The deletion of the distal CCAAT box region of the Aγ-globin gene in Mack HPFH abolishes the binding of the erythroki specific protein NFE3 and of the CCAAT displacement protein

Abstract

Non-deletion Hereditary Persistence of Fetal Hemoglobin (HPFH) is characterized by great elevation of the synthesis, in adult age, of fetal hemoglobin (HbF), of either the A gamma or G gamma type. Strong genetic evidence indicates point mutations in the G gamma- or A gamma-globin promoter as responsible for overexpression of the mutated gene. Here we report that a 13 nucleotides deletion in the CCAAT box region of the A gamma-globin promoter, associated with greater than 100 fold overexpression of the gene, abolishes the in vitro binding of the ubiquitous factors CP1 and CDP (CCAAT displacement protein) and of the erythroid specific protein NFE3. Loss of NFE3 binding is consistent with a similar effect of the -117 G greater than A HPFH mutation, suggesting a possible role of NFE3 as a negatively acting factor. In addition, loss of CDP binding indicates that this alteration might also contribute to the HPFH phenotype in this particular case, suggesting possible heterogeneity of the mechanisms causing HPFH.

NF-Y Organizes the γ-Globin CCAAT Boxes Region

“…1), with the ␣ (lanes 4 -6), (lanes 8 -10), h␥P (human ␥ proximal, lanes 16 -18), g␥P (prosimian Galago crassicaudatus ␥ proximal, see Ref. 36, lanes 28 -30), h␥D (human ␥ distal, lanes 20 -22) and g␥D (G. crassicaudatus ␥ distal, lanes [32][33][34], one predominant shifted band is visible; these complexes are supershifted by specific anti-YB and antiYAc antibodies (12). In each case we ran parallel migrations of the Y box oligo incubated with recombinant NF-YA and purified NF-YB/NF-YC; as shown in Fig.…”

“…Purified NF-Y was run in parallel with the MHC Ea Y box oligo (lanes 7, 11, 15, 19, 23, 27, 31, and 35). Asterisks refer to the NF-E3 band visible on the ⑀-h␥ D and g␥ P CCAAT boxes, as described previously (32,36) bin reporter gene (plasmid pAG1) and generated mutants in the proximal or in the distal CCAAT boxes (see scheme in Fig. 3A).…”

“…Several lines of reasoning point to CCAAT sequences as important elements in globin gene expression as follows: (i) they are present in all globin promoters; (ii) they have been remarkably conserved in different species at a fixed distance from the cap site; (iii) genomic footprinting of all globin promoters in erythroid cells showed invariable protection of CCAAT sequences in vivo, indicating binding of activators (31); (iv) the functional importance has been documented in the ␤-, ␣-, -, and ⑀-globin promoters (2-5, 15); (v) the ␥-globin duplicated CCAAT boxes are the target of mutations affecting developmental silencing of ␥-globin expression in adult life. In particular, strong genetic evidence suggests that point mutations in the CCAAT box region are causative of the HPFH syndromes, characterized by increased fetal globin levels in adults (32)(33)(34)(35)(36)(37).Several studies investigated factors binding to CCAAT; CDP, NFE3, and c/EBP appear to recognize different globin promoters (3, 32-36). One CCAAT-binding protein, ␣-CP1, was purified to homogeneity using an ␣-globin CCAAT box affinity * This work was supported in part by grants from Murst (to R. M. and S.…”

“…One CCAAT-binding protein, ␣-CP1, was purified to homogeneity using an ␣-globin CCAAT box affinity column and found to be a trimeric factor most likely identical to NF-Y (15). Moreover, competition and supershift EMSA experiments performed with nuclear extracts determined that a CCAAT binding activity similar to NF-Y (termed CP1 in the globin field) binds to the ␥-globin promoters (32,33). However, identification of CCAAT binding activities in other promoters is less certain; therefore, we wished to definitely determine which of the globin CCAAT boxes represents a bona fide NF-Y-binding site and establish a hierarchy of affinities.…”

Native highland and lowland populations differ in .GAMMA.-globin gene promoter polymorphisms related to altered fetal hemoglobin levels and delayed fetal to adult globin switch after birth

“…The 4-bp deletion is a common polymorphism in Americans of European and African ancestry (Coleman et al, 1991). By contrast, an elevated expression of the A γ globin has been associated with a G→A substitution at position −117 adjacent to the distal CAAT box of the A γ gene promoter, possibly by inhibiting binding of a repressor (Mantovani et al, 1989;Pistidda et al, 1995). This polymorphism has been found in Mediterranean populations.…”