UNDERSTANDING FETAL GLOBIN GENE EXPRESSION: A STEP TOWARDS EFFECTIVE HbF REACTIVATION IN HAEMOGLOBINOPATHIES

By the turn of the millennium, 7% of the earth’s population will carry a significant globin gene mutation. These mutations, known collectively as the haemoglobinopathies, are the most prevalent monogenic disorders worldwide. They affect the synthesis of gene products from two multigene loci, the aand b-globin clusters. The globin loci act in concert to produce differing subtypes of haemoglobin in response to changing oxygen requirements during human development. The most significant of these ‘switches’ in subtype occurs at birth, with the change from fetal (HbF) to adult (HbA) haemoglobin production. At the level of gene transcription this equates to a switch solely in the b-cluster, with silencing of the fetal g-genes and dramatic up-regulation of adult b-gene transcription (Stamatoyannopoulos & Nienhuis, 1994). The fetal/adult switch heralds the onset of the devastating haemoglobinopathies, b-thalassaemia and sickle cell disease (SCD). The b-thalassaemias are characterized by dyserythropoiesis, transfusion dependence, iron overload while in SCD, painful crises and organ infarction predominate. Amelioration of the symptoms of these haemoglobinopathies has been documented in patients with elevated levels of HbF (Bunn, 1993). In b-thalassaemia elevated levels of g-chains combine with the redundant a-chains leading to reduced dyserythropoiesis and transfusion requirements. In SCD, HbF is a potent inhibitor of the polymerization of deoxyhaemoglobin S, thus reducing sickling and vascular occlusion (Bunn, 1997). These observations suggest that treatment strategies aimed at preserving or reactivating fetal haemoglobin production after birth should be explored for these diseases. Elevated levels of fetal haemoglobin are observed in adult humans in two significantly different contexts. The first is in the setting of a diverse group of genetic mutations which are unified phenotypically by persistent synthesis of HbF after birth, known collectively as hereditary persistence of fetal haemoglobin (HPFH) (Poncz et al, 1989). The second involves the reactivation of the quiescent fetal genes in adult erythroid progenitors as seen in stress erythropoiesis, drug induction and various other disease states. This paper examines the molecular and cellular mechanisms underlying the production of fetal haemoglobin during normal erythroid development and differentiation, and in the context of HPFH or g-gene reactivation. In addition, we discuss how these mechanisms could be potentially harnessed as therapeutic strategies for the haemoglobinopathies. The b-globin cluster: a myriad of cis-acting sequences and trans-acting factors The human b-globin locus consists of five genes arranged on chromosome 11 in the order in which they are expressed developmentally (e, g, g, d, b). Gene expression is restricted to the erythroid lineage and governed by a precise developmental programme. Embryonic haemoglobin (eglobin) is synthesized in the yolk sac until week 5 of gestation when g-globin gene activation occurs in the fetal liver and expression of the e-gene is silenced. At birth, the second haemoglobin switch occurs when b-globin gene expression commences in the bone marrow with a concomitant silencing of g-gene expression. This highly specific pattern of gene expression is regulated by cis-acting sequences within the genes themselves, distal sequences which provide enhancer activity and trans-acting factors which bind to these regulatory elements (Stamatoyannopoulos & Nienhuis, 1994; Orkin, 1995a; Jane & Cunningham, 1996). The ability to modify fetal globin expression necessitates that we understand the normal mechanisms governing globin gene regulation. To this end, studies in mice transgenic for the human globin genes have provided a number of seminal observations. From these studies it is evident that sequences within or immediately flanking the genes confer the appropriate developmental and tissue specificity of expression. However, expression levels are low in this context and subject to position effects (Starck et al, 1994). Restoration of high-level positionindependent expression is observed when a 14 kb segment of DNA 6–20 kb 5 of the e-gene is included in transgenic constructs (Grosveld et al, 1987). This region, characterized by the presence of four erythroid-specific DNAse I hypersensitivity sites (HS1–4), is known as the locus control region (LCR) (Tuan et al, 1985; Grosveld et al, 1987). The importance of the LCR is highlighted by the thalassaemia phenotype observed in the Hispanic deletion of HS2–4. These patients produce no g-, dor b-chains despite the fact that the individual globin genes remain intact (Driscoll et al, 1989). The LCR appears to influence globin gene expression by predominantly acting as a tissue-specific transcriptional activator (Grosveld et al, 1987). However, roles in insulation of the genes, chromatin opening and locus replication have also been proposed (Fraser et al, 1990; Forrester et al, 1990; Aladjem et al, 1995). The cis-acting sequences in the individual globin genes and the LCR serve as templates for binding of transcription factors. The functions of these proteins are diverse and include transcriptional activation, mediation of the British Journal of Haematology, 1998, 102, 415–422