The human B-globin locus control region (LCR) is a complex and powerful DNA regulatory element located from 8 to 22 kbp upstream of the embryonic E-globin gene. The LCR is composed of five subregions that reveal strong sensitivity to deoxyribonuclease I in erythroid cells (hypersensitive sites HS1 to HS5). The results of many previous studies provide strong evidence supporting the hypothesis that the LCR is able to confer high level, tissue-specific expression to the human globin genes in a position-independent and copy-number dependent manner in transgenic mice. This activity is likely based on two activities intrinsic to the LCR. First the LCR is able to dominantly open the chromatin structure regardless of the position of the integrated globin locus in transgenic mice. Second, the LCR mediates high-level expression of the globin genes probably by direction communication with individual globin gene promoters. None of the previous studies, however, addressed the function of the LCR in a defined heterochromatic environment in the mouse genome. To clearly demonstrate that the LCR has intrinsic and dominant chromatin opening activity it is important to analyze LCR function in the context of a closed and repressive chromatin environment.
The aim of this proposal is to integrate the complete human p-globin locus into the mouse hypoxanthine-guanine phosphoribosyltransferase gene (Hprt) using a yeast artificial chromosome based target construct and to then analyze LCR activity within the active and inactive X chromosome. By flanking the LCR with /loxP sites, Cre/lox mediated recombination will be used to analyze chromatin structure and globin gene expression in the presence and absence of the LCR. Furthermore, this model system will be used to examine the effect of the LCR on chromatin structure of the targeted Hprt gene on the inactive X chromosome. These studies could provide insight into the mechanisms by which certain domains on the inactive X chromosome escape inactivation.
