T to C substitution at-175 or-173 of the γ-globin promoter affects GATA- and Oct-1 binding in vitro differently but can independently reproduce the hereditary persistence of fetal hemoglobin phenotype in transgenic mice

The T to C substitution at position - 175 of the gamma-globin gene has been identified in some individuals with non-deletion hereditary persistence of fetal hemoglobin ( HPFH). In this study, the HPFH phenotype was reestablished in transgenic mice carrying the mu'LCRAgamma(-175)psibetadeltabeta construct, which contained a 3.1-kb mu'LCR cassette linked to a 29-kb fragment from the Agamma- to gamma-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 mu'LCRAgamma(-173)psibetadeltabeta construct, in which the - 175 T to C Agamma gene was substituted with the - 173 T to C Agamma 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 gamma-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 gamma-globin gene in adult erythrocytes.