ReviewInsight of fetal to adult hemoglobin switch: Genetic modulators and therapeutic targets
Introduction
Way back in 1866, Korber et al., discovered that the hemoglobin obtained from umbilical cord was far more resistant to alkali denaturation than the normal adult hemoglobin (HbA, α2β2) (Chromosome location: α globin gene-16p13.3; β globin gene-11p15.4) [1]. After almost 60 years of this discovery, in 1934 Brinkman et al., established that the variation in the alkali resistant characteristic of hemoglobin was due to the globin portion of the molecule and thus the alkali resistant hemoglobin was termed as fetal hemoglobin (HbF, α2γ2) (Chromosome location: γ globin gene-11p15.4) since it formed the major hemoglobin fraction of the growing fetus [2]. During the same time period, Drescher and Kiinzer, & Huehns et al., identified that the embryonic hemoglobin in small human embryos (gestational age: 7 weeks-12 weeks) had an alkali denaturation between HbA and HbF [3]. These series of discoveries left behind a perplexing question; does the hemoglobin in humans during the ontogeny undergo successive switches? And if yes, is there a perspective for molecular cure by reactivating fetal globin genes in hemoglobinopathy patients?
The possibility that hemoglobinopathies can be cured by induction of fetal hemoglobin was realized as the symptoms are seen in 6 months of age after birth when there is a gradual reduction in the HbF levels. In β-thalassemia, the main cause of the disease is an imbalance in the globin chain ratio, wherein the excess unbound α globin chain precipitates, inhibiting the maturation of the erythroid precursors, ultimately leading to ineffective erythropoiesis and anemia [4]. Thus, the continued γ-globin gene expression in β-thalassemics, may lead to a reduction in the globin chain imbalance as γ-globin synthesized will combine with the excess unbound α-globin chain [5]. Similarly, the appreciating role of fetal hemoglobin in sickle cell disease started more than 60 years ago when Janet Watson confirmed that infants with sickle cell disease with high HbF level had few symptoms and that their deoxygenated erythrocytes took a longer time to sickle and did not deform as extensively as did their sickle cell trait-carrying mother's cells [6]. The pathophysiology of sickle cell disease is dependent on the polymerization of deoxy sickle hemoglobin under low oxygen condition due to altered biophysical property, which is retarded with increased HbF concentration. Both HbF and its mixed hybrid tetramer (α2βSγ) cannot enter the deoxy sickle hemoglobin polymer phase, thus circumventing the cellular damage evoked by HbS polymers [7]. In both the hemoglobinopathy conditions, increased γ-globin gene expression acts as a well-known disease modifier. Hence understanding the molecular mechanism of hemoglobin switching and identification of molecular targets, for reversing this switch, is a subject of intense research [4].
During the ontogeny, globin genes (ε, Gγ, Aγ, δ, β) in the β-globin cluster are sequentially expressed, leading to the production of different hemoglobin molecules with distinct physiological properties. The site for primary hemoglobin synthesis is the embryonic yolk sac and the first wave of hemoglobin switch (primitive to definitive) occurs in the fetal liver after 5 weeks of gestation, wherein there is a switch from embryonic (ζ2ε2) to fetal hemoglobin (α2γ2) [8]. Towards the end of 3rd trimester, there is a gradual change from fetal (α2γ2) to adult hemoglobin (α2β2). This second major hemoglobin switch occurs in the bone marrow and lasts until 6 months of age after birth [8]. Fig. 1 shows a schematic representation of different hemoglobin progressively expressed during distinct stages of development. With the dawn of molecular era, various models for gene switching have been described. These include (i) Gene competition: In this model, the γ and β globin genes during the ontogeny compete for interaction with the LCR in stage specific manner for their expression. (ii) Chromosomal looping: Here, the hypersensitive sites in LCR of the β globin cluster, loops out to come in close proximity with the appropriate gene within the cluster, for promoting its transcription. (iii) Autonomous gene silencing: It is a self-governed process where all the elements for gene silencing are located within or in near proximity to the target gene that has to be repressed. (iv) Polymerase tracking model: It is based on the idea that the enhancer-bound protein complex consisting of RNA polymerase II scrutinizes the DNA sequence until a promoter region is confronted to activate gene expression. It is hypothesized that these models collectively bring about the process of globin gene switching [8,9]. Further, the hemoglobin switching process involves a complex interaction between the cis-acting elements (locus control region of the beta-globin gene cluster) and trans-acting transcription factors which co-ordinately carry out chromatin remodelling activities [9].
The β-globin gene cluster contains a 5′ distal regulatory element known as Locus Control Region (LCR), which has 5 active DNAase I hypersensitive sites (HS) 1–5, that is required for appropriate globin gene expression. This is followed with the series of globin genes (ε, Gγ, Aγ, δ, β) arranged sequentially in their order of expression (Fig. 2). To the downstream of the β globin gene is another HS site 3’ HS1. The β-globin cluster is flanked with olfactory receptors at both 5′ and 3′ ends [10]. Wijgerde et al., proposed a competitive model for globin gene regulation, wherein the LCR participates in the long-range looping interaction, which leads to only one productive LCR-globin gene interaction [10]. The potent transcriptional activation of the LCR is also due to the clustering of various erythroid transcription factors that bind to the hypersensitivity sites which act in concert to facilitate long-range looping activity. The cis-acting promoter sequences in the individual globin genes and the LCR serve as a template for the binding of various transcription factors [10]. It is observed that naturally occurring mutations in γ globin promoter region is associated with the hereditary persistence of fetal hemoglobin phenotype (HPFH). The mechanisms underlying the continued expression of HbF with these mutations are thought to involve alterations in the protein binding motifs that ultimately ablates the binding of repressors or enhancers affecting the gene expression [11]. The most extensively studied variation in several population groups is the XmnI polymorphism (C → T) residing at −158 position (HBG2 c.-211C → T) in the Gγ globin promoter region. The T allele of XmnI polymorphism is linked to the raised HbF levels and milder hemoglobinopathy conditions [12]. Though T allele in its homozygous state seems to have little effect on HbF levels in normal individuals, it has been associated with an increase of HbF especially during the erythropoietic stress, suggesting its role in HbF induction [13]. In clinically milder hemoglobinopathy patients where no mutation is detected in the γ-globin promoter region, the other genetic modulators [HBS1L-MYB intergenic region, BCL11A, KLF1] leading to raised HbF phenotype could play some role [7,8].
Section snippets
Role of factors involved in the hemoglobin switch and new therapeutic targets
More recently recognised variability in HbF expression both in non-anemic individuals and patients with β-hemoglobin disorders stimulated efforts to detect the genetic factors that lead to this deviation. Genome-wide association mapping studies have identified quantitative trait loci in the transcription factor encoding genes that play a role in the globin gene regulation and hemoglobin switching. Hence in this section we discuss the role of major transcription factors and complexes: the
Newly identified genes associated with gamma globin gene regulation
Recently, multiple studies highlighting the fetal to adult hemoglobin switch process has identified the new transcription factors that regulate the γ-globin gene expression. Carrocini et al., 2015 used phylogenetic footprinting between different species, to determine the binding sites of various transcription factors in HBG1 and HBG2 genes noncoding regions, to understand their role in HbF expression. They observed that GATA2 and TAL1 may act as positive regulators of γ-globin gene. GATA2 is
Interplay of epigenetic modifiers and transcription factors in the HbF regulation
The fetal to adult hemoglobin switch process is also actively governed by various epigenetic modifications that are linked to hyper cytosine methylation, histone deacetylation, and alteration in the chromatin structure [51]. In recent years, the epigenetic enzymes regulating these processes have become the pharmacological targets for the induction of γ-globin gene expression [52].
Association of DNA polymorphisms with raised HbF levels and amelioration of the clinical severity
Over past 2 decades numerous association studies have been published and the role of HbF modulators have been proposed with a notable impact on amelioration of disease severity of β-hemoglobinopathy. Among these are the variations located in the highly conserved regulatory regions of γ-globin gene promoter which acts as a binding site for transcription factors bringing about changes in the γ-globin gene expression (Table 1). In a recent study by Li-Ren et al., have shown that −175 T → C
Targeted translational research for increasing the HbF levels: opportunities and challenges!
The common transcription factors that govern the developmental switches execute their action by complex inter-dependent networks. The mechanisms underlying the function of these regulators are so stringent, that any deviations in their expression or protein-protein interaction may evoke disastrous phenotype leading to anemia, leukemia or lymphomas [76]. Hence it is pivotal to elucidate their mechanisms, with the basic step of placing these factors in a hierarchy in which they function. With
Conclusions and future directions
An increase in HbF levels has potential therapeutic implications as it ameliorates the clinical severity of β-hemoglobinopathies. With this review, we conclude that SNPs located at various quantitative trait loci are associated with meagre elevation of HbF levels in the range of 3–5%, nevertheless, they can increase the HbF output to clinically beneficial levels when co-inherited with hemoglobinopathies. Many recent studies have tried to dissect the mechanisms and transcription factors involved
Practice points
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The γ-globin gene promoter variations (cis-acting factors) have been shown to have a direct effect on increasing the HbF levels. This hypothesis is strengthened by the identification of the mutant T allele of the XmnI polymorphism (−158C → T) in milder hemoglobinopathy patient group.
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The HbF boosting alleles of γ-globin promoter variations, BCL11A polymorphisms, HBS1L-MYB polymorphisms, KLF1 variations may synergistically increase the HbF levels.
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Studies have shown that patients inheriting more
Research agenda
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The key genetic regulators of fetal hemoglobin may be considered as therapeutic targets for up-regulating HbF levels in hemoglobinopathy patients.
Declaration of Competing Interest
Both the authors have read and approved the manuscript. None of the authors had a conflict of interest related to this manuscript.
Acknowledgements
We thank Indian Council of Medical Research (ICMR), New Delhi and University of Mumbai for their support. PH collected the data and wrote the manuscript. AN guided the manuscript preparation.
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