We previously demonstrated that USF activates the expression of the adult β-globin gene in erythroid cell lines, while a dominant-negative mutant of USF (A-USF) inhibits expression (13
). Targeted deletions of either the USF1 or USF2 coding region in mice do not result in obvious hematopoietic defects, suggesting that USF1 and USF2 are able to partially compensate for each other during mouse embryonic development (8
). This is supported by the observation that the compound homozygous mutations lead to early embryonic lethality (8
). We thus hypothesized that the expression of A-USF exclusively or preferentially in erythroid cells interferes with the function of both USF1 and USF2 without affecting vital functions of these proteins in other tissues and organs. A-USF contains the USF-specific dimerization domain and sufficiently inhibits the function of both USF1 and USF2 (45
). As shown in Fig. , the A-USF coding region is under the control of human β-globin LCR elements HS2 and HS3, the human β-globin promoter, and the human β-globin downstream enhancer element. The expression cassette also is flanked by two copies of the chicken HS4 insulator sequence to protect the transgene from position effects (Fig. ). We previously demonstrated that this construct is able to express a β-globin gene at high levels at various integration sites in transgenic mice, including a region close to the centromere (29
). However, it should be noted that the construct may be subject to autoregulation by A-USF itself because it contains two USF recognition sites: one in HS2 and one in the β-globin promoter. This also may limit the potential deleterious effect of expressing the dominant-negative mutant.
FIG. 1. Generation and analysis of mice expressing A-USF. (A) DNA construct pITRp543f2A-USF4 used to generate transgenic mice expressing dominant-negative USF (A-USF). The A-USF coding region is under the control of the human β-globin gene promoter (β-P) (more ...)
Three transgenic founders were generated with this construct (founders I, II, and III), although two of these founders did not transmit the transgene (founders I and III). A-USF was expressed in the spleen of all transgenic mice but not in the liver (Fig. and data not shown). The analysis of A-USF in the spleen of three F1 females from founder II revealed that the expression of A-USF varied between littermates (Fig. ). The founder of line II is male, and we failed to obtain transgenic male offspring; we thus reason that the transgene integrated into the X chromosome and that the expression of A-USF in all erythroid cells is not compatible with survival. Females showed a somewhat variegated phenotype, likely because of differences in the silencing of the transgene on the X chromosome.
To analyze the effect of expressing A-USF on βmaj-globin gene expression, we treated transgenic (founder I and three F1 females from founder II) and four control wild-type (WT) littermates with phenylhydrazine, which induces hemolytic anemia and increases the number of nucleated red blood cells in the spleen. We found that βmaj-globin gene expression was reduced by 50% in the nontransmitting transgenic mouse (founder I) compared to expression in a wild-type control mouse (Fig. ). The expression of A-USF in the spleen of phenylhydrazine-treated F1 animals from the transmitting line varied (Fig. ), and it resulted in a two- to fivefold decrease in βmaj-globin gene expression in three transgenic littermates (line II samples 1 to 3) (Fig. ) compared to that of three wild-type littermates. Five transgenic female mice but none of the wild-type mice died as a result of the phenylhydrazine treatment, indicating a possible defect in erythropoiesis. Consistently with this observation is the fact that 4-week-old transgenic female mice weighed 2 to 3 g less than wild-type littermates (23 ± 1 and 26 ± 1 g, respectively).
The results shown in Fig. demonstrate that USF is required for the high-level expression of the adult βmaj-globin gene. We also observed a reduction in α-globin gene expression that was comparable to a reduction in βmaj-globin (data not shown). The expression of A-USF does not globally affect gene expression; we did not observe a change in β-actin or GAPDH gene expression (data not shown). We next analyzed the recruitment of RNA Pol II at the murine βmaj-globin gene in mice expressing or not expressing A-USF (Fig. ). The expression of A-USF in erythroid cells led to a reduction in USF2 and Pol II binding to the βmaj-globin promoter. The recruitment of RNA Pol II to the control β-actin gene was not affected in A-USF-expressing mice (data not shown).
Because we were unable to obtain transgenic males, we examined embryos at different stages of development. At 14.5 dpc, the fetal liver is the major site of erythropoiesis. In two different litters at 14.5 dpc, which were obtained by mating a transgenic female (line II) with a wild-type male, we detected several reabsorbed and pale embryos. Genotyping with primers specific for the A-USF transgenic construct and for the Y chromosome revealed that the reabsorbed embryos were transgenic males (data not shown). We next examined embryos at earlier stages: 10.5, 11.5, and 12.5 dpc. At 10.5 and 11.5 dpc, all embryos appeared to be alive and normally developed; however, several of the embryos were pale, and these were identified by PCR as transgenic males (Fig. ). At day 12.5, the male transgenic embryos ceased to develop further, demonstrating that the male transgenic embryos did not survive beyond 11.5 dpc.
FIG. 2. Analysis of transgenic mouse embryos at different stages of development. Male embryos were isolated at the indicated time points of development from A-USF transgenic females (F1 females from line II) mated with wild-type (WT) males. Embryos were placed (more ...)
We next examined the expression of globin genes in 10.5- and 11.5-dpc embryos. The expression of A-USF caused a reduction in the expression of all globin genes compared to that of wild-type littermates (Fig. ). In 10.5-dpc yolk sac samples, the expression of the embryonic
γ and βH1
genes as well as that of the Hba α1- and βmin
-globin genes was reduced 5- to 10-fold, and in 11.5-dpc fetal liver samples there was a 5- to 10-fold reduction in the expression of the adult βmaj
-globin gene. We also examined the effect of A-USF on the expression of other erythroid cell-specific genes, including those encoding transcription factors regulating erythropoiesis, like GATA-1, EKLF, Tal-1, NF-E2 (p45), and HoxB4 (Fig. ). HoxB4, a homeobox transcription factor expressed in primitive hematopoietic stem cells, previously has been shown to be regulated by USF in K562 cells (22
). We found that the expression of HoxB4 and GATA-1 is reduced by only about twofold in the yolk sac of transgenic males. In contrast, the expression of transcription factors EKLF, Tal-1, and NF-E2 (p45) was reduced by 5- to 10-fold, suggesting that USF is required for the expression of these genes during primitive erythropoiesis. The expression of another well-characterized erythroid cell-specific gene, Band3, also was reduced by more than fivefold in the transgenic yolk sac samples. The expression of A-USF did not affect the transcription of USF1 or USF2 (Fig. and data not shown) or that of the housekeeping genes GAPDH and β-actin (data not shown).
FIG. 3. Effects of A-USF expression on the expression of erythroid genes and erythroid cell-specific transcription factors. RNA was extracted from 10.5- or 11.5-dpc embryos, reverse transcribed, and subjected to qRT-PCR performed in triplicate. (A) qRT-PCR analysis (more ...)
To exclude the possibility that the phenotype we observed in the male transgenic embryos is due to the integration of the transgene and the consequent disruption of a specific cellular function, we generated and analyzed two transient transgenic embryos at 10.5 dpc. Both embryos appeared pale, expressed A-USF as measured by RT-PCR (Fig. ), and revealed reductions in the expression of α- and β-globin genes as well as that of EKLF and Band3 (Fig. ). The expression of USF1 was not affected in these mice. The data demonstrate that the expression of A-USF in erythroid cells of transgenic mice leads to consistent defects in erythropoiesis in multiple independent transgenic embryos. Therefore, the erythroid phenotype observed in the transmitting line (line II) is unlikely to be due to the disruption of gene expression patterns at the site of transgene integration.
FIG. 4. Generation and analysis of transient transgenic mouse embryos expressing A-USF. Fertilized oocytes were injected with the A-USF expression construct and implanted into the uterus of a pseudopregnant foster mother. Embryos (11.5 dpc) were isolated and (more ...)
To verify that the expression of A-USF affects the binding of USF in transgenic embryos, we examined the binding of USF1 to LCR element HS2 in yolk sac samples taken from 10.5-dpc transgenic embryos (line II) and wild-type litter mates using the μChIP assay, which allows the detection of protein-chromatin interactions with a small number of cells. The binding of USF1 to the LCR was reduced in transgenic embryos compared to that of wild-type littermates (Fig. ). The interaction of RNA Pol II with LCR element HS2 also was reduced in 10.5-dpc yolk sac samples from transgenic mice compared to that of littermates (Fig. ), whereas there was no change in the association of RNA Pol II with the GAPDH gene between wild-type and transgenic embryos (Fig. ).
FIG. 5. μChIP analysis of RNA Pol II and USF1 association with LCR element HS2 and the GAPDH gene in the yolk sac of wild-type and A-USF transgenic embryos. Embryos (10.5 dpc) were taken from an A-USF transgenic female (mated to a wild-type male). Yolk (more ...)
Because EKLF, Tal-1, and NF-E2 (p45) failed to be expressed at high levels in the hematopoietic tissue of transgenic mice, we examined the possibility that USF directly regulates these genes. We performed ChIP to examine the interaction of USF with the gene loci encoding these transcription factors during the differentiation of murine erythroleukemia (MEL) cells. One of the multiple DNA regulatory elements in the EKLF gene locus contains an E-box, which previously has been shown to interact with Tal-1 (1
). Both subunits of USF associated with the E-box-containing regulatory region of the EKLF gene in MEL cells (Fig. ). We also observed interactions of USF with the Tal-1 gene locus, which also contains an E-box motif in a regulatory element. Interestingly, the interaction of USF with the Tal-1 gene decreased during DMSO-induced MEL cell differentiation. USF binding also was detectable at the GATA-1 gene locus (Fig. ). There are no previous data concerning E-box elements regulating the GATA-1 gene. We failed to detect significant interactions of USF1 with the NF-E2 (p45) gene; however, the recovery of USF2-precipitated p45 gene fragments was higher than that of the IgG control. The data suggest that the Tal-1 and EKLF genes are direct targets of both USF1 and USF2 in differentiating erythroid cells. There was no significant binding of USF to the control Necdin gene, which is not expressed in erythroid cells (Fig. ). We confirmed the interactions of USF2 with the erythroid cell-specific gene loci in primary erythroid cells taken from 16.5-dpc mouse fetal liver samples (Fig. ). The ChIP results demonstrated that USF2 interacts with the EKLF, GATA-1, and Tal-1 gene loci but not with the Necdin gene locus. We observed a reproducible interaction of USF2 with the NF-E2 (p45) gene locus in fetal liver cells.
FIG. 6. Interaction of USF with regulatory elements of genes encoding hematopoietic-specific transcription factors. ChIP analysis of the interaction of USF1 and USF2 with regulatory elements of the EKLF, GATA-1, Tal-1, and NF-E2 (p45) genes as well as with the (more ...)
We next examined the possibility that the expression of A-USF in erythroid cells impairs their differentiation potential. We began these studies by examining 10.5-dpc yolk sac cells from transgenic embryos and wild-type littermates for the expression of the transferrin receptor CD71, which is expressed at high levels in developing erythroid cells and serves as a marker for erythroid progenitors (40
). The CD71-mediated sorting of yolk sac cells revealed that the number of CD71+
cells was about threefold lower in the transgenic embryos than that of the wild-type embryos (Fig. ). Furthermore, we observed a decrease in the number of cells that express high levels of Ter-119 (Fig. ), which is a marker for more differentiated erythroid cells (32
). The number of benzidine-positive cells also was reduced by three to fourfold in the yolk sac cells from transgenic embryos compared to those taken from wild-type littermates (data not shown). Taken together, these results demonstrate that USF is an important contributor to erythroid cell differentiation and mediates the high-level expression of erythroid transcription factors and the expression of the globin genes.
FIG. 7. Transgenic (TG) A-USF embryos reveal a reduction in the number of CD71-positive and Ter-119-positive erythroid cells. Yolk sac cells from 10.5-dpc male embryos were isolated and subjected to flow cytometry against CD71 or Ter-119. Hatched areas indicate (more ...)
To examine whether USF not only regulates the differentiation of erythroid cells but also functions within the context of differentiating cells, we analyzed the expression of the βH1-globin gene in Ter-119-sorted cells obtained from transgenic or wild-type embryos (Fig. ). The expression of the βH1-globin gene was reduced in Ter-119+ cells isolated from two transgenic embryos compared to that of their wild-type littermates.