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.     

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.     

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.     

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.     

Phylogenetic origins and adaptive evolution of avian and mammalian haemoglobin genes

Recent years have seen rapid growth in amino acid sequence data on globins and nucleotide sequence data on haemoglobin genes and pseudogenes, and cladistic analysis of these data continues to reveal new facets of globin evolution. Our present findings demonstrate: (1) avian and mammalian embryonic alpha genes (pi and xi, respectively) had a monophyletic origin involving an alpha locus duplication about 400 Myr ago soon after the duplication which separated alpha and beta genes; (2) much later in phylogeny, independent beta-gene duplications produced the embryonic rho locus of birds and embryonic epsilon and fetal gamma loci of mammals. This parallels the earlier finding that myoglobins evolved more than once from generalized globin ancestors. Here we support the view that such globin evolution resulted from natural selection acting on mutations in duplicated genes. Thus, our evidence contradicts the neutralist view in which almost all amino acid substitutions in descent to extant globins evaded positive selection.     

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.     

Characterisation of deletions which affect the expression of fetal globin genes in man.

Deletions in the DNA of individuals with hereditary persistence of fetal haemoglobin (HPFH) and 8 beta-thalassaemia have been mapped as a means of identifying regulatory sequences involved in the switch from fetal to adult globin gene expression. The end points of these deletions have been precisely located with respect to restriction endonuclease cleavage sites within and surrounding the gamma-, delta- and beta-globin genes in normal human DNA and the deletion maps were used to obtain definitive evidence for the physical linkage of the fetal and adult beta-like globin genes in the order 5'Ggamma-Agamma-delta-beta 3'. Correlation of haematological data and the location of deletions in two cases of HPFH and one case of deltabeta-thalassaemia suggest that a region of DNA located near the 5'-end of the delta-globin gene may be involved in the suppression in cis of gamma-globin gene expression in adults. The interpretation of a second case of deltabeta-thalassaemia is complicated by the fact that the deletion removes the Agamma-gene in addition to the region near the 5'-end of the delta-globin gene.     

Diversity of murine gamma genes and expression in fetal and adult T lymphocytes

The search for the genes encoding the T-cell receptor alpha and chains revealed a third gene, T gamma (ref. 1), which shares with t T alpha (refs 2-7) and T beta (refs 8-15) genes a number of structure features, including somatic rearrangement during T-cell development. T gamma gene expression appears to be unnecessary in son mature T cells and is at its greatest in fetal thymocytes encouraging speculation that T gamma has a role in T-cell development and may be involved in the recognition of polymorphic major histocompatibility complex (MHC) products during thymic education. One argument against the participation of T gamma in such a process has been its apparently limited diversity, due to the small number of gene segments available for rearrangement. We here describe the identification of additional T gamma V-gene segments and demonstrate that they can be rearranged to previously identified J- and C-gene segments and are expressed in fetal thymocytes. In addition we describe a variety of patterns of T gamma mRNA processing which may be significant for T gamma gene regulation.     

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.     

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.     

The gene for theta-globin is transcribed in human fetal erythroid tissues

A new gene like the alpha-globin gene has been identified in higher primates at the 3' end of the alpha-globin gene cluster. There is some controversy as to whether this gene, theta, is a functional globin gene or a non-functional pseudogene. The high degree of sequence conservation displayed by theta between primates and various mammals, such as horse and rabbit, suggests that this gene is functional in some species. Furthermore, theta encodes a 141-amino-acid polypeptide in sequence similar to alpha-globin and appears to possess functional RNA-processing signals. But the promoter region of theta is unlike the other globin genes because its CCAAT and ATA box sequences are displaced from the coding sequence by the insertion of a 200-base-pair GC-rich sequence. We demonstrate here the presence of theta-globin messenger RNA in human fetal erythroid tissue, but not in adult erythroid or other non-erythroid tissues. Furthermore, theta-globin mRNA is detectable in significant amounts in a human erythroleukaemic cell line. These results predict that theta-globin protein will be found in the early stages of human fetal development. Surprisingly, the promoter sequence of theta-globin does not correspond to the CCAAT and ATA box sequences of the gene but rather lies within the adjacent GC-rich sequence, resulting in a heterogeneous series of mRNA 5' ends 50-10 base pairs to 5' of the initiation codon. This type of promoter is reminiscent of that found in housekeeping genes such as adenine deaminase and hypoxanthine-guanine phosphoribosyl-transferase.     

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.     

Haemoglobin switching in human embryos: asynchrony of zeta----alpha and epsilon----gamma-globin switches in primitive and definite erythropoietic lineage

Haemoglobin switching in humans provides a unique model for investigating the mechanisms underlying expression of a developmentally regulated gene family. Numerous studies have focused on the switch from fetal to adult (that is, gamma----beta) globin, but little is known about the embryonic----fetal (that is, zeta----alpha and epsilon----gamma) switches, as well as the transition from 'primitive' yolk sac to 'definitive' liver erythropoiesis. Here we have studied the embryonic----fetal haemoglobin switches in yolk sac, liver and circulating blood erythroblasts from 25 embryos and 6 fetuses. Globin synthesis was also evaluated in purified 'primitive' and 'definitive' erythroblasts. Primitive erythroblasts synthesize essentially zeta and epsilon chains at 5 weeks and alpha- and epsilon-globin with a minor aliquot of zeta and gamma chains at 6-7 weeks, whereas definitive erythroblasts produce alpha and epsilon + gamma + beta-globin at 6 weeks but only alpha and gamma + beta chains from 8 weeks onward. In both lineages the zeta----alpha and the epsilon----gamma switches are asynchronous, the former preceding the latter. Furthermore, zeta- and beta-globin synthesis is restricted to primitive and definitive erythroblasts respectively. These findings are discussed in terms of a monoclonal model for haemoglobin switching in early human ontogeny.     

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.     

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.