Effects of human locus control region elements HS2 and HS3 on human β-globin gene expression in transgenic mouse

Abstract

The locus control region (LCR) is the most important cis-element in the regulation of β-globin gene expression. DNaseI-hypersensitive site (HS) 2 and HS3 are two significant components of β-LCR. To examine the effect of HS2, HS3, and HS2–HS3 (combination of HS2 and HS3) on the spatial and temporal expression of the human β-globin gene, we have produced transgenic mice with constructs, in which the gene encoding enhanced green fluorescent protein (EGFP) is driven by β-globin promoter and under the control of HS2, HS3, and HS2–HS3, respectively. The results showed that HS2 and HS3 each had the same enhancement activity in regulation of β-globin gene expression in transgenic mice. When HS2 and HS3 were in combination (HS2–HS3), the two cis-elements showed a marked synergy in regulating β-globin gene spatial and temporal expression as well as its expression level in transgenic mice although the EGFP expression varied largely among different transgenic mouse litters. The results also showed that HS2 was able to confer β-globin gene expression in embryonic yolk sac, fetal liver, and adult bone marrow, which was not developmentally stage-specific, while HS3 could confer the same β-globin gene expression in the adult. Thus, HS3 was different from HS2, the former being more important for specific expression of β-globin gene in the developmental stages and the switch of γ→β-globin genes. Our results indicate that the mechanism of γ→β switch could be best explained by the “divided model.”

Introduction

Human β-globin gene locus contains five functional genes. They are arranged 5′ε-Gγ-Aγ-ψβ-δ-β-3′ from the centromere to the telomere on chromosome 11 (11p15.5) and they are expressed in erythroid tissues in this order at several developmental stages. ε-Globin gene is expressed embryonically, γ-globin genes are expressed fetally, and β-globin gene is expressed in adults. Thus, there are two developmental stages in β-globin locus expression. One is embryonic-to-fetal (ε→γ) switch, and the other is fetal-to-adult (γ→β) switch. The mechanisms underlying these two switching processes offer solid insight into the study of the regulation of eukaryotic gene expression.

The expression of human β-globin locus is under the control of the coregulation of proximal elements (such as promoters, enhancers) and distal elements (known as locus control region, LCR) [1], [2], [3], [4], [5]. LCR is the most important cis-element for the regulation of β-globin locus expression. LCR is located 5 to 22-kilobases (kb) upstream of ε-globin and consists of five DNaseI-hypersensitive sites (HS1–5) [6], [7]. LCR controls the chromatin structure, transcription, and replication properties of β-globin locus [8], [9], [10], [11], [12]. LCR is required for domain-opening, enhancement of β-like globin gene expression, and insulation from position effects [13], [14], [15].

Individual HSs have a core fragment of 200–400 bp, which is conserved and required for their functions. However, the flanking sequences of individual HSs are also required for their functions and interaction among them [13], [16], [17]. HS1 has no appreciable activity in most transfection assays and transgenic mice [18], [19]. Human HS5 may have some function as an insulator [20]. But in the erythroid background, the deletion of HS5 has only a small effect on transcription of the β-globin genes and no effect on the formation of neighboring HSs [11]. HS4 has a stronger effect in transgenic mice than in stably or transiently transfected cells, and can contribute an increased expression in combination with other HSs [21], [22]. HS2 can enhance expression of β-like globin gene both transiently from integrated constructs and after integration into a chromosome in transfected erythroid cell lines [22], [23]. HS2 will confer position independent and high-level expression in transgenic mice [24]. In addition, HS2 may confer some β-like globin gene developmental stage-specific expression [25]. HS3 has weak or no enhancer activity in transiently transfected cells. However, HS3 has the activity of enhancement and chromatin domain opening when stably integrated into chromosome in transfected cells or transgenic mice [26], [27]. These results show that HS2 and HS3 are two significant components of β-LCR. However, it is not yet clear how these HSs elements act in regulating spatial and temporal expression of β-like globin genes, how their activities lead to the full activity of LCR, and how these individual HSs interact.

In order to examine the effect of human LCR elements HS2, HS3, and HS2–HS3 (combination of HS2 and HS3) on human β-globin gene spatial and temporal expression, three erythroid-specific recombinant plasmids were constructed and their expressions in transgenic mice were measured. We found that HS2 and HS3 in combination showed a marked synergy in regulating β-globin gene spatial and temporal expression and its expression level in transgenic mice, and HS3 was more important than HS2 for specific expression of β-globin gene in the developmental stages and the switch of γ→β-globin genes.