Deletion of the core region of 5' HS2 of the mouse beta-globin locus control region reveals a distinct effect in comparison with human beta-globin transgenes

Abstract

The beta-globin locus control region (LCR) is a large DNA element that is required for high-level expression of beta-like globin genes from the endogenous mouse locus or in transgenic mice carrying the human beta-globin locus. The LCR encompasses 6 DNaseI hypersensitive sites (HSs) that bind transcription factors. These HSs each contain a core of a few hundred base pairs (bp) that has most of the functional activity and exhibits high interspecies sequence homology. Adjoining the cores are 500- to 1000-bp "flanks" with weaker functional activity and lower interspecies homology. Studies of human beta-globin transgenes and of the endogenous murine locus show that deletion of an entire HS (core plus flanks) moderately suppresses expression. However, human transgenes in which only individual HS core regions were deleted showed drastic loss of expression accompanied by changes in chromatin structure. To address these disparate results, we have deleted the core region of 5'HS2 from the endogenous murine beta-LCR. The phenotype was similar to that of the larger 5'HS2 deletion, with no apparent disruption of chromatin structure. These results demonstrate that the greater severity of HS core deletions in comparison to full HS deletions is not a general property of the beta-LCR.

Figure 1.

Generating HS2 core deletion. (A) Strategies for the murine β-globin LCR 5′HS2 deletions. (i) Schematic representation of HS2 and the homologous recombination (HR)-targeting construct with the CMV-HyTK gene flanked by 2 inverted loxP sites (arrows) replacing the 5′HS2 full site. (ii) Structure of the HR product with the RMCE site replacing the full 5′HS2. (iii) RMCE constructs. (iv) RMCE products showing the full deletion and the core deletion with the 5′HS2 flanks in their normal or inverted orientation. (B) Sequence comparison of the human and mouse 5′HS2 core deletions, including the binding sites of transcription factors most likely to be bound to the core.

Figure 2.

Identification of HS2 core, HS2 core inverted, and HS2 full deletion clones. (A) Map shows the Southern strategies to screen the modified ES clones. N indicates cutting sites for NheI; the short line under the maps, the probe; and , the CMV-HyTK gene after homologous recombination. The expected band sizes are noted. (B) ΔHS2 full deletion Southern blot. Lanes 1 and 5, ΔHS2 full/S; lane 2, D/S; and lanes 3 and 4, HS2(HR)/S. (C) ΔHS2 core deletion Southern blot. Lanes 1 and 3, ΔHS2c/S; lane 2, ΔHS2c(inv)/S; lane 4, HS2(HR)/S; and lane 5, D/S.

Figure 3.

β-Like globin gene expression after in vitro erythroid differentiation of 5′HS2 mutant ES clones. Sample gels are shown. S indicates the single allele, and D, the diffuse allele on which mutations are made; ΔHS2c, the core deletion; and ΔHS2full, the full site deletion. Wild-type (WT) values are the WT D/S ratio normalized to 1.0. The mutant/S ratio is divided by WT D/S ratio before normalization of WT D/S. Error bars represent the standard deviation of the mean for 10 individual primitive (Bh1 assay) or definitive (Bmajor and Bminor assay) erythroid colonies before normalization.

Figure 4.

β-Like globin gene expression in 5′HS2 core deletion mice. (A) Sample gels of Ey and Bh1 expression in yolk sac primitive erythroid cells. Graph of composite data from assays of Ey and Bh1 mRNA in WT/S and ΔHS2c/S yolk sac. WT values are the WT D/S ratio divided by itself to normalize to 1.0. The mutant/S ratio is divided by the prenormalization WT D/S ratio. Error bars represent the standard deviation of the mean for multiple individual embryos before normalization. (B) Sample gels of Bmaj+min expression in embryonic day 15.5 fetal liver of WT D/S and ΔHS2c/S mice. Graph of composite data from assay of Bmaj+min mRNA in WT D/S and ΔHS2c/S yolk sac (YS), fetal liver (FL), and adult peripheral blood (PB). Data normalization is done as described for Figure 3. Five or more embryos and 3 or more adult mice were assayed. ΔHS2c or ΔHS2core indicates the core deletion.

Figure 5.

DNaseI hypersensitive site formation in the HS2 core deletion mouse. Left panel is the Southern blot showing HS formation in WT mice. M indicates the molecular marker lane. The DNaseI concentration is 0 in the first lane and increases as shown by the triangle. The right panel is the Southern blot of HS formation in ΔHS2core mice. Expected size bands for HS sites or parent band are labeled.