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730 Part VI: The Erythrocyte Chapter 48: The Thalassemias: Disorders of Globin Synthesis 731

the nucleus, it undergoes a good deal of processing that entails capping cluster, its action must be fundamentally different from that of the
the 5′ end and polyadenylation of the 3′ end, both of which probably β-globin LCR because the chromatin structure of the α-gene cluster is
serve to stabilize the transcript (Chap. 10). The intervening sequences in an open conformation in all tissues.
are removed from the mRNA precursor in a complex two-stage process Some forms of thalassemia result from deletions involving these
that relies on certain critical sequences at the intron–exon junctions. regulatory regions. In addition, the phenotypic effects of deletions of
The method by which globin gene clusters are regulated is impor- these gene clusters are strongly positional, which may reflect the relative
tant to understanding the pathogenesis of the thalassemias. Many distance of particular genes from the LCR and HS40.
details remain to be determined, but studies performed over the last few
years have provided at least an outline of some of the major mechanisms Developmental Changes in Globin Gene Expression
of globin gene regulation. 7,9,40–42 One particularly important aspect of human globin genes is regulation
Most of the DNA within cells that is not involved in gene tran- of the switch from fetal to adult hemoglobin. Because many of the tha-
scription is packaged into a compact form that is inaccessible to lassemias and related disorders of the β-globin gene cluster are asso-
transcription factors and RNA polymerase. Transcriptional activity ciated with persistent γ-chain synthesis, a full understanding of their
is characterized by a major change in the structure of the chromatin pathophysiology must include an explanation for this important phe-
surrounding a particular gene. These alterations in chromatin struc- nomenon, which plays a considerable role in modifying their pheno-
ture can be identified by enhanced sensitivity to exogenous nucleases. typic expression.
Erythroid lineage-specific nuclease-hypersensitive sites are found at The complex topic of hemoglobin switching has been the subject
7,42
several locations in the β-globin gene cluster, which vary during dif- of several extensive reviews. β-Globin synthesis commences early
ferent stages of development. In fetal life, these sites are associated with during fetal life, at approximately 8 to 10 weeks’ gestation. β-Globin
the promoter regions of all four globin genes. In adult erythroid cells, synthesis continues at a low level, approximately 10 percent of the
the sites associated with the γ genes are absent. The methylation state total non–α-globin chain production, up to approximately 36 weeks’
of the genes plays an important role in their ability to be expressed. In gestation, after which it is considerably augmented. At the same time,
human and other animal tissues, the globin genes are extensively meth- γ-globin chain synthesis starts to decline so that, at birth, approximately
ylated in nonerythroid organs and are relatively undermethylated in equal amounts of γ- and β-globin chains are produced. Over the first
hematopoietic tissues. Changes in chromatin configuration around the year of life, γ-chain synthesis gradually declines. By the end of the
globin genes at different stages of development are reflected by altera- first year, γ-chain synthesis amounts to less than 1 percent of the total
tions in their methylation state. non–α-globin chain output. In adults the small amount of hemoglobin
In addition to the promoter elements, several other important F is confined to an erythrocyte population called F cells.
regulatory sequences have been identified in the globin gene clusters. How this series of developmental switches is regulated is not clear.
For example, several enhancer sequences thought to be involved with The process is not organ specific but is synchronized throughout the
tissue-specific expression have been identified. Their sequences are developing hematopoietic tissues. Although environmental factors may
similar to the upstream activating sequences of the promoter elements. be involved, the bulk of experimental evidence suggests some form of
Both consist of a number of “modules,” or motifs, that contain bind- “time clock” is built into the hematopoietic stem cell. At the chromo-
ing sites for transcriptional activators or repressors. The enhancer somal level, regulation appears to occur in a complex manner involv-
sequences are thought to act by coming into spatial apposition with the ing both developmental stage-specific trans-activating factors and the
promoter sequences to increase the efficiency of transcription of partic- relative proximity of the different genes of the β-globin gene cluster to
ular genes. It now is clear that transcriptional regulatory proteins may LCR. Some of the elements involved in the stage-specific regulation of
bind to both the promoter region of a gene and to the enhancer. Some of human globin genes have been identified. KLF1 (erythroid Kruppel-
these transcriptional proteins, GATA-1 and NFE-2, for example, appear like factor), a developmental stage–enriched protein, activates human
40
to be largely restricted to hematopoietic tissues. These proteins may β-globin gene expression and is involved in human γ- to β-globin gene
43
bring the promoter and the enhancer into close physical proximity, per- switching. More recently BCL11A and MYB have also been identified
mitting transcription factors bound to the enhancer to interact with the as being involved in this process. 42
transcriptional complex that forms near the TATA box. At least some Fetal hemoglobin synthesis can be reactivated at low levels in
of these hematopoietic gene transcription factors likely will be develop- states of hematopoietic stress and at higher levels in certain hematologic
mental-stage specific. malignancies, notably juvenile myeloid leukemia. However, high levels
Another set of erythroid-specific nuclease-hypersensitive sites is of hemoglobin F production are seen consistently in adult life only in
located upstream from the embryonic globin genes in both the α- and the hemoglobinopathies.
β-gene clusters. These sites mark the regions of particularly important
control elements. In the case of the β-globin gene cluster, the region
is marked by five hypersensitive sites to DNase I treatment (HS) (an MOLECULAR BASIS OF THE
40
enzyme used to detect DNA-protein interaction). The most 5′ site THALASSEMIAS
(HS5) does not show tissue specificity. HS1 through HS4, which together
form the locus control region (LCR), are largely erythroid-specific. Each Once cloning and sequencing of globin genes from patients with many
of the regions of the LCR contains a variety of binding sites for erythroid different forms of thalassemia were possible, the wide spectrum of muta-
transcription factors. The precise function of the LCR is not known, but tions underlying these conditions became clear. A picture of remarkable
it is undoubtedly required to establish a transcriptionally active domain heterogeneity has emerged. For more extensive coverage of this topic,
spanning the entire globin gene cluster. The α-globin gene cluster also the reader is referred to several monographs and reviews. 7,9,10,44–46
has a major regulatory element of this kind, in this case HS40. This
41
forms part of four highly conserved noncoding sequences, or multispe- β-THALASSEMIA
cies conserved sequences (MCSs), called MSC-R1-R4; of these elements β-thalassemia is extremely heterogeneous at the molecular level.
7
only MSC-R2, that is HS40, is essential for α-globin gene expression. More than 200 different mutations have been found in association
Although deletions of this region inactivate the entire α-globin gene with the β-thalassemia phenotype. Broadly, they fall into deletions
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