Elsevier

Blood Cells, Molecules, and Diseases

Volume 41, Issue 3, November–December 2008, Pages 244-251
Blood Cells, Molecules, and Diseases

BP1 motif in the human β-globin promoter affects β-globin expression during embryonic/fetal erythropoiesis in transgenic mice bearing the human β-globin gene

https://doi.org/10.1016/j.bcmd.2008.05.008Get rights and content

Abstract

The binding of the transcription factor BP1 (β protein 1) to its site on the promoter of the adult β-globin gene has a silencing effect on β-globin transcription in vitro. To better understand the mechanism of BP1's negative regulation of β-globin expression, we developed transgenic mice bearing a human β-globin locus-containing cosmid. Specifically, we introduced a mutated BP1 binding site (mtBP1) into the promoter of the β-globin gene sequence of this cosmid construct. In the mtBP1 mice, we detected a more than a 20-fold increase in human β-globin expression in the yolk sac-derived blood at E10.5, a 3-fold increase in fetal livers at E13.5, and an approximately 1.4-fold increase in adult reticulocytes compared with control mice bearing the human β-globin gene with the wild-type BP1 binding site sequence (wtBP1). Our in vivo observations support the contention that the BP1 binding site of the β-globin promoter plays an important role in the regulation of transcription of the adult β-globin gene.

Introduction

During erythroid cell development, globin gene expression within the β-globin cluster is achieved through a precise balance of negative and positive regulation. Current hypotheses about the possible mechanisms for negative regulation include competition between genes for interaction with the locus control region (LCR) [1] and autonomous gene silencing involving sequences located in the proximal and distal promoters [2], [3], [4]. BP1 (β protein 1) plays an important role in β-globin gene regulation, because binding of BP1 to its site on the promoter of the adult β-globin gene has a silencing effect on β-globin transcription in vitro [5], [6], [7], [8], [9]. The adult β-globin gene includes two upstream silencer regions (silencer I and II), and BP1 binds to both of these regions [5], [8]. In our earlier studies of BP1 function in K562 cells, an erythroleukemia cell line with an embryonic–fetal phenotype, we have shown that introduction of multiple mutations in two BP1 binding sites enhances β-globin promoter activity [6]. Inhibition of BP1 expression also has been demonstrated to increase levels of β-globin mRNA in K562 cells [9]. To better understand the effects of the negative regulation of β-globin expression at the level of the whole organism, we have developed a transgenic mouse expressing the human β-globin gene mutated at the BP1 binding site. Because the silencer II element does not interfere with high mobility group (HMG) protein binding sites as silencer I does [7], we mutated the silencer II BP1 binding site in a cosmid construct that we subsequently used to create transgenic mice. To study human β-globin gene expression during the transgenic mouse embryo development, we analyzed embryonic and fetal blood, and adult reticulocytes of the transgenic mice. We have also studied the role of BP1 in β-globin expression in human and mouse cell lines. In mouse cells, we analyzed effects of Dlx4 expression, which is the most probable mouse ortholog of BP1 [8], on human β-globin expression.

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Cell cultures and transfection

K562 and U937 cells (ATCC, Manassas, VA) were grown in RPMI 1640 culture medium supplemented with 10% fetal calf serum, 2mM glutamine, 100U/ml penicillin, and 100μg/ml streptomycin. Murine erythroleukemia cell line (MEL) were grown in IMEM culture medium supplemented with 10% fetal calf serum, 2mM glutamine, and 50μg/ml gentamicin. Cells were regularly subcultured to maintain exponential growth. Transfection of cells was performed using an electroporation apparatus (Nucleofector System, Amaxa,

Transgenic mice with cosmid gene constructs

Three transgenic mouse lines (designated as A, B, and C) were generated with the cosmid construct containing the mutated BP1 binding site sequence (mtBP1). Real-time PCR analysis showed that these lines contained between two and eight copies of the cosmid. After correction for copy number, the mean human β-globin gene mRNA levels in adult reticulocytes from mtBP1 mice and wtBP1 mice were 102% and 81% relative to the level of mouse α-globin mRNA, respectively (Table 2).

TaqMan PCR analysis of β-globin expression

The mRNA levels of human

Discussion

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 the murine ortholog of BP1, Dlx4, is also

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