Abstract
During development the human β-globin gene cluster undergoes two switching processes at the embryo-fetal and fetal-adult stages, respectively, involving changes in chromatin remodeling and in transcriptional regulatory networks. In particular, during the perinatal period, the switch from fetal-to-adult globin gene expression leads to fetal globin genes silencing and progressive decline of fetal hemoglobin (HbF). Impaired hemoglobin switching is associated with hereditary persistence of HbF (HPFH), a condition in which the fetal globin genes fail to be completely silenced in adult red blood cells. This condition, when co-inherited with hemoglobinopathies, has great therapeutic potential because elevated HbF levels can ameliorate β-thalassemia and sickle cell anemia. Therefore, there is a growing interest about the complex network of factors that regulate fetal globin genes expression. Here we discuss the activity of transcriptional repressors of fetal globin genes and their potential role as novel therapeutic targets in β-thalassemia.
Keywords
- globin gene switching
- HbF silencing
- LCR
- transcriptional repressors
- genome editing
1. Introduction
The human β-globin locus consists of five functional genes,
In the last decades, large research efforts have been devoted to disclose the molecular mechanisms underlying this
Fetal hemoglobin levels are regulated by complex mechanisms involving factors linked or not to the β-globin gene (
Below we discuss the contribution of some of the main cis- and trans-acting determinants so far identified in silencing fetal globin gene expression and the most recent therapeutic advances based on fetal globin gene reactivation in the treatment of hemoglobinopathies.
2. Regulatory elements and transcriptional factors involved in fetal globin gene silencing
2.1 The role of the locus control region (LCR) in chromatin remodeling and globin gene switching
The locus control region (LCR), located upstream of the
The LCR contains five DNAse I hypersensitive sites (HS 1–5), four of which (HS1–HS4) acting as erythroid cell-specific enhancers that are required for proper order of activation of the globin genes during ontogeny. However, differently from classical enhancer elements, LCR activity is both orientation- and distance-dependent and prevents position effects, therefore also
It is now generally accepted that globin gene switching can occur through not mutually exclusive mechanisms involving direct competition between stage-specific gene promoters for LCR enhancer elements, gene expression activation, and/or silencing mechanisms promoted by specific activators and repressors as well as by epigenetic modifications. By chromatin conformation capture (3C) experiments, it has been shown that LCR exerts long-distance transcriptional enhancement through dynamic organization of the locus in stage-specific chromatin loops that allow the LCR enhancers to be positioned in close proximity of the genes that are to be activated [12]. This mechanism is also consistent with the evidence that the LCR can interact with only one promoter at a time. These interactions also strictly depend on the availability of specific trans-acting factors, non-DNA-binding factors linked to transcription factors and chromatin facilitators bound to regulatory regions of genes to be transcribed (Figure 1).
It has also been shown that epigenetic mechanisms at the
Chromatin looping, transcriptional protein complexes, and epigenetic modifications require a large array of protein factors whose list grows continuously, thus providing deeper insights into these mechanisms. The role of some of the most relevant of them will be herein discussed.
2.2 Cold-shock domain protein A (CSDA)
Cold-shock domain (CSD) proteins have been reported to be largely involved in a variety of gene expression regulation processes such as transcriptional activation and repression or posttranscriptional mechanisms including mRNA packaging, transport, localization, and stability. In these proteins the CSD domain does not bind to unique DNA sequence motifs, but it is able to recognize intramolecular triplex H-DNA structures, commonly generated by CT-rich sequences. The cold-shock domain protein A (CSDA), also known as DNA-binding protein A (dbpA), is a protein with a molecular weight of about 60 kDa [14]. The CSDA gene is located at position 12p13.1 and comprises 10 exons spanning a 24-kb genomic region. CSDA consists of three structural domains: an alanine/proline (AP)-rich N-terminal domain followed by a central cold-shock domain (CSD) and a C-terminal domain, containing four alternating clusters of basic and acid amino acids. The DNA-binding domain is encoded by exons 1–5. Within this region, the cold-shock domain, comprising about 70 amino acid residues, is encoded by exons 2–5 [14]. A RNP1 motif [GA] – [FY] – [GA] – [FY] – [IVA], conserved in this protein family [15], is present in the 3′ end of exon 2. The exon 6, encoding 69 amino acids, is alternatively spliced. The C-terminus (exons 6–9) C-terminal domain mediates protein-protein interactions [16]. Alternative splicing of exon 6 results in two main isoforms, namely, CSDA isoform
CSDA
2.3 B-cell lymphoma/leukemia 11A (BCL11A)
The
In the last years, many efforts have been aimed at clarifying the repression mechanism exerted by BCL11A at the
2.4 Krüppel-like factor 1 (KLF1)
The transcription factor KLF1 (Krüppel-like factor 1), formerly known as
The
In the HBB cluster, KLF1 exerts a dual role in fetal-to-adult globin gene switching by different mechanisms. On one hand, as also evidenced by studies on (+/−) KLF1 transgenic mice [12], KLF1 contributes to directly activate the
2.5 Myb
Myb is a DNA-binding transcriptional regulator of approximately 75 kDa that plays a crucial role in hematopoiesis and erythropoiesis. The
A large variety of proteins have been identified so far as Myb interactors: DNA-binding transcription factors that directly bind Myb, transcriptional coactivators that mediate interactions with the transcriptional machinery, and protein factors that are able to modify Myb activity. Each of these of protein factors can act either as activator or repressor of Myb function, depending both on cell type and protein microenvironment [43].
In the last decade, a GWAS approach led to the identification of a set of single nucleotide polymorphisms at the
Notably, further evidence of the repressive role of Myb on fetal globin gene expression has also been provided by clinical observations of higher HbF levels in patients with trisomy 13. It has been demonstrated that in this condition, the high HbF levels are related to the dysregulation of two microRNAs, miR-15a and miR-16-1, both localized on chromosome 13q14, which mediate Myb downregulation through the binding to its 3′-UTR region [47].
Nevertheless, in spite of all these evidences, the mechanism of repression of fetal globin genes exerted by Myb is not fully understood. It has been proposed that Myb could regulate HbF expression by two not mutually exclusive mechanisms: by activating the expression levels of fetal globin gene repressors as BCL11A and KLF1 or by controlling the kinetics of erythroid differentiation. In fact, low Myb levels have been found associated with reduced levels of these
3. Novel therapeutic approaches for β-thalassemia
Currently, clinical management of β-thalassemia and SCD patients is largely dependent on regular blood transfusions associated with chelating agents for the treatment of systemic iron overload. Indeed, except for allogeneic transplantation strategies, whose favorable outcome is anyway restricted to the availability of a well-matched donor, or for the more recent haploidentical transplantation option that however carries substantial risks, no other curative option is currently available for hemoglobinopathies. Therefore, due to the relevant global health burden of these diseases, there is still a great need for effective and definitive large-scale treatments.
In the last decades, given the ameliorative effects of elevated HbF levels that can lead to decreased hemoglobin polymerization in SCD as well as to reduced α-chain imbalance in β-thalassemia, a great deal of effort has been devoted to the development of new therapies aimed at reactivating fetal globin gene expression. Therefore, according to the evidences that hypo-methylated DNA and hyper-acetylated histones are well-known epigenetic marks of transcriptionally active regions, inhibitors of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) have been developed as novel pharmacologic approaches to support transfusion therapy. Among the first drugs to be identified, 5-azacytidine and decitabine were found to induce increased HbF levels through DNA hypo-methylation process. Subsequently, the use of hydroxyurea (HU) was also investigated as a promising HbF inducer agent in adults with severe SCD. Although the mechanism through which HU induces HbF reactivation is still not completely understood, HU treatment was found to increase HbF levels and reduce HbS polymerization, thus improving clinical symptoms and quality of life of these patients. Another epigenetic approach was also exploited through the use of HDAC inhibitors, including butyrate and its derivatives that have been demonstrated to stimulate HbF production in β-thalassemia patients [49].
More recently, many hopes were directed toward the development of gene therapy procedures designed to introduce a normal copy of the β-globin gene by viral vectors that offer the advantages of long-term benefits and, even more importantly, being based on autologous transplant, do not require a matched donor. However, in spite of great expectations for a rapid development of this approach, for many years it proved a difficult goal, and only recently it has been successfully introduced in clinical practice. Notably, during the last years, additional promising therapeutic options have been provided by progress in gene-editing technologies, including the clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR-associated 9 (Cas9) (CRISPR-Cas9) tool, designed to introduce highly specific genome modifications for either gene disruption or correction [48, 50, 51]. At the same time, progress in understanding the molecular mechanisms underlying fetal globin gene silencing has contributed to identify several potential molecular targets, thus paving the way to novel therapeutic approaches for hemoglobinopathies that, besides correcting the defective β-globin gene, are now aimed at editing genomic segments that regulate fetal hemoglobin synthesis in hematopoietic stem cells (HSCs).
Interestingly, some of the most promising editing strategies for hemoglobinopathies that are being pursued include the reactivation of fetal globin genes that could be achieved either by silencing fetal globin gene repressors or, alternatively, by introducing known HPFH mutations in the HBB cluster. Since the beginning, targeting Myb or KLF1 appeared to be rather problematic given the pleiotropic roles played by these two factors in hematopoiesis and in erythropoiesis, respectively, whereas BCL11A emerged as one of the most appropriate candidate to be silenced, given its well-recognized role in HbF repression [48, 50, 51]. However, ubiquitous BCL11A knockdown rapidly turned out to be an unsuccessful strategy, given its role in other biological pathways including neuronal and B-lymphocyte development [28]. Consequently, alternative strategies are being underway focused on reducing its expression selectively in erythroid cells that could be achieved by disruption of its intronic erythroid-specific enhancer without affecting non-erythroid cells [51]. A similar approach could also be used to tune down Myb expression levels by editing SNPs at the
4. Conclusions
The research in this field should be immediately focused on the development of safe, effective drug therapies that can be accomplished through fetal globin gene induction and at the same time on the prospect of cures through bone marrow transplantation using the promise of genome-editing strategies that will bring a vastly improved quality and quantity of life to patients who suffer from these devastating disorders.
Acknowledgments
This work was supported by the Regione Campania SATIN grant, grant/award number 2018–2020.
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