Exportin 7 (RanBP16) Plays An Essential Role In Terminal Erythroid Differentiation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3877-3877
Author(s):  
Shilpa Hattangadi ◽  
Karly Burke ◽  
Harvey Lodish
Keyword(s):  
Cellular Proliferation ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Chromatin Condensation ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Activating Transcription Factor 4 ◽  
Activating Transcription Factor ◽  
Terminal Erythroid Differentiation

Abstract Abstract 3877 Members of the nuclear transport receptor family of importins and exportins regulate the passage of proteins between the nucleus and cytoplasm. Although evolutionarily conserved across several species, Exportin 7 (Xpo7 or RanBP16) and its cargo are not well understood. In our study, we find that Xpo7 is highly erythroid-specific, as all other exportins are downregulated during terminal erythroid differentiation, a process including the induction of a highly specialized erythroid expression program, a set number of 3–5 terminal cell divisions, and chromatin condensation and eventual enucleation. Xpo7, in contrast, is highly induced during terminal erythropoiesis. Using retroviral shRNA knockdown of Xpo7 in in vitro fetal liver erythroid cell cultures, we demonstrate that exportin 7 is necessary for normal cellular proliferation and terminal erythroid differentiation, specifically for normal enucleation. Through microarray and biocomputational analysis of mRNA isolated from the knockdown cultures, we have found that the promoters of genes that are dysregulated after Xpo7 knockdown are enriched for binding sites for the activating transcription factor 4 (ATF4). Given that the erythroid phenotype of the ATF4 knockout mouse is very similar to the specific erythroid defects we observe in our in vitro knockdown analysis, our data suggests that either ATF4 or its binding protein may be Xpo7's cargo during terminal erythroid differentiation. Ongoing studies aimed at confirming this mechanism, the interaction between ATF4 and Xpo7, and the role and cargo of Xpo7 in terminal erythroid differentiation, are underway. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Erythroid-Specific Variant of the Nuclear Exportin Xpo7 Conserved Only in Mammals May Explain Functional Differences Between Mammalian Definitive and Lower Vertebrate (or Primitive) Erythropoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2440-2440
Author(s):  
Sandra Martinez-Morilla ◽  
Srividhya Venkatesan ◽  
Seibel Katharina ◽  
Jeffrey D. Cooney ◽  
Barry H. Paw ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Chromatin Condensation ◽  
Erythroid Differentiation ◽  
Erythroid Progenitor ◽  
Lower Vertebrate ◽  
Primary Mouse ◽  
Lower Vertebrates

Abstract Exportin 7 (Xpo7) was previously found to be crucial for late murine erythroid nuclear maturation when its knockdown significantly inhibited chromatin condensation and ultimate enucleation in a primary mouse erythroblast culture [1]. Additionally, Xpo7 was found to have an erythroid-specific isoform, transcribed from an erythroid-specific transcription start site (TSS) in the first intron of Xpo7 (named Xpo7-1B in contrast to the ubiquitously expressed variant -1A) [1]. Xpo7-1B differs from Xpo7-1B in 8 amino acid residues in the first exon and 9 residues in the 5th exon, and these differences are not conserved either in lower vertebrates or in primitive mouse erythropoiesis. It is intriguing that both lower vertebrate and primitive mammalian erythroblasts do not enucleate. In order to understand the in vivo function of Xpo7 and determine if these differences in the two isoforms confer differential functions to the proteins or are simply regulatory, we examined the definitive fetal liver erythropoiesis (considered adult "stress erythropoiesis") and adult steady state erythropoiesis of transgenic mouse models lacking either Xpo7-1A, Xpo7-1B, or both. We had previously knocked down Xpo7 in zebrafish using morpholinos (zebrafish only express the Xpo7-1A homologue) and found no effect on adult erythroid differentiation or proliferation. Adult Xpo7-1A knockout (KO) mice are born in normal Mendelian ratios, are not anemic, and express normal Xpo7-1B only in the blood. Closer examination reveals that fetal liver erythroid proliferation is actually higher in Xpo7-1A KO mice than either heterozygous (HET) or wild type (WT) while differentiation is normal. The double KO (Xpo7-1AB KO) appears to be embryonic lethal as only 4 live knockout pups were born out of 71 live births and even these 4 pups appear to be partial chimeras. We characterized the Xpo7-1AB HET mice in more detail and fetal liver erythroblasts of HET mice showed normal proliferation and differentiation compared to WT, and adults showed no differences in red blood cell (RBC) counts, hematocrit or hemoglobin. No complete live Xpo7-1AB KO has been born so we are now evaluating embryos at E3, E9.5, and E14.5. Given the possibility of embryonic lethality, in order to evaluate the erythroid-specific isoform Xpo7-1B, we have crossed a mouse floxed for the Xpo7-1B TSS/1st exon with a tamoxifen-inducible Cre-recombinase mouse. These mice have been given tamoxifen after reaching adulthood and we are currently evaluating their hematopoietic parameters. We have also set up HET-HET timed matings and administered tamoxifen to pregnant females to evaluate fetal liver erythropoiesis in Xpo7-1B HET, WT, and KO embryos. Not only will this work further our understanding of the process of global chromatin condensation during erythropoiesis, data from this in vivo study may reveal differences in erythropoiesis between mammals and lower vertebrates, which may also reflect intrinsic differences in mammalian primitive versus definitive erythropoiesis. Based on our in vitro knockdown studies, we anticipate that the phenotype of the Xpo7-1B KO mice may be similar to congenital dyserythropoietic anemia (CDA), a human disorder involving abnormalities in erythroid progenitor chromatin condensation and nuclear membrane substructure, so examination of these mice may also uncover a novel mechanism for this form of ineffective erythropoiesis. [1] Hattangadi, SM, et al. Blood (2014) 124, 1931-40. Disclosures No relevant conflicts of interest to declare.

Impairment of human terminal erythroid differentiation by histone deacetylase 5 deficiency

Blood ◽  
2021 ◽  
Author(s):  
Yaomei Wang ◽  
Wei Li ◽  
Vince Schulz ◽  
Huizhi Zhao ◽  
Xiaoli Qu ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Molecular Mechanisms ◽  
Chromatin Condensation ◽  
Erythroid Differentiation ◽  
Chromatin Accessibility ◽  
Erythroid Cell ◽  
Global Changes ◽  
Human Cd34 ◽  
Wide Range ◽  
Terminal Erythroid Differentiation

Histone deacetylases (HDACs) are a group of enzymes catalyzing the removal of acetyl groups from histone and non-histone proteins. HDACs have been shown to play diverse functions in a wide range of biological processes. However, their roles in mammalian erythropoiesis remain to be fully defined. We show here that of the eleven classic HDAC family members, six of them (HDAC 1,2,3 and HDAC 5,6,7) are expressed in human erythroid cells with HDAC5 most significantly up regulated during terminal erythroid differentiation. Knockdown of HDAC5 by either shRNA or siRNA in human CD34+ cells followed by erythroid cell culture led to increased apoptosis, decreased chromatin condensation, and impaired enucleation of erythroblasts. Biochemical analyses revealed that HDAC5 deficiency resulted in activation of p53 in association with increased acetylation of p53. Furthermore, while acetylation of histone 4 (H4) is decreased during normal terminal erythroid differentiation, HDAC5 deficiency led to increased acetylation of H4 (K12) in late stage erythroblasts. This increased acetylation was accompanied by decreased chromatin condensation, implying a role for H4 (K12) deacetylation in chromatin condensation. ATAC-seq and RNA-seq analyses revealed that HDAC5 knockdown leads to increased chromatin accessibility genome wide and global changes in gene expression. Moreover, pharmacological inhibition of HDAC5 by the inhibitor LMK235 also led to increased H4 acetylation, impaired chromatin condensation and enucleation. Taken together, our findings have uncovered previously unrecognized roles and molecular mechanisms of action for HDAC5 in human erythropoiesis. These results may provide insights into understanding the anemia associated with HDAC inhibitor treatment.

Klf1 Regulatory Networks in Primary Erythroid Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1462-1462
Author(s):  
Michael Tallack ◽  
Thomas Whitington ◽  
Brooke Gardiner ◽  
Eleanor Wainwright ◽  
Janelle Keys ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Es Cells ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Gene Promoters ◽  
Erythroid Cells ◽  
Red Cell Membrane ◽  
Sequencing Platform

Abstract Abstract 1462 Poster Board I-485 Klf1/Eklf regulates a diverse suite of genes to direct erythroid cell differentiation from bi-potent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which Klf1 works, we performed Klf1 ChIP-seq using the SOLiD deep sequencing platform. We mapped more than 10 million unique 35mer tags and found ∼1500 sites in the genome of primary fetal liver erythroid cells are occupied by endogenous Klf1. Many reside within well characterised erythroid gene promoters (e.g. b-globin) or enhancers (e.g. E2f2 intron 1), but some are >100kb from any known gene. We tested a number of Klf1 bound promoter and intragenic sites for activity in erythroid cell lines and zebrafish. Our data suggests Klf1 directly regulates most aspects of terminal erythroid differentiation including synthesis of the hemoglobin tetramer, construction of a deformable red cell membrane and cytoskeleton, bimodal regulation of proliferation, and co-ordination of anti-apoptosis and enucleation pathways. Additionally, we suggest new mechanisms for Klf1 co-operation with other transcription factors such as those of the gata, ets and myb families based on over-representation and spatial constraints of their binding motifs in the vicinity of Klf1-bound promoters and enhancers. Finally, we have identified a group of ∼100 Klf1-occupied sites in fetal liver which overlap with Klf4-occupied sites in ES cells defined by Klf4 ChIP-seq. These sites are associated with genes controlling the cell cycle and proliferation and are Klf4-dependent in skin, gut and ES cells, suggesting a global paradigm for Klfs as regulators of differentiation in many, if not all, cell types. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mitochondria Clearance Mechanisms Via Interaction with Macrophages in Erythropoiesis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2320-2320
Author(s):  
Chong Yang ◽  
Toshio Suda ◽  
Xiaoxuan Lin ◽  
Mitsuhiro Endo
Keyword(s):  
Large Scale ◽  
Conflicts Of Interest ◽  
Terminal Differentiation ◽  
Erythroid Differentiation ◽  
Transcriptional Pattern ◽  
Mitochondria Transfer ◽  
Erythroid Maturation ◽  
Terminal Erythroid Differentiation ◽  
Late Stages

Abstract Adult erythropoiesis involves a series of well-coordinated events resulting in the production of mature red blood cells. One of such events is the mitochondria clearance, which is known to occur cell-autonomously via autophagy-dependent mechanisms. Interestingly, we identified a sequential changes in the transcriptional pattern during terminal erythroid differentiation based on the expression of several macroautophagy (e.g. Atg3, Atg5, Atg7 and Atg10) and non-canonical mitophagy (e.g. Pink1, Park2, Bnip3l/Nix, P62 and Ulk1) genes. Hence we hypothesize that the progressive reduction in mitochondria during terminal erythroid differentiation is directed by distinct transcriptionally-regulated programs. Notably, we revealed a gradual reduction of the expression of lysosome related genes (e.g. Lamp1, CD63, and Atp6v) and lysosomal activities from early to late stages of terminal differentiation. On the other hand, P62-Pink1-Parkin mediated ubiquitin proteasome degradation of mitochondria proteins seems to be more prominent during late stage erythropoiesis. Hence our data suggest that mitochondria clearance is predominantly mediated by canonical autophagy during early stages of terminal differentiation, whereas non-canonical mitophagy pathway seem to play a more predominant role to regulate late stages erythroid maturation. Next, we discovered mitochondria transfer activities from erythroblasts to their niche. In the context of erythropoiesis, macrophages are known to interact closely with erythroblasts to provide a specialized niche for erythroid precursors to proliferate, differentiate and enucleate. We showed defective erythropoiesis after macrophage depletion in the bone marrow. Subsequently, we identified a tendency for early erythroblasts to associate with macrophages and isolated those erythroblasts from mito-dendra2 mice with trackable mitochondria to establish a murine primary cell co-culture system. Then we report mitochondria transfer activities in the erythroid niche via different modes including direct uptake, micro-vesicle transfer and tunnelling nanotubes (TNT). Interestingly, interchangeable structures between micro-vesicles and TNTs have been observed, suggesting an interplay between cytoskeleton and membrane lipid molecules in the mitochondria transfer mechanisms. Furthermore, mitochondria transfer activities have also been observed in the co-culture of mito-dendra2 erythroid cells with a macrophage cell line, RAW cells, and are significantly enhanced by the activation of the RAW cells via Tfe3 activation. In summary, our findings may provide insight into the mitochondria clearance machineries that mediates erythroid maturation to fulfil the clinical demand for large scale transfusable blood cell production in vitro. Disclosures No relevant conflicts of interest to declare.

scholarly journals Homeodomain-interacting protein kinase 2 plays an important role in normal terminal erythroid differentiation

Blood ◽  
2010 ◽  
Vol 115 (23) ◽  
pp. 4853-4861 ◽  
Author(s):  
Shilpa M. Hattangadi ◽  
Karly A. Burke ◽  
Harvey F. Lodish
Keyword(s):  
Cell Cycle Progression ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Cycle Progression ◽  
Interacting Protein ◽  
Primary Mouse ◽  
Proliferation And Apoptosis ◽  
Hemoglobin Biosynthesis ◽  
Terminal Erythroid Differentiation

Abstract Gene-targeting experiments report that the homeodomain-interacting protein kinases 1 and 2, Hipk1 and Hipk2, are essential but redundant in hematopoietic development because Hipk1/Hipk2 double-deficient animals exhibit severe defects in hematopoiesis and vasculogenesis, whereas the single knockouts do not. These serine-threonine kinases phosphorylate and consequently modify the functions of several important hematopoietic transcription factors and cofactors. Here we show that Hipk2 knockdown alone plays a significant role in terminal fetal liver erythroid differentiation. Hipk1 and Hipk2 are highly induced during primary mouse fetal liver erythropoiesis. Specific knockdown of Hipk2 inhibits terminal erythroid cell proliferation (explained in part by impaired cell-cycle progression as well as increased apoptosis) and terminal enucleation as well as the accumulation of hemoglobin. Hipk2 knockdown also reduces the transcription of many genes involved in proliferation and apoptosis as well as important, erythroid-specific genes involved in hemoglobin biosynthesis, such as α-globin and mitoferrin 1, demonstrating that Hipk2 plays an important role in some but not all aspects of normal terminal erythroid differentiation.

scholarly journals Hsp70 Is a New Major Regulator of Erythropoiesis by Preventing Caspase-3-Mediated Cleavage of GATA-1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 581-581 ◽  
Author(s):  
Jean-Antoine X. Ribeil ◽  
Yael Zermati ◽  
Joelle Kersual ◽  
Michael Dussiot ◽  
Francoise Audat ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Caspase 3 ◽  
Morphological Changes ◽  
Chromatin Condensation ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Direct Role ◽  
Vitro Assay ◽  
Terminal Erythroid Differentiation

Abstract Red blood cell production depends on apoptosis rate of erythroid progenitors and precursors. This process is mainly regulated by the glycoprotein Erythropoietin (Epo), which positively regulates through the JAK/STAT5 pathway, in synergy with the transcription factor GATA-1, the expression of the antiapoptotic protein Bcl-XL. Thus, Epo starvation results in caspase-3 activation through the intrinsic mitochondrial pathway, and as a consequence GATA-1 is cleaved and apoptosis occurs. Recently we have shown that caspase-3 activation is also absolutely required for the morphological changes that occur during normal human terminal erythroid differentiation, including chromatin condensation as well as nucleus and cell size reduction. In this context, although activated caspase-3 colocalizes in the nucleus with GATA-1, apoptosis and GATA-1 cleavage do not occur (Zermati et al J exp med, 2001, 193; 247–254). Heat Shock Proteins (Hsp) are chaperons that play a major role as a modulator of apoptosis. Here, we report by western blot and confocal analysis that inducible Hsp70 is constituvely expressed during human terminal eythroid differentiation, in both nuclear and cytoplasmic erythroblast compartments. Hsp70 co-localizes and co-immunoprecipitates with GATA-1 in the nucleus at the onset of caspases activation during terminal erythroid differentiation. Rapidly (<6h) after Epo starvation, while Hsp70 nuclear localization is lost, GATA-1 is cleaved. In contrast, in the presence of the nuclear export inhibitor Leptomycin B, Hsp70 remains in the nucleus and GATA-1 is not cleaved. In order to demonstrate the direct role of Hsp70 in GATA-1 protection, in an in vitro assay, we have shown that in a dose dependant manner recombinant Hsp70, but not Hsp90, inhibits recombinant caspase-3-mediated proteolysis of GATA-1. In human erythroid CD36+GPA+ progenitors, transfection of siRNA Hsp70, but not of a scramble siRNA, reduces significantly (80% vs 0%of inhibition) Hsp 70 nuclear and cytoplasmic expression, and is associated with GATA-1 cleavage at the onset of caspase-3 activation occuring during erythroid differentiation. Hsp70 inhibition leads to significant decrease of GATA-1 regulated genes expression (hemoglobinization (8% vs 50% of benzidine positive cells) ; Bcl-XL expression ( 80% inhibition) and apoptosis (8% vs 40%trypan blue positive cells). Interestingly, only mature cells are affected by siRNA Hsp70 and exhibit apoptotic features as assessed by topro3 stain and nuclear fragmentation (acidophils (90%) vs basophilic erythroblasts (10%)). Therefore, Hsp70 exerts a new critical antiapoptotic role during terminal erythropoiesis by preventing caspase-3-mediated cleavage of GATA-1. We propose a model in which, Epo determines the fate of erythroblasts (apoptotsis vs differentiation) downstream of caspase-3 activation by regulating the localization of Hsp70 (cytoplasmic vs nuclear and cytoplasmic).

A High Throughput Screening System for the Identification of Novel Genes in the Regulation of Mammalian Erythropoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2093-2093
Author(s):  
Peng Ji ◽  
Piu Wong ◽  
Harvey F. Lodish
Keyword(s):  
Cell Differentiation ◽  
High Throughput ◽  
High Throughput Screening ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Megaloblastic Anemia ◽  
Chromatin Condensation ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Novel Genes

Abstract Abstract 2093 Mammalian erythropoiesis is globally regulated by erythropoietin (Epo). Epo binds to its receptor on the cell surface of erythroid precursors. This induces a series of downstream pathways that promote cell differentiation and inhibit apoptosis. A recent genome wide transcriptional profile study demonstrated that over 600 genes are up-regulated during erythropoiesis. Surprisingly, the roles that most of the identified genes play in erythroid cells are still unknown. Understanding the functions of these unknown genes in the erythroid cells is necessary to elucidate the pathogenesis of red cell disorders such as congenital dyserythropoietic anemias, fanconi anemia, aplastic anemia, megaloblastic anemia, as well as leukemia and myelodysplastic syndromes with leukemic or dysplastic erythroid features. The goal of our study is to identify novel genes involved in different stages of erythropoiesis. To achieve this goal, we developed a high-throughput flow cytometry based assay that simultaneously detects erythroid cell differentiation and enucleation. In this assay, mouse fetal liver erythroblasts were purified and infected with lentiviruses containing a mammalian shRNA knockdown library obtained from the Broad Institute. The infected cells were cultured in a 96-well plate. Over the following two days the unaffected cells fully differentiate with approximately 60% of the cells enucleated. However, those cells in which shRNAs have knocked down genes critical for erythropoiesis are expected to show alterations in differentiation and/or enucleation. The system was validated using lentiviruses expressing shRNAs against Gata1 and mDia2, known proteins that are involved in the early and late stages of erythropoiesis, which showed inhibitions of differentiation and enucleation, respectively. We have pre-screened the most up-regulated 100 genes that play unknown functions in the erythroid cells. Future studies will be focused on the identified novel genes on their functions in erythroid lineage commitment, Epo mediated signaling pathways, hemoglobin enrichment, chromatin condensation, and enucleation. Disclosures: No relevant conflicts of interest to declare.

Exportin 7 (RanBP16) Plays An Essential Role in Terminal Erythroid Chromatin Condensation and Enucleation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 178-178 ◽  
Author(s):  
Shilpa Hattangadi ◽  
Karly Burke ◽  
Jennifer Eng ◽  
Jeffrey D. Cooney ◽  
Junxia Wang ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Chromatin Condensation ◽  
Protein Composition ◽  
Nuclear Proteins ◽  
Erythroid Cell ◽  
Mitotic Chromosomes ◽  
Erythroid Progenitors ◽  
Surface Marker Expression

Abstract Abstract 178 In contrast to compacting chromatin into highly condensed mitotic chromosomes, the quite distinct process of global chromatin condensation culminating in enucleation that occurs during terminal erythroid development is still poorly understood. By examining the protein composition of the erythroid nucleus from early erythroblast to ultimate extrusion, I observed that extruded nuclei are largely depleted of all nuclear proteins. Given my previous observations that the highly-regulated but reportedly nonspecific nuclear export protein, Xpo7 or RanBP16, is highly induced during erythropoiesis and highly erythroid-specific, I hypothesized that its role may be to remove almost all nuclear proteins in order to allow the terminal erythroid chromatin to condense. Knockdown of Xpo7 using shRNA in primary fetal liver erythroid progenitors resulted in severe inhibition of chromatin condensation and enucleation but had little effect on hemoglobin accumulation or erythroid cell surface marker expression. As expected based on my hypothesis, proteomic analysis of nuclei from Xpo7-knockdown cells revealed largely all nuclear proteins, some of which may be responsible for the process of histone redistribution during chromatin condensation. Xpo7 is also highly regulated: besides its promoter being bound directly by the erythroid master regulators GATA1 and Klf1 (unpublished data), it is also the target of a miRNA whose level decreases during erythropoiesis, miR-181, and whose overexpression has been shown to result in the inhibition of terminal enucleation. Because chromatin condensation occurs in lower vertebrates without subsequent enucleation, I have also explored the localization and function of Xpo7 in zebrafish using in situ hybridization and morpholinos, respectively, and found that this export function is specific to mammalian chromatin condensation, providing evidence that condensation and enucleation are inextricably linked processes in mammals. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Introduction of Two Simultaneous Mutations By Genome Editing Greatly Enhances the Accumulation of the Endogenous Fetal Hemoglobin in Human Normal Erythroid Cells

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 947-947
Author(s):  
Nikoleta Psatha ◽  
Chang Li ◽  
Hongjie Wang ◽  
Demetri Dalas ◽  
George Stamatoyannopoulos ◽  
...  
Keyword(s):  
Genome Editing ◽  
Conflicts Of Interest ◽  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Nuclease Activity ◽  
Erythroid Cell ◽  
Erythroid Progenitors ◽  
Knock Out ◽  
Gamma Globin

Abstract Thalassemia or sickle cell patients with increased fetal hemoglobin have an ameliorated clinical picture up to transfusion independency. Genome editing with targeted nucleases of the γ-globin silencer BCL11a or of the HBG promoter has been shown to reenact an HPFH-like phenotype by significantly increasing the endogenous fetal globin (HbF) expression. In order to achieve higher efficiency in HbF reactivation we developed an innovative approach, mediated through a highly customizable helper dependent-adenoviral vector (HD-Ad5/35++) with nuclease activity. This vector bares the Cas9 gene and two sequential gRNAs, permitting simultaneous targeting of two different DNA loci, specifically, the erythroid enhancer of BCL11a and the HBG promoter to recreate the -114 to -102 HPFH deletion. This double targeting vector was compared to the respective single gRNA vectors. We observed that disruption of the BCL11a-enhancer increased Gγ globin, whereas disruption of the HBG promoter had a greater impact on the Αγ globin expression. Simultaneous disruption of both loci in human erythroid progenitors increased the overall HbF expression from less than 1% in the control to more than 20%, through a possibly synergistic effect of the two mutations (HbF:2.2% in BCL11a-enhancer only knock-out, 10.8% in HBG-only knock out) by affecting both gamma globin chains. The editing efficiency per locus was similar between the single gRNA and double gRNA vectors. Erythroid cell morphology and phenotypic profile of the double knock-out cells did not differ compared to the single knock-out cells. In addition to our in vitro experiments, we observed that mobilized peripheral blood CD34+ cells transduced and edited by the double gRNA vector, can efficiently engraft in NSG mice. Furthermore, the engrafted edited cells after erythroid differentiation expressed significantly higher levels of gamma globin compared to the control. This strategy has the potential to induce higher levels of HbF reactivation with a clinical benefit in patients with beta globin disorders. Disclosures No relevant conflicts of interest to declare.

Targeted disruption of the activating transcription factor 4 gene results in severe fetal anemia in mice

Blood ◽  
2002 ◽  
Vol 99 (3) ◽  
pp. 736-745 ◽  
Author(s):  
Howard C. Masuoka ◽  
Tim M. Townes
Keyword(s):  
Transcription Factor ◽  
Leucine Zipper ◽  
Cellular Proliferation ◽  
Fetal Liver ◽  
Embryonic Stem ◽  
Adenosine Monophosphate ◽  
Basic Leucine Zipper ◽  
Activating Transcription Factor

Abstract Activating transcription factor (ATF) 4 is a ubiquitous basic leucine-zipper transcription factor that is a member of the ATF/cyclic adenosine monophosphate responsive element–binding (CREB) protein family. To determine the in vivo function of ATF4, the ATF4 gene in murine embryonic stem cells was deleted and homozygous mutant mice were generated. ATF4 null fetuses were severely anemic because of an impairment in fetal-liver definitive hematopoiesis; the hematocrit in 15.5-day mutant fetuses was 0.15, whereas that in controls was 0.35. The fetal livers in homozygous ATF4 mutants were pale and hypoplastic. In vitro culture of fetal-liver cells showed fewer hematopoietic progenitors per embryo and a dramatic decrease in the size of progenitor colonies. Culture of primary murine embryonic fibroblasts showed a proliferative defect. These results suggest that ATF4 is critical, in a cell-autonomous manner, for normal cellular proliferation, especially for the high-level proliferation required during fetal-liver hematopoiesis.

Sign in / Sign up

Export Citation Format

Share Document