Hypersensitive site 2 specifies a unique function within the human beta-globin locus control region to stimulate globin gene transcription

The human beta-globin locus control region (LCR) harbors both strong chroma tin opening and enhancer activity when assayed in transgenic mice. To under stand the contribution of individual DNase I hypersensitive sites (HS) to t he function of the human beta-globin LCR, we have mutated the core elements within the context of a yeast artificial chromosome (YAC) carrying the ent ire locus and then analyzed the effect of these mutations on the formation of LCR HS elements and expression of the genes in transgenic mice. In the p resent study, we examined the consequences of two different HS2 mutations. We first generated seven YAC transgenic lines bearing a deletion of the 375 -bp core enhancer of HS2, Single-copy HS2 deletion mutants exhibited severe ly depressed HS site formation and expression of all of the human beta-glob in genes at every developmental stage, confirming that HS2 is a vital, inte gral component of the LCR We also analyzed four transgenic lines in which t he core element of HS2 was replaced by that of HS3 and found that while HS3 is able to restore the chromatin-opening activity of the LCR, it is not ab le to functionally replace HS2 in mediating high-level globin gene transcri ption, These results continue to support the hypothesis that HS2, HS3, and HS4 act as a single, integral unit to regulate human globin gene transcript ion as a holocomplex, but they can also be interpreted to say that formatio n of a DNase I hypersensitive holocomplex alone is not sufficient for media ting high-level globin gene transcription. We therefore propose that the co re elements must productively interact with one another to generate a uniqu e subdomain within the nucleoprotein holocomplex that interacts in a stage- specific manner with individual globin gene promoters.