Abstract
Hereditary persistence of fetal hemoglobin (HPFH) is a result of mutations that prevent the silencing of the g-globin genes during the adult stage of definitive erythropoiesis. Two types of HPFH are recognized, deletional HPFH and non-deletional HPFH. Mutations in the later class have been identified in the proximal promoters of the Ag- and Gg-globin genes. Individuals homozygous for sickle cell disease or certain b-thalassemia mutations, that have in addition a HPFH mutation, do not suffer the deleterious effects of these diseases. These subjects provide the natural evidence supporting the clinical effort to reactivate fetal hemoglobin as the major treatment for SCD and b-thalassemias. Thus, understanding the molecular mechanisms regulating the g-globin genes is essential for identification of points of therapeutic intervention. Although the number of point mutations causing HPFH has grown over the years, the biochemical mechanisms affected by these alterations remains elusive. In addition, it is unlikely that all potential mutations have been identified in humans. A complete catalog of all potential HPFH point mutations, coupled with knowledge of the transcriptional processes affected by them will be an invaluable step towards effectively treating these diseases. We recently identified a novel T>A HPFH mutation in a GATA site at position -566 of the Ag-globin promoter, the most distal in the promoter to date, that affects binding of a GATA-1-FOG-1-Mi2 repressor complex. Since this study utilized mutated human b-globin locus yeast artificial chromosome (b-YAC) transgenic mice, where a second copy of the Ag-globin gene was introduced near the locus control region, we produced b-YAC transgenic mice containing the -566 mutation at the normally located Ag-globin gene. These mice display a mild HPFH phenotype, an approximately 3% increase in g-globin gene expression, compared to wild-type b-YAC mice. Chromatin immunoprecipitation (ChIP) studies demonstrated that this mutation prevents GATA-1 binding when g-globin is repressed in post-conception day 18 (E18) fetal liver, whereas recruitment was observed in wild-type b-YAC transgenic samples from the same developmental stage. These data are consistent with the presence of a GATA-1-mediated repressor complex at this GATA site when g-globin is not expressed. GATA-1-mediated repression may be a general mechanism of g-globin silencing. To begin testing this hypothesis, we utilized previously generated Ag-globin -117 G>A Greek HPFH b-YAC transgenic mice, which show a 5–8% increase in g-globin synthesis in adult erythropoiesis. Published data suggested that this mutation affects nearby GATA-1 binding. Our ChIP data confirmed these results, however the GATA-1 multi-protein complex that is affected may differ from that recruited to the -566 GATA binding site. Finally, we have developed a cell-based selection that is being used to identify a comprehensive set of Ag-globin HPFH promoter mutations. Chemical inducer of dimerization (CID)-dependent Ag-globin promoter-eGFP b-YAC bone marrow cells were derived from transgenic mice and mutagenized with N-ethyl, N-nitrosourea (ENU). These cells are normally GFP−; treatment with g-globin-inducers or the presence of the -117 Greek HPFH mutation results in GFP+ cells. GFP+ cells were collected by FACS and individual cell clones expanded so that genomic DNA could be isolated. Promoter proximal regions were amplified by four PCR primer sets and subjected to heteroduplex analysis with the corresponding wild-type Ag-globin promoter PCR products as the control amplicons. Twenty three heteroduplexes have been detected among 158 mutant clones screened. Most are clustered in the proximal promoter. These data suggest that we have produced HPFH mutations, likely consisting of those known in human populations, as well as novel sites that affect repressor binding or enhance recruitment of transcriptional activators.
A fetal globin gene mutation in A gamma nondeletion hereditary persistence of fetal hemoglobin increases promoter strength in a nonerythroid cell.