Developmental regulation of the human beta-globin locus and the role of the O and P regions in beta-like globin gene expression

The human beta-globin locus is a highly complex genetic system that has become a classical model for studying the regulation of eukaryotic gene expression. The locus consists of five functional beta-like globin genes, epsilon- Ggamma-Agamma-delta-beta and a powerful upstream regulatory element, the locus control region (LCR) that is physically composed of five DNase-I hypersensitive sites. The globin genes are regulated in a tissue and developmental stage-specific manner and are arranged spatially in the order in which they are expressed during development. During development two switches in globin gene expression occur to accommodate the changing oxygen needs of the fetus. The first switch from embryonic epsilon- to fetal gamma-globin occurs at six weeks of gestation and the second switch from gamma-globin to adult beta- and delta-globin occurs shortly after birth. Intense studies of globin gene expression over the past two decades have enhanced our understanding considerably of the molecular mechanisms that together ensure a highly specialized, tissue- and stage-specific transcription pattern of the beta-like genes during development. From these studies many of the key players at the human beta-globin locus have been identified. They include the cis-acting regulatory elements, such as the LCR and gene-proximal regulatory sequences, involved in establishing and maintaining specific chromatin conformations and histone modification patterns throughout the locus, and transcriptional activation, as well as trans-acting factors, both erythroid-specific and general. Much of the current research on the human beta-globin locus has been focused on further elucidating the mechanisms involved in the regulation of the fetal gamma-globin genes in an effort to develop potential therapies that will enhance fetal hemoglobin expression in individuals with sickle cell disease or beta-thalassemia. Therefore, two regions, O and P, located upstream of the delta-globin gene, that had previously been shown to function as silencers of the gamma-globin genes in transient transfection assays were investigated. We hypothesized that if the O and P regions do in fact function to silence the gamma-globin genes during development, then the deletion of these regions from the human beta-globin locus would result in the persistence of gamma-globin in the adult erythroid cells To test this hypothesis, the O and P regions were deleted from a 213 kb human beta-globin locus yeast artificial chromosome (beta-YAC). Transgenic mice were produced with the modified beta-YAC and the effects of this OP deletion on expression of the human beta-like globin genes were analyzed. Deletion of these regions resulted in the abolishment of epsilon-globin gene expression during embryonic erythropoiesis and a substantial decrease in beta-globin globin gene expression during definitive erythropoiesis. Conversely, expression of the gamma-globin genes was only slightly affected during fetal definitive erythropoiesis. Although these results do not indicate that the O and P regions are involved in silencing the gamma-globin genes during development, they do provide some insight into the role of these regions in the regulation of the beta-like globin genes.