Analysis of Mice Containing a Targeted Deletion of β-Globin Locus Control Region 5′ Hypersensitive Site 3

Abstract

To examine the function of murine ␤-globin locus control region (LCR) 5 hypersensitive site 3 (HS3) in its native chromosomal context, we deleted this site from the mouse germ line by using homologous recombination techniques. Previous experiments with human 5 HS3 in transgenic models suggested that this site independently contains at least 50% of total LCR activity and that it interacts preferentially with the human ␥-globin genes in embryonic erythroid cells. However, in this study, we demonstrate that deletion of murine 5 HS3 reduces expression of the linked embryonic y-and ␤H 1-globin genes only minimally in yolk sac-derived erythroid cells and reduces output of the linked adult ␤ (␤major plus ␤minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-neo cassette was left within the HS3 region of the LCR, a much more severe phenotype was observed at all developmental stages, suggesting that PGK-neo interferes with LCR activity when it is retained within the LCR. Collectively, these results suggest that murine 5 HS3 is not required for globin gene switching; importantly, however, it is required for approximately 30% of the total LCR activity associated with adult ␤-globin gene expression in adult erythrocytes.The human ␤-globin gene cluster consists of a set of five functional genes and one pseudogene. The cluster is active exclusively in erythroid cells, and the individual ␤-like globin genes are expressed during specific developmental stages (33). Sequences local to the individual globin genes are sufficient to direct developmental stage-specific and tissue-specific expression (3,4,6,24,37,38), but high-level expression of the globin genes depends on distant regulatory sequences collectively called the locus control region (LCR) (33). The LCR is composed of a series of erythroid cell-specific, developmentally stable DNase I-hypersensitive sites (HSs) (5, 12, 39) that contain the sequences essential for LCR activity (7,11,13,16,24,29,36).Recent efforts have been directed toward determining the essential sequences within the LCR. Homology comparisons between the LCRs of humans, mice, goats, and rabbits have revealed conservation of the HS cores but not the intervening sequences (17-19, 22, 23, 25, 31). This finding suggests that the conserved sequences define the HS; functional assays have confirmed this hypothesis (12-14, 17, 18, 29, 30, 36). The HSs have been tested individually in transient and stable assays in tissue culture cells and in transgenic mice; 5Ј HS2, -3, and -4 do indeed contain the information that directs high-level expression of linked genes.Recent studies have focused on the activities of the individual HSs within the context of the entire ␤-globin cluster. These approaches have been undertaken to avoid the complications of studying small transgenes in which distant or undefined regulatory influences may be missing. Transgenic animals have now been made by using large DNA fragments derived from the ␤-globin cluster (1,8,15,27,28,35); although these tra...

Transcription of the HS2 Enhancer toward a cis-Linked Gene Is Independent of the Orientation, Position, and Distance of the Enhancer Relative to the Gene

“…On the other hand, previous studies of targeted replacement of HS2 and HS3 in the murine LCR (15,21) suggest that the transcription of a foreign gene at the location of the HS2 or the HS3 site disrupts proper LCR function. In this study we have shown that the HS2 enhancer is transcribed predominantly in a direction toward the cis-linked gene apparently from defined DNA motifs within the HS2 sequence.…”

“…In the human ␤-LCR, inserting an independent transcription unit at a location between the HS2 enhancer and the downstream globin genes also disrupted LCR function (23) and caused the distantly downstream ␤-globin gene to be transcriptionally turned off. The mechanism by which the transcription of a foreign gene within the LCR might disrupt LCR function is not clearly understood (21).…”

“…Surprisingly, similar targeted deletion of HS3 also did not cause significant changes in the expression of the ␤-like globin genes (22). These findings suggest that LCR function is due not to the contribution of any specific HS site but to a series of interactions among its functional components, including the enhancer elements underlying the HS sites.In the above-described targeted deletion studies, deleting the HS2 or the HS3 sequence from the endogenous murine LCR and inserting in its place an independent transcription unit-a neomycin-resistant gene driven by a strong promoter-has been found to disrupt LCR function and cause severe anemia in and the deaths of homozygous transgenic mice (15,21). In the human ␤-LCR, inserting an independent transcription unit at a location between the HS2 enhancer and the downstream globin genes also disrupted LCR function (23) and caused the distantly downstream ␤-globin gene to be transcriptionally turned off.…”

“…In the above-described targeted deletion studies, deleting the HS2 or the HS3 sequence from the endogenous murine LCR and inserting in its place an independent transcription unit-a neomycin-resistant gene driven by a strong promoter-has been found to disrupt LCR function and cause severe anemia in and the deaths of homozygous transgenic mice (15,21). In the human ␤-LCR, inserting an independent transcription unit at a location between the HS2 enhancer and the downstream globin genes also disrupted LCR function (23) and caused the distantly downstream ␤-globin gene to be transcriptionally turned off.…”

A Targeted Mutation at the T-Cell Receptor α/δ Locus Impairs T-Cell Development and Reveals the Presence of the Nearby Antiapoptosis Gene Dad1

“…Because targeted deletions allow a direct comparison with the activity of the endogenous locus, it is the more valid approach when trying to test the necessity of a particular regulatory element and may reveal unexpected redundancy. For example, targeting experiments with the ␤-globin LCR have contributed significant understanding to that gained from transgenic data (9,15). Here we describe a deletion of the endogenous TCR␣ LCR by gene targeting which leads to incomplete TCR␣ expression.…”