The structure genetic control and synthesis of normal hemoglobin

Structure and function

The varying oxygen requirements during embryonic, fetal and adult life are reflected in the synthesis of different structural hemoglobins at each stage of human development. They all have the same general tetrameric structure, however, consisting of two different pairs of globin chains, each attached to one heme molecule. Adult and fetal hemoglobins have a chains combined with P chains (Hb A, a2P2), 8 chains (Hb A2, a282) and y chains (Hb F, a2y2). In embryos, a-like chains called Z chains combine with y chains to produce Hb Portland (Z2Y2), or with £ chains to make Hb Gower 1 (Z2£2), while a and £ chains form Hb Gower 2 (a2£2). Fetal hemoglobin is heterogeneous; there are two varieties of y chain that differ only in their amino acid composition at position 136, which may be occupied by either glycine or alanine; y chains containing glycine at this position are called G^ chains, those with alanine, A^ chains (Figure 1.1).

The synthesis of hemoglobin tetramers consisting of two unlike pairs of globin chains is absolutely essential for the effective function of hemoglobin as an oxygen carrier. The classical sigmoid shape of the oxygen dissociation curve, which reflects the allosteric properties of the hemoglobin molecule, ensures that, at high oxygen tensions in the lungs, oxygen is readily taken up and later released effectively at the lower tensions encountered in the tissues. The shape of the curve is quite different to that of myoglobin, a molecule which consists of a single globin chain with heme attached to it, which, like abnormal hemoglobins that consist of ho-motetramers of like-chains, has a hyperbolic oxygen dissociation curve.

1 Kb

31 32 99 100

31 32 99 100

30 31

Z2Y2 Hb Portland

Hb Gower 2

a2Y2 HbF

Z2e2 Hb Gower 1

Z2Y2 Hb Portland

Hb Gower 2

a2Y2 HbF

1 Kb

HbA2

Embryo Fetus Adult

Fig. 1.1 The genetic control of human hemoglobin production in embryonic, fetal and adult life a2ô2

The transition from a hyperbolic to a sigmoid oxygen dissociation curve, which is absolutely critical for normal oxygen delivery, reflects cooperativity between the four heme molecules and their globin subunits. When one of them takes on oxygen, the affinity of the remaining three increases markedly; this happens because hemoglobin can exist in two configurations, deoxy(T) and oxy(R), where T and R represent the tight and relaxed states, respectively. The T configuration has a lower affinity than the R for ligands such as oxygen. At some point during the addition of oxygen to the hemes, the transition from the T to the R configuration occurs and the oxygen affinity of the partially liganded molecule increases dramatically. These allosteric changes result from interactions between the iron of the heme groups and various bonds within the hemoglobin tetramer, which lead to subtle spatial changes as oxygen is taken on or given up.

The precise tetrameric structures of the different human hemoglobins, which reflect the primary amino acid sequences of their individual globin chains, are also vital for the various adaptive changes that are required to ensure adequate tissue oxygenation. The position of the oxygen dissociation curve can be modified in several ways. For example, oxygen affinity decreases with increasing CO2 tension (the Bohr effect). This facilitates oxygen loading to the tissues, where a drop in pH due to CO2 influx lowers oxygen affinity; the opposite effect occurs in the lungs. Oxygen affinity is also modified by the level of 2,3-biphosphoglycerate (2,3-BPG) in the red cell. Increasing concentrations shift the oxygen dissociation curve to the right, that is, they reduce oxygen affinity, while diminishing concentrations have the opposite effect. 2,3-BPG fits into the gap between the two P chains when it widens during deoxygenation, and interacts with several specific binding sites in the central cavity of the molecule. In the deoxy configuration the gap between the two P chains narrows and the molecule cannot be accommodated. With increasing concentrations of 2,3-BPG, which are found in various hypoxic and anemic states, more hemoglobin molecules tend to be held in the deoxy configuration and the oxygen dissociation curve is therefore shifted to the right, with more effective release of oxygen.

Fetal red cells have greater oxygen affinity than adult red cells, although, interestingly, purified fetal hemoglobin has an oxygen dissociation curve similar to that of adult hemoglobin. These differences, which are adapted to the oxygen requirements of fetal life, reflect the relative inability of Hb F to interact with 2,3-BPG compared with Hb A. This is because the y chains of Hb F lack specific binding sites for 2,3-BPG.

In short, oxygen transport can be modified by a variety of adaptive features in the red cell that include interactions between the different heme molecules, the effects of CO2 and differential affinities for 2,3-BPG. These changes, together with more general mechanisms involving the cardiorespira-

tory system, provide the main basis for physiological adaptation to anemia.

Genetic control of hemoglobin

The a- and P-like globin chains are the products of two different gene families which are found on different chromosomes (Figure 1.1). The P-like globin genes form a linked cluster on chromosome 11, spread over approximately 60 kb (kb = kilobase or 1000 nucleotide bases). The different genes that form this cluster are arranged in the order 5'-£-Gy-AY-yP-5-P-3'. The a-like genes also form a linked cluster, in this case on chromosome 16, in the order 5'-Z-yZ-ya1-a2-a1-3'. The yP, yZ and ya genes are pseudogenes; that is, they have strong sequence homology with the P, Z and a genes but contain a number of differences that prevent them from directing the synthesis of any products. They may reflect remnants of genes that were functional at an earlier stage of human evolution.

The structure of the human globin genes is, in essence, similar to that of all mammalian genes. They consist of long strings of nucleotides that are divided into coding regions, or exons, and non-coding inserts called 'intervening sequences' (IVS), or introns. The P-like globin genes contain two introns, one of 122-130 between codons 30 and 31 and one of 850-900 base pairs between codons 104 and 105 (the exon codons are numbered sequentially from the 5' to the 3' end of the gene; that is, from left to right). Similar, though smaller, introns are found in the a and Z globin genes. These introns and exons, together with short non-coding sequences at the 5' and 3' ends of the genes, represent the major functional regions of the particular genes. However, there are also extremely important regulatory sequences that subserve these functions, which lie outside the genes themselves.

At the 5' non-coding (flanking) regions of the globin genes, as in all mammalian genes, there are blocks of nucleotide homology. The first, the ATA box, is about 30 bases upstream (to the left) of the initiation codon; that is, the start word for the beginning of protein synthesis (see below). The second, the CCAAT box, is about 70 base pairs upstream from the 5' end of the genes. About 80-100 bases further upstream there is the sequence GGGGTG, or CACCC, which may be inverted or duplicated. These three highly conserved DNA sequences, called 'promoter elements', are involved in the initiation of transcription of the individual genes. Finally, in the 3' non-coding region of all the globin genes there is the sequence AATAAA, which is the signal for cleavage and polyA addition to RNA transcripts (see below: Gene action and globin synthesis).

The globin gene clusters also contain several sequences that constitute regulatory elements, which interact to promote erythroid-specific gene expression and coordination of the changes in globin gene activity during development. These include the globin genes themselves and their promoter elements—enhancers (regulatory sequences that increase gene expression despite being located at a considerable distance from the genes) and 'master' regulatory sequences called, in the case of the P globin gene cluster, the 'locus control region' (LCR); and, in the case of the a genes, HS40 (a nuclease-hy-persensitive site in DNA 40 kb from the a globin genes). Each of these sequences has a modular structure made up of an array of short motifs that represent the binding sites for tran-scriptional activators or repressors.

Gene action and globin synthesis

The flow of information between DNA and protein is summarized in Figure 1.2. When a globin gene is transcribed, messenger RNA (mRNA) is synthesized from one of its strands, a process which begins with the formation of a transcription complex consisting of a variety of regulatory proteins together with an enzyme called RNA polymerase (see below). The primary transcript is a large mRNA precursor which contains both intron and exon sequences. While in the nucleus, this molecule undergoes a variety of modifications. First, the introns are removed and the exons are spliced together. The intron/exon junctions always have the same sequence: GT at their 5' end, and AG at their 3' end. This appears to be essential for accurate splicing; if there is a mutation at these sites this process does not occur. Splicing reflects a complex series of intermediary stages and the interaction of a number of different nuclear proteins. After the exons are joined, the mRNAs are modified and stabilized; at their 5' end a complex CAP structure is formed, while at their 3' end a string of adenylic acid residues (polyA) is added. The mRNA processed in this way moves into the cytoplasm, where it acts as a template for globin chain production. Because of the rules of base pairing—that is, cytosine always pairs with thymine, and guanine with adenine—the structure of the mRNA reflects a faithful copy of the DNA codons from which it is synthesized; the only difference is that, in RNA, uracil (U) replaces thymine (T).

Amino acids are transported to the mRNA template on carriers called transfer RNAs (tRNAs); there are specific tRNAs for each amino acid. Furthermore, because the genetic code is redundant (that is, more than one codon can encode a particular amino acid), for some of the amino acids there are several different individual tRNAs. Their order in the globin chain is determined by the order of codons in the mRNA. The tRNAs contain three bases, which together constitute an anticodon; these anticodons are complementary to mRNA codons for particular amino acids. They carry amino acids to the template, where they find the appropriate positioning by codon-anticodon base-pairing. When the first tRNA is in po-

Flanking

Transfer RNA

Amino ^ acid Growing chain

Processed chain

Finished chain

_Flanking

Mechanism Globin Chain Synthesis

Excision of introns Splicing of exons

Processed mRNA

Translation

Transfer RNA

Amino ^ acid Growing chain

Processed chain

Finished chain

Gene mRNA precursor

Excision of introns Splicing of exons

Processed mRNA

Translation

Fig. 1.2 The mechanisms of globin gene transcription and translation

sition, an initiation complex is formed between several protein initiation factors together with the two subunits which constitute the ribosomes. A second tRNA moves in alongside and the two amino acids that they are carrying form a peptide bond between them; the globin chain is now two amino acid residues long. This process is continued along the mRNA from left to right, and the growing peptide chain is transferred from one incoming tRNA to the next; that is, the mRNA is translated from 5' to 3'. During this time the tRNAs are held in appropriate steric configuration with the mRNA by the two ribosomal subunits. There are specific initiation (AUG) and termination (UAA, UAG and UGA) codons. When the ribosomes reach the termination codon, translation ceases, the completed globin chains are released, and the ribosomal subunits are recycled. Individual globin chains combine with heme, which has been synthesized through a separate pathway, and then interact with one like chain and two unlike chains to form a complete hemoglobin tetramer.

Regulation of hemoglobin synthesis

The regulation of globin gene expression is mediated mainly at the transcriptional level, with some fine tuning during translation and post-translational modification of the gene products. DNA that is not involved in transcription is held tightly packaged in a compact, chemically modified form that is inaccessible to transcription factors and polymerases and which is heavily methylated. Activation of a particular gene is reflected by changes in the structure of the surrounding chromatin, which can be identified by enhanced sensitivity to nucleases. Erythroid lineage-specific nuclease-hypersensitive sites are found at several locations in the P globin gene cluster. Four are distributed over 20 kb upstream from the e globin gene in the region of the P globin LCR (Figure 1.3). This vital regulatory region is able to establish a transcriptionally active domain spanning the entire P globin gene cluster. Several enhancer sequences have been identified in this cluster. A variety of regulatory proteins bind to the LCR, and to the promoter regions of the globin genes and to the enhancer sequences. It is thought that the LCR and other enhancer regions become opposed to the promoters to increase the rate of transcription of the genes to which they are related.

These regulatory regions contain sequence motifs for various ubiquitous and erythroid-restricted transcription factors. Binding sites for these factors have been identified in each of the globin gene promoters and at the hypersensitive-site regions of the various regulatory elements. A number of the factors which bind to these areas are found in all cell types. They include Sp1, Yy1 and Usf. In contrast, a number of transcription factors have been identified, including GATA-1, EKLF and NF-E2, which are restricted in their distribution to erythroid cells and, in some cases, megakaryocytes and mast cells. The overlapping of erythroid-specific and ubiquitous-factor binding sites in several cases suggests that competitive binding may

60 kb

Chromosome 11

40 kb

HS-40

Chromosome 16

z a2 a1

Fig. 1.3 The positions of the major regulatory regions in the P and a globin gene clusters

The arrows indicate the position of the erythroid lineage-specific nuclease-hypersensitive sites. HS = hypersensitive.

play an important part in the regulation of erythroid-specific genes. Another binding factor, SSP, the stage selector protein, appears to interact specifically with e and y genes.

The binding of hematopoietic-specific factors activates the LCR, which renders the entire P globin gene cluster transcriptionally active. These factors also bind to the enhancer and promoter sequences, which work in tandem to regulate the expression of the individual genes in the clusters. It is likely that some of the transcriptional factors are developmental-stage-specific, and hence may be responsible for the differential expression of the embryonic, fetal and adult globin genes. The a globin gene cluster also contains an element, HS40, which has some structural features in common with the P LCR, although it is different in aspects of its structure. A number of enhancer-like sequences have also been identified, although it is becoming clear that there are fundamental differences in the pattern of regulation of the two globin gene clusters.

In addition to the different regulatory sequences outlined above, there are also sequences which may be involved specifically with 'silencing' of genes, notably those for the embryonic hemoglobins, during development.

Some degree of regulation is also mediated by differences in the rates of initiation and translation of the different mRNAs, and at the post-transcriptional level by differential affinity for different protein subunits. However, this kind of post-tran-scriptional fine tuning probably plays a relatively small role in determining the overall output of the globin gene products.

Regulation of developmental changes in globin gene expression

During development, the site of red cell production moves from the yolk sac to the fetal liver and spleen, and thence to bone marrow in the adult. Embryonic, fetal and adult hemoglobin synthesis is approximately related in time to these changes in the site of erythropoiesis, although it is quite clear that the various switches, between embryonic and fetal and between fetal and adult hemoglobin synthesis, are beautifully synchronized throughout these different sites. Fetal hemoglobin synthesis declines during the later months of gestation and Hb F is replaced by Hbs A and A2 by the end of the first year of life.

Despite a great deal of research, very little is known about the regulation of these different switches from one globin gene to another during development. Work from a variety of different sources suggests that there may be specific regions in the a and P globin gene clusters that are responsive to the action of transcription factors, some of which may be developmental-stage-specific. However, proteins of this type have not yet been isolated, and nothing is known about their regulation and how it is mediated during development.

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