A haemoglobin switching activity modulates hereditary persistence of fetal haemoglobin

During human development there is a switch from fetal to adult haemoglobin formation, reflecting the differential expression of fetal (G gamma and A gamma) and adult (beta and delta) globin genes. Mutations that inhibit this switch produce variants of the syndrome of hereditary persistence of fetal haemoglobin (HPFH). Adult heterozygotes for these mutants produce 15-30% fetal haemoglobin (HbF) in their red cells. The general assumption is that the mutations result in a permanent switching on of gamma-globin genes. Here, however, we show that fetal globin expression can be turned off in cultures of HPFH cells by an uncharacterized factor in fetal sheep serum. This is the first demonstration that mutations affecting the developmental expression of globin genes can be modulated by exogenous factors. The findings raise the possibility that the phenotype of HPFH is not simply the direct result of mutations in or around globin genes but the consequence of the mutations on the interaction of globin genes with trans-acting regulatory factors.     

A point mutation in the A gamma-globin gene promoter in Greek hereditary persistence of fetal haemoglobin

Hereditary persistance of fetal haemoglobin (HPFH) is a benign condition characterized by the production in adulthood of more than 1% fetal haemoglobin (HbF, alpha 2 gamma 2) in the absence of erythropoietic stress. Several genetic types have been discerned based on the level of HbF produced, the relative contributions of the duplicated fetal (G gamma and A gamma) globin genes, and the presence or absence of deletions involving the beta and delta genes in cis to the mutation. Greek HPFH is a non-deletion variety in which heterozygotes produce 10-20% HbF, predominantly due to overproduction of the A gamma chain. We have cloned a 40-kilobase (kb) region of the beta-globin cluster from a Greek HPFH allele and report here that a point mutation (G----A) occurs 117 base pairs (bp) 5' to the cap site of the A gamma-globin gene, just upstream of the distal CCAAT sequence. The corresponding region of the G gamma-globin gene is normal. We discuss the implications of this finding for the developmental regulation of globin gene expression.     

Human gamma-globin genes silenced independently of other genes in the beta-globin locus.

Erythropoiesis during human development is characterized by switches in expression of beta-like globin genes during the transition from the embryonic through fetal to adult stages. Activation and high-level expression of the genes is directed by the locus control region (LCR), located 5' to the epsilon gene. The location of the LCR and its role in directing high-level expression of the globin genes has led to the suggestion that competition from the beta gene for interaction with the LCR has a major role in silencing the fetal gamma genes during adult life. We have now constructed lines of transgenic mice containing the human A gamma globin gene linked to the LCR. We observe high-level expression of the transgene in the embryonic stages but silencing of the gene in adult animals. We conclude that the gamma gene is not deregulated by the presence of the LCR and that competition from the beta gene is not required for silencing of the gamma genes in adult life. The silencing is therefore likely to be mediated by stage-specific factors binding to sequences immediately flanking the genes.     

G to A substitution in the distal CCAAT box of the A gamma-globin gene in Greek hereditary persistence of fetal haemoglobin

The sequence 5'TTGGPyCAAT 3' (the 'CCAAT box') is a constituent of the promoter region of many eukaryotic and prokaryotic genes and is believed to play a part in promoter function. A characteristic of the two fetal human globin genes (A gamma and G gamma) is a duplication of a 12-base pair (bp) sequence containing the CCAAT box. Here we report a G----A substitution in the TTG sequence of the distal CCAAT box of the A gamma-globin gene in an individual with the A gamma (Greek) type of hereditary persistence of fetal haemoglobin (HPFH). This represents the first report of a natural mutation of the CCAAT box in a eukaryotic gene. The fact that this transition is associated with inappropriate expression of the A gamma gene in adult life suggests that the CCAAT box (or its surrounding sequences) may have a role in the developmental control of gamma-globin genes.     

gamma-beta-Thalassaemia studies showing that deletion of the gamma- and delta-genes influences beta-globin gene expression in man

In gamma-beta-thalassaemia, human gamma- and beta-globin gene expression is suppressed; this results in a severe anaemia in newborns which subsequently develops into a beta-thalassaemia syndrome in adult life. This hereditary disease is now shown to be the result of a deletion of at least 40,000 base pairs of the gammadeltabeta-globin gene locus. The gamma- and delta-globin genes are deleted in the affected chromosome but, surprisingly, the beta-globin gene is still present, together with a large segment of the DNA sequences flanking the gene on its 5'-side and the entire region on the 3'-side of the gene. Hence, a deletion of DNA far from the beta-globin gene results in the suppression of its activity.     

Increased gamma-globin expression in a nondeletion HPFH mediated by an erythroid-specific DNA-binding factor

In man, a shift from gamma- to beta-globin gene expression in erythroblasts underlies a switch from fetal to adult haemoglobin during development. In hereditary persistence of fetal haemoglobin (HPFH), inappropriately high gamma-globin expression in adult life is associated with deletions in the beta-globin cluster or with single-base changes upstream of the gamma-globin genes. To account for enhanced gamma-gene expression in HPFH of the non-deletion type, we tested the nuclear proteins of human erythroleukaemia cells that bind gamma-promoter sequences in vitro by correlating specific mutations in their binding sites with promoter activity. An erythroid-specific factor (GF-1) binds as a single molecule to the -195 to -170 region and contacts two TATCT(AGATA) motifs, but not the conserved octamer (ATGCAAAT) that separates them. We observe that a single change (at -175, T----C) found in HPFH leads to increased promoter activity only in erythroid cells. This effect is mediated by GF-1, the human counterpart of the chicken erythroid factor Eryf 1. The form of HPFH we studied here is an inherited disorder which can be ascribed to the action of a cell-specific DNA-binding factor on a mutant promoter.     

Developmental regulation of human fetal-to-adult globin gene switching in transgenic mice

Transgenic mice containing a human fetal (gamma-) or adult (beta-) globin gene linked to the beta-globin gene locus activation region (LAR) express the gene throughout development. By contrast, transgenic mice containing LAR linked to both a fetal and an adult globin gene display the normal developmental switch from fetal to adult gene expression. This suggests that the human fetal-to-adult globin gene switch is controlled through a mutually exclusive interaction between LAR and either the gamma- or beta-globin gene, resulting in the expression of only one gene at any given moment.     

Developmental regulation of a cloned adult beta-globin gene in transgenic mice

At different stages of mammalian development, distinct embryonic, fetal and adult haemoglobins are synthesized in erythroid cells, a process termed haemoglobin switching. The cellular and molecular mechanisms controlling haemoglobin switching have been intensively studied, but remain poorly understood. To study the developmental regulation of globin gene expression, we have produced transgenic mice in which cloned globin genes are present in erythroid cells throughout development. Recently, we reported that adult mice in several transgenic lines carrying a hybrid mouse/human adult beta-globin gene, expressed the gene in a correct tissue-specific manner. This finding raised the question of whether an exogenous globin gene could also be subject to appropriate stage-specific regulation. We report here that the hybrid beta-globin gene, like the endogenous adult beta-globin genes, is inactive in yolk sac-derived embryonic erythroid cells and is expressed for the first time in fetal liver erythroid cells. Our results indicate that a stage-specific pattern of expression can be conferred by cis-acting regulatory elements closely linked to an adult beta-globin gene. They also suggest that the embryonic and adult beta-globin genes in the mouse are activated (or repressed) by distinct trans-acting regulatory factors present in embryonic, fetal and adult erythroid cells.     

An embryonic pattern of expression of a human fetal globin gene in transgenic mice

During the evolution of the beta-globin family gene in vertebrates, different globin genes acquired different developmental patterns of expression. In mammals, specific 'embryonic' beta-like globins are synthesized in the earliest erythroid cells, which differentiate in the yolk sac of the embryo. In most mammals the embryonic globin chains are replaced by 'adult' beta-globins in fetal and adult erythrocytes, which arise in the liver and bone marrow, respectively. However, in simian primates (including humans), a distinct 'fetal' type of beta-like globin chain predominates in fetal erythroid cells. Based on the pattern of DNA sequence homologies between different mammalian species, these fetal globin genes, G gamma and A gamma, are thought to have descended from an ancestral gene, 'proto-gamma', which was embryonic in its pattern of expression. In the mouse, as well as in most other mammalian species, the descendants of the proto-gamma gene continue to function as embryonic genes. To investigate the evolutionary changes that led to the 'fetal recruitment' of the gamma-globin genes in primates, we have introduced the cloned human G gamma-globin gene into the mouse germ line. We report here that the human G gamma gene reverts to an embryonic pattern of expression in the developing mouse. This observation suggests that during evolution a shift occurred in the timing of expression of a trans-acting signal controlling the proto-gamma gene.     

Structure of the human fetal globin gene locus.

We have derived a 'map' of restriction enzyme sites in and around the human gamma-globin genes. This has enabled us to show that there are two gamma-globin genes per haploid set, that the genes contain 'introns' within the same regions of DNA as the human beta and delta-globin genes, and that the genes are 3,500 base pairs apart. We conclude that the correct gene organisation of the human beta-like globin locus is GgammaAgammadeltabeta.     

Specific expression of a foreign beta-globin gene in erythroid cells of transgenic mice

The globin gene family represents an attractive system for the study of gene regulation during mammalian development, as its expression is subject to both tissue-specific and temporal regulation. While many aspects of globin gene structure and expression have been described extensively, relatively little is known about the cis-acting DNA sequences involved in the developmental regulation of globin gene expression. To begin to experimentally define these regulatory sequences, we have taken the approach of introducing cloned globin genes into the mouse germ line and examining their expression in the resulting transgenic animals. Here we describe a series of transgenic mice carrying a hybrid mouse/human adult beta-globin gene, several of which express the gene exclusively or predominantly in erythroid tissues. These studies demonstrate that regulatory sequences closely linked to the beta-globin gene are sufficient to specify a correct pattern of tissue-specific expression in a developing mouse, when the gene is integrated at a subset of foreign chromosomal positions.     

Preferential deletion of exons in Duchenne and Becker muscular dystrophies

Duchenne and Becker muscular dystrophy (DMD and BMD) genes are located in Xp21 on the short arm of the X chromosome. DMD patients display a much more severe clinical course than BMD patients, and yet about 10% of cases of each have been reported to have deletions for parts of the gene. Using a complementary DNA subclone of the DMD gene we have screened 66 DMD and BMD patients who had not previously shown deletions with the probes then available. Fifteen patients have a deletion of this part of the gene, indicating a higher deletion frequency in this region (22%). Exons were deleted in five severely affected DMD patients and in ten BMD patients. Significantly, most of these deletions begin in the same region of the cDNA, which implies that there is a common mechanism for the generation of many of these mutations. An apparently identical deletion in one family gave classical BMD in two brothers (presenting in their teens) and only very mild muscle weakness in their 86-year-old great-great-uncle. Taking these data together with data using the probes previously published, we are able to detect deletions directly in 40% of our families requiring antenatal diagnosis or carrier detection.     

Control of haemoglobin switching by a developmental clock?

The pattern of haemoglobin production changes at the embryonic, fetal and postnatal stages of human development, reflecting the expression of different globin genes in both the alpha-like and beta-like gene clusters. Recent studies have identified alterations in the state of DNA methylation and sensitivity to nuclease digestion associated with developmental expression of the globin genes in red blood cell precursors, but the mechanism initiating these changes remains unknown. Despite the screening of large numbers of blood samples from newborn infants, no mutants have been found which affect the timing of these changes (with one possible exception involving a chromosomal translocation), thus necessitating alternative approaches to analysing the cellular basis for the timing of haemoglobin switching. Although many mechanisms are possible, the initiation of the switch from fetal to adult haemoglobin could be regulated essentially either by a developmental clock inherent to haematopoietic stem cells or by an inductive environment, and in an attempt to distinguish between these possibilities, we have transplanted sheep fetal haematopoietic tissue into adult animals. Although previous experiments of this type produced conflicting results, the accumulated results presented here demonstrate that the pattern of haemoglobin production after transplantation is determined largely by the gestational age of the fetal donor cells.     

Model for antenatal diagnosis of beta-thalassaemia and other monogenic disorders by molecular analysis of linked DNA polymorphisms

Polymorphisms of DNA restriction sites within the human fetal globin genes have been used to identify chromosomes that carry beta-thalassaemia genes in individuals heterozygous for this disease. This has allowed an antenatal diagnosis for beta-thalassaemia to be carried out by observation of the pattern of the inherited polymorphism of a linked DNA sequence not involved in the genetic pathogenesis of the disease. In the populations we have investigated there is no constant pattern of polymorphism that segregates with the beta-thalassaemia gene. The use of linked polymorphisms should, therefore, be applicable to antenatal diagnosis both of beta-thalassaemia and of any other single-gene defect for which there is a DNA probe specific for a sequence linked to the affected locus.