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.     

Distal CCAAT box deletion in the A gamma globin gene of two black adolescents with elevated fetal A gamma globin.

Point mutations in G gamma and A gamma globin gene promoters are associated with increased production of G gamma and A gamma globin, respectively. To determine whether an upstream promoter mutation could account for elevated A gamma in a Black adolescent with A gamma-beta+-HPFH and sickle cell trait, we cloned the 13 kb BglII fragment containing both gamma genes into phage lambda vector EMBL3. For one clone, the A gamma upstream promoter showed no hybridization to a 19 bp oligonucleotide whose sequence centered at -117. A gamma promoter sequence data for this mutant clone revealed a 13 bp deletion which eliminated the A gamma distal CCAAT box. Amplified A gamma genomic DNA of this and a similar case showed hybridization to both deletion-mutant and normal oligonucleotide probes. We propose that this 13 bp deletion removes part of the binding site for a repressor protein which is abundant in adult erythroid cells.     

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.     

Increased erythroid-specific expression of a mutated HPFH gamma-globin promoter requires the erythroid factor NFE-1.

The -175 T greater than C mutation in the promoter of the A gamma- or G gamma-globin gene causes a 50-100 fold increase of the expression of the respective gene in adult erythroid cells (Hereditary Persistence of Fetal Hemoglobin). We show here that this mutation increases 3-9 fold the expression of a gamma-CAT reporter plasmid transfected into the erythroid cells K562, but not that of the same plasmid in non erythroid cells. The overexpression of the mutant is abolished by the mutation of the binding site for the erythroid specific factor NFE1; inactivation of the adjacent binding site for the ubiquitous factor OTF1 does not cause overexpression of the normal gamma-globin promoter. Previous results demonstrated that the -175 mutation slightly increases the in vitro binding of NFE1 and almost abolishes that of OTF1; the present functional data indicate that altered binding of NFE1, but not of OTF1, is responsible for the observed overexpression of the mutated promoter.     

Roles of fetal G gamma-globin promoter elements and the adult beta-globin 3'' enhancer in the stage-specific expression of globin genes.

The human fetal G gamma-globin and adult beta-globin genes are expressed in a tissue- and developmental stage-specific pattern in transgenic mice: the G gamma gene in embryonic cells and the beta gene in fetal and adult erythroid cells. Several of the cis-acting DNA sequences thought to be responsible for these patterns of expression are located 5' to the G gamma-globin gene and 3' to the beta-globin gene. To further define the locations and functional roles of these elements, we examined the effects of 5' truncations on the expression of the G gamma-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene. We found that sequences between -201 and -136 are essential for expression of the G gamma-globin gene, whereas those upstream of -201 have little effect on the level or tissue or stage specificity of G gamma-globin expression. The G gamma-globin upstream sequences from -201 to -136 were, furthermore, capable of activating a linked beta-globin gene in embryonic blood cells; however, a G gamma-globin fragment from -383 to -206 was similarly active in this assay, and the complete fragment from -383 to -136 was considerably more active than either of the smaller fragments, suggesting the presence of multiple cis-acting elements for embryonic blood cells. Our data also suggested the possibility of a negative regulatory element between -201 and -136. These results are discussed in relation to several DNA elements in the G gamma-globin upstream region, which have been shown to bind nuclear factors in erythroid cells. Finally, we observed that removal of the beta-globin 3'-flanking sequences, including the 3' enhancer, from the G gamma-globin upstream-beta-globin hybrid gene resulted in a 25-fold reduction in expression in embryonic blood cells. This suggests that the beta-globin 3' enhancer is potentially active at the embryonic stage and thus cannot be solely responsible for the fetal or adult specificity of the beta-globin gene.     

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.     

Heterogeneity of the gamma-globin gene sequences in Japanese individuals: implication of gene conversion in generation of polymorphisms

The linked fetal globin genes (the G gamma- and A gamma-globin genes) were cloned from Japanese individuals with three different haplotypes of the HindIII polymorphisms within the gamma-globin genes. Determination of nucleotide sequences of the segment spanning from IVS2 to the 3' flanking region of each gamma-globin gene revealed that nucleotide differences are located at 43 positions and a stretch of simple GT or GC sequences. Almost half of the nucleotide changes could be accounted for by gene conversion between the G gamma- and A gamma-globin genes. We found that gene conversion had created the SacI polymorphic site just downstream of the A gamma-globin coding region. Association of the SacI polymorphic site with the HindIII polymorphic site suggests that the region containing these two sites was derived from that of the linked G gamma-globin gene through a gene conversion event. The nucleotide sequences obtained here are identical to those of the Caucasoid fetal globin genes of the same haplotypes, with the exception of some sequence changes in the hot spots of mutations. These results indicate that the sequence heterogeneity of the gamma-globin genes can be classified into three major categories according to HindIII haplotypes. The possible mechanisms of generation of the heterogeneity of the gamma-globin gene sequences are discussed.     

Two novel arrangements of the human fetal globin genes: G gamma-G gamma and A gamma-A gamma.

We describe two novel arrangements of the human fetal globin gene region: one chromosome with two linked A gamma genes (A gamma-A gamma) and two chromosomes with two linked G gamma genes (G gamma-G gamma). The gamma genes of these three chromosomes were cloned and the unusual 5' A gamma gene and one of the unusual 3' G gamma genes were partially sequenced. Both of these unusual genes differ from the genes normally found at their respective locations by a nucleotide substitution at the site of the single coding region difference between normal G gamma and A gamma genes. In both cases, the substitution is identical to the nucleotide found at that position in the normal neighboring gene. The unusual 3' G gamma gene also differs from normal A gamma genes at two other nucleotide positions, but both differences appear to be "private" or exclusive to this particular gene. These unusual fetal globin gene arrangements could have arisen from point mutations or from gene conversions of limited extent, the boundaries of which have been determined for all three chromosomes.     

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.     

A gene controlling fetal hemoglobin expression in adults is not linked to the non-alpha globin cluster.

The possible linkage between a gene causing heterocellular hereditary persistence of fetal hemoglobin (HPFH) and human non-alpha globin loci has been studied in a large Sardinian family. In this family a homozygous beta o-thalassemic patient was found, with an unusually mild form of this disease, which was ascribed to the co-existence of a gene causing heterocellular HPFH. DNA polymorphisms in the non-alpha globin cluster were analyzed by restriction enzyme digestion with HincII, HindIII and BamHI and with epsilon-, gamma-and beta-globin probes; the pattern of inheritance of these polymorphisms indicates that the HPFH gene is transmitted with one beta o-thalassemic gene in a single instance, with the second beta o-thalassemic gene in three instances and with a normal beta-globin gene in two cases. These data indicate that this HPFH gene is not linked to the non-alpha globin gene cluster, in contrast to previous observations with different HPFH genes, and suggest that this gene might code for diffusible substances acting, directly or indirectly, on gamma-globin gene expression.     

A composite transposon 3'' to the cow fetal globin gene binds a sequence specific factor.

Two unusual sequence organizations were found within the beta-globin locus of the cow. Each was a composite, consisting of closely linked Alu-type repeats with a short stretch of genomic non-repetitive sequence, called a lagan, sandwiched between. One lagan was found 3' to the fetal globin gene, while the second lay between the adult globin gene and a globin pseudogene. Southern blot analysis indicated that both lagans appeared twice within the cow haploid genome, with the second copies lying outside the cow beta-globin locus. One of these non-globin locus homologues was cloned and subjected to sequence analysis. Comparison of the DNA sequence data showed that the lagan-Alu composite was transposed as a unit. The lagan 3' to the cow fetal globin gene contains the recognition site for a sequence specific DNA binding factor. This factor was present in extracts from fetal, but not from adult cow tissues.     

A protein factor binding to an octamer motif in the gamma-globin promoter disappears upon induction of differentiation and hemoglobin synthesis in K562 cells.

Using the electrophoretic mobility shift assay and the footprinting technique, we studied the binding of nuclear proteins from erythroid and non erythroid human cells to the promoter region of the human gamma-globin gene. Two regions (A and B) of the promoter are bound by proteins present in uninduced K562 cells, but not in induced K562 cells nor in fetal liver erythroblasts; a protein binding to region A is also present in a variety of lymphoid and myeloid cells. Region B is centered on an octamer sequence identical to that present in immunoglobulin promoter and enhancers and other eukaryotic promoters; a B region binding protein common to K562 and other cells efficiently binds the octamer containing region of the histone H2B gene, while different B region proteins are more specific for uninduced K562 cells and the gamma-globin octamer containing fragment. The possible role of these nuclear proteins in gamma-globin gene regulation and/or cell differentiation is discussed.     

CCAAT/enhancer-binding protein-beta participates in insulin-responsive expression of the factor VII gene

Expression of the human coagulation factor VII (FVII) gene by hepatoma cells was modulated in concert with levels of glucose and insulin in the culture medium. In low glucose medium without insulin, amounts of both FVII mRNA and secreted FVII protein were coordinately increased; in the presence of glucose with insulin, both were decreased. Analysis of the FVII promoter showed that these effects could be reproduced in a reporter-gene system, and a small promoter element immediately upstream of the translation start site of the gene, which mediated these effects, was identified. Mutation of this element largely abrogated the glucose/insulin-responsive change in expression of the reporter gene. Several members of the CCAAT/enhancer-binding protein family were found to be capable of binding the identified sequence element but not the mutated element. The expression of a FVII minigene directed by a segment of the native FVII promoter responded to co-expressed activating and inhibiting forms of CCAAT/enhancer-binding protein beta.