Regulation of the human fetal gamma globin genes.

Abstract

The purpose of this report was to study fetal gamma globin gene regulation. In the non-deletion form of hereditary persistence of fetal hemoglobin (HPFH), point mutations occur within the promoter regions of the paired gamma globin genes. Since individuals with HPFH continue to express fetal globin in adult life, these point mutations may highlight sequences which contribute to the fetal to adult hemoglobin switch. Five HPFH mutations occur within seven base pairs in the $-$200 region of the gamma globin promoters. By gel retardation and footprinting assays, several proteins were shown to bind this region; at least one of the DNA-protein complexes results from Sp1 protein binding. Experiments with purified Sp1 protein and Sp1 antibody identified three regions within the gamma globin promoters which bind to Sp1. The transcriptionally important CACCC site at $-$140 has a high affinity for Sp1. The $-$50 and $-$200 regions constitute weak Sp1 binding sites. Additional proteins also bind to each of these sites. Expression studies in Drosophila SL2 cells demonstrated that these Sp1 binding sites allow Sp1 mediated transactivation of the gamma globin promoter. The Sp1-dependent promoter strength was reduced 57% by a point mutation within the CACCC sequence. The HPFH point mutation at $-$198 dramatically increases the binding of Sp1. As a result of this increased affinity, the $-$198 HPFH promoter is transactivated to a greater extent than the wild type promoter. Finally, transient expression assays were done in an attempt to demonstrate that the point mutations are responsible for the overproduction of gamma globin in adult cells. However, the constructs used failed to recapitulate the overexpression seen in vivo. This may be due to the need for additional cis sequences in the construct or the need to conduct these experiments in a cell line which can be induced to switch. In conclusion, the data presented here increases the understanding of the trans-factors which interact with the gamma globin promoters and suggests that Sp1 may play an important role in the regulation of the fetal gamma globin genes.