Globin Synthesis

Steven Fiering, Dartmouth Medical School, New Hampshire, USA David IK Martin, Victor Chang Cardiac Research Institute, Sydney, Australia Eric E Bouhassira, Albert Einstein College of Medicine, New York, USA
Globin synthesis refers to the expression of the a-like and b-like globin genes in red blood cells. Mutations of these genes cause the haemoglobinopathies, the most common genetic disorders in humans. The regulation of the globin genes is not only an important model system for understanding gene expression in mammals, but a fuller understanding of this problem will lead to novel clinical approaches to the haemoglobinopathies.

Secondary article
Article Contents
. What is Haemoglobin? . Globin Gene Switching is a Model of Transcriptional Regulation . Haemoglobinopathies . Regulatory DNA Elements Close to the Genes, and the Proteins that Bind to Them . Posttranscriptional Regulation . Future Prospects

What is Haemoglobin?
Simply stated, haemoglobin is what makes blood red. it is a protein complex that is produced in copious amounts exclusively in red blood cells (erythrocytes), and it functions to carry oxygen from the lungs to the tissues and carbon dioxide from the tissues to the lungs, and so is essential for life in vertebrates. That much has been known for a long time, but more recently it has been revealed that haemoglobin also plays an important role in the regulation of blood ow to the tissues through its ability to bind and release nitric oxide. Haemoglobin molecules are tetramers and the protein components of these tetramers are known as the globins. Each tetramer is composed of two a (alpha)-like and two b (beta)-like globin chains and each tetramer is associated with four haem molecules. During haemoglobin synthesis, newly formed a-like and b-like chains assemble as stable ab heterodimers that then reversibly associate in tetramers. The structure of the haemoglobin tetramer produces the allosteric properties that are critical for the delivery of oxygen to the tissues and have long been cited as the classical example of an allosterically regulated molecule. In mammals, the globin genes are organized as a cluster of closely related genes encoding a-like proteins and a separate cluster of closely related genes encoding b-like
5 (a) 10 KB 5 (b)
Figure 1 Maps of the b-globin locus (a) and the a-globin locus (b). Expressed genes are shown as red boxes, pseudogenes are shown as white boxes and cis-regulatory elements are shown as blue boxes. Arrows denote the transcriptional direction of expressed genes. Both maps have the same scale.

LCR

G A

3HS

proteins (Figure 1). In humans, the multiple genes encoding the family of a-globin proteins are grouped together on chromosome 16 (position 16p13.3), while the multiple genes encoding the family of b-globin proteins are found at the b-globin locus on chromosome 11 (position 11p15.5). During mammalian development, red cell production (erythropoiesis) takes place at dierent anatomical sites. Production of red cells successively occurs in the yolk sac during embryonic life, in the fetal liver during fetal life, and in the spleen and bone marrow, shortly before and after birth. Finally, in humans, the bone marrow becomes the exclusive site of erythropoiesis except when conditions of severe anaemia, which stimulates extraordinary production of red cells, reactivates production of red cells outside the bone marrow. In most other mammals such as mice, the spleen retains an erythropoietic role throughout life. Roughly, but not precisely, coincident with the changes of location for red cell development, is a series of changes among the genes that are expressed from the a-globin and b-globin loci. These changes in gene expression, called the globin switches, are regulated at the transcriptional level. One reason for interest in the regulation of globin synthesis is that several very common genetic diseases are due to mutations in the a- and b-globin genes and their regulatory regions. These haemoglobinopathies are, as a group, among the most common severe genetic disorders among humans. A full understanding of the regulation of gene expression at these loci might permit manipulation of expression for therapeutic benet.

HS-40

Globin Gene Switching is a Model of Transcriptional Regulation

The globin gene switch has for many years been a model for the study of complex transcriptional regulation in mammals. Study of the tissue specicity of globin expression and its developmental regulation started long before the modern era of molecular genetics because globin chains
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and globin-encoding messenger RNA (mRNA) are abundant molecules that are readily available for study. Since the dierent types of globin proteins could be distinguished on simple gels, the basic outlines of the globin switch were apparent as one of the earliest molecular biology ndings. The globin mRNAs were among the rst human complementary DNAs (cDNAs) to be cloned and sequenced. In time this led to the cloning of the genes and the sequencing of the entire a- and b-globin gene clusters.

Haematopoietic stem cell Other haematopoietic lineages Erythroid lineage

BFU-E

CFU-E

Proerythroblast

Organization of the a- and b-globin loci

As shown in Figure 1, the human a- and b-globin loci have much in common organizationally. Both loci contain several expressed globin genes as well as pseudogenes. Both loci have a distinct directionality: all genes are expressed from the same strand. Therefore all transcription occurs in the same direction, giving these loci a 5 and 3 orientation. Both loci undergo a switch of expressed genes with the genes expressed at the earliest stages of development at the 5 end of the locus and both loci also contain major positive regulatory elements at their 5 extremity. The major regulatory element in the human a-globin locus is HS-40, so named because it was rst recognized as a site that is hypersensitive (HS) in a DNAaseI assay of the local chromatin structure and it is roughly 40 kb from the a genes. HS-40 enhances expression of globin genes in red cells in experimental constructs in transgenic mice. Similarly, at the far 5 end of the b-globin locus, there is a powerful positive regulatory element named the b-globin locus control region (LCR). This element, larger than HS40, contains ve major HS sites that have varying degrees of ability to increase expression from linked b-globin genes in transgenic mice. While it is clear that the b-LCR and the a-HS-40 are required for normal expression of the globin genes, their mechanism of action is not yet clear.

Basophilic erythroblast

Erythrocyte

Reticulocyte

Orthochromatic Polychromatophilic erythroblast erythroblast

Figure 2 Development of erythrocytes. The colours of the cells reflect colours seen with Wright Giemsa staining and the cell sizes reflect sizes relative to each other.

Red cell development (erythropoiesis)

Red cells arise from haematopoietic stem cells that give rise to all the blood cells. These stem cells are very rare and therefore poorly characterized morphologically (Figure 2). During erythropoiesis, some of these progenitors dierentiate into erythroid precursors such as BFU-E and CFUE, two cell types that are still rather rare and best identied functionally by their ability to form erythroid colonies in vitro in response to erythropoietin. At the pro-erythroblast and subsequent stages in erythroid development, cells are classied by their morphology in WrightGiemsa stained bone marrow. The pro-erythroblasts proliferate rapidly and initiate transcription of the globin genes. During the ensuing stages of maturation, the basophilic and polychromatophilic erythroblasts continue to transcribe the globin genes and to assemble haemoglobin. During the following stage, orthochromatic erythroblasts condense
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the chromatin in the nucleus; all transcription stops and then these cells lose their nuclei (enucleate). In the nal stage of erythropoiesis, reticulocytes enter the circulation; although they have no nucleus they continue to translate globin mRNA and assemble haemoglobin. Finally, translation stops and all mRNA is degraded in the mature erythrocyte. Erythrocytes have a lifespan of 120 days in humans and 40 days in mice, so the developmental process of erythropoiesis continues throughout life. Erythrocytes are removed from circulation and destroyed when they are no longer functional, generally when the plasma membrane loses its exibility and is unable to deform readily on passage through small vessels. Haemoglobin constitutes more than 90% of the protein in mature erythrocytes and therefore erythrocytes appear remarkably simple, being essentially packages of haemoglobin, but they are not inert. They cannot transcribe or translate mRNA, but have simple apparatuses for generating adenosine triphosphate (ATP), transporting molecules across the cell membrane, and protecting themselves against oxidative damage. The three major sites of erythropoiesis correlate with the three recognizable types of mature red cells. Primitive erythrocytes formed in the yolk sac are the largest mature red cells. They contain a nucleus that is tightly condensed and no longer actively expressing any genes, but unlike denitive erythrocytes formed later in development, primitive erythrocytes do not enucleate. Prior to the condensation of the nucleus, these cells express predominantly e (epsilon) from the b-globin locus and z (zeta), a2 and a1 from the a-globin locus. The progenitor cells which give rise to primitive erythrocytes are distinct from the progenitors that give rise to denitive erythrocytes (and all

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Globin Synthesis

other blood cell types) found in the postnatal circulatory system. The red cells produced in the fetal liver are the fetaldenitive erythrocytes. Like denitive erythrocytes found in the adult, these cells lose their condensed nucleus at the nal stage of their formation. However, the fetal-denitive cells can be distinguished from erythrocytes from an adult because of their larger size and dierent haemoglobin types. The fetal-denitive cells express predominantly g (gamma)-globin (both Gg and Ag) from the b-globin loci, and both a2 and a1 but little or no z-globin from the aglobin loci. The progenitor cells of these erythrocytes are true haematopoietic stem cells, able to give rise to all cells of the adult haematopoietic system. Bone marrow-derived adult-denitive erythrocytes also enucleate, and are the familiar and easily recognized biconcave red cells. These cells express predominantly b and a small amount of d(delta) from the b-globin loci, and both a2 and a1 from the a-globin loci. The various haemoglobins produced during development have dierent oxygen anities and are probably adapted to life either in utero or after birth. Nevertheless, the various a-like chains appear to be functionally interchangeable, as do the various b-like chains. This point is critical in understanding some of the strategies for treating haemoglobinopathies.

Haemoglobinopathies
The haemoglobinopathies are mendelian recessive genetic diseases and are by far the most frequent severe genetic disorders among humans. The haemoglobinopathies fall into two broad categories: the thalassaemias, caused by mutations that lead to a reduction in the amount of either a- or b-globin, and structural mutations in which an abnormal protein is produced. Thalassaemias aecting either the b-globin locus or the a-globin locus are common, but severe b-thalassaemia is by far the most common severe thalassaemia. b-Thalassemia major is fatal unless treated with frequent blood transfusions, which themselves lead to potentially fatal complications. The common structurally abnormal globin is the bS-globin, which has a glutamic acid to valine substitution at position 6, making an abnormal protein which polymerizes at low oxygen tension and damages the erythrocyte. This polymerization deforms the erthrocytes and produces a condition known as sickle cell disease. The pathology of sickle cell disease is the result of an interaction between the damaged erythrocytes and blood vessels which leads to occlusion of the vessels and consequent hypoxic damage to tissues. While generally less severe than thalassaemia major, sickle cell disease still results in progressive damage to multiple organs that can be fatal even early in life.

Although prenatal diagnosis is now available, haemoglobinopathies are a major healthcare burden in many areas, and result in greatly shortened lifespan and reduced quality of life even when the best medical treatments are available. No denitive therapies for these disorders exist, other than bone marrow transplantation which is expensive and potentially fatal. A great deal of attention has therefore been focused on developing therapies for these diseases. The prevalence of the haemoglobinopathies in many populations presents an excellent example of mutations that are detrimental in the homozygous state but are enriched in the population because they confer a selective advantage on heterozygous carriers. The geographic distribution of these extraordinarily prevalent mutant alleles coincides with areas where malaria is, or has been, endemic. The parasites that cause malaria spend a critical portion of their life cycle in the erythrocyte. It was rst postulated by J. B. S. Haldane in 1948 that heterozygous carriers of a haemoglobinopathy mutation, which have only mildly abnormal red cells, have some protection against malaria, leading to selection for the mutations in areas where the disease is endemic. This hypothesis is now widely accepted, but the molecular basis for the protection of heterozygotes is still not completely understood and probably involves multiple mechanisms. The currently favoured hypothesis regarding the antimalarial eects of a heterozygous sickle cell genotype is that glycolysis by the malarial parasite leads to a decrease in the intracellular pH of red blood cells and that pH drop induces polymerization of Hb S (haemoglobin containing b-globin with the sickle cell mutation). Polymer formation leads to the removal of the infected cells by the spleen and also prevents replication of the parasite because Hb S polymer is not digestible by the parasite. In addition, adhesion of infected red cells to vascular endothelial cells, which is a normal part of the life cycle of malaria parasites, causes vaso-occlusion and therefore deoxygenation and associated polymerization of Hb S with similar antimalarial consequences. For thalassaemia, the currently favoured hypothesis is that heterozygotes are more susceptible as young children to the less virulent malarial parasite Plasmodium vivax, and infection by P. vivax leads to increased resistance to subsequent infection by the more dangerous P. falciparum (Clegg and Weatherall, 1998).

The relationship of globin switching to haemoglobinopathies

The molecular basis of the switch of globin expression from embryonic to fetal and then adult genes is of major interest not only because it presents a fascinating example of complex gene regulation, but also because a thorough understanding of this phenomenon might result in improved treatment of the haemoglobinopathies. The
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occasional occurrence of adults with signicant expression of the fetal stage g-globin chains in association with bthalassaemia or sickle cell disease has led to the realization that g-globin can substitute for b-globin, and that its expression ameliorates the severe pathology of the bhaemoglobinopathies. Since most patients with b-haemoglobinopathies have a fully functional set of g-globin genes, reactivation of these genes would cure the disease. Expression of unusually high levels of g-globin in postnatal life in rare individuals, referred to as hereditary persistence of fetal haemoglobin (HPFH), is thus of considerable interest, since it provides clues to the mechanism of gglobin silencing which must be overcome. The clinically important questions relating to b-globin switching in humans can be summarized as: Why are the g-globin genes expressed at very low levels in postnatal life (less than 1% of total b-like globin expression in most people after 2 years of age)? Why do some people express much higher levels of g-globin (20% of total b-like globin or higher)? Can expression of the g-globin genes be activated pharmacologically during postnatal life?

Control of globin gene transcription: DNA elements at a distance from the genes and transcription factors binding to those elements
We have mentioned above that both the a- and the b-globin loci have powerful positive regulatory DNA elements that are 550 kb away from the genes that they inuence. The bglobin LCR and the a-HS-40 are critical for regulation of the globin loci in their native genomic sites, since deletions of either the b-LCR (Driscoll et al., 1989) or HS-40 (Liebhaber et al., 1990) that leave all the genes and their promoters intact result in severe reduction or complete loss of gene expression in the aected locus. The distant regulatory elements are also eective in regulating transgenes in mice, and transgenic mice have been the main approach used to study them. Without these elements, globin genes are often expressed very poorly when placed at randomly selected genomic sites in transgenic mice. Their inclusion in a construct results in expression that is so robust that almost every mouse shows signicant expression of the transgenic globin genes in red cells, regardless of the integration site. The b-LCR contains a series of hypersensitive sites that have been conserved during the evolution of mammalian species (suggesting an important function) and are the binding sites for transcription factors. The HS-40 element, with only one hypersensitive site, appears to bind the same group of factors that bind the b-LCR. One obvious question is why the b-globin locus has a much larger and more complex distal regulatory element than the a-globin locus. The answer may lie in the dierence between the neighbourhoods of these two loci, although the point is by
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no means settled. The a-globin locus is anked on both sides by constitutively expressed genes, and in fact HS-40 is in an intron of one of these genes, so that the chromatin structure of the a-globin locus may be generally permissive for transcription (Vyas et al., 1995). The b-globin locus has no constitutively expressed genes nearby, and instead is anked by olfactory receptor genes that are expressed only in the nasal epithelium (Bulger et al., 1999). This suggests that the chromatin structure of the region anking the bglobin locus is less permissive for transcription, so that the locus needs a more extensive set of regulatory elements to activate expression. Comparison of the sequences of the b-globin LCRs of various mammals shows that there is considerable similarity between their hypersensitive sites, particularly in the central part of the site termed the core, where the interspecies sequence homology runs at 90% or better over 100300 bases. Adjoining the cores are larger anking regions (approximately 1 kb on each side) in which there is recognizable interspecies homology that averages 60 70%. There is no recognizable similarity between species in the regions of the LCR outside the hypersensitive site cores and their anking regions. The cores of the HSs contain binding sites for a group of erythroid-specic and general transcription factors. All the HSs have binding sites for GATA-1, and all have CACC motifs that are recognized by Sp1 and related proteins like EKLF (erythroid Kru ppel-like factor). Some of the HSs have one or two sites that bind members of the NF-E2 and AP-1 families of factors. The cores can be categorized as rather simple arrangements that bind the same factors, although some evidence suggests that the spacing between binding sites within the cores could be important in the activity of the HS. Perhaps surprisingly, there appear to be no proteins that bind only to the HSs. All proteins found to bind the LCR also bind in multiple sites throughout the bglobin locus, and in the areas around other erythroidspecic genes. Although it is quite clear that the b-globin LCR and the a-globin HS-40 are required in their respective loci for high-level expression, the mechanism by which they inuence expression is still not clearly established. It is probably a combination of inuences which rst creates structural changes appropriate for expression, and then also somehow stimulates transcription. There has been a great deal of investigation of the b-globin LCR in particular, and some conclusions from a variety of transgenic mouse experiments with the human locus are: (1) proximity of a globin gene to the LCR favours expression; (2) the e and g genes are autonomously regulated, since they are expressed at the proper developmental stages in LCR-containing transgenes. The b gene, however, seems to require other globin genes between it and the LCR for proper developmental regulation. Transgenes containing the LCR and only b are deregulated and expressed throughout development, but transgenes

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Globin Synthesis

with the LCR and both g and b have proper b regulation when the g gene is placed between the b gene and the LCR. The latter result, along with studies done with the chicken b-globin locus (Choi and Engel, 1988), supports the idea that there is a competition for the activity of the LCR, but the details of this competition and its role in switching in the endogenous (rather than transgenic) locus is still unclear. Studies of individuals with mutations that cause HPFH have been a productive source of information about regulatory sequences that can aect g gene silencing. The HPFH mutations can be subdivided according to whether the upregulation of g is from both Gg and Ag or from only one of them. Mutations that cause increased adult expression of both g genes are usually associated with deletions that remove the d and b genes and more or less sequence past these genes. The smaller size deletions that remove the d and b genes and result in HPFH leave intact an enhancer region called the 3b enhancer that is just past the b gene (Figure 1). It is postulated that the juxtaposition of the 3b enhancer close to the g genes is responsible for the HPFH phenotype in that situation. Other larger deletions that remove the d and b genes plus signicantly more sequence past the b gene are also thought to introduce positive regulatory elements close to the g genes and thereby activate g expression in the adult. It is clear from analysis of many deletions of the d and b genes that simply deleting those genes is not enough to cause HPFH, the deletion must also bring a putative positive regulatory region close to the g genes.

Regulatory DNA Elements Close to the Genes, and the Proteins that Bind to Them
The haemoglobin switch is thought to be caused by transcription factors that act at specic stages of development, and there has been considerable eort devoted to nding these factors. This has produced a picture of the factors binding, especially the g-globin and b-globin promoters, but this work has not yet produced a clear understanding of the role of any of these factors in the switch. The promoters of the globin genes are all relatively similar. They contain TATA boxes, which are bound by proteins that nucleate the complex that initiates transcription. Upstream of the TATA boxes are CCAAT elements, GATA-1-binding sites, and CACC elements similar to those found in the LCR. GATA-1 was the rst erythroid transcription factor to be cloned, and one of the early studies implicated GATA-1 in a nondeletional HPFH caused by a single base change in the g-globin promoter. CACC elements play a role in globin expression, since

mutations in these elements in the human b gene are associated with thalassaemia. One factor binding to the CACC element of the b gene is known as EKLF (Miller and Bieker, 1995). It is required for expression of the b gene, since in mice that have been engineered to be decient for EKLF, very little of the mouse fetal/adult genes (b-major and b-minor) are expressed, even though the embryonic genes (Ey and bH1) are expressed normally. When such mice are transgenic for the human b-globin locus, the b gene is not expressed but the e and g genes are expressed and the g genes are partially resistant to silencing (Perkins et al., 1996). However, EKLF alone is not sucient for activation of b-globin expression, since the protein is expressed in yolk sac derived erythrocytes even though b is not expressed by those cells in humans. One current hypothesis is that the EKLF protein is covalently modied by phosphorylation or acetylation, which alters its activity exclusively in the b-globin-expressing cells. Another possibility is that EKLF needs a stage-specic partner that participates in activating b expression. CACC boxes are also potentially bound by other Sp1 family members expressed in erythrocyte precursors and these other proteins might be playing a role in globin gene expression. Models of globin switching hypothesize stage-specic factors that either activate or inactivate specic genes at specic stages. One positive regulator that appears to activate the g-globin genes at the correct developmental stage has been cloned and named the stage-selector protein (Jane et al., 1995). This is a heterodimer of a ubiquitously expressed transcription factor, CP2, and a novel partner that has not yet been fully characterized. There are also reports of cis-elements and factors binding to them that act to silence the early genes to allow the switch to proceed. These eorts have not revealed any proteins that are expressed in a stage-specic manner so their mechanisms are unclear. Surprisingly, some reports suggest that GATA-1 may also act to suppress expression of the embryonic genes during later development, which suggests that GATA-1 may play a role as both a stimulatory and a repressive factor (Raich et al., 1995). There are also point mutations in the g gene promoters that are associated with HPFH. The basic model of how these mutations aect switching is that the mutations change the factors binding at these sites by either removing existing binding sites for negative regulatory factors or producing novel binding sites for positive regulatory factors. Such mutations only aect the g gene with the mutation, unlike the large deletions that cause HPFH which aects both g genes. The promoters of Gg and Ag are very similar and these point mutations are clustered in three distinct regions of these promoters. One region is a G/ C-rich sequence that lies about 200 base pairs 5 of the transcriptional initiation site. This region appears to be binding Sp1 or members of that family. A second site in which point mutations are associated with HPFH is at 175 base pairs upstream of the initiation site. In this area a
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variety of factors could be binding, including OCT-1, GATA-1 and HMG(I/Y). The third region is found at a site that is centred 114 base pairs upstream of the initiation site in a region called the CCAAT box. The CCAAT box is known to bind ubiquitous factors CP1 (CCAAT-binding protein 1) and CDP (CCAAT displacement protein) and the erythroid-specic factor NF-E3. Although in vitro studies of these binding sites reveal that these mutations do mediate changes in anity of factors thought to bind to these sites, alterations in binding of these factors in vivo remain to be demonstrated.

Besides the potential therapies that could arise from basic research into globin switching, continued research into the regulation of globin synthesis is likely to continue to broaden our understanding of gene regulation in mammals. This in turn will probably have an impact on a variety of other pathological processes in humans.

References
Bulger M, VonDoorninck JH, Saitoh N et al. (1999) Conservation of sequence and structure anking the mouse and human b-globin loci: the b-globin genes are embedded within an array of odorant receptor genes. Proceedings of the National Academy of Sciences of the USA 96: 51295134. Choi OR and Engel JD (1988) Developmental regulation of b-globin gene switching. Cell 55: 1726. Clegg JB and Weatherall DJ (1998) Thalassemia and malaria: insights into an old problem. Proceedings of the Association of American Physicians 111: 278282. Driscoll C, Dobkin DS and Alter B (1989) gdb thalassemia due to a de novo mutation deleting the 5 b-globin locus activating region hypersensitive sites. Proceedings of the National Academy of Sciences of the USA 86: 74707474. Jane SM, Nienhuis AW and Cunningham JM (1995) Hemoglobin switching in man and chicken is mediated by a heteromeric complex between the ubiquitous transcription factor CP2 and a developmentally specic protein. EMBO Journal 14: 97105. Liebhaber SA, Griese EU, Weiss I et al. (1990) A major positive regulatory region is located far upstream of the human a-globin gene locus. Proceedings of the National Academy of Sciences of the USA 87: 94319435. Liebhaber SA, Wang Z, Cash FE, Monks B and Russell JE (1996) Developmental silencing of the embryonic z-globin gene: concerted action of promoter and 3 anking region combined with stage-specic silencing by the transcribed segment. Molecular and Cellular Biology 16: 26372646. Miller IJ and Bieker JJ (1993) A novel, erythroid cell-specic murine transcription factor that binds to the CACCC element and is related to the Kruppel family of nuclear proteins. Molecular and Cellular Biology 13: 27762786. Perkins AC, Gaensler KM and Orkin SH (1996) Silencing of human fetal globin expression is impaired in the absence of the adult beta-globin gene activator protein EKLF. Proceedings of the National Academy of Sciences of the USA 93: 1226712271. Raich N, Clegg CH, Grofti J, Romeo PH and Stamatoyannopoulos G (1995) GATA-1 and YY1 are developmental repressors of the human e-globin gene. EMBO Journal 14: 801809. Vyas P, Vickers MA, Picketts DJ and Higgs DR (1995) Conservation of position and sequence of a novel, widely expressed gene containing the major human alpha-globin regulatory element. Genomics 29: 679689.

Posttranscriptional Regulation
It is likely that globin synthesis is also regulated by posttranscriptional mechanisms and this is most clearly documented by studies of the a locus. Studies of the switch at the a-globin locus in which z-globin is only expressed from the yolk sac-derived cells suggest that one contributing factor to this switch is that z-globin mRNA is severely destabilized in bone marrow-derived erythrocytes (Liebhaber et al., 1996). This is in stark contrast to the impressive stability of the a-globin and b-globin mRNAs in maturing bone marrow-derived erythrocytes. The a and b mRNAs are so stable that they can be found abundantly in reticulocytes that have already enucleated.

Future Prospects
Most treatments for the haemoglobinopathies are still focused on managing the pathology and there has not been signicant progress in therapies that will cure these diseases. One approach that has been tried and has met with mixed success is pharmacological induction of expression of the g genes. The pharmacological agents that have had the most success, butyrate and hydroxyurea, are agents with broad biological activities and therefore are dicult to use successfully. Surprisingly, hydroxyurea appears to improve the clinical picture of sickle cell disease patients even without a concomitant increase in fetal haemoglobin. Hopefully, as the molecular mechanism of switching is unravelled, more specic pharmacological agents that activate g expression more reliably and with fewer side eects will be found. Gene therapy, in which appropriate genes are introduced into cells in order to correct a defective function, holds great promise for curing haemoglobinopathies and other genetic disorders. However there are signicant hurdles that have yet to be overcome before this will be a practical cure for the haemoglobinopathies. The most important of these hurdles are establishing proper regulation of the introduced gene and eciently introducing the gene of interest into the haematopoietic stem cells where it can be properly utilized as the erythrocytes develop.
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Further Reading
Forget BG (1998) Molecular basis of hereditary persistence of fetal hemoglobin. Annals of the New York Academy of Science 850: 3845. Hardison R, Slightom JL, Gumucia DL et al. (1997) Locus control regions of mammalian b-globin gene clusters: combining phylogenetic analyses and experimental results to gain functional insights. Gene 205: 7394. Orkin SH (1995) Regulation of globin gene expression in erythroid cells. European Journal of Biochemistry 231: 271281.

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