Effect of LCR 5′HS4 and 5′HS1 Deletions on β-Like Globin Gene Expression in β-Yac Transgenic Mice.

A 213 Kb human β-globin locus yeast artificial chromosome (β-YAC) was modified by homologous recombination to delete 2.9 Kb of cross-species conserved sequence similarity encompassing the LCR 5′HS4 (Δ5′HS4 β-YAC). Three transgenic mouse lines were established; each contained two intact copies of the β-globin locus as determined by long range restriction enzyme mapping (LRRM) and Southern blot hybridization analyses. Human ε-, γ- and β-globin, and mouse α- and ζ-globin mRNAs were measured by RNAse protection in hematopoietic tissues derived from staged embryos, fetuses and adult mice. No difference in the temporal pattern of globin transgene expression was observed between Δ5′HS4 β-YAC mice and wild-type β-YAC mice. In addition, quantitative per-copy human β-like globin mRNA levels were similar between Δ5′HS4 and wild-type β-YAC transgenic lines, although γ-globin gene expression was slightly increased in the fetal liver, while β-globin gene expression was slightly decreased in Δ5′HS4 β-YAC mice. These data are in contrast to data obtained from β-YAC mice containing a deletion of the 280 bp 5′HS4 core. In these mice, γ- and β-globin gene expression was significantly decreased during fetal definitive erythropoiesis and β-globin gene expression was decreased during adult definitive erythropoiesis. However, these data are consistent with the observation that deletion of the 5′HS core elements is more deleterious than large deletions of the 5′HSs. Together, the compiled deletion data supports the hypothesis that the LCR exists as a holocomplex in which the 5′HS cores form an active site and the flanking 5′HS regions constrain the holocomplex conformation. In this model, 5′HS core mutations are dominant negative, whereas larger deletions allow the LCR to fold into alternate holocomplex structures that function normally, albeit less efficiently.

To complete the study on the contribution of the individual 5′HSs to LCR function, a 0.8 Kb 5′HS1 fragment was deleted in the 213 Kb β-YAC by homologous recombination. Two ΔHS1 β-YAC transgenic lines have been established; four additional founders were recently identified. Of the two lines, one contains two intact copies of the globin locus; the other contains four deleted copies, one of which extends from the LCR through just 5′ to the β-globin gene. For both lines, ε-globin gene expression was markedly reduced, approximately 5–10 fold, during primitive erythropoiesis. Developmental expression profiles and levels of the γ- and β-globin genes (in the line that contains loci including the β-globin gene) were unaffected by deletion of 5′HS1. Breeding of the remaining four founders to obtain F1 and F2 progeny for similar structure/function studies is in progress. Decreased expression of the β-globin gene is the first phenotype ascribed to a 5′HS1 mutation, suggesting that this HS does indeed have a role in LCR function.