The major finding of this study is that transgenic mice bearing the human β-globin gene with a mutated BP1 binding site (mtBP1) have significantly higher human β-globin transcript levels in E10.5-13.5 blood cells as compared with control transgenic mice bearing the human β-globin gene with a wild-type BP1 binding site (wtBP1). The differences in human β-globin expression between the mutant and control mice may be due to the fact that mRNA of murine ortholog of BP1, Dlx4, is also predominantly expressed in embryonic blood at E10.5.
Specifically, we have made several observations. First, mutating the DNA sequence of silencer II (in the BP1 binding site of the human β-globin promoter, ) increased β-globin expression. Expression of the human β-globin mRNA was significantly increased (more than 20-fold) in yolk sac-derived embryonic blood of mtBP1 transgenic mice at E10.5 as compared with expression in control (wtBP1) transgenic mice. We also detected a three-fold elevation of mRNA levels of human β-globin in E13.5 fetal liver of mtBP1 mice as compared to wtBP1 mice, and found that human β-globin expression in adult reticulocytes of mtBP1 mice was up to 1.4-fold higher than in adult reticulocytes of wtBP1 mice. Second, the expression of mouse Dlx4 in E10.5 yolk sac-derived embryonic blood was higher than its expression in E13.5 fetal liver or adult erythroid cells. Our PCR data indicated that yolk sac blood was a primary site of Dlx4 expression, because the total RNA isolated from whole 11-day embryos had less Dlx4 mRNA expression than that from embryonic blood.
Finally, we performed in vitro analysis of β-globin promoter activity of EGFP-reporter constructs in MEL, U937, and K562 cells. When MEL cells were transfected with an EGFP-reporter construct driven by a β-globin promoter with either a wtBP1 or a mtBP1 binding site, elevated activities on a similar scale were observed. In contrast, K562 cells transfected with an EGFP-reporter construct containing the β-globin promoter with the mtBP1 binding site demonstrated an increase in EGFP expression as compared with cells tranfected with an EGFP-reporter construct containing the wtBP1 binding site β-globin promoter. We found that mouse Dlx4 was not expressed in the MEL cell line, which can explain the difference in results obtained using MEL and K562 cells. While the MEL cell line did not express Dlx4, it expressed mouse β-major mRNA at high levels. It is very likely that the absence of Dlx4 and, therefore, the inability of the negative regulator of β-globin transcription to bind its site, allowed not only higher mouse endogenous β-major expression but also human β-globin-EGFP-reporter construct expression. In this in vitro set of experiments, we also transfected the BP1-expressing U937 human cell line with the two constructs, and found higher expression of the β-globin promoter’s activities in EGFP constructs with the mtBP1 construct when compared with the wtBP1 construct.
Previous experiments from our and other laboratories have shown that BP1 represses the β-globin gene [5
]. The BP1 protein binding site in both silencers has been determined by DNase I footprint analysis [5
] and its consensus sequence, (A/T)T(A/C)(A/T)ATATG, has been deduced [8
]. It has previously been shown that BP1 is an isoform of human DLX4 [8
]. The DLX4 gene belongs to the Distal-less (DLX) family of homeobox genes, which encode transcription factors that are essential for early development. The Distal-less family of genes comprises at least six different members, DLX1-DLX6 [11
]. Murine Dlx4 shares 88% DNA sequence homology with BP1 thus raising the possibility that the murine Dlx4 actually corresponds to BP1 [8
]. The mRNAs of BP1 and murine Dlx4 are highly homologous (80% homology), and their homeobox sequences encode almost identical protein DNA-binding domains. The differences in the mRNA expression of human β-globin between mtBP1 and wtBP1 mice may be due to the fact that murine Dlx4 mRNA is also predominantly expressed in embryonic blood at E10.5. We do not know whether Dlx4 binds to the silencer II sequence directly, or through a partner protein. To our knowledge, the mechanism has not yet been established. Future experiments would be needed to confirm if mouse Dlx4 also binds to the silencer II element as BP1 does.
A possible mechanism for the regulation of human adult β-globin was described in our earlier publications [9
]. Briefly, BP1 binding to its silencer regions induces HMG-mediated DNA bending that impairs the binding of positive regulators, such as EKLF and GATA-1 (). When a DNA silencing motif is mutated, BP1 cannot bind to its target sequence. This destabilizes the DNA-protein and protein-protein interactions of the entire silencing complex, thereby allowing the positive regulators access to the promoter to induce gene expression (). To this end, BP1 might work in concert with other transcription factors involved in the regulation of human adult β-globin, but as of yet have not been identified.
Model of the potential mechanism for the regulation of β-globin gene expression. LCR, Locus Control Region; HMG, High Mobility Group proteins.