as potential regulators of the glycophorin genes (49). Using a probe encompassing a longer segment of the GPA promoter (GPA-84), we demonstrate the existence of a large protein complex containing SCL and its partners. We show that within the GPA proximal promoter, the most crucial determinant for binding of the SCL complex is the G1 motif (followed by the Sp1, G2, and E elements) and that all these sites are required for optimal binding. We further complement these findings with the demonstration (through ChIP) that partners of the SCL complex indeed occupy the GPA promoter in vivo in hematopoietic cells. These findings further underscore the importance of Sp1 within the SCL complex, since Lécuyer et al. previously demonstrated that activation of the c-kit promoter by the SCL complex is critically dependent on the presence of a consensus GC-box and that Sp1 physically interacts with multiple partners of the complex (31).
Involvement of LMO2 and Ldb1 in erythroid gene regulation. The results presented in this report reveal (using transient transactivation assays, ChIP, and AS-mediated loss of function in TF-1 cells) the importance of LMO2 and Ldb1 as essential partners within the SCL complex and establish their requirement for the appropriate regulation of an erythrocyte-specific gene. These findings contrast with those of a previous study suggesting that LMO2 and Ldb1 are negative regulators of erythropoiesis, as their enforced expression was shown to hinder terminal erythroid differentiation of G1ER cells (65), a GATA-1-deficient cell line blocked at the proerythroblast stage of differentiation. While these studies may appear contradictory at face value, previous analyses of Chip, the Drosophila orthologue of Ldb1, may help to reconcile these findings. Chip is a widely expressed regulator of several crucial processes, including embryonic segmentation (38), neuronal development (50), and dorsoventral patterning of the Drosophila wing (18). During wing morphogenesis, Chip associates within complexes containing the LIM-homeodomain protein Apterous (as well as the Drosophila LMO protein) and maintaining the appropriate stoichiometry of these complexes is crucial for proper wing development (18, 37, 51, 64). In this context, both overexpression and loss-of-function mutations of the Chip gene lead to the same phenotypic abnormalities in wing morphogenesis (18). Therefore, if the stoichiometry characteristics of LMO2- and Ldb1-containing complexes are similarly tightly regulated during erythropoiesis, enforced expression of these factors (as performed by Visvader et al.) (65) might interfere with endogenous complexes through sequestration mechanisms and lead to the same outcome as loss-of-function approaches, which were utilized in the present study. In support of this hypothesis, a recent report by Xu and colleagues (70), who identified the protein 4.2 gene as a erythroid target of SCL and its partners, showed that enforced expression of wild-type or a dominant-negative version of Ldb1 perturbed activation of the protein 4.2 gene (consistent with the view that the stoichiometry of these complexes is indeed important for erythroid gene regulation).
In Drosophila, Chip was initially identified as an important regulator of enhancer-promoter communication (38), a property that seemingly relies on its ability to self-dimerize and to interact with several families of regulators, including LIM domain- and homeodomain-containing proteins (58). Furthermore, it has recently been shown that proper patterning of the Drosophila nervous system depends on the ability of Chip to interact physically with Pannier, a Drosophila orthologue of GATA-1, and with bHLH factors of the Achaete/Scute complex (50). Our results extend these findings to show that mammalian Ldb1 also directly interacts with GATA family members and bHLH factors. Interestingly, Ldb1 gene ablation in mice results in severe patterning defects and, among other phenotypes, compromises the development of yolk sac blood islands (39). This hematopoietic phenotype is most likely caused by defects in gene regulation by the SCL complex at the onset of hematopoiesis. We also provide evidence that LMO proteins can self-associate (in similarity to the homodimerization of the LIM proteins CRP and MLP) (4, 19). Therefore, this network of interactions most likely modulates the assembly, targeting, and activity of the SCL complex at different levels in the hematopoietic hierarchy.
Functional specialization within the SCL complex. SCL is an important regulator at several positions in the hematopoietic hierarchy. Whether its molecular mode of action differs in different hematopoietic lineages or populations remains ill defined. In the present study, we found differences in the mechanisms by which SCL regulates the gene GPA versus our previous analysis of the gene c-kit, which constitute erythroid and stem or progenitor cell targets of SCL, respectively. First, we found that maximal GPA promoter activation (at lower concentrations of expression vector) and assembly of the SCL complex on GPA promoter sequences requires an E-box motif and SCL DNA binding activity, although at higher concentrations of SCL (as observed following enforced expression in TF-1 cells) SCL DNA binding mutants are active. In contrast, Lécuyer et al. previously showed that activation of the c-kit promoter by the SCL complex was E-box independent and did not require SCL DNA binding (31). This mechanistic difference is consistent with the observation that SCL DNA binding-defective mutants rescue hematopoietic cell commitment in SCL-/- embryonic stem cells although they are unable to restore the proper maturation of definitive erythroid cells (44). Therefore, the requirement for SCL DNA binding is one characteristic that might delineate SCL function at the onset of hematopoiesis and during erythropoiesis. It is possible that a higher level of affinity of DNA binding by the SCL complex is required for the proper activation of the erythroid program, which would be provided in part by SCL itself and by other partners of the complex, such as GATA and Sp/XKLF family members. It will be possible to assess whether the necessity of SCL DNA binding is a broad difference that distinguishes erythroid and stem cell targets of SCL through the identification and molecular characterization of additional SCL target genes.
Unlike the results seen with SCL, the bHLH domain of E47 is unable to replace the function of the full-length protein during GPA promoter activation. Moreover, this truncated protein is dominant negative over wild-type E47 both in transient assays and in chromatin, as it likely competes for DNA binding with endogenous E proteins. This finding contrasts with that of a previous study of the POMC promoter, in which E47-bHLH was shown to form a functional tripartite complex with NeuroD and Pitx-1 (46). Besides the C-terminally located bHLH domain, the E47 protein harbors two distinctive activation domains in its N terminus (designated AD1 and AD2) which are absent from E47-bHLH. These domains are highly conserved in other ubiquitously expressed bHLH factors such as E12, E2-2, and HEB, and E. Lécuyer and T. Hoang have observed that HEB can functionally replace E47 within the SCL complex (unpublished data). Therefore, it is likely that the transactivation domains of E47 are required for the proper function of the SCL complex (although our results do not exclude the possibility that an unknown function of the N-terminal moiety of E47 might be involved). Interestingly, it has recently been shown that the AD1 domain serves as a recruitment motif for the SAGA histone acetyltransferase complex (32). Since infection of TF-1 cells with E47-bHLH causes an important shift in the mobility of the SCL complex, it is possible that this mutant hinders the recruitment of additional regulatory factors (such as elements of the SAGA complex) to SCL target genes. Further investigation will be required to evaluate this possibility.
Transcription regulation by the SCL complex in different hematopoietic compartments. During differentiation, transcription factor complexes may undergo dynamic changes in composition, a view described as a cocktail party scenario by Sieweke and Graf (56). For example, our observations identified a requirement for Sp1 as a member of the SCL complex in c-Kit-expressing cells and in erythroid cells (reference 31 and the present study) and there is no evidence for the involvement of Sp1 in T cells (41). By evolving in such a manner, the activity and target gene specificity of such multifactorial complexes might be modulated by environmental cues and favor differentiation towards particular cell fates. This type of mechanism would seem energetically cost effective for an organism, as it would bypass the requirement for major dismantling events as a prerequisite to the commencement and shutdown of different programs of gene expression. In this respect, our finding that FOG can inhibit promoter activation by the SCL complex demonstrates how cofactors can modulate the activity of higher-order transcription factor complexes through interactions with specific components. Since the GATA-FOG interaction is essential for erythroid and megakaryocytic cell differentiation (13, 61), our findings suggest that during differentiation into these lineages a proportion of the GATA factor pool is recruited into FOG-containing complexes, thus enabling GATA factors to exert functions that are independent of SCL complexes. Furthermore, our observation that SCL complexes containing GATA-1 or GATA-2 demonstrate preferential activation efficiency for erythroid or stem cell targets, respectively, suggests that SCL-containing complexes may evolve dynamically during hematopoiesis to favor the maintenance of pluripotency or to consolidate differentiation towards specific lineages.
At the onset of hematopoiesis, when GATA-2 is the predominant GATA family member, SCL complexes would be required for the activation of stem cell targets such as c-kit (31), which would favor the maintenance of an undifferentiated phenotype. At this stage, SCL complexes may also start to weakly activate the expression of erythroid targets such as GPA, GATA-1, and EKLF, which would help to prime stem and progenitor cells for their eventual commitment towards the erythroid-megakaryocytic pathways. The activity of such a complex would account for the multilineage gene expression priming that is thought to precede the commitment of hematopoietic stem cells into different lineages (28). Since the level of GATA-1 expression increases in progenitors of the erythroid lineage and that of GATA-2 is down regulated, the replacement of GATA-2 by GATA-1 within the SCL complex might delineate a point at which the activation of erythrocyte-specific genes is engaged more robustly. In the T lineage, however, GATA-3 is preferentially expressed and may substitute for GATA-2 within the SCL complex to drive the expression of T-cell-specific genes (41). Therefore, subtle variations in composition seem to modulate the specificity of action of SCL-containing complexes and may also account for the differential requirements for SCL DNA binding activity in different hematopoietic compartments. This type of mechanism is most likely a recurrent theme in cell fate determination in many other tissues.
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ACKNOWLEDGMENTS |
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We thank Peter D. Aplan for SIL-SCL transgenic mice as well as Stuart H. Orkin, Catherine Porcher, John D. Crispino, and Jacques Drouin for providing expression vectors for SCL mutants, the GATA-1 V205R mutant, FOG, and E47-bHLH, respectively.
This work was supported in part by a grant from the Canadian Institute for Health Research (CIHR) to T.H.; by a studentship from the CIHR (E.L.), and by a fellowship from the Leukemia Research Fund of Canada (S.H.).
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FOOTNOTES |
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REFERENCES |
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, distribution from WT colonies; •, SCLtg mouse colonies). The coefficient of correlation between SCL and GPA mRNA levels (r) is shown. There was no correlation with lysozyme mRNA levels (r = 0.05). (H) Increased GPA and SCL levels in colony cells from SCLtg mice. Data represent the medians of the distributions shown in panel F for mRNA expression in individual colonies from bone marrow cells of SCLtg and age-matched control mice.
