gene hematopoietic enhancer, G1HE, is a powerful activator of transcription in a transgenic-mouse assay in vivo (17
). In this study, we have demonstrated that G1HE acts as a classic enhancer element in a reporter transfection assay. Results of a deletion analysis with both the transfection and transgenic-mouse systems indicate that the core 149-bp region within G1HE contains a strong GATA-1
gene regulatory activity for the erythroid lineage. A consensus GATA box, an atypical GAT box, and two E boxes reside within the core region. Mutational analyses of these cis
-acting elements have revealed that while the consensus GATA box is critical for the activity of G1HE in both primitive and definitive hematopoiesis, the other elements are not. Hematopoietic GATA factors GATA-1, GATA-2, and GATA-3 were all demonstrated to bind to the consensus GATA box of G1HE. This result is quite intriguing, since duplicated GATA boxes have been found in the IT promoter (16
) and the upstream region of the IE promoter (3
). These GATA boxes are required for reporter gene expression in transfection analyses. Taken together, these results demonstrate that a network of GATA factors and binding sites regulate GATA-1
gene expression in hematopoietic cells and suggest that GATA-1 and/or other GATA factors work as key regulators in this network through direct interaction with the cis
-acting GATA boxes in multiple regulatory regions. This network seems to be the molecular basis for the erythroid cell-specific expression of the GATA-1
We also found that while the core 149-bp region of G1HE was sufficient to drive lacZ reporter gene expression in erythroid cells, the region was inadequate to generate reporter gene expression in megakaryocytes of E15 fetal liver. To attain megakaryocytic expression of the reporter gene, a slightly longer region of G1HE (i.e., bp 1 to 235) was required. Since disruption of the GATA box at nt 133 in the G1HE reporter resulted in loss of megakaryocytic expression of the reporter gene, the core region was also required for the enhancer activity of G1HE in the fetal liver megakaryocytes. These results thus indicate that G1HE consists of multiple cis-acting elements and that these elements have distinct functions in erythroid and megakaryocytic lineage-specific gene expression.
G1HE can activate transcription in a position-independent manner from a cognate promoter in both the transfection and transgenic-mouse assays. Unlike in the transfection assay, however, the presence of the IE promoter was necessary for the reporter genes to recapitulate the endogenous GATA-1 gene expression profile in the transgenic-mouse assay. Whereas G1HE activated the SV40 promoter in the transfection analysis, it could not do so in the transgenic mice. These discrepancies in G1HE function between the transfection and transgenic-mouse assays may be due to the transgene configuration. In the reporter transfection assay, the reporter gene exists transiently in the nucleus as an episome outside the host genome. In contrast, the reporter gene is integrated into the transgenic mouse genome, where chromatin structure and the influences of regulatory sequences surrounding the integration site can affect reporter gene expression. Thus, the transgenic-mouse assay is a more stringent test for correct gene-regulatory activity than is the DNA transfection assay.
While inversion of G1HE relative to the IE promoter had only a quantitative effect on reporter gene transcription in the transfection assay, proper orientation of G1HE is absolutely required for its activity in the transgenic mice. We do not know the basis for the strict orientation dependence of the G1HE activity in vivo. One plausible explanation is that the three-dimensional structure of the protein complex interacting with the core enhancer (and/or promoter) region may be crucial for transcriptional activation in a chromatin configuration. In fact, a high-molecular-weight complex containing GATA-1 was detected in nuclear extracts of MEL cells with the GGE probe of G1HE. An alternative explanation is to assume that transcription initiation at the GATA-1 locus is regulated by a tracking mechanism and that the inverted G1HE may disturb the synthesis of long RNA transcripts initiated from far upstream. These issues remain to be resolved.
A transcription factor complex containing GATA-1, SCL, and E2A assembled by Lmo2 and Lbd1 was proposed to associate with DNA through an E box and then to be stabilized by interaction with a GATA box in an in vitro reconstitution analysis (32
). Importantly, in G1HE, E box 2 resides 10 bp 3′ to the consensus GATA box, similar to the arrangement of the DNA binding site used in the reconstitution assay. Because this model has never been tested in vivo, we examined the activity of each cis
element in G1HE in detail. While mutation of E box 2 did not affect the enhancer activity at all, mutations in the consensus GATA box severely affected reporter gene expression in yolk sacs, definitive erythrocytes, and megakaryocytes, demonstrating that the GATA box is essential for the activity of G1HE but E box 2 is not. In addition, a high-molecular-weight complex was detected with the GGE probe and the GGEm (E-box-mutated) probe in MEL cell nuclear extracts, while its level was markedly decreased when the GGmE (GATA box-mutated) probe was used. Our results also suggest that the high-molecular-weight complex generated with the GGE probe may be distinct from the complex formed with the consensus GE probe, in that E2A is not involved in the GGE-based high-molecular-weight complex. Thus, GATA-1 or other hematopoietic GATA factors bind to the GATA box in G1HE and serve as an anchoring factor, as well as contributing to the formation of the high-molecular-weight protein complex which regulates GATA-1
An attractive model proposes that GATA-2 binds the GATA motif in the early stage of differentiation whereas GATA-1 replaces GATA-2 in the late stage. Three lines of evidence support this notion. First, we previously demonstrated that G1HE is active in the GATA-1 knockdown environment (26
), suggesting that some other GATA factors can substitute at the GATA box. Second, our recent experiment shows that the GATA-1 knockdown mice can be rescued by GATA-2, and the GATA box in G1HE is indeed occupied by GATA-2 in the mouse cells (Takahashi and Yamamoto, unpublished). This indicates that other GATA factors can actually substitute for GATA-1 in vivo. Third, GATA-1, GATA-2, and GATA-3 can bind to the GATA boxes in the G1HE core (C. D. Trainor, unpublished observation; see above); these sequences actually form a GATA-pal configuration similar to other double GATA sites, to which both GATA-1 and GATA-2 can bind (28
). It has also been shown that the expression of GATA-2 precedes that of GATA-1 in the hematopoietic lineage (8
SCL-null mice were reported to die by E10.5 due to severe anemia (19
). mRNAs for GATA-1 and EKLF were not detected in E9.5 yolk sacs and embryos, suggesting that SCL is necessary for GATA-1
gene expression. These findings thus appear inconsistent with the results of our E-box mutation. To resolve this discrepancy, we carried out an additional experiment, in which SCL−/−
mice were crossed with IE3.9intGFP transgenic mice. The latter mice express GFP reporter in yolk sac hematopoietic cells under the regulatory influence of G1HE. The results unequivocally demonstrated that GFP reporter is expressed in yolk sac cells in the absence of SCL, thus excluding the possibility that SCL is essential for initiation of G1HE activity. This data is in good agreement with the recent rescue experiment of SCL−/−
embryos by using a transgene that expresses SCL under the influence of regulatory sequences from the GATA-1
). In the latter mouse embryos, the GATA-1
gene regulatory region drove the expression of SCL cDNA in the absence of endogenous SCL protein, proving that SCL is not required for transcription from the GATA-1 enhancer/promoter.
The reason for the marked decrease in the number of GFP-positive cells in the yolk sacs of SCL−/−::GFP+ embryos is not clear. One plausible explanation is that SCL is essential for the growth and/or differentiation of early primitive myeloerythroid progenitors, so that without SCL, the hematopoietic compartment cannot expand in the yolk sac. If this is the case, the GFP-positive cells may correspond to the yolk sac progenitors of hematopoiesis. However, it is technically not feasible to analyze the properties of the GFP-positive cells in SCL−/−::GFP+ embryos, and this point remains to be addressed.
Recently, the zebrafish GATA-1
gene-regulatory region was examined in a transgenic-fish assay, and distal double GATA sites were found to promote and maintain GATA-1 transcription (10
). In the zebrafish GATA-1
gene, a proximal CACCC box is also critical for the initiation of GATA-1
gene expression in hematopoietic cells. However, since the zebrafish GATA-1
gene sequence has diverged substantially from those of the mouse and human GATA-1
genes, the cis
-acting regulatory elements are difficult to compare. For instance, we previously identified a regulatory region in the first intron that specifies GATA-1
gene expression in definitive erythroid cells (17
), but this intronic regulatory element does not appear to be conserved in the zebrafish GATA-1
Our preliminary data suggests that the erythroid-cell specificity of the GATA-1 gene also depends on the contribution of a regulatory element in the upstream promoter region (S. Nishimura, S. Takahashi, and M. Yamamoto, unpublished observation). Identification of cis-acting elements that organize the lineage specificity of GATA-1 gene expression and elucidation of the intricate relationships among the factors interacting with these elements are apparently the focus of our future research.