Nuclear proteins that bind the human gamma-globin gene promoter: alterations in binding produced by point mutations associated with hereditary persistence of fetal hemoglobin.

Abstract

The molecular mechanisms responsible for the human fetal-to-adult hemoglobin switch have not yet been elucidated. Point mutations identified in the promoter regions of y-globin genes from individuals with nondeletion hereditary persistence of fetal hemoglobin (HPFH) may mark cis-acting sequences important for this switch, and the trans-acting factors which interact with these sequences may be integral parts in the puzzle of y-globin gene regulation. We have used gel retardation and footprinting strategies to define nuclear proteins which bind to the normal -y-globin promoter and to determine the effect of HPFH mutations on the binding of a subset of these proteins. We have identified five proteins in human erythroleukemia cells (K562 and HEL) which bind to the proximal promoter region of the normal -y-globin gene. One factor, yCAAT, binds the duplicated CCAAT box sequences; the -117 HPFH mutation increases the affinity of interaction between yCAAT and its cognate site. Two proteins, -yCAC1 and -yCAC2, bind the CACCC sequence. These proteins require divalent cations for binding. The -175 HPFH mutation interferes with the binding of a fourth protein, yOBP, which binds an octamer sequence (ATGCAAAT) in the normal 'y-globin promoter. The HPFH phenotype of the -175 mutation indicates that the octamer-binding protein may play a negative regulatory role in this setting. A fifth protein, EF'ya, binds to sequences which overlap the octamer-binding site. The erythroid-specific distribution of EFya and its close approximation to an apparent repressor-binding site suggest that it may be important in y-globin regulation.The human fetal-to-adult hemoglobin switch, which normally occurs at birth, is characterized by reduced -y-globin gene expression and increased activity of the adult ,B-and 8-globin genes. Despite major advances in the understanding of the structure of the human hemoglobin genes, the mechanism controlling this developmental switch has remained elusive (28).Clues to the location of important regulatory sequences in the globin genes might be gleaned from the study of naturally occurring mutations associated with abnormal expression of these genes. Such a model is provided by nondeletion hereditary persistence of fetal hemoglobin (HPFH). This syndrome is characterized by elevated expression (5-to 80-fold) of the -y-globin gene in adult life. By sequencing of DNA from affected individuals, several point mutations have been identified in the promoter regions of the overexpressed y-globin genes ( Table 1). The concordance of these point mutations with the HPFH phenotype (9, 61) suggests that they mark important regulatory sequences in the -y-globin genes. In fact, when cloned into expression vectors containing the chloramphenicol acetyltransferase (cat) gene and tested in K562 cells, the mutant promoters direct three-to eightfold overexpression of chloramphenicol acetyltransferase relative to a normal promoter (F. S. Collins, D. M. Bodine, W. K. Lockwood, J. L. Cole, L. Mickley, and T. Ley, Clin. Res. 34:454a, 198...

Discrimination among potential activators of the beta-globin CACCC element by correlation of binding and transcriptional properties.

“…During this process, adult globin genes and other erythroid cell-specific genes are transcriptionally activated (36). We have found that several CACCC-binding activities occur in MEL nuclear extracts, in agreement with previous reports (9,22,35). Although these DNA-binding activities can be distinguished by their mobilities in native gels, they share many other properties.…”

Role of NF-Y in In Vivo Regulation of the γ-Globin Gene

“…Three typical GATA-1 motifs are present in the proximal promoter: one in Ϫ220 ␥ and two around Ϫ175 ␥. In addition, gel shift assays have shown that GATA-1 binds weakly to the duplicated CCAAT box region (3,15,21). A suggestion that GATA-1 binding to the duplicated CCAAT box region functions as ␥ gene repressor (3,21) was not supported by later experiments (29).…”

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

“…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 .…”