Production and Characterization of K562 Cellular Clones Hyper-Expressing the Gene Encoding α-Globin: Preliminary Analysis of Biomarkers Associated with Autophagy.

One of the most relevant pathophysiological hallmarks of ß-thalassemia is the accumulation of toxic α-globin chains inside erythroid cells, which is responsible for their premature death (hemolysis). In this context, the availability of an experimental model system mimicking the excess in α-globin chain production is still lacking. The objective of the present study was to produce and characterize K562 cellular clones forced to produce high amounts of α-globin, in order to develop an experimental model system suitable for studies aimed at the reduction of the accumulation of toxic α-globin aggregates. In the present study, we produced and characterized K562 cellular clones that, unlike the original K562 cell line, stably produced high levels of α-globin protein. As expected, the obtained clones had a tendency to undergo apoptosis that was proportional to the accumulation of α-globin, confirming the pivotal role of α-globin accumulation in damaging erythroid cells. Interestingly, the obtained clones seemed to trigger autophagy spontaneously, probably to overcome the accumulation/toxicity of the α-globin. We propose this new model system for the screening of pharmacological agents able to activate the full program of autophagy to reduce α-globin accumulation, but the model may be also suitable for new therapeutical approaches targeted at the reduction of the expression of the α-globin gene.

MicroRNAs in ß-thalassemia.

ß-thalassemia is a lethal inherited disease resulting from ß-globin gene mutations. Severe ß-thalassemia requires regular blood transfusions. Other active interventions, including iron chelating, stem cell transplantation and gene therapy, have remarkably improved the quality of life and prolonged the survival of patients with transfusion-dependent ß-thalassemia, but all with significant limitations and complications. MicroRNAs (miRNAs), encoded by a class of endogenous genes, are found to play important roles in regulating globin expression. Among the miRNAs of particular interest related to ß-thalassemia, miR-15a/16-1, miR-486-3p, miR-26b, miR-199b-5p, miR-210, miR-34a, miR-138, miR-326, let-7, and miR-17/92 cluster elevate γ-globin expression, while miR-96, miR-146a, miR-223-3p, and miR-144 inhibit γ-globin expression. A couple of miRNAs, miR-144 and miR-150, repress α-globin expression, whereas miR-451 induces α-, ß- and γ-globin expression. Single nucleotide polymorphism in miRNA genes or their targeted genes might also contribute to the abnormal expression of hemoglobin. Moreover, changes in the expression of miR-125b, miR-210, miR-451, and miR-609 reflect the severity of anemia and hemolysis in ß-thalassemia patients. These results suggest that miRNAs are potential biomarkers for the diagnosis and prognosis of ß-thalassemia, and miRNA-based therapeutic strategy might be used as a coordinated approach for effectively treating ß-thalassemia.

Mechanisms mediating suppression of globin gene transcription in Danio rerio nonerythroid cells.

We studied the repression of adult and embryo-larval genes of the major globin gene locus in D. rerio fibroblasts. The results obtained suggest that at least some of the globin genes are repressed by Polycomb, similarly to human α-globin genes. Furthermore, within two α/ß globin gene pairs, repression of α-type and ß-type genes appears to be mediated by different mechanisms, as increasing the level of histone acetylation can activate transcription of only ß-type genes.

Synthesis of Recombinant Human Hemoglobin With NH2 -Terminal Acetylation in Escherichia coli.

The development of new technologies for the efficient expression of recombinant hemoglobin (rHb) is of interest for experimental studies of protein biochemistry and the development of cell-free blood substitutes in transfusion medicine. Expression of rHb in Escherichia coli host cells has numerous advantages, but one disadvantage of using prokaryotic systems to express eukaryotic proteins is that they are incapable of performing post-translational modifications such as NH2 -terminal acetylation. One possible solution is to coexpress additional enzymes that can perform the necessary modifications in the host cells. Here, we report a new method for synthesizing human rHb with proper NH2 -terminal acetylation. Mass spectrometry experiments involving native and recombinant human Hb confirmed the efficacy of the new technique in producing correctly acetylated globin chains. Finally, functional experiments provided insights into the effects of NH2 -terminal acetylation on O2 binding properties. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Gene synthesis and cloning the cassette to the expression plasmid Basic Protocol 2: Selection of E. coli expression strains for coexpression Basic Protocol 3: Large-scale recombinant hemoglobin expression and purification Support Protocol 1: Measuring O2 equilibration curves Support Protocol 2: Mass spectrometry to confirm NH2 -terminal acetylation.

Synergistic silencing of α-globin and induction of γ-globin by histone deacetylase inhibitor, vorinostat as a potential therapy for ß-thalassaemia.

ß-Thalassaemia is one of the most common monogenic diseases with no effective cure in the majority of patients. Unbalanced production of α-globin in the presence of defective synthesis of ß-globin is the primary mechanism for anaemia in ß-thalassaemia. Clinical genetic data accumulated over three decades have clearly demonstrated that direct suppression of α-globin and induction of γ-globin are effective in reducing the globin chain imbalance in erythroid cells hence improving the clinical outcome of patients with ß-thalassaemia. Here, we show that the histone deacetylase inhibitor drug, vorinostat, in addition to its beneficial effects for patients with ß-thalassaemia through induction of γ-globin, has the potential to simultaneously suppress α-globin. We further show that vorinostat exhibits these synergistic beneficial effects in globin gene expression at nanomolar concentrations without perturbing erythroid expansion, viability, differentiation or the transcriptome. This new evidence will be helpful for the interpretation of existing clinical trials and future clinical studies that are directed towards finding a cure for ß-thalassaemia using vorinostat.

A cytosine-rich splice regulatory determinant enforces functional processing of the human α-globin gene transcript.

The establishment of efficient and stable splicing patterns in terminally differentiated cells is critical to maintenance of specific functions throughout the lifespan of an organism. The human α-globin (hα-globin) gene contains 3 exons separated by 2 short introns. Naturally occurring α-thalassemia mutations that trigger aberrant splicing have revealed the presence of cryptic splice sites within the hα-globin gene transcript. How cognate (functional) splice sites are selectively used in lieu of these cryptic sites has remained unexplored. Here we demonstrate that the preferential selection of a cognate splice donor essential to functional splicing of the hα-globin transcript is dependent on the actions of an intronic cytosine (C)-rich splice regulatory determinant and its interacting polyC-binding proteins. Inactivation of this determinant by mutation of the C-rich element or by depletion of polyC-binding proteins triggers a dramatic shift in splice donor activity to an upstream, out-of-frame, cryptic donor. The essential role of the C-rich element in hα-globin gene expression is supported by its coevolution with the cryptic donor site in primate species. These data lead us to conclude that an intronic C-rich determinant enforces functional splicing of the hα-globin transcript, thus acting as an obligate determinant of hα-globin gene expression.

Molecular Basis and Genetic Modifiers of Thalassemia.

Thalassemia is a disorder of hemoglobin characterized by reduced or absent production of one of the globin chains in human red blood cells with relative excess of the other. Impaired synthesis of ß-globin results in ß-thalassemia, whereas defective synthesis of α-globin leads to α-thalassemia. Despite being a monogenic disorder, thalassemia exhibits remarkable clinical heterogeneity that is directly related to the intracellular imbalance between α- and ß-like globin chains. Novel insights into the genetic modifiers have contributed to the understanding of the correlation between genotype and phenotype and are being explored as therapeutic pathways to cure this life-limiting disease.

Computational construction of 3D chromatin ensembles and prediction of functional interactions of alpha-globin locus from 5C data.

Conformation capture technologies measure frequencies of interactions between chromatin regions. However, understanding gene-regulation require knowledge of detailed spatial structures of heterogeneous chromatin in cells. Here we describe the nC-SAC (n-Constrained-Self Avoiding Chromatin) method that transforms experimental interaction frequencies into 3D ensembles of chromatin chains. nC-SAC first distinguishes specific from non-specific interaction frequencies, then generates 3D chromatin ensembles using identified specific interactions as spatial constraints. Application to α-globin locus shows that these constraints (∼20%) drive the formation of ∼99% all experimentally captured interactions, in which ∼30% additional to the imposed constraints is found to be specific. Many novel specific spatial contacts not captured by experiments are also predicted. A subset, of which independent ChIA-PET data are available, is validated to be RNAPII-, CTCF-, and RAD21-mediated. Their positioning in the architectural context of imposed specific interactions from nC-SAC is highly important. Our results also suggest the presence of a many-body structural unit involving α-globin gene, its enhancers, and POL3RK gene for regulating the expression of α-globin in silent cells.

[Main regulatory element (MRE) of the Danio rerio α/ß-globin gene domain exerts enhancer activity toward the promoters of the embryonic-larval and adult globin genes].

In warm-blooded vertebrates, the α- and ß-globin genes are organized in domains of different types and are regulated in different fashion. In cold-blooded vertebrates and, in particular, the tropical fish Danio rerio, the α- and ß-globin genes form two gene clusters. A major D. rerio globin gene cluster is in chromosome 3 and includes the α- and ß-globin genes of embryonic-larval and adult types. The region upstream of the cluster contains c16orf35, harbors the main regulatory element (MRE) of the α-globin gene domain in warm-blooded vertebrates. In this study, transient transfection of erythroid cells with genetic constructs containing a reporter gene under the control of potential regulatory elements of the domain was performed to characterize the promoters of the embryonic-larval and adult α- and ß-globin genes of the major cluster. Also, in the 5th intron of c16orf35 in Danio reriowas detected a functional analog of the warm-blooded vertebrate MRE. This enhancer stimulated activity of the promoters of both adult and embryonic-larval α- and ß-globin genes.

Understanding α-globin gene regulation and implications for the treatment of ß-thalassemia.

Over the past three decades, a vast amount of new information has been uncovered describing how the globin genes are regulated. This knowledge has provided significant insights into the general understanding of the regulation of human genes. It is now known that molecular defects within and around the α- and ß-globin genes, as well as in the distant regulatory elements, can cause thalassemia. Unbalanced production of globin chains owing to defective synthesis of one, and the continued unopposed synthesis of another, is the central causative factor in the cellular pathology and pathophysiology of thalassemia. A large body of clinical, genetic, and experimental evidence suggests that altering globin chain imbalance by reducing the production of α-globin synthesis ameliorates the disease severity in patients with ß-thalassemia. With the development of new genetic-based therapeutic tools that have a potential to decrease the expression of a selected gene in a tissue-specific manner, the possibility of decreasing expression of the α-globin gene to improve the clinical severity of ß-thalassemia could become a reality.

Apoptosis-inducing Factor, Mitochondrion-associated 2, Regulates Klf1 in a Mouse Erythroleukemia Cell Line.

BACKGROUND/AIM: Apoptosis-inducing factor, mitochondrion-associated 2 (Aifm2), is a DNA-binding oxoreductase protein that promotes apoptosis. To assess its potential role in erythropoiesis we analyzed the effects of Aifm2 loss-of-function in the murine erythroleukemia line (MEL). MATERIALS AND METHODS: MEL cells were transfected with siRNA targeting Aifm2 for 24 h and evaluated by cell counting, flow cytometry with annexin V and PI staining and gene expression analysis. RESULTS: Aifm2 knockdown did not affect the apoptotic status of MEL cells. However, Aifm2 knockdown significantly increased expression of the erythropoietic transcription factor Klf1 (2.9±0.2-fold, p<0.05) and decreased α- and ß-globin expression (0.6±0.2-fold, p<0.05 and 0.5±0.2-fold, p<0.01). CONCLUSION: Aifm2 may function in differentiation of erythroid MEL cells in vitro.

Autoinduction, purification, and characterization of soluble α-globin chains of crocodile (Crocodylus siamensis) hemoglobin in Escherichia coli.

We have established a method to express soluble heme-bound recombinant crocodile (Crocodylus siamensis) α-globin chain holo-protein in bacteria (Escherichia coli) using an autoinduction system without addition of exogenous heme. This is the first time that heme-bound crocodile α-globin chains have been expressed in bacteria without in vitro heme reconstitution. The observed molecular mass of purified recombinant α-globin is consistent with that calculated from the primary amino acid sequence of native crocodile (C. siamensis) α-globin. Both the monomeric and the dimeric protein configuration formed by intermolecular disulfide bond could be purified as soluble protein. Spectroscopic characterization [UV-visible, circular dichroism (CD), and electron paramagnetic resonance (EPR)] of purified recombinant α-globin demonstrates nearly identical properties as reported for hemoglobin and myoglobin isolated from other organisms. For comparison, cyanide and nitric oxide binding of purified α-globin was also investigated. These results suggested that C. siamensis α-globin expressed in E. coli was folded correctly with proper incorporation of the heme cofactor. The expression method we now describe can facilitate production and isolation of individual globin chains in order to further study the mechanism and assembly of crocodile hemoglobin.

PIPKIIα is widely expressed in hematopoietic-derived cells and may play a role in the expression of alpha- and gamma-globins in K562 cells.

Characterized for the first time in erythrocytes, phosphatidylinositol phosphate kinases (PIP kinases) belong to a family of enzymes that generate various lipid messengers and participate in several cellular processes, including gene expression regulation. Recently, the PIPKIIα gene was found to be differentially expressed in reticulocytes from two siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIα and the production of globins. Here, we investigated PIPKIIα gene and protein expression and protein localization in hematopoietic-derived cells during their differentiation, and the effects of PIPKIIα silencing on K562 cells. PIPKIIα silencing resulted in an increase in α and γ globins and a decrease in the proliferation of K562 cells without affecting cell cycle progression and apoptosis. In conclusion, using a cell line model, we showed that PIPKIIα is widely expressed in hematopoietic-derived cells, is localized in their cytoplasm and nucleus, and is upregulated during erythroid differentiation. We also showed that PIPKIIα silencing can induce α and γ globin expression and decrease cell proliferation in K562 cells.

Comparisons between RT-PCR, real-time PCR, and in vitro globin chain synthesis by α/ß ratio calculation for diagnosis of α- from ß-thalassemia carriers.

BACKGROUND: Thalassemia, which may be due to point mutations, translocations, and deletions involving the α or ßglobin gene, is the most prevalent single gene disorder in Iran.This study aims to calculate the α/ß ratio in normal cases, α- and ß-thalassemia carriers by RT-PCR, real-time PCR, and in vitro globin chain synthesis (GCS) in order to establish the most accurate technique to distinguish between α- and ß-thalassemia carriers in suspicious cases. METHODS: The α/ß ratios were calculated in all samples by RT-PCR, real-time RT-PCR, and in vitro GCS. RESULTS: Using RT-PCR, the ratios were 1.09 ± 0.07 in normal samples, 1.2 ± 0.17 in ß-thalassemia, 1.08 ± 0.19 in mild α-thalassemia, and 0.96 ± 0.19 in severe α-thalassemia carriers. In real-time RT-PCR, the ratios were 2.21 ± 1.36 in normal samples, 5.12 ± 1.83 in ß-thalassemia, 2.88 ± 0.81 in mild α-thalassemia, and 1.18 ± 0.52 in severe α-thalassemia carriers. With GCS, the ratios were 1.03 ± 0.1 in normal samples, 1.9 ± 0.37 in ß-thalassemia, 0.8 ± 0.13 in mild α-thalassemia, and 0.59 ± 0.12 in severe α-thalassemia carriers. CONCLUSION: To determine the most accurate technique, we statistically analyzed the α/ß ratios obtained from the three standard methods. The ratio obtained by GCS and real-time PCR were helpful in distinguishing between α and ß carriers in suspicious patients in whom the mutation detection was limited and the risk for offspring was not clear. The use of this technique is more obvious when time is restricted (i.e. during the pregnancy period).

A multifunctional 5-aminolevulinic acid derivative induces erythroid differentiation of K562 human erythroleukemic cells.

Anemia is a major clinical symptom of a wide variety of pathological conditions a common related to reduced erythropoiesis. Whereas erythropoietin treatment showed an improvement in the patients' condition, it revealed increased risks of thromboembolic and cardiovascular events. Herein we describe stimulation of erythropoiesis by the multifunctional 1-(butyryloxy)ethyl-5-amino-4-oxopentanoate, (AlaAcBu), a 5-aminolevulinic-acid (ALA) derivative, which undergoes metabolic hydrolysis yielding two erythroid differentiation inducers, ALA and butyric acid (BA), each acting through a different mechanism. ALA, the first precursor in the heme biosynthesis, accelerates heme synthesis and BA, a histone deacetylase inhibitor (HDACI) that activates the transcription of globin mRNA. Our results show that the AlaAcBu mutual prodrug is a potent chemical differentiation inducer of K562 human erythroleukemia cells manifested by augmentation of heme and globin synthesis and assembly of hemoglobin. Exposure of K-562 cells to AlaAcBu resulted in an increase in heme synthesis and globin expression. Stimulation of the heme pathway was evident by the over-expression of porphobilinogen deaminase (PBGD) and ferrochelatase. AlaAcBu promoted cellular erythroid differentiation depicted by the expression of the marker glycophorin A and cellular maturation characterized by cytoplasm hemoglobinization, polar arrangement of mitochondria and a developed central vacuolar system preceding nuclear extrusion. The ability of AlaAcBu to promote differentiation along the erythroid lineage and to dramatically induce hemoglobin synthesis presented in this report.

Stress granules contribute to α-globin homeostasis in differentiating erythroid cells.

Hemoglobin is the major biosynthetic product of developing erythroid cells. Assembly of hemoglobin requires the balanced production of globin proteins and the oxygen-carrying heme moiety. The heme-regulated inhibitor kinase (HRI) participates in this process by phosphorylating eIF2α and inhibiting the translation of globin proteins when levels of free heme are limiting. HRI is also activated in erythroid cells subjected to oxidative stress. Phospho-eIF2α-mediated translational repression induces the assembly of stress granules (SG), cytoplasmic foci that harbor untranslated mRNAs and promote the survival of cells subjected to adverse environmental conditions. We have found that differentiating erythroid, but not myelomonocytic or megakaryocytic, murine and human progenitor cells assemble SGs, in vitro and in vivo. Targeted knockdown of HRI or G3BP, a protein required for SG assembly, inhibits spontaneous and arsenite-induced assembly of SGs in erythroid progenitor cells. This is accompanied by reduced α-globin production and increased apoptosis suggesting that G3BP+ SGs facilitate the survival of developing erythroid cells.

A transgenic mouse model expressing exclusively human hemoglobin E: indications of a mild oxidative stress.

Hemoglobin (Hb) E (ß26 Glu→Lys) is the most common abnormal hemoglobin (Hb) variant in the world. Homozygotes for HbE are mildly thalassemic as a result of the alternate splice mutation and present with a benign clinical picture (microcytic and mildly anemic) with rare clinical symptoms. Given that the human red blood cell (RBC) contains both HbE and excess α-chains along with minor hemoglobins, the consequence of HbE alone on RBC pathophysiology has not been elucidated. This becomes critical for the highly morbid ß(E)-thalassemia disease. We have generated transgenic mice exclusively expressing human HbE (HbEKO) that exhibit the known aberrant splicing of ß(E) globin mRNA, but are essentially non-thalassemic as demonstrated by RBC α/ß (human) globin chain synthesis. These mice exhibit hematological characteristics similar to presentations in human EE individuals: microcytic RBC with low MCV and MCH but normal MCHC; target RBC; mild anemia with low Hb, HCT and mildly elevated reticulocyte levels and decreased osmotic fragility, indicating altered RBC surface area to volume ratio. These alterations are correlated with a mild RBC oxidative stress indicated by enhanced membrane lipid peroxidation, elevated zinc protoporphyrin levels, and by small but significant changes in cardiac function. The C57 (background) mouse and full KO mouse models expressing HbE with the presence of HbS or HbA are used as controls. In select cases, the HbA full KO mouse model is compared but found to be limited due to its RBC thalassemic characteristics. Since the HbEKO mouse RBC lacks an abundance of excess α-chains that would approximate a mouse thalassemia (or a human thalassemia), the results indicate that the observed in vivo RBC mild oxidative stress arises, at least in part, from the molecular consequences of the HbE mutation.

Correction of ß654-thalassaemia mice using direct intravenous injection of siRNA and antisense RNA vectors.

Although the therapeutic efficacy of ß(654)-thalassaemia treatment using a combination of RNAi and antisense RNA to balance the synthesis of α- and ß-globin chains has been demonstrated previously, and the safety of lentiviral delivery remains unclear. Herein, we used the same ß(654)-thalassaemia mouse model to develop a therapy involving direct delivery of siRNA and antisense RNA plasmids via intravenous injection to simultaneously knock down α-globin transcript levels and restore correct ß-globin splicing. The amount of α-globin mRNAs in siRNA-treated MEL cells decreased significantly, and the properly spliced ß-globin mRNA was restored in HeLaß(654) cells transfected with pcDNA-antisense plasmid. Furthermore, treatment of ß(654)-thalassaemic mice with siRNA and antisense RNA plasmids resulted in significant reduction of poikilocytosis and reticulocyte counts in blood samples, decreased nucleated cell populations in bone marrow, and reduced intrasinusoidal extramedullary haematopoiesis loci and iron accumulation in liver. RT-PCR analysis revealed that treatment resulted in down-regulation of α-globin mRNA synthesis by ~50% along with an increase in the presence of normally spliced ß-globin transcripts, indicating that the phenotypic changes observed in ß(654)-thalassaemic mice following treatment resulted from restoration of the balance of α/ß-globin biosynthesis.