TARGETED DELETION OF 5'HS2 OF THE MURINE BETA-GLOBIN LCR REVEALS THATIT IS NOT ESSENTIAL FOR PROPER REGULATION OF THE BETA-GLOBIN LOCUS

The beta-globin locus control region (LCR) is a complex regulatory ele ment that is essential for the appropriate red cell-specific expressio n of all cis-linked beta-globin genes. Of the five hypersensitive site s that define the LCR, only 5'HS2 has been shown to augment gene expre ssion in vitro in both transient and stable assays, as well as in tran sgenic mice. Thus, 5'HS2 has been assumed to be an important element f or the function of the LCR in vivo. We have utilized homologous recomb ination in murine embryonic stem (ES) cells and phenotypic analysis in derived mice to investigate the function of 5'HS2 in its normal chrom osomal position in the murine beta-globin locus. Replacement of 5'HS2 with a selectable marker gene (Delta HS2+neo) causes a 2-5-fold reduct ion in expression of all of the genes in the locus, and a more pronoun ced effect (10-12-fold) on the most 5' embryonic globin gene, Ey, when expression of this gene is first detectable during embryogenesis. The mutation produces no alterations in the developmental timing of expre ssion of the globin genes. When homozygous, the deletion/replacement m utation is lethal in utero, with the embryos dying during the stage of yolk sac and early fetal liver erythropoiesis. To distinguish phenoty pic effects resulting from the deletion of 5'HS2 from those attributab le to insertion of the selectable marker, the selectable marker was re moved by expressing the FLP site-specific recombinase in ES cells harb oring the homologous recombination event. Mice derived from these ES c ells (Delta HS2 Delta neo) demonstrated nearly full expression of all the beta-like globin genes on the mutated chromosome. These results in dicate that although 5'HS2 demonstrates significant regulatory activit ies in a variety of assays, deletion of this element from the endogeno us beta-globin locus has no significant effect on the timing or extent of expression of the locus. In addition, this result emphasizes that when using homologous recombination to analyze complex regulatory elem ents in vivo, the inserted selectable marker must be removed to avoid influencing the phenotype of the mutation.