Abstract
Defects in the developmental changes of human hemoglobin production characterized by the continued expression of fetal globin during adult life are classified as hereditary persistence of fetal hemoglobin (HPFH). Among the various molecular lesions associated with this phenotype, the non-deletion forms with point mutations in the promoter region are thought to provide mechanistic clues for gamma-globin gene regulation. The natural occurrence of four different base substitutions mapping within six nucleotides of a homopurine.homopyrimidine motif in the upstream promoter region demarcate a potential control element. To assess its importance for transcriptional activity, we compared the -202 (C----G), -198 (T----C) and -196 (C----T) HPFH mutations with the normal sequence in binding studies with nuclear proteins from erythroid and non-erythroid cells. Wildtype DNA and HPFH mutations at -202 or -196 showed only a weak protein interaction of unclear functional significance. In contrast, -198 (T----C) generated overlapping, high-affinity binding sites for two ubiquitous nuclear proteins. One cognate protein was identified as the transcription factor Sp1. The second one was termed NF-G.C as it interacted strongly with the homopolymer poly(dG).poly(dC). The generation of additional recognition sites for trans-acting factors by the -198 HPFH mutation correlated with a modest increase in promoter activity in vitro specifically with nuclear extracts from erythroid cells. The activation appears to be mediated by binding of Sp1, but it requires interaction with an erythroid-specific factor, most likely GF-1. Templates containing the -196 HPFH mutation showed a transcriptional activity identical to wildtype. This suggests that despite the topological proximity of the mutations, the HPFH phenotype may be established by different mechanisms.