Hypoxic and nitrosative stress conditions modulate expression of myoglobin genes in a carcinogenic hepatobiliary trematode, Clonorchis sinensis

Despite recent evidence suggesting that adult trematodes require oxygen for the generation of bioenergy and eggshells, information on the molecular mechanism by which the parasites acquire oxygen remains largely elusive. In this study, the structural and expressional features of globin genes identified in Clonorchis sinensis, a carcinogenic trematode parasite that invades the hypoxic biliary tracts of mammalian hosts, were investigated to gain insight into the molecules that enable oxygen metabolism. The number of globin paralogs substantially differed among parasitic platyhelminths, ranging from one to five genes, and the C. sinensis genome encoded at least five globin genes. The expression of these Clonorchis genes, named CsMb (CsMb1—CsMb3), CsNgb, and CsGbX, according to their preferential similarity patterns toward respective globin subfamilies, exponentially increased in the worms coinciding with their sexual maturation, after being downregulated in early juveniles compared to those in metacercariae. The CsMb1 protein was detected throughout the parenchymal region of adult worms as well as in excretory-secretory products, whereas the other proteins were localized exclusively in the sexual organs and intrauterine eggs. Stimuli generated by exogenous oxygen, nitric oxide (NO), and nitrite as well as co-incubation with human cholangiocytes variously affected globin gene expression in live C. sinensis adults. Together with the specific histological distributions, these hypoxia-induced patterns may suggest that oxygen molecules transported by CsMb1 from host environments are provided to cells in the parenchyma and intrauterine eggs/sex organs of the worms for energy metabolism and/or, more importantly, eggshell formation by CsMb1 and CsMb3, respectively. Other globin homologs are likely to perform non-respiratory functions. Based on the responsive expression profile against nitrosative stress, an oxygenated form of secreted CsMb1 is suggested to play a pivotal role in parasite survival by scavenging NO generated by host immune cells via its NO dioxygenase activity.

Recent Approaches for Manipulating Globin Gene Expression in Treating Hemoglobinopathies

Tissue oxygenation throughout life depends on the activity of hemoglobin (Hb) one of the hemeproteins that binds oxygen in the lungs and secures its delivery throughout the body. Hb is composed of four monomers encoded by eight different genes the expression of which is tightly regulated during development, resulting in the formation of distinct hemoglobin tetramers in each developmental stage. Mutations that alter hemoglobin structure or its regulated expression result in a large group of diseases typically referred to as hemoglobinopathies that are amongst the most common genetic defects worldwide. Unprecedented efforts in the last decades have partially unraveled the complex mechanisms that control globin gene expression throughout development. In addition, genome wide association studies have revealed protective genetic traits capable of ameliorating the clinical manifestations of severe hemoglobinopathies. This knowledge has fueled the exploration of innovative therapeutic approaches aimed at modifying the genome or the epigenome of the affected cells to either restore hemoglobin function or to mimic the effect of protective traits. Here we describe the key steps that control the switch in gene expression that concerns the different globin genes during development and highlight the latest efforts in altering globin regulation for therapeutic purposes.

Epigenetic Insights and Potential Modifiers as Therapeutic Targets in β–Thalassemia

Thalassemia, an inherited quantitative globin disorder, consists of two types, α– and β–thalassemia. β–thalassemia is a heterogeneous disease that can be asymptomatic, mild, or even severe. Considerable research has focused on investigating its underlying etiology. These studies found that DNA hypomethylation in the β–globin gene cluster is significantly related to fetal hemoglobin (HbF) elevation. Histone modification reactivates γ-globin gene expression in adults and increases β–globin expression. Down-regulation of γ–globin suppressor genes, i.e., BCL11A, KLF1, HBG-XMN1, HBS1L-MYB, and SOX6, elevates the HbF level. β–thalassemia severity is predictable through FLT1, ARG2, NOS2A, and MAP3K5 gene expression. NOS2A and MAP3K5 may predict the β–thalassemia patient’s response to hydroxyurea, a HbF-inducing drug. The transcription factors NRF2 and BACH1 work with antioxidant enzymes, i.e., PRDX1, PRDX2, TRX1, and SOD1, to protect erythrocytes from oxidative damage, thus increasing their lifespan. A single β–thalassemia-causing mutation can result in different phenotypes, and these are predictable by IGSF4 and LARP2 methylation as well as long non-coding RNA expression levels. Finally, the coinheritance of β–thalassemia with α–thalassemia ameliorates the β–thalassemia clinical presentation. In conclusion, the management of β–thalassemia is currently limited to genetic and epigenetic approaches, and numerous factors should be further explored in the future.

Genetic variation in haemoglobin alters breathing in high-altitude deer mice

ABSTRACTPhysiological systems often have emergent properties but the effects of genetic variation on physiology are often unknown, which presents a major challenge to understanding the mechanisms of phenotypic evolution. We investigated the in vivo effects on respiratory physiology of genetic variants in haemoglobin (Hb) that contribute to hypoxia adaptation in high-altitude deer mice (Peromyscus maniculatus). We created F2 inter-population hybrids of highland and lowland deer mice to test the phenotypic effects of α- and β-globin variants on a mixed genetic background. High-altitude genotypes were associated with breathing phenotypes that enhance O2 uptake in hypoxia, including a deeper more effective breathing pattern and an augmented hypoxic ventilatory response. These effects could not be explained by erythrocyte Hb-O2 affinity or globin gene expression in the brainstem. Therefore, adaptive variation in haemoglobin can have unexpected effects on physiology that are distinct from the canonical function of this protein in circulatory O2 transport.

Effect of Nicotinamide, 1-Methylnicotinamide, and N'-Methylnicotinamide on Erythroid Colony Formation and γ-Globin Expression in Cultured Baboon CD34+ Cells

Pharmacological treatments designed to increase Fetal Hemoglobin (HbF) levels offer great promise to alleviate the symptoms and improve the lifespan of the vast numbers of patients afflicted with sickle cell disease (SCD) and β-thalassemia. Hydroxyurea can increase HbF, but a large fraction of patients with SCD do not respond to the drug. DNMT1 and LSD1 inhibitors are the most powerful drugs to increase HbF but are limited by side effects that include neutropenia, thrombophilia and/or thrombocytopenia. The development of new, more effective, and safer pharmacological strategies to augment HbF levels in the blood thus continues to be an important goal. Previous studies have shown that γ-globin gene expression is dynamically regulated during erythroid differentiation (Papayannopoulou et al PNAS 74:2923-2927, 1977). The proportion of γ-globin gene expression is higher at earlier stages (BFUe) of erythroid differentiation than at more advanced stages (CFUe). Therefore, we suggest the hypothesis that expansion of primitive, less differentiated progenitors might favor increased γ-globin, particularly when combined other HbF-inducing drugs. To investigate this hypothesis, we have tested whether nicotinamide (NAM), the major NAM metabolite 1-methylnicotinamide (1-mNAM), and N'-methylnicotinamide (N'-mNAM), a chemical derivative of NAM, can foster expansion of erythroid colony-forming cells (BFUe and CFUe) and increase γ-globin expression of cultured baboon CD34+ cells. Previous observations have shown that NAM facilitates in vitro expansion of cord blood CD34+ cells and enhanced long term engraftment in transplanted recipients (Horwitz et al J Clin Invest 124:3121, 2014). Contrasting effects of NAM, 1-mNAM, and N'-mNAM on differentiation and proliferation of the murine erythroleukemia cell line (MEL) have been previously reported (Terada et al PNAS 76:6414, 1979; Kuykendall et al Toxicol In Vitro 21:1656, 2007). While both NAM and N'-mNAM induced MEL cell differentiation, N'-mNAM was far more potent. In contrast, 1-mNAM increased cell proliferation, reduced spontaneous differentiation, and blocked differentiation induced by NAM and N'-mNAM. The effect of all three forms of NAM was examined using bone marrow (BM) CD34+ cells from a pre-clinical non-human primate large animal model. To test the effect of NAM, 1-mNAM, and N'-mNAM on expansion of erythroid colony-forming cells, the agents (5mM) were added to liquid cultures of baboon CD34+ bone marrow cells previously expanded for 5 days in serum-free expansion media (SFEM). Colony assays were performed on d8. In two experiments total erythroid colonies (BFUe and CFUe) were 2 fold higher in cultures treated with 1-mNAM compared to untreated controls (p<0.05) while no effect was observed in cultures treated with NAM. No colonies were observed in cultures treated with N'-mNAM (Figure 1A). Observation of Wright's stained cytospin preparations showed extensive erythroid differentiation on d8 in cells treated with N'-mNAM (Figure 1B). The effect of NAM, 1-mNAM, and N'-NAM on γ-globin expression was tested in baboon CD34+ cells grown in co-culture with the AFT024 cell line. NAM, 1-mNAM, or N'-mNAM (5mM) were added to cultures on d7. Expression of γ- and β-globin mRNA was measured by RT-PCR on d17. Increased γ-globin expression (0.57±0.04 γ/γ+β)) was observed in cells treated with N'-mNAM on d7 compared to untreated controls (0.20±0.09 γ/γ+β; p<0.001). NAM and 1-mNAM had no significant effect on γ-globin gene expression (Figure 1C). These results thus show that while erythroid colonies are increased by 1-mNAM, N'-mNAM is a potent inducer of erythroid differentiation and increases γ-globin expression in primary cultures of baboon CD34+ cells. In conclusion, 1-mNAM and N'-mNAM have contrasting effects on erythroid differentiation in primary baboon CD34+ cell cultures, confirming previous experiments in the MEL cell line. Future experiments are planned to test the effect of these agents on HbF in the baboon. Disclosures Saunthararajah: EpiDestiny: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.