T to C Substitution at –175 or –173 of the γ-Globin Promoter Affects GATA-1 and Oct-1 Binding in Vitro Differently but Can Independently Reproduce the Hereditary Persistence of Fetal Hemoglobin Phenotype in Transgenic Mice

Abstract

The T to C substitution at position ؊175 of the ␥-globin gene has been identified in some individuals with nondeletion hereditary persistence of fetal hemoglobin (HPFH). In this study, the HPFH phenotype was reestablished in transgenic mice carrying the LCRA␥ ؊175 ␤␦␤ construct, which contained a 3.1-kb LCR cassette linked to a 29-kb fragment from the A␥-to ␤-globin gene with the natural chromosome arrangement but with the ؊175 mutation, which provided evidence for this single mutation as the cause of this form of HPFH. The HPFH phenotype was also reproduced in transgenic mice carrying the LCRA␥ ؊173 ␤␦␤ construct, in which the ؊175 T to C A␥ gene was substituted with the ؊173 T to C A␥ gene. In vitro experiments proved that the ؊175 mutation significantly reduced binding of Oct-1 but not GATA-1, whereas the ؊173 mutation dramatically decreased binding of GATA-1 but not Oct-1. These results suggest that abrogation of either GATA-1 or Oct-1 binding to this promoter region may result in the HPFH phenotype. An in vivo footprinting assay revealed that either the ؊175 mutation or the ؊173 mutation significantly decreased overall protein binding to this promoter region in adult erythrocytes of transgenic mice. We hypothesize that a multiprotein complex containing GATA-1, Oct-1, and other protein factors may contribute to the formation of a repressive chromatin structure that silences ␥-globin gene expression in normal adult erythrocytes. Both the ؊173 and ؊175 T to C substitutions may disrupt the complex assembly and result in the reactivation of the ␥-globin gene in adult erythrocytes.The human fetal globin genes (G␥ and A␥) are mainly expressed in fetal liver. The switch from fetal to adult (␦ and ␤) globin gene expression occurs in the perinatal period concomitantly with the establishment of the bone marrow as the main site of erythropoiesis. In some individuals, however, this switching is incomplete, and ␥-globin expression persists in adult erythrocytes. Such a condition, found in syndromes of hereditary persistence of fetal hemoglobin (HPFH) 1 and (␦␤) 0 -thalassemias, can result from deletions within the ␤-globin gene cluster (1). However, in some syndromes known as nondeletion HPFH, point mutations in the ␥-globin gene promoter seem to be responsible for the continuance of ␥-globin expression (1). It has been revealed that most of the point mutations lie in or near the trans-acting factor binding motifs within the promoter and that the mutations usually affect the binding of some trans-acting factors in vitro (2).Studies in transgenic mice carrying either Ϫ117 A␥ HPFH cosmid or Ϫ117 A␥ HPFH yeast artificial chromosome showed that transgenic expression could mimic the developmental expression program observed in HPFH, providing powerful proof that the single point mutation was the cause of the Greek form of HPFH (3, 4). Berry et al. (3) speculated that this change of ␥-globin expression during development could be correlated with the loss of GATA-1 binding to the ␥-promoter region around Ϫ117 and deduced that ...

Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin

“…2f). In addition, we performed ChIP experiments for GATA1, an erythroid transcription factor previously demonstrated to bind the À 175 region in vitro 9,20 , in both MEL: A g WT and À 175T4C: A g cells. There was a modest 1.7-fold increase in GATA1 binding to the g-globin promoter when the mutation was present but this increase was not statistically significant (P ¼ 0.19 as determined by unpaired twotailed t-test; Supplementary Fig.…”

“…Yet in ChIP experiments, we were unable to observe significant differences in GATA1 binding to the WT and À 175T4C sequences. We have not examined the role of OCT1/POU2F1, which has also been reported to bind to this region of the g-globin promoter in vitro 20,33,34 .…”

Fetal hemoglobin chemical inducers for treatment of hemoglobinopathies

“…The functional role of these point mutations was supported through the analysis of transgenic mice carrying the −200, −175, and −115 mutations [18][19][20]. Some of the mutations modify binding sites, present in the γ-globin promoter regions, for the transcription factors GATA-1 or NF-E4 or occur at the level of the distal CCAAT box upstream.…”

“…The T to C substitution of the γ-globin gene has been identified in some individuals with non-deletion HPFH. In vitro experiments provided evidence that −175 mutations significantly reduced binding of the transcription factor Oct-1, but not GATA-1, whereas −173 mutations markedly decreased binding of GATA-1 but not of Oct-1 [20].…”

“…As mentioned above, the T to C mutation at position −175 of the γ-globin gene has been identified in some individuals with nondeletion HPFH. Studies on WT and mutant −175 γ-globin promoter region showed that this promoter region binds a multiprotein complex containing GATA-1, Oct-1, and other transcription factors, contributing to the formation of a repressive chromatin structure that silences γ-globin expression in adult erythroid cells [73].…”