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

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 gamma-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 gamma-globin gene regulation. We have used gel retardation and footprinting strategies to define nuclear proteins which bind to the normal gamma-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 gamma-globin gene. One factor, gamma CAAT, binds the duplicated CCAAT box sequences; the -117 HPFH mutation increases the affinity of interaction between gamma CAAT and its cognate site. Two proteins, gamma CAC1 and gamma CAC2, bind the CACCC sequence. These proteins require divalent cations for binding. The -175 HPFH mutation interferes with the binding of a fourth protein, gamma OBP, which binds an octamer sequence (ATGCAAAT) in the normal gamma-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 gamma a, binds to sequences which overlap the octamer-binding site. The erythroid-specific distribution of EF gamma a and its close approximation to an apparent repressor-binding site suggest that it may be important in gamma-globin regulation.     

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.     

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.     

Expression of the human gamma-globin gene after retroviral transfer to transformed erythroid cells.

Regulation of the human fetal (gamma) globin gene and a series of mutant gamma-globin genes was studied after retroviral transfer into erythroid cells with fetal or adult patterns of endogenous globin gene expression. Steady-state RNA from a virally transferred A gamma-globin gene with a normal promoter increased after induction of erythroid maturation of murine erythroleukemia cells and comprised from 2% to 23% of the mouse beta maj-globin RNA level. RNA expression from the virally transferred A gamma-globin gene comprised 23% of the endogenous G gamma- + A gamma-globin expression in K 562 cells after treatment with hemin. Expression from a virally transferred gamma- or beta-globin gene exceeded endogenous gamma- or beta-globin expression by a factor of 6 or more in the human erythroleukemia line KMOE, in which the endogenous globin genes are weakly inducible. In these experiments, no difference in expression was observed between the gene with the normal promoter and an A gamma-globin gene with a point mutation in its promoter (-196 C-to-T) that has been associated with hereditary persistence of fetal hemoglobin (HPFH). To test for cis-acting determinants located within the introns of the gamma-globin gene, expression was measured from a set of gamma-globin genes configured with either intron alone or with neither intron. In contrast to an intronless beta-globin gene, which is not expressed in MEL cells, the intronless gamma-globin gene was expressed in MEL cells at 24% of the level of an intron-containing gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

A novel C-T transition within the distal CCAAT motif of the G gamma-globin gene in the Japanese HPFH: implication of factor binding in elevated fetal globin expression.

Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by the continued expression of the fetal globin gene in adulthood. Both deletional and nondeletional forms have been described. We studied one Japanese family with two different nondeletional forms of HPFH. Analysis of polymorphic restriction sites in the beta-globin gene cluster suggested that one affecting both G gamma and A gamma globin expression in two members of the family could be associated with unknown conditions not linked to the beta-globin gene loci. Characterization by the polymerase chain reaction (PCR) of another form producing a G gamma-HPFH phenotype in two other members demonstrated a novel C-T transition at the nucleotide -114 within the distal CCAAT motif of the G gamma-globin gene. Using gel retardation assays on various nuclear extracts, we also demonstrated that this novel mutation abolishes the binding of the ubiquitous CCAAT binding factor, CP1 to the distal CCAAT motif of the gamma-globin gene but does not affect the binding of any erythroid specific factor, thereby suggesting a possible role for CP1 in the developmental regulation of fetal globin expression.     

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.     

Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice.

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.     

Analyses of linked beta-globin genes suggest that nondeletion forms of hereditary persistence of fetal hemoglobin are bona fide switching mutants.

The analysis of nondeletion forms of hereditary persistence of fetal hemoglobin (ndHPFH) has led to the identification of cis-acting elements, located in the promoter regions of the fetal genes, that appear to be involved in the process of fetal to adult hemoglobin switching. Individuals with these disorders demonstrate elevated levels of fetal hemoglobin and lowered levels of adult hemoglobin during adult life. This phenotype could be either the result of an abnormality in the switching process or the result of two independent mutations: one mutation increasing the level of fetal (gamma) gene expression and another mutation decreasing the level of adult (beta) globin gene expression. Here we demonstrate that the adult beta genes linked to two different forms of ndHPFH, G gamma beta + HPFH and Greek ndHPFH, produce normal levels of correctly processed mRNA in transient-expression systems. We also report that the nucleotide sequences of the beta genes are normal. These results indicate that these gamma gene promoter mutations are linked to functionally normal beta-globin genes and are consistent with the hypothesis that these mutations interfere with the normal switching process.     

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.     

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.     

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.