Center for Tsukuba Advanced Research Alliance and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8577, Japan
*Author for correspondence (e-mail: masi{at}tara.tsukuba.ac.jp)
Accepted March 19, 2001
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SUMMARY |
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Key words: Autoregulation, First intron, GATA1, GFP reporter gene, Hematopoiesis, Transactivation, Transgenic, Zebrafish, Zinc finger
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INTRODUCTION |
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Detailed deletion analyses indicated that the core 149 bp in G1HE, which contains the double GATA-related motif (GATT + GATA), contributes to the activity of the regulatory domain to express the reporter gene in the erythroid lineage (Vyas et al., 1999; Nishimura et al., 2000). The conservation of GATA motifs within the gata1 gene regulatory regions of various experimental animals suggests the importance of cis-acting GATA motifs for the hematopoietic lineage-specific expression of the gata1 gene.
Gel retardation analyses have shown that mouse GATA1 can bind to the GATA sites both in the proximal double GATA motif and in the G1HE (Nicolis et al., 1991; Tsai et al., 1991; Schwartzbauer et al., 1992; Vyas et al., 1999; Nishimura et al., 2000), suggesting that gata1 gene expression is maintained by an autoregulatory mechanism during hematopoietic cell development. However, the transcriptional activation of the gata1 gene by GATA1 has been examined only in the fibroblast transfection systems: the magnitude of activation was small and it is likely that the choice of fibroblasts is a limiting factor (Hannon et al., 1991; Tsai et al., 1991). Systematic analysis of gata1 gene autoregulation has not been previously conducted in vivo in reporter transgenic systems.
The zebrafish is advantageous for analyzing in vivo mechanisms of transcriptional regulation. As zebrafish development is relatively quick and the transparent embryos develop outside the mother, three important transcription factor analyses can be conducted using the zebrafish system. First, spatial and temporal expression profiles of the transcription factor and its target genes during embryogenesis can be easily studied in live embryos using green fluorescence protein (GFP) as the reporter gene (Higashijima et al., 1997; Long et al., 1997). Second, the effects of either overexpression or ectopic expression of transcription factors can be examined with ease by injecting the necessary synthetic RNA into early stage embryos (Kobayashi et al., 1998; Kobayashi et al., 2001). Third, various developmental events, such as embryonic hematopoiesis, can be observed within a couple of days (Amatruda and Zon, 1999).
In this study, we have used zebrafish to clarify whether GATA1 can activate its own gene expression. We prepared transgenic fish containing the GFP reporter linked to the zebrafish gata1 gene hematopoietic regulatory domain (HRD). Importantly, the expression of GFP was induced ectopically by the overexpression of GATA1 in a GATA site-dependent manner from the gata1-HRD-GFP transgene. We also prepared stable transgenic fish lines with the same reporter transgene construct and performed functional domain analysis. The results clearly indicate that an intact GATA1 function is required for the ectopic reporter gene expression. These results, thus, provide the first in vivo evidence for the existence of an autoregulatory mechanism in hematopoietic gata1 gene expression.
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MATERIALS AND METHODS |
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Southern blot and PCR analyses
Genomic zebrafish DNA was prepared from the whole adult bodies of AB strains by a standard method that was modified for zebrafish DNA as described previously (Westerfield, 1995). Isolated genomic DNA was digested with restriction enzymes and analyzed by agarose gel electrophoresis. DNA fragments were transferred to ZetaProbe membrane (BioRad) and hybridized at 55°C to an AlkPhos Direct-labeled probe, which corresponds to the first intron of the zebrafish gata1 gene. Membranes were washed at 60°C in buffer containing a blocking reagent according to the manufacturers instruction (Amersham Pharmacia Biotech). PCR was carried out using primers corresponding to the 3'-end of the first exon (5'-GATAAGCAAGCAAACAGGCG) and to the 5'-end of the second exon (5'-TATAGGACGACGAGGCTCGG).
5'-Rapid amplification of cDNA ends (5' RACE) assay
Total zebrafish RNA was prepared using RNAzol B (TEL-TEST) from either whole embryos at 18 hours or from adult hematopoietic tissues (kidney, spleen plus liver). 5' RACE assay was carried out using the 5' RACE System (GIBCO BRL). Briefly, 4 µg each of total RNA was reverse transcribed using the antisense primer 5'-GCAGTGTTCTGGTAGATGG, which is specific for the gata1 third exon. The product was amplified using the 5' RACE abridged anchor primer and a gata1 third exon-specific antisense primer 5'-TACTGGACCAGACCGTGG. The resulting cDNA was further amplified using the abridged universal amplification primer and another gata1 third exon-specific antisense primer 5'-TGACCTGCAGAGTTGTCTAGCC. 5' RACE products were subcloned into pBluescript II SK and their sequences were determined.
Fish embryos and larvae
Zebrafish embryos and larvae were obtained by natural mating (Westerfield, 1995) and staged accordingly (Kimmel et al., 1995). Germline transgenic fish were identified under the fluorescent microscope by their expression of GFP. Whole-mount in situ hybridization was performed, as described previously (Kobayashi et al., 2001).
Microinjection of zebrafish embryos
p8.1G1-eGFP and its derivatives described below were linearized by digesting the vector backbone with either KpnI or SacI. Digested DNA was resuspended in water and injected into the blastomere of early one-cell stage embryos (see Fig. 5A). For RNA injection, synthetic capped RNA was made with the SP6 mMESSAGE mMACHINE in vitro transcription kit (Ambion) using linearized DNA of the pCS2 derivatives described below. RNA was injected into the yolk at the one-cell stage for expression in whole bodies. For spatially localized gene overexpression, two- to eight-cell stage embryos were injected into a single blastomere, along with 200 µg/ml mRNA for DsRed (Clontech) or 0.125% tetramethyl-rhodamine dextran as cell lineage markers (see Fig. 5A).
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Plasmid construction
A DNA fragment corresponding to the 8.1-kb upstream region of the translational initiation site of zebrafish gata1 and the eGFP fragment of pCS2-eGFP (kindly provided by Dr J. J. Breen) were ligated together into pBluescript IISK, and the resulting plasmid was named p8.1kG1-eGFP. For construction of p8.1kG1dl1-eGFP, a 0.2 kb DNA fragment containing the first exon and translational initiation site, but not the first intron, was prepared by PCR and ligated into p8.1kG1-eGFP. For construction of the 5'-deleted gata1 mutants, p8.1kG1-eGFP was linearized with KpnI and ClaI, and incubated with exonuclease III, followed by blunting with mung bean nuclease and the self-ligation. Selected constructs were sequenced and named p5.7kG1-eGFP, p5.1kG1-eGFP and p3.9kG1-eGFP, according to the positions of their 5' ends from the translational initiation site. For construction of p8.1G1m1-eGFP, mutations in a distal double GATA motif were introduced by PCR into p8.1G1-eGFP.
To construct pCS2zGATA1, pCS2zGATA1dN56, pCS2zGATA1dN80, pCS2zGATA1dCF and pCS2zGATA1dNF, cDNA fragments corresponding to 1Met-418Val, 56S-418Val, 80L-418Val, 1Met-282Leu plus 325Val-418Val, and 1Met-233Pro plus 283Ile-418Val, respectively, were prepared by PCR and subcloned into pCS2+ (Rupp et al., 1994). Constructs pCS2HAzGATA1, pCS2HAzGATA1dCF and pCS2HAzGATA1dNF were generated by inserting a cDNA fragment for the HA peptide (MEYPYDVPDYAA) just upstream of the first ATG site of GATA1 in pCS2zGATA1, pCS2zGATA1dCF and pCS2zGATA1dNF, respectively. All constructs were verified by a DNA sequencing.
Immunoblot analysis
Embryos were homogenized with a pestle in buffer A (20 mM Hepes (pH 7.6), 1.5 mM MgCl2, 10 mM NaCl, 20% glycerol, 0.2 mM EDTA, 1 mM dithiothreitol, 1 mM p-amidinophenylmethanesulfonyl fluoride, 1xprotease inhibitor cocktail (Roche)) and incubated at 0°C for 5 minutes in buffer B (buffer A plus 0.1% Triton X-100), and nuclei were collected from resulting homogenate by centrifugation at 600 g for 10 minutes. In vitro translated proteins were generated by TNT Coupled Wheat Germ Extract Systems (Promega) using pCS2 derivatives as DNA templates. Immunoblot analysis using anti-HA antibody (12CA5, Roche) was performed as described previously (Kobayashi et al., 2001).
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RESULTS |
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To our surprise, a 1.5-kb intron just upstream of the translational initiation site was identified in the isolated genomic clones (Fig. 1A). As this intron was not present in the clone reported by Long et al. (Long et al., 1997), we carried out several additional analyses for confirmation. First, Southern blot analysis with zebrafish genomic DNA was performed along with cloned phage DNA using a fragment corresponding to the first intron as a probe (Fig. 1B). The lengths of the positive fragments were identical in genomic DNA and cloned phage DNA in three independent digestions: XbaI (expected fragments are 3.8 kb + 1.6 kb), SpeI (2.9 kb) and XbaI/SpeI (1.4 kb). The result also correlated well with the restriction enzyme site map of the cloned DNA.
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Third, we isolated two gata1 BAC clones from a zebrafish genomic DNA BAC library (Genome Systems) and confirmed the existence of the first intron in both clones (data not shown). Taken together, these results unequivocally demonstrate that zebrafish gata1 gene contains an intron disrupting the 5'-untranslated region (UTR) of the gene. This intron corresponds to the first intron found in other vertebrate Gata1 genes cloned and characterized to date, such as human, mouse, rat and chicken (Hannon et al., 1991; Nicolis et al., 1991; Tsai et al., 1991; Onodera et al., 1997b), indicating that the structure and organization of the gata1 genes is well conserved among vertebrates.
Finally, we examined the mRNA sequences of gata1 in embryos or adult hematopoietic tissues by 5' RACE assay. For this purpose, we amplified cDNAs prepared from the total RNA of either 18-hour-old embryos or adult hematopoietic tissues, and determined the sequences of selected clones. Sequences corresponding to the first and second exons, but not to the first intron, were found in all of the cDNAs prepared from embryos (eight out of eight) or from hematopoietic tissues (16 out of 16). Additional first exons, such as the IT exon (Ito et al., 1993) or the exon 1b (Tsai et al., 1991) in the mouse Gata1 gene, were not found in the zebrafish gata1 gene analyzed in this study. These results thus indicate that the first intron of the zebrafish gata1 gene is spliced out in the gata1 transcripts in embryonic and adult hematopoietic tissues.
The first intron enhances gata1 gene regulatory activity in zebrafish larvae
The first intron of the mouse Gata1 gene is required for its efficient expression in definitive erythroid cells (Onodera et al., 1997a). Similarly, expression of the CAT reporter gene driven by the chicken GATA1 gene regulatory region was four-fold greater in the presence of the first intron in 10-day-old chicken definitive erythroid cells (Hannon et al., 1991). Consistent with these results, the present identification of the first intron in the zebrafish gata1 gene suggests the importance of the first intron in the hematopoietic expression of the gata1 gene, especially at the late stage of development.
To further verify this contention, GFP reporter constructs fused to the zebrafish gata1 gene regulatory region, either with (8.1kG1-eGFP) or without (8.1kG1dI1-eGFP) the first intron, were prepared and their promoter activity was examined in zebrafish embryos (Fig. 2A). Each construct was microinjected into one-cell-stage embryos and expression of the GFP reporter gene was monitored using a fluorescence microscope at distinct stages of development. We first analyzed the embryos injected with 8.1kG1-eGFP. In these embryos, the GFP expression was observed in the lateral plate mesoderm (LPM) at 15 hours (data not shown) and in the intermediate cell mass (ICM) at 24 hours (day 1 in Fig. 2B), where prospective hematopoietic cells occur (Detrich et al., 1995). The expression of GFP was highly analogous to the gata1 gene expression profile determined by in situ hybridization (Fig. 2B; data not shown). These results thus demonstrate that the genomic region used for 8.1kG1-eGFP is sufficient to recapitulate the hematopoietic gata1 gene expression profile in zebrafish embryos. We, thus, named this genomic region the gata1 gene hematopoietic regulatory domain (gata1-HRD).
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Within the region containing parts of the aorta and vein (Fig. 2C), we counted the number of cells expressing GFP in the blood circulation at days 2, 4, 6 and 10. The ratios of GFP-positive cell number in 8.1kG1dI1-eGFP-injected embryos to that in 8.1kG1-eGFP injected embryos were calculated at each larval stage (Fig. 2D). The ratio was 61% on day 2 for the DNA injection at concentration of 50 µg/ml (approximately 50 pg per embryo). This value was not significantly altered when the DNA concentration was decreased to 25 or 10 µg/ml (51% and 75%, respectively). However, the ratios were reduced considerably according to the larval development for all DNA concentrations. These results indicate that the first intron only affects gata1 gene expression at the larval stage and may be required for the maintenance of late phase gata1 expression.
In order to compare the structure of the first intron of zebrafish gata1 with those of other vertebrate gata1 genes, we determined the entire nucleotide sequence of the 1.5 kb first intron. The sequence has been deposited in DDBJ/EMBL/GenBank Database under Accession Number AB052888. Neither the GATA repeat nor the AP-1 repeat were found in the zebrafish first intron, both of which exist in the first intron of mouse gata1 and were demonstrated to be critical for GFP reporter activity in erythroid SKT6 cells (Seshasayee et al., 2000). The hormone-responsive element in the first intron of chicken GATA1, which was recognized by the thyroid hormone receptor and the chicken ovalbumin upstream promoter (COUP) transcription factor (Trainor et al., 1995), was not found. To date, entire sequence of the chicken first intron (161 bp; Hannon et al., 1991) and only 2.4 kb of the 4.4 kb sequence of the mouse first intron (DDBJ/EMBL/GenBank Database, X57530) have been reported. From the cross-species comparison, an AGxxAATGxxG sequence located at nucleotide position -319 was identical among the first introns of zebrafish, mouse and chicken. Although a double GATA motif surrounded by several E-boxes at nucleotide position -125 is interesting, it was not found in the known sequences of the gata1 first intron of other species.
A distal double GATA motif is necessary for GFP expression driven by gata1-HRD
A distal double GATA motif is located in approximately 6.4 kb upstream from the translational initiation site of the zebrafish gata1 gene. This motif is necessary for the hematopoietic expression of the zebrafish gata1 gene, as demonstrated by reporter transgenic analyses using constructs lacking the first intron (Meng et al., 1999). In order to examine whether the first intron containing another double GATA motif can replace the activity of the distal motif, we prepared a set of deletion constructs based on 8.1kG1-eGFP and analyzed their activity in zebrafish embryos (Fig. 3). Strong GFP expression was observed in the ICM at 22 hours in 37% of injected embryos with full-length construct. Deletion of the upstream region, including the double GATA motif, reduced the number of cells expressing GFP, while removal of the first intron caused only a weak effect. As the number of GFP-positive cells reflects the gene regulatory activity of the template, the distal GATA motif seemed important for the activity of gata1-HRD during the early phase of hematopoiesis.
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According to the criterion that positive embryos express GFP in more than six cells in the ICM (right column in Fig. 3), all constructs, including the shortest 3.9kG1-eGFP construct, were found to have some activity in promoting specific GFP expression. These results support the contention that the distal double GATA motif is important in enhancing the gata1 promoter activity, but not essential for specific gene expression in hematopoietic tissues. Using a similar criterion for identifying GFP-positive embryos, Meng et al. reported that less than 2% of embryos injected with the double GATA motif deletion construct 4623GM2 (corresponding to -6.2 kb in our constructs) showed specific GFP expression (Meng et al., 1999). At present, we do not have any plausible explanation for the discrepancy, except for the presence of the first intron in our reporter constructs.
Overexpression of GATA1 induces ectopic expression of GFP from gata1-HRD
To examine whether GATA1 regulates its own gene expression, we produced stable zebrafish lines containing the 8.1kG1-eGFP transgene in the chromosome (8.1kG1-eGFP fish) and used their progeny for analyzing how GATA1 overexpression affects gata1-HRD activity. Second generation (F2) embryos or larvae of the 8.1kG1-eGFP fish showed potent GFP expression in the LPM and blood cells at 15 hours and at 7 days, respectively (Fig. 4A).
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Both N- and C-terminal zinc-finger domains are required for the inducible expression of the GFP reporter
Mutation studies of mouse and chicken GATA1 showed that proteins lacking the N-terminal zinc finger (NF) could bind to DNA and activate expression of the reporter gene, whereas proteins lacking the C-terminal zinc finger (CF) were inactive (Martin and Orkin, 1990; Yang and Evans, 1992). NF stabilizes GATA1 binding to clusters of GATA sites, such as the double GATA motif in gata1-HRD (Martin and Orkin, 1990; Trainor et al., 1996). To elucidate whether GATA1-mediated ectopic expression of GFP requires an intact GATA1 function or not, we designed GATA1 constructs without NF or CF, and examined their ability to induce ectopic GFP expression in the embryos of 8.1kG1-eGFP fish (Fig. 4C). Injection of CF-deleted GATA1 (GATA1dCF) mRNA resulted in negligible induction of ectopic GFP expression, even when the mRNA concentration was increased to 200 µg/ml (Fig. 4D), indicating that CF is crucial for activity. Similarly, overexpression of NF-deleted GATA1 (GATA1dNF) showed negligible induction of ectopic GFP expression, suggesting that NF is also indispensable for activity. We did not analyze GATA1dNF mRNA injected embryos at a high dose (>150 µg/ml), as embryonic development ceased at the gastrula stage, probably because of the toxic effects of GATA1dNF (data not shown). To confirm that reduction in GFP-inducing activity of GATA1 by zinc-finger deletion was not due to instability of these mutant proteins, we overexpressed HA-tagged GATA1 constructs in embryos and analyzed their expression at protein level by immunoblot analysis. GFP-inducing activities of the HA-tagged and untagged constructs were comparable for each GATA1 proteins (data not shown). Expression level of overexpressed proteins was similar between NF- or CF-deleted GATA1 and wild-type GATA1 (Fig. 4E), indicating that both NF and CF were in fact required for gata1-HRD-directed GFP expression.
The N-terminal (NT) domain of 66 amino acid residues in the mouse GATA1 is necessary for transactivation in transfection assays using COS or NIH3T3 cells. NT also confers transactivational activity upon fusion to heterologous DNA-binding domains (Martin and Orkin, 1990). Strikingly, deletion of 71 amino acid residues of the chicken GATA1 NT region reduces the level of transactivation in QT6 fibroblasts (Yang and Evans, 1992). Owing to the analogy with mammalian and avian GATA1, we examined the function of the zebrafish GATA1 NT domain in ectopic GFP expression. GATA1 constructs deleted the NT domain of 56 or 80 residues (GATA1dN56 and GATA1dN80, respectively) were prepared and their activity to induce GFP was examined in 8.1kG1-eGFP embryos (Fig. 4C). The induction of ectopic GFP expression was observed with the NT deletion mutants. The magnitude of induction was weaker than that of wild-type GATA1 mRNA at a low mRNA concentration range, but stronger than both the NF or CF deletion mutants (Fig. 4D). The GATA1dN56 and GATA11dN80 constructs gave reproducible results. These results indicate that the NT domain also contributes to the GATA1 transactivational activity for gata1-HRD-directed ectopic expression of GFP. The results also suggest that the presence of excessive amounts of GATA1dNT protein can compensate the NT activity.
Distal double GATA motif is required for the induction of ectopic GFP expression
In order to identify target sites for GATA1 in gata1-HRD, we set up a successive-injection system of GFP reporter DNA and synthetic capped RNA providing trans-acting factors (Fig. 5A). After injecting 8.1kG1-eGFP at the early one-cell stage, GATA1 mRNA was injected into a single blastomere at 2 to 8 cell stage, together with tetramethyl-rhodamine dextran as a cell lineage marker. In these embryos, GATA1 was randomly overexpressed in some parts of the body and identified as rhodamine positive cells. The results showed that 31% of the GATA1 mRNA-injected embryos showed a strong ectopic expression of GFP at 15 hours. The GFP-positive area was also positive for rhodamine (Fig. 5B, thick arrow). Such GFP induction was not observed in embryos injected with the cell lineage marker alone (Fig. 5B, thin arrow).
The important observation here is that induction of GFP expression was significantly reduced when 5.7kG1-eGFP or shorter constructs were used as template DNA (Fig. 5C). The double GATA motif was localized in this 2.4 kb sequence deleted from 8.1kG1-eGFP, which was identified as a requirement for gata1-HRD activity in blood cells (see Fig. 3). Therefore, to determine whether the double GATA motif is required for induction of ectopic GFP expression, we examined the response to the GATA1 overexpression using the point mutant construct 8.1kG1mG-eGFP as a reporter. When we injected the 8.1kG1mG-eGFP reporter and the GATA1 mRNA successively, only 11% of embryos showed ectopic GFP expression (Fig. 5C). This frequency was comparable with that of the 5.7kG1-eGFP construct. These results indicate that the distal double GATA motif in gata1-HRD actually mediates the response to the exogenous GATA1, implying that gata1-HRD may be a direct target for GATA1 transactivation.
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DISCUSSION |
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Recent progress in transgenic and gene targeting technologies has allowed the direct confirmation that transcription factor genes undergo a positive autoregulatory control loop. For example, the following genes may be governed by this regulatory mechanism: Pit1 in the anterior pituitary cells (DiMattia et al., 1997); Hoxa4, Hoxb4, Hoxd4 and their Drosophila homolog, deformed, in anteroposterior patterning (Bergson and McGinnis, 1990; Gould et al., 1997; Packer et al., 1998); and glial cells missing in the glial cells (Miller et al., 1998); and fushi tarazu in the segmentation (Schier and Gehring, 1992). Indeed, a pioneering study demonstrated that the nematode elt-2 gene, a gene encoding a single finger GATA factor, is positively autoregulated (Fukushige et al., 1998); ectopic expression of Elt-2 induced the expression of lacZ from a transgenic elt-2 promoter-lacZ reporter construct. In common with the zebrafish gata1 gene, autoregulation appears to work directly, since functional Elt-2-GFP fusion proteins co-localized exclusively with Elt-2 binding sites in cell nuclei (Fukushige et al., 1999).
First intron of the gata1 gene
In this study, we have identified the first intron of the zebrafish gata1 gene. As this intron was not found during the previous zebrafish gata1 gene analysis (Long et al., 1997), it was necessary to confirm that the first intron is indeed present. The existence of this intron was established on the basis of three criteria. First, two independent phage clones and two independent BAC clones contained the intron sequence. Second, Southern blotting and PCR analyses of zebrafish genomic DNA indicated the presence of a 1.5 kb intron between the first and second exons. Third, this 1.5 kb sequence does not exist in the GATA1 mRNA. Discovery of the zebrafish first intron proves that a strong cross species conservation occurs in the structure of the gata1 gene, as all currently characterized gata1 genes contain a first intron that disrupts the 5'-UTR. It is intriguing to note that not only the gata1 gene, but other family members of the hematopoietic GATA factor family, gata2 and gata3, contain the first intron, which disrupts the 5'-UTR at a similar site (George et al., 1994; Labastie et al., 1994; Nagai et al., 1994; Brewer et al., 1995; Minegishi et al., 1998; Nony et al., 1998). Furthermore, an intron disrupting the 5'-UTR also exists in the genes encoding the cardiac GATA factors, gata4, gata5 and gata6 (Soudais et al., 1995; MacNeill et al., 1997; Brewer et al., 1999). Thus, the first intron is a structure common to the vertebrate GATA factor genes.
The general conservation of the first intron among GATA factor genes suggests that it is functionally significant in the regulation of the these genes. One plausible possibility is that it may contribute to the control of temporal or spatial gene expression profile during development and/or cell differentiation, as is the case for gata1 (Hannon et al., 1991; Onodera et al., 1997a; Seshasayee et al., 2000). An alternative possibility is that the first intron may contribute to the selection of first exons/promoters used in the GATA genes. In the testis, mouse Gata1 mRNA is mainly transcribed from the IT exon, a first exon distinct from hematopoietic IE exon (Ito et al., 1993; Onodera et al., 1997b). Alternative first exons/ promoters have also been identified in the gata2, gata5 and gata6 genes (Minegishi et al., 1998; MacNeill et al., 1997; Brewer et al., 1999; Pan et al., 2000). In this regard, we were unable to find alternative first exon in the zebrafish gata1 gene through 5' RACE analysis using mRNA derived from hematopoietic tissues or testis (K. N., M. K. and M. Y., unpublished). However, although the possibility still remains that some zebrafish tissues retain an alternative form of GATA1 mRNA containing a first exon that is distinct from the one identified in this study.
Role of NF in the positive autoregulation
GATA1 NF has been demonstrated to be essential for GATA1 function in hematopoietic tissue development. Although dispensable in the induction of megakaryocytic differentiation (Visvader et al., 1995), NF is strictly required for terminal erythroid differentiation (Weiss et al., 1997). In the present study, we have shown that NF is required for the GATA1 function in the inducible expression of GFP reporter, implying a role for NF in the maintenance of gata1 gene expression. NF contributes to the stability of DNA binding when GATA1 binds to a double rather than to a single GATA site (Martin and Orkin, 1990; Trainor et al., 1996). Likewise, our present results indicate that the distal double GATA motif in gata1-HRD is important for transactivation by GATA1. In addition to the DNA-binding activity, NF interacts with FOG1, an essential co-factor of GATA1 (Tsang et al., 1997). NF has also been implicated in the formation of a GATA1 dimer, through an NF-CF interaction (Mackay et al., 1998), which is noteworthy as overexpression of GATA1 alone induces ectopic GFP reporter gene expression in zebrafish embryos.
A double GATA-related motif in the G1HE of the mouse Gata1 gene was required for its expression in yolk-sac hematopoietic cells, and definitive erythroid and megakaryocytic cells (Vyas et al., 1999; Nishimura et al., 2000). Gel retardation analyses using nuclear extracts from mouse erythroleukemia cells have shown that a multi-protein complex including GATA1, SCL/Tal-1, E2A, Lmo2 and Ldb1 binds to this motif (Vyas et al., 1999; Nishimura et al., 2000). Lmo2 was demonstrated to interact directly with the fingers in GATA1 and is assumed to act as a bridging molecule for GATA1, SCL/Tal-1, and Ldb1 (Osada et al., 1995; Wadman et al., 1997). Homologs of SCL/Tal-1, Lmo2 and Ldb1 have been cloned from zebrafish and the expression of these genes in hematopoietic cells has been confirmed (Gering et al., 1998; Liao et al., 1998; Thompson et al., 1998; Toyama et al., 1998). It would be intriguing to test whether a multi-protein complex can also bind to the distal double GATA motif in the zebrafish gata1-HRD and play a role in the positive autoregulation of gata1 gene expression.
Zebrafish system for transcription studies
Both the spatial and temporary activities of particular gene regulatory regions are easily detected in zebrafish embryos using GFP as a reporter gene. Therefore, various GATA1 deletion mutants were expressed in the embryos of 8.1G1-eGFP transgenic fish lines in order to examine their effects on the activity of gata1-HRD. Indeed, there has been an increase in the number of studies using stable transgenic zebrafish lines and GFP as a reporter gene (Amsterdam et al., 1995; Higashijima et al., 1997; Higashijima et al., 2000; Long et al., 1997; Jessen et al., 1999; Linney et al., 1999; Halloran et al., 2000). A convenient technique for analyzing the function of transcription factors in vivo is to overexpress the factor in embryos carrying the target sequence fused to a reporter gene. So far, this technique has been applied only to Drosophila, nematode and Xenopus embryonic models. Compared with the Xenopus system (Latinkic et al., 1997; Laurent et al., 1997; Mochizuki et al., 2000), however, zebrafish embryos have several advantages in gene regulation studies, such as rapid development, body transparency and existence of a large number of mutants that can change the genetic background of the GFP reporter fish. Thus, we consider that zebrafish to represent an excellent model system for studying the in vivo function of transcription factors.
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ACKNOWLEDGMENTS |
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