Analysis of the mechanism of action of non-deletion hereditary persistence of fetal hemoglobin mutants in transgenic mice

Abstract

Transgenic mice carrying an Aγ gene construct containing a −382 5′ truncation of the Aγ gene promoter have a phenotype of hereditary persistence of fetal hemoglobin (HPFH) but, when the CACCC box of the −382Aγ promoter is deleted, there is no γ gene expression in the adult mice. We used this system to investigate the mechanism whereby human HPFH mutations result in γ gene expression in the adult. Introduction of the −198 T→C HPFH mutation into the CACCC‐less Aγ gene construct re‐established the HPFH phenotype, indicating that this mutation increases promoter strength, most probably by establishing a novel CACCC box sequence in the −198Aγ region. The HPFH phenotype was also re‐established when the −117 C→T HPFH mutation was introduced into a −141Aγ promoter with a destroyed CACCC box, indicating that this mutation increases γ promoter strength in the absence of the CACCC motif. The T→A −175 HPFH mutation failed to re‐establish the HPFH phenotype when the CACCC box was deleted, indicating that γ gene expression in this mutation is CACCC box dependent. These results provide the first in vivo experimental evidence in support of mechanistic heterogeneity of the non‐deletion HPFH mutants.

Purification and Identification of Proteins That Bind to the Hereditary Persistence of Fetal Hemoglobin –198 Mutation in the γ-Globin Gene Promoter

“…1B, lane 1, arrows a-c) that were competed away by the unlabeled HPFH Ϫ198 oligonucleotide (lane 4), but not by the WT Ϫ198 oligonucleotide (lane 5). These bands were partially competed away with a 100-fold excess of unlabeled WT CACCC oligonucleotide (lane 2), but not by the mutant CACCC oligonucleotide (lane 3), suggesting that the proteins forming these retarded bands are associated with the CACCC family, as reported previously (3)(4)(5)9). Below the three bands, there were several fast migrating bands (bracket d).…”

“…We have shown previously that the HPFH Ϫ198 mutation is able to retain ␥ gene expression in adult transgenic mice when the CACCC box is disrupted (9). Because the CACCC box is indispensable for ␥ gene expression in the adult, these results suggested that the HPFH Ϫ198 mutation creates a new element that is able to substitute for the function of the CACCC box in adults.…”

Silencing of Aγ-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the −566 GATA Site

“…An understanding of the molecular basis underlying interaction of these regulatory motifs is not complete. Although autonomous silencing of ␥-globin transcription during development has been documented (17,19,29,39,66), the sequences encoding the silencer element(s) and the mechanism(s) of repression have only recently been explored (64,68,69).…”

Transcriptional regulation of FKLF-2 (KLF13) gene in erythroid cells

“…4). In addition two CCAAT boxes could be a potential GATA-1-binding site [16,17]. We, however, failed to demonstrate GATA-1-binding to these sequences by gel shift experiments, in which there was a distinct GATA-1-binding to the canonical GATA sequence (data not shown).…”

“…There are two possibilities: either GATA-1 binds to the promoter region but the binding is too weak to be detected; or the activation of the promoter by GATA-1 is indirect, a case such as transcription factor(s) regulated by GATA-1 activate the KLF13 gene promoter through the CCAAT or neighboring sequences. The former possibility may be supported by the evidence that the binding to CCAAT element is very unstable [17]. In addition, the non-canonical GATA-1 binding sequence, GATT that overlaps with the distal CCAAT motif of the KLF13 gene promoter (Fig.…”