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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Gene expression profiles of the developing human retina

Retinaplaysimportantrolesintheperception,proc-essandtransmissionofvisualsignalsandthefunctionsoftheretinadepend,toalargeextent,onitshighlyorganizedstructure.During3—6weeksinhumanembryogenesis,theneuralectodermgrowsoutfromthediencephalonstoformtheopticvesicleandtheninvaginatestoformtheopticcup.Theouterlayeroftheopticcupbecomesthenon-neuralretinalpigmentepithelium(RPE)andtheinnerlayerbecomestheneuralretina.RPEcellsproliferateslowlyandappeardifferentiatedandpigmentedasearlyas6—8weeksandremain…     

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.