T to C substitution at -175 or -173 of the gamma-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

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 beta-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.     

The effects of HPFH mutations in the human gamma-globin promoter on binding of ubiquitous and erythroid specific nuclear factors.

Genetic evidence indicates that single point mutations in the gamma-globin promoter may be the cause of high expression of the mutated gene in the adult period (Hereditary Persistence of Fetal Hemoglobin, HPFH). Here we show that one of these mutations characterized by a T----C substitution at position -175 in a conserved octamer (ATGCAAAT) sequence, abolishes the ability of a ubiquitous octamer binding nuclear protein to bind a gamma-globin promoter fragment containing the mutated sequence; however, the ability of two erythroid specific proteins to bind the same fragment is increased three to five fold. DMS interference and binding experiments with mutated fragments indicate that the ubiquitous protein recognizes the octamer sequence, while the erythroid specific proteins B2, B3 recognize flanking nucleotides. Competition experiments indicate that protein B2 corresponds to an erythroid-specific protein known to bind to a consensus GATAG sequence present at several locations in alpha, beta and gamma-globin genes. Although the distal CCAAT box region of the gamma-globin gene shows a related sequence, an oligonucleotide including this sequence does not show any ability to bind the above mentioned erythroid protein; instead, it binds a different erythroid specific protein, in addition to a ubiquitous protein. The -117 G----A mutation also known to cause HPFH, and mapping two nucleotides upstream from the CCAAT box, greatly decreases the binding of the erythroid-specific, but not that of the ubiquitous protein, to the CCAAT box region fragment.     

The -117 mutation in Greek HPFH affects the binding of three nuclear factors to the CCAAT region of the gamma-globin gene.

The Greek form of hereditary persistence of fetal hemoglobin (HPFH) is associated with a point mutation immediately upstream of the distal of the two CCAAT elements of the A gamma-globin gene. Three proteins present in nuclear extracts of erythroleukemia cells bind to this CCAAT region and contact the nucleotide mutated in Greek HPFH. The ubiquitous CCAAT-binding factor CP1 interacts preferentially with the proximal CCAAT sequence. An erythroid cell-specific factor, referred to as NF-E, binds with a higher affinity to the distal CCAAT region and interacts only with sequences flanking the CCAAT motif. The third protein is the vertebrate homologue of the sea urchin CCAAT displacement protein and recognizes sequences in both CCAAT elements and their flanking sequences. While the point mutation in Greek HPFH slightly strengthens the binding of CP1 and the CCAAT displacement protein, the same base change strongly reduces the binding of NF-E to the distal CCAAT region, suggesting a possible role of NF-E in the repression of gamma-globin genes in adult erythroid cells.     

High levels of human gamma-globin gene expression in adult mice carrying a transgene of deletion-type hereditary persistence of fetal hemoglobin.

Persistent expression of the gamma-globin genes in adults with deletion types of hereditary persistence of fetal hemoglobin (HPFH) is thought to be mediated by enhancer-like effects of DNA sequences at the 3' breakpoints of the deletions. A transgenic mouse model of deletion-type HPFH was generated by using a DNA fragment containing both human gamma-globin genes and HPFH-2 breakpoint DNA sequences linked to the core sequences of the locus control region (LCR) of the human beta-globin gene cluster. Analysis of gamma-globin expression in six HPFH transgenic lines demonstrated persistence of gamma-globin mRNA and peptides in erythrocytes of adult HPFH transgenic mice. Analysis of the hemoglobin phenotype of adult HPFH transgenic animals by isoelectric focusing showed the presence of hybrid mouse alpha2-human gamma2 tetramers as well as human gamma4 homotetramers (hemoglobin Bart's). In contrast, correct developmental regulation of the gamma-globin genes with essentially absent gamma-globin gene expression in adult erythroid cells was observed in two control non-HPFH transgenic lines, consistent with autonomous silencing of normal human gamma-globin expression in adult transgenic mice. Interestingly, marked preferential overexpression of the LCR-distal (A)gamma-globin gene but not of the LCR-proximal (G)gamma-globin gene was observed at all developmental stages in erythroid cells of HPFH-2 transgenic mice. These findings were also associated with the formation of a DNase I-hypersensitive site in the HPFH-2 breakpoint DNA of transgenic murine erythroid cells, as occurs in normal human erythroid cells in vivo. These results indicate that breakpoint DNA sequences in deletion-type HPFH-2 can modify the developmentally regulated expression of the gamma-globin genes.     

The deletion of the distal CCAAT box region of the A gamma-globin gene in black HPFH abolishes the binding of the erythroid specific protein NFE3 and of the CCAAT displacement protein.

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.     

Increased Sp1 binding mediates erythroid-specific overexpression of a mutated (HPFH) gamma-globulin promoter.

The -198 T----C mutation in the promoter of the A gamma-globin gene increases 20-30 fold the expression of this gene in adult erythroid cells of patients (Hereditary Persistence of Fetal Hemoglobin, HPFH). We show here that this mutation creates a strong binding site, resembling a CACCC box, for two ubiquitous nuclear proteins, one of which is Sp1. The mutated promoter is four to five-fold more efficient than a normal gamma-globin promoter in driving expression of a CAT reporter plasmid when transfected into erythroid cells. The overexpression of the mutant is abolished by the introduction of an additional mutation disrupting the new binding site. No overexpression of the mutant is observed in non-erythroid cells, indicating that the ubiquitous factors bound on the mutated sequence must cooperate with erythroid specific factors.     

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

Transgenic mice carrying an (A)gamma gene construct containing a -382 5' truncation of the (A)gamma gene promoter have a phenotype of hereditary persistence of fetal hemoglobin (HPFH) but, when the CACCC box of the -382(A)gamma promoter is deleted, there is no gamma gene expression in the adult mice. We used this system to investigate the mechanism whereby human HPFH mutations result in gamma gene expression in the adult. Introduction of the -198 T-->C HPFH mutation into the CACCC-less (A)gamma 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 -198(A)gamma region. The HPFH phenotype was also re-established when the -117 C-->T HPFH mutation was introduced into a -141(A)gamma promoter with a destroyed CACCC box, indicating that this mutation increases gamma 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 gamma 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.     

Restriction endonuclease mapping of gamma-delta-beta-globin region in G gamma (beta)+ HPFH and a Chinese A gamma HPFH variant.

Restriction endonuclease mapping of the beta-globin genomic region was used for studying the molecular basis of two variants of hereditary persistence of fetal hemoglobin (HPFH): an African G gamma (beta)+ HPFH and a Chinese HPFH variant with predominant synthesis of A gamma chains. HPFH and control DNA samples were digested with a battery of restriction enzymes, and the fragments were identified by hybridization to a family of discrete probes. DNA fragments from the A gamma HPFH (Chinese) and the G gamma (beta)+ HPFH individuals were identical with those of the normal controls. These findings suggest that the two mutants are the result of small structural anomalies of DNA sequences that play a role in the regulation of the expression of gamma-globin genes.     

Role of the duplicated CCAAT box region in gamma-globin gene regulation and hereditary persistence of fetal haemoglobin.

Hereditary persistence of fetal haemoglobin (HPFH) is a clinically important condition in which a change in the developmental specificity of the gamma-globin genes results in varying levels of expression of fetal haemoglobin in the adult. The condition is benign and can significantly alleviate the symptoms of thalassaemia or sickle cell anaemia when co-inherited with these disorders. We have examined structure-function relationships in the -117 HPFH gamma promoter by analysing the effect of mutating specific promoter elements on the functioning of the wild-type and HPFH promoters. We find that CCAAT box mutants dramatically affect expression from the HPFH promoter in adult blood but have little effect on embryonic/fetal expression from the wild-type promoter. Our results suggest that there are substantial differences in the structure of the wild-type gamma promoter expressed early in development and the adult HPFH promoter. Together with previous results, this suggests that gamma silencing is a complex multifactorial phenomenon rather than being the result of a simple repressor binding to the promoter. We present a model for gamma-globin gene silencing that has significant implications for attempts to reactivate the gamma promoters in human adults by pharmacological means.