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.     

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.     

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.     

Detection of a major gene for heterocellular hereditary persistence of fetal hemoglobin after accounting for genetic modifiers.

"Heterocellular hereditary persistence of fetal hemoglobin" (HPFH) is the term used to describe the genetically determined persistence of fetal hemoglobin (Hb F) production into adult life, in the absence of any related hematological disorder. Whereas some forms are caused by mutations in the beta-globin gene cluster on chromosome 11, others segregate independently. While the latter are of particular interest with respect to the regulation of globin gene switching, it has not been possible to determine their chromosomal location, mainly because their mode of inheritance is not clear, but also because several other factors are known to modify Hb F production. We have examined a large Asian Indian pedigree which includes individuals with heterocellular HPFH associated with beta-thalassemia and/or alpha-thalassemia. Segregation analysis was conducted on the HPFH trait FC, defined to be the percentage of Hb F-containing cells (F-cells), using the class D regressive model. Our results provide evidence for the presence of a major gene, dominant or codominant, which controls the FC values with residual familial correlations. The major gene was detected when the effects of genetic modifiers, notably beta-thalassemia and the XmnI-G gamma polymorphism, are accounted for in the analysis. Linkage with the beta-globin gene cluster is excluded. The transmission of the FC values in this pedigree is informative enough to allow detection of linkage with an appropriate marker(s). The analytical approach outlined in this study, using simple regression to allow for genetic modifiers and thus allowing the mode of inheritance of a trait to be dissected out, may be useful as a model for segregation and linkage analyses of other complex phenotypes.     

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.     

One haplotype is associated with the Swiss type of hereditary persistence of fetal hemoglobin in the Yugoslavian population

Summary Blood samples from normal adults and from members of seven families with the Swiss type of hereditary persistence of fetal hemoglobin (HPFH) from Yugoslavia were analyzed for their fetal hemoglobin (Hb F) and ^G levels, while haplotyping defined the chromosomes at eight or nine polymorphic restriction sites. The data indicate that Swiss-HPFH, characterized by slightly elevated Hb F and ^G levels and no recognizable hematological abnormality, is associated with a chromosome whose restriction enzyme haplotype is identical to the no. 3 (Senegal) haplotype found in black sickle cell (SS) patients. Many adults with this chromosome have high ^G but normal Hb F levels. It is suggested that the Swiss-HPFH phenotype results from the action of more than one factor; one is linked to the -globin gene cluster and causes high ^G values, while others result in an increased Hb F production and are perhaps of different origins.     

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.     

Analysis of the recombination zone in hereditary persistence of fetal hemoglobin with fetal gene rearrangement of the G gamma-G gamma-A gamma type

An increased synthesis of fetal hemoglobin in adult life is a common feature of the genetically determined severe disorders like beta thalassemia and sickle cell anemia. A continued synthesis of fetal hemoglobin in adults is also characteristic of clinical or subclinical syndromes like respectively delta beta thalassemia or hereditary persistence of fetal hemoglobin (HPFH). These disorders are highly heterogeneous with respect to their molecular defects as well as to the composition of Hb F. We report here a novel case of hereditary persistence of fetal hemoglobin in heterozygous state discovered by chance, in a young perfectly healthy french man. The gamma chain of his fetal hemoglobin was almost entirely composed of G gamma chains. Molecular analysis of the DNA revealed the existence of triplicated gamma genes on one chromosome with the genotype arrangement of G gamma-G gamma-A gamma. A polymorphic Xmn I restriction site (at position -158 5' to the cap site) was present in 5' of both of these G gamma genes. The presence of this site in front of G gamma gene had previously been shown to be associated both with high G gamma phenotype constitutively and also with high fetal hemoglobin level only in case of anemic stress. In the absence of any anemic stress in this individual, the constitutive increase of both fetal hemoglobin and G gamma chains could be due to the presence of a chromosome with triplicated arrangement of gamma genes. The classical triplication (G gamma-A gamma-G gamma-A gamma) does not result in HPFH phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Targeted deletion of beta 1 integrins in F9 embryonal carcinoma cells affects morphological differentiation but not tissue-specific gene expression

The integrin superfamily of heterodimeric transmembrane adhesion receptors mediates many cell-cell and cell-matrix interactions whose functions are believed to be critical for normal morphogenesis and differentiation. By eliminating the beta 1 integrin gene through homologous recombination, we have assessed the role of the beta 1 integrin family in the F9 embryonal carcinoma model for endodermal differentiation. F9 cells were unexpectedly found to maintain three copies of the beta 1 gene and complete elimination required three sequential rounds of targeting to generate triple knockout lines (beta 1 TKO). Elimination of the beta 1 integrin family of adhesion receptors from F9 cells resulted in reduced adhesion to fibronectin, laminin and collagen, but strongly enhanced adhesion to vitronectin. The absence of beta 1 integrins did not promote significant compensatory upregulation of either beta 3 or beta 5 subunits, both of which are known to act as vitronectin receptors when associated with alpha v. The loss of beta 1 integrins severely affected morphological differentiation when the beta 1-deficient cells were induced to differentiate to either parietal or visceral endoderm. Parietal endoderm derived from beta 1-deficient cells retained a rounded morphology and migrated poorly on both fibronectin and vitronectin. Visceral endoderm derived from beta 1- deficient cells were also unable to form a normal, confluent epithelial monolayer; instead, a non-contiguous layer containing clumps of disorganized cells was observed. However, loss of beta 1 integrins did not interfere with induction by differentiating agents of tissue- specific gene products for either visceral or parietal endoderm. These results suggest that beta 1 integrins mediate morphological differentiation (migration and epithelial formation) but not tissue- specific gene expression in induced F9 cells, and that these two processes are not necessarily linked in this system.     

Deletion of the PAT1 gene affects translation initiation and suppresses a PAB1 gene deletion in yeast

The yeast poly(A) binding protein Pab1p mediates the interactions between the 5' cap structure and the 3' poly(A) tail of mRNA, whose structures synergistically activate translation in vivo and in vitro. We found that deletion of the PAT1 (YCR077c) gene suppresses a PAB1 gene deletion and that Pat1p is required for the normal initiation of translation. A fraction of Pat1p cosediments with free 40S ribosomal subunits on sucrose gradients. The PAT1 gene is not essential for viability, although disruption of the gene severely impairs translation initiation in vivo, resulting in the accumulation of 80S ribosomes and in a large decrease in the amounts of heavier polysomes. Pat1p contributes to the efficiency of translation in a yeast cell-free system. However, the synergy between the cap structure and the poly(A) tail is maintained in vitro in the absence of Pat1p. Analysis of translation initiation intermediates on gradients indicates that Pat1p acts at a step before or during the recruitment of the 40S ribosomal subunit by the mRNA, a step which may be independent of that involving Pab1p. We conclude that Pat1p is a new factor involved in protein synthesis and that Pat1p might be required for promoting the formation or the stabilization of the preinitiation translation complexes.     

Sequence of the region downstream of theVitreoscilla hemoglobin gene:vgb is not part of a multigene operon

The 1668 base pairs (bp) downstream of theVitreoscilla hemoglobin gene were sequenced in the hope of finding related genes that might be part of an operon. Instead, a sequence was found that constituted an open reading frame (ORF) of 569 amino acids (apparently the carboxy-terminal part of a larger ORF), in the direction opposite to the hemoglobin gene. This sequence was found to have 64% similarity with the 1685 by at the 3 end of theEscherichia coli uvrA gene. The inferred amino acid sequence of the Vitreoscilla DNA has 69% similarity with the corresponding sequence of theE. coli uvrA protein, with similarities of 90, 100, and 85% in the helix-turn-helix, C-terminal ATP binding, and C-terminal zinc finger domains, respectively. The distance between the 3 ends of theVitreoscilla hemoglobin anduvrA genes is 63 bp.     

Homozygotes for the hereditary persistence of fetal hemoglobin: The ratio of Gγ to Aγ chains and biosynthetic studies

Two sons of a previously reported Ghanaian homozygote for the hereditary persistence of fetal hemoglobin (HPFH) (Ringelhann et al., 1970) also are HPFH homozygotes. In addition, another unrelated adult Ghanaian homozygote has been detected. All of these Ghanaian homozygotes as well as three American Black HPFH homozygotes have the G gamma A gamma type of HPFH with a G gamma to A gamma ratio of about 3:2, in contrast to an Asiatic Indian homozygote who has the G gamma type. Globin chain synthesis in HPFH homozygotes is unbalanced, with a gamma/alpha ratio of 0.6 or less, whereas it is balanced in heterozygotes according to most reports.