Hemoglobin switching is an example of tissue specific and temporal regulation of gene expression. In humans, embryonic globin chains are expressed during the first trimester of gestation followed by a switch to the expression of fetal globin chains early in the second trimester and adult globin chains after birth. Previous worked performed in the Laboratory of Chemical Biology has identified a silencer 200 to 400 bp upstream of the epsilon (e) globin gene which has been implicated in suppressing epsilon globin gene expression during the fetal and adult stages. Our work has focused on characterizing proteins binding to the silencer and the functional activity of the binding of trans-acting factors to the silencer. We are in th process of elucidating the mechanism(s) that trans- acting factors interacting with the silencer utilize in order to regulate e-globin transcription. We are also studying the interactions of the silencer with other regulatory elements controlling e-globin gene expression. Using DNase I footprinting, we have identified several sites of protein binding to the silencer. The major protected region shares a high percentage of homology with the binding sites of an erythroid specific transcription factor GATA-1 and a ubiquitous silencer binding protein, YY1. Using gel mobility shift assays with K562 nuclear extract, YY1 protein and a probe bracketing the major protected region and competitor DNA with mutations in the sites homologous to GATA-1 an YY1 binding sites we have demonstrated that a protein, likely GATA-1 binds to the GATA site in the silencer and that another protein, likely YY1, binds to the silencer. We are currently in the process of examining the activity of GATA-1 and YY1 on the expression of beta-like globin genes and determining the mechanism by which GATA-1 and YY1 binding to the silencer inhibits epsilon globin gene expression. Using transient expression assays we have demonstrated that the epsilon globin silencer inhibits expression of a heterologous promoter and that this repression requires an intact YY1 binding site. We have also demonstrated that GATA proteins activate expression of e-globin in erythroid cells but in a non-erythroid cell type represses expression. These data suggest that the mechanism of silencing of the e-globin gene in adult erythroid cells may involve unusual transcriptional repression by both GATA and YY1 proteins, probably mediated by one or more other transcriptional factors.
