In this study, we examined how the stage-specific expression of the Gata1 gene is attained during erythroid differentiation using the G1BAC-GFP transgenic mouse. We found that the G1BAC-GFP transgenic-mouse system gave rise to stable expression of the GFP reporter in vivo. Therefore, by exploiting this system, we identified the two important erythroid progenitor populations BREP and CREP, containing mainly BFU-E stage progenitors and CFU-E stage progenitors, respectively. Deletion of the G1HE from the G1BAC-GFP construct (i.e., ΔG1HE) resulted in a significant reduction in the GFP-positive progenitor population in the BREP fraction. In contrast, GFP-positive progenitors in the CREP fraction and Ter119-positive erythroblasts were practically preserved in the ΔG1HE mice, albeit the GFP intensity was reduced to almost half of the G1BAC-GFP level. We also found that targeted deletion of G1HE from the chromosomal Gata1 locus (the ΔneoΔHS mutant) markedly suppressed the GATA1 mRNA level in the BREP fraction. These results demonstrate that the G1HE significantly contributes to Gata1 gene activation in BREP stage cells and to the differentiation of BREP cells to CREP stage cells and later erythroblasts.
To further dissect the mechanisms for this decrease of Gata1 gene expression in GFP-positive BREP cells, we deleted the G1HE core from the G1BAC-GFP construct or introduced mutations into the GATA box therein. Transgenic-mouse lines harboring these two mutant BAC constructs showed quite similar decreases in GFP-positive cells in the BREP fraction compared to that in the control mice, indicating that the GATA box is an indispensable motif in the G1HE. Thus, the Gata1 gene is regulated distinctly in each differentiation stage of erythroid cell development. This mode of regulation is summarized in Fig. .
In order to monitor the endogenous Gata1
gene expression profile, in this study we established a novel reporter transgenic-mouse system using a 196-kbp G1BAC. We previously developed and analyzed the plasmid-based Gata1
gene vector G1HRD, which covers approximately 8 kbp of the Gata1
). Except for the difficulty in achieving copy number-dependent transgene expression, which is inherent in the plasmid-based transgenic-mouse approach, the G1HRD substantially recapitulated Gata1
gene expression in the mouse (23
). Furthermore, GATA1 cDNA expressed under the regulatory influence of the G1HRD nicely rescued the GATA1-deficient mouse from embryonic lethality (33
). These results strongly supported the G1HRD containing virtually all the essential regulatory elements for hematopoietic Gata1
gene expression. However, we noticed that the G1HRD had an apparent limitation in fully recapitulating Gata1
gene expression and that the G1HRD could not direct reporter gene expression in BREP or BFU-E stage progenitors (38
). To this end, we examined the G1BAC-based reporter system.
In this study, we found that G1BAC-GFP recapitulated the endogenous Gata1
gene expression in the BREP stage more faithfully than the G1HRD. Importantly, each transgenic-mouse line exhibited reproducible copy number-dependent GFP reporter expression. Thus, the G1BAC construct retained sufficient regulatory information for Gata1
gene expression during erythroid differentiation. We envisage that the G1BAC may harbor an insulator activity somewhere outside the G1HRD region that acts to circumvent the chromosomal position effect variegation at the transgene integration site (reviewed in reference 10
). In this regard, the recent identification of DNase I-hypersensitive sites located beyond the G1HRD region is noteworthy and may provide clues for further study of Gata1
gene regulation (43
GATA1 plays multiple roles during erythroid cell development (3
). We surmise that the molecular basis of these differential contributions of GATA1 to erythropoiesis may be due, at least in part, to the differences in the threshold set by each gene for activation by GATA1. By comparing the expression profiles of GATA1 target genes in erythroid cells from wild-type, Gata1
knockdown, and Gata1
knockout mice, we noticed that these graded levels of GATA1 expression actually influenced the decision of erythroid progenitors to undergo apoptosis, proliferation, or terminal differentiation (28
). We found here that the GFP level, and hence the GATA1 expression level, changes in erythroid cells at each stage. We surmise that this stage-specific expression of GATA1 may be essential for erythropoiesis. Indeed, a sixfold overexpression of GATA1 in differentiating erythroblasts led to blocked erythropoiesis (48
) and decreased GATA1 expression to approximately 5% of the wild-type level, resulting in erythroleukemia in mice (31
). These accumulating lines of evidence support our contention that the stage-specific fine-tuning of Gata1
gene expression is critical to the mechanisms orchestrating erythropoiesis.
We have provided convincing evidence that the conserved GATA box in the G1HE core is required for G1HE activity, especially at the BREP stage. Our next question concerned which GATA factor actually binds to this GATA box at the BREP stage. In the present study, we found that GFP intensity was not decreased in BREP cells harboring the GATA1 knockdown mutation (Fig. and ), suggesting that other GATA factors might contribute to the Gata1
gene activation at the BREP stage. In this regard, it is interesting that GATA2 is expressed in immature hematopoietic progenitors and that this GATA2 expression is repressed by GATA1 during erythroid differentiation (7
). GATA2 is expressed more abundantly in the BREP fraction than in the CREP fraction or later erythroblasts (data not shown). Moreover, the expression level of GATA2 was higher in BREP cells isolated from the Gata1G1.05/+
mice than in those from wild-type mice (data not shown). It was also reported that GATA2 binds to this GATA box in the early stage of erythroid differentiation (23
). Thus, it seems plausible that, in G1HE-mediated Gata1
gene activation, GATA2 works during the early stage of erythroid differentiation or in BREP stage cells but that there is a switch over to GATA1 in the later stage. Considering this situation, it would be intriguing to examine the nature of the transcription factor complex containing GATA2 and its binding to the G1HE core using sorted BREP cells.
One of the important applications of the new cell-sorting strategy for EEP or BREP cells based on the expression of G1BAC-GFP is characterization of the normal erythroid differentiation pathway and disorder of the pathway. We observed an abnormal accumulation of BREP stage cells in the bone marrow of Gata1.05
knockdown heterozygous (Gata1G1.05/+
) mice, as well as Gata1ΔneoΔHS/Y
mutant mice, in which Gata1
gene expression is markedly suppressed in the BREP stage. These observations suggest that in BREP stage cells, GATA1 is essential for driving normal erythroid differentiation and for suppressing the abnormal proliferation of BREP stage cells. Consistent with this notion, it was recently reported that differentiation of GATA1-deficient ES cells was partially arrested at the MEP stage and that these cells are also contained in the BREP population (35
). Since Gata1G1.05/+
knockdown mice develop c-Kit-positive leukemia (31
), the abnormally accumulated BREP population might be predisposed to c-Kit-positive leukemogenesis.
To simplify, the molecular basis of cell differentiation can be defined as the inducible changes in gene expression profiles that are controlled by lineage-restricted transcription factors. Therefore, in order to understand the mechanisms by which the cell differentiation process is regulated, it is crucial to delineate the regulation in terms of the expression of lineage-restricted transcription factors. We have described here how the use of Gata1
BAC transgenic mice, coupled with homologous-recombination-based mutation analysis, proved to be an outstanding approach leading to the identification and dissection of GATA regulatory domains, as well as to a detailed understanding of their activities. The present analysis unequivocally demonstrates a novel function of the G1HE, which is essential for BREP stage-specific Gata1
expression. We speculate that the other Gata1
gene regulatory elements also possess such regulatory functions. As genetic mutations in the human GATA1
locus have been linked to human diseases (4
), the possibility exists that mutations in the regulatory region might also be responsible for human diseases by affecting the expression level of GATA1. Only through the identification of the enhancer functions and an understanding of their precise activities will we be able to elucidate the complex network of regulatory control underlying erythroid differentiation.