The Acute Promyelocytic Leukemia (APL)-Associated Fusion Proteins PML/RAR and PLZF/RAR Directly Bind to and Inhibit GATA-1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1929-1929
Author(s):  
Anita Seshire ◽  
Claudia Oancea ◽  
Dieter Hoelzer ◽  
Martin Ruthardt ◽  
Elena Puccetti
Keyword(s):  
Transcription Factors ◽  
Fusion Proteins ◽  
Binding Capacity ◽  
Globin Gene ◽  
Direct Interaction ◽  
Erythroid Differentiation ◽  
Beta Globin ◽  
Hematopoietic Stem ◽  
All Trans Retinoic Acid

Abstract The pathogenesis of acute myeloid leukemia (AML) is strictly related to a block of terminal differentiation. The APL is a well characterized subtype of AML, which is related in the 95% of the cases by the presence of the t(15;17) and in 2% by the presence of t(11;17). In several cell models the resulting PML/RAR and PLZF/RAR fusion proteins (X-RAR) recapitulate the leukemic phenotype by inducing a state of refractoriness to various inducers of myeloid differentiation. Accordingly, expression in animal models of both PML/RAR and PLZF/RAR leads to the development of leukemia. The treatment with all trans retinoic acid (t-RA) is able to overcome the block of differentiation of PML/RARα- but not that of PLZF/RAR-positive-blasts. These fusion proteins block differentiation through several mechanisms such as aberrant chromatin modeling by aberrant recruitment of histone deacetylase activity or the deregulation of differentiation-relevant transcription factors such as PU.1, VDR or C/EBPalpha. The deregulated function of these transcription factors can be due to their transcriptional down-regulation or to a sequester by direct interaction. Nothing is known about how the X-RAR block erythroid and megacaryocytic differentiation. Therefore we investigated whether and how the X-RAR interfere with the functionality of the differentiation-relevant transcription factor GATA-1. It has been recently reported that the lack of GATA-1 severely impairs erythroid differentiation and contributes to the accumulation of immature megakaryocytic blasts. He we report thatGATA-1 directly interacts with X-RAR in vivo as revealed by co-immunoprecipitation and mammalian two hybrid assays;GATA-1 expression was not transcriptionally deregulated by the X-RAR;the GATA-1 binding capacity to the H2S beta-globin locus was severely inhibited by the presence of the X-RAR as revealed by ChIP experiments in K562, whereas the transactivation of the GATA-target promotor alpha-IIb was not impaired by the the X-RAR in classical transient promoter studies;treatment with t-RA restored GATA-1 binding to the H2S locus of the beta-globin gene;the overexpression of GATA-1 in the presence of EPO reduced the the colony forming units of PLZF/RAR-positive Sca1+/lin− hematopoietic stem cells (HSC) and diminished the replating efficiency of PML/RAR-positive HSC, but did not increase erythroid differentiation monitored by TER 119 expression. Taken together our data demonstrate that the X-RAR interfere with the functionality of GATA-1 by direct interaction with GATA-1. It remains to definitively clarify whether the X-RAR inhibit the access of GATA-1 to its target promoters or whether they interfere with the accessibility of the GATA-1 for transcriptional co-activators. The fact that the overexpression of GATA-1 did not increase differentiation of HSC and the X-RAR were unable to inhibit the transactivation of a GATA-1 target promoter strongly suggests that the X-RAR interfere with the GATA-1 functionality in the context of the chromatin.

scholarly journals A human beta-globin gene fused to the human beta-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1384-1392 ◽  
Author(s):  
I Plavec ◽  
T Papayannopoulou ◽  
C Maury ◽  
F Meyer
Keyword(s):  
Bone Marrow Cells ◽  
Globin Gene ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
In Erythroid Cells

Abstract Retroviral-mediated gene transfer of human beta-globin provides a model system for the development of somatic gene therapy for hemoglobinopathies. Previous work has shown that mice receiving a transplant of bone marrow cells infected with a retroviral vector containing the human beta-globin gene can express human beta-globin specifically in erythroid cells; however, the level of expression of the transduced globin gene was low (1% to 2% per gene copy as compared with that of the endogenous mouse beta-globin gene). We report here the construction of a recombinant retrovirus vector encoding a human beta- globin gene fused to the 4 major regulatory elements of the human beta- globin locus control region (LCR). The LCR cassette increases the level of expression of the globin gene in murine erythroleukemia cells by 10- fold. To study the level of expression in vivo, mouse bone marrow cells were infected with virus-producing cells and the transduced cells were injected into lethally irradiated recipients. In the majority of provirus-containing mice (up to 75%), expression of human beta-globin in peripheral blood was detected at least 3 to 6 months after transplantation. Twelve animals representative of the level of expression of the transduced gene in blood (0.04% to 3.2% of the endogenous mouse beta-globin RNA) were selected for further analysis. A range of 0.4% to 12% of circulating erythrocytes stained positive for human beta-globin protein. Based on these values, the level of expression of the transduced gene per cell was estimated to be 10% to 39% of the endogenous mouse beta-globin gene. These data demonstrate that fusion of the LCR to the beta-globin gene in a retroviral vector increases the level of beta-globin expression in murine erythroleukemia cells and suggest that high-level expression can be obtained in erythroid cells in vivo after transduction into hematopoietic stem cells.

Targeted Gene Modification In Hematopoietic Stem Cells: A Potential Treatment For Thalassemia and Sickle Cell Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 434-434
Author(s):  
Andreas Reik ◽  
Kai-Hsin Chang ◽  
Sandra Stehling-Sun ◽  
Yuanyue Zhou ◽  
Gary K Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Target Gene ◽  
Ex Vivo ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Beta-thalassemia (β-thal) and sickle cell disease (SCD) are monogenic diseases caused by mutations in the adult β-globin gene. A bone marrow transplant (BMT) is the only curative treatment, but its application is limited since (i) HLA-matched donors can be found for <20% of cases, and (ii) the allogeneic nature of the transplant involves the significant risk of graft vs host disease (GvHD). Elevated levels of fetal γ-globin proteins observed in a subset of individuals carrying β-thal and SCD mutations ameliorate the clinical picture or prevent the development of disease complications. Thus, strategies for the selective and persistent upregulation of γ-globin represent an attractive therapeutic approach. Recent insights into the regulation of γ-globin transcription by a network of transcription factors and regulatory elements both inside and outside the β-globin locus have revealed a set of new molecular targets, the modulation of which is expected to elevate γ-globin levels for potential therapeutic intervention. To this end, we and others have established that designed zinc finger nucleases (ZFNs) transiently introduced into stem cells ex vivo provide a safe and efficient way to permanently ablate the expression of a specific target gene in hematopoietic stem cells (HSC) by introduction of mutations following target site cleavage and error-prone DNA repair. Here we report the development and comparison of different ZFNs that target various regulators of γ-globin gene transcription in human HSCs: Bcl11a, Klf1, and specific positions in the γ-globin promoters that result in hereditary persistence of fetal hemoglobin (HPFH). In all cases these target sites / transcription factors have previously been identified as crucial repressors of γ-globin expression in humans, as well as by in vitro and in vivo experiments using human erythroid cells and mouse models. ZFN pairs with very high genome editing activity in CD34+ HSCs were identified for all targeted sites (>75% of alleles modified). In vitro differentiation of these ZFN-treated CD34+ HSCs into erythroid cells resulted in potent elevation of γ-globin mRNA and protein levels without significant effects on erythroid development. Importantly, a similar and specific elevation of γ-globin levels was observed with RBC progeny of genome-edited CD34+ cells obtained from SCD and β-thal patients. Notably, in the latter case a normalization of the β-like to α-globin ratio to ∼1.0 was observed in RBCs obtained from genome-edited CD34s from two individuals with β-thalassemia major. To deploy this strategy in a clinical setting, we developed protocols that yielded comparably high levels of target gene editing in mobilized adult CD34+ cells at large scale (>108 cells) using a clinical-grade electroporation device to deliver mRNA encoding the ZFN pair. Analysis of modification at the most likely off-target sites based on ZFN binding properties, combined with the maintenance of target genome editing observed throughout erythroid differentiation (and in isolated erythroid colonies) demonstrated that the ZFNs were both highly specific and well-tolerated when deployed at clinical scale. Finally, to assess the stemness of the genome-edited CD34+ HSCs we performed transplantation experiments in immunodeficient mice which revealed long term engraftment of the modified cells (>16 weeks, ∼25% human chimerism in mouse bone marrow) with maintenance of differentiation in vivo. Moreover, ex vivo erythroid differentiation of human precursor cells isolated from the bone marrow of transplanted animals confirmed the expected elevation of γ-globin. Taken together, these data suggest that a therapeutic level of γ-globin elevation can be obtained by the selective disruption, at the genome level, of specific regulators of the fetal to adult globin developmental switch. The ability to perform this modification at scale, with full retention of HSC engraftment and differentiation in vivo, provides a foundation for advancing this approach to a clinical trial for the hemoglobinopathies. Disclosures: Reik: Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Lee:Sangamo BioSciences: Employment. Truong:Sangamo BioSciences: Employment. Wood:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Luong:Sangamo BioSciences: Employment. Chan:Sangamo BioSciences: Employment. Liu:Sangamo BioSciences: Employment. Miller:Sangamo BioSciences: Employment. Paschon:Sangamo BioSciences: Employment. Guschin:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Giedlin:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Urnov:Sangamo BioSciences: Employment.

scholarly journals A human beta-globin gene fused to the human beta-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1384-1392
Author(s):  
I Plavec ◽  
T Papayannopoulou ◽  
C Maury ◽  
F Meyer
Keyword(s):  
Bone Marrow Cells ◽  
Globin Gene ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
In Erythroid Cells

Retroviral-mediated gene transfer of human beta-globin provides a model system for the development of somatic gene therapy for hemoglobinopathies. Previous work has shown that mice receiving a transplant of bone marrow cells infected with a retroviral vector containing the human beta-globin gene can express human beta-globin specifically in erythroid cells; however, the level of expression of the transduced globin gene was low (1% to 2% per gene copy as compared with that of the endogenous mouse beta-globin gene). We report here the construction of a recombinant retrovirus vector encoding a human beta- globin gene fused to the 4 major regulatory elements of the human beta- globin locus control region (LCR). The LCR cassette increases the level of expression of the globin gene in murine erythroleukemia cells by 10- fold. To study the level of expression in vivo, mouse bone marrow cells were infected with virus-producing cells and the transduced cells were injected into lethally irradiated recipients. In the majority of provirus-containing mice (up to 75%), expression of human beta-globin in peripheral blood was detected at least 3 to 6 months after transplantation. Twelve animals representative of the level of expression of the transduced gene in blood (0.04% to 3.2% of the endogenous mouse beta-globin RNA) were selected for further analysis. A range of 0.4% to 12% of circulating erythrocytes stained positive for human beta-globin protein. Based on these values, the level of expression of the transduced gene per cell was estimated to be 10% to 39% of the endogenous mouse beta-globin gene. These data demonstrate that fusion of the LCR to the beta-globin gene in a retroviral vector increases the level of beta-globin expression in murine erythroleukemia cells and suggest that high-level expression can be obtained in erythroid cells in vivo after transduction into hematopoietic stem cells.

Differential Role of Myeloid Transcription Factors, C/EBPα and PU.1 In Leukemogenesis by MLL-Fusion Oncogenes.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1568-1568
Author(s):  
Yumi Fukuchi ◽  
Kana Kuroda ◽  
Ken Sadahira ◽  
Ryouichi Ono ◽  
Daniel G. Tenen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Fusion Proteins ◽  
Conditional Knockout ◽  
Leukemic Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Immortalized Cells

Abstract Abstract 1568 MLL translocations found in acute leukemia possess unique clinical characteristics. They have over 50 different fusion partners and show poor prognosis. These MLL fusion proteins lost H3K4 methyltransferase activity of wild-type MLL, but gained the ability to induce aberrant expression of HoxA cluster genes. Moreover, these proteins are able to transform hematopoietic stem/progenitor cells into leukemic stem cells (LSCs). Previous studies have shown that C/EBPα and PU.1, well-known myeloid specific transcription factors, were common molecular targets of myeloid malignancies. We and others have recently shown that C/EBPα and PU.1 are negative regulators of hematopoietic stem cells, suggesting that these transcription factors may play a role in the generation of LSCs. Because we have little knowledge on the role of C/EBPα and PU.1 in MLL-leukemia, we asked whether these key myeloid transcription factors were involved in the leukemogenesis by MLL-fusion proteins, especially in the stages of leukemia initiation and/or progression. First, we investigated the role of C/EBPα and PU.1 by in vitro self-renewal capacity and in vivo leukemia formation by MLL-fusion oncogenes. Bone marrow (BM) cells were harvested from C57BL/6J mice treated with 5-FU (150 mg/kg), and pre-stimulated with recombinant mouse (rm) SCF, rmIL-6, rhFL, rhTPO (50 ng/ml each). Cells were then transduced with pMYs-IG-MLL-ENL or pMXs-IG-MLL-Septin6, serially replated in methylcellulose, and transferred to rmIL-3 (10 ng/ml) containing liquid culture (immortalized cells), or were transplanted into lethally irradiated recipients (primary leukemic cells). MLL-ENL (or MLL-Septin6) immortalized cells or MLL-ENL primary leukemic cells were transduced with pMXs-IRES-DsRed-C/EBPα-ER or pMXs-IRES-DsRed-PU.1-ER. GFP+DsRed+ cells were sorted and serially replated in methylcellulose with or without 4-hydroxytamoxifen (4-HT) (1 mM), or were treated with or without 4-HT (1 mM) for 5 days followed by transplantion into sublethally irradiated secondary recipients. The results showed that overexpression of PU.1, but not C/EBPα, completely suppressed the serial replating capacity of MLL-ENL- and MLL-Septin6-immortalized cells. Moreover, activation of PU.1 suppressed propagation of MLL-ENL leukemic cells in the secondary recipients. In contrast, activation of C/EBPα did not eradicate leukemic cells in the same settings. To elucidate the role of PU.1 in the initiation of leukemia by MLL-ENL, we took PU.1+/− BM cells, or E14.5 fetal liver (FL) cells from PU.1-/- or +/− mice, and examined their capability to initiate leukemia when they were transduced with MLL-ENL. The result showed that leukemia did not develop in the absence of PU.1, and PU.1 haploinsufficiency prolonged survival of the recipients. A role of PU.1 in leukemia progression/maintenance by MLL-ENL was also tested using PU.1 conditional knockout mice. BM cells from PU.1flox/flox or flox/- mice were transduced with pMYs-IG-MLL-ENL, and transplanted into lethally irradiated recipients. PU.1-flox allele was conditionally deleted in primary leukemia cells by induction of Cre recombinase, whose effect was assessed by transplanting Cre-treated cells into secondary recipients. The result showed that conditional inactivation of PU.1 perturbed propagation of MLL-ENL leukemic cells, indicating that PU.1 is absolutely required not only for initiation, but also for maintenance of MLL-leukemia. Taken together, these results suggest that the dosage of PU.1 activity has profound impact on the self-renewal of LSCs and in vivo leukemia formation induced by MLL-fusion oncogenes. Therefore, PU.1 may serve as a potential therapeutic target for MLL-leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mediator complex subunit Med19 binds directly GATA transcription factors and is required with Med1 for GATA-driven gene regulation in vivo

2020 ◽  
Vol 295 (39) ◽  
pp. 13617-13629
Author(s):  
Clément Immarigeon ◽  
Sandra Bernat-Fabre ◽  
Emmanuelle Guillou ◽  
Alexis Verger ◽  
Elodie Prince ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Target Genes ◽  
Direct Interaction ◽  
Mediator Complex ◽  
Loss Of Function ◽  
Transcriptional Responses ◽  
Rna Pol Ii ◽  
Evolutionarily Conserved

The evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? We have previously established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. Here, we observe mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. We further show that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp-dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, we show a direct interaction between Med19 and Med1 by GST pulldown experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit–TF partnerships are probably oversimplified models. We propose several mechanisms to account for the transcriptional regulation of GATA-targeted genes.

scholarly journals The inactive beta globin gene on a gamma delta beta thalassemia chromosome has a normal structure and functions normally in vitro

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 766-770
Author(s):  
PT Curtin ◽  
YW Kan
Keyword(s):  
Globin Gene ◽  
Large Deletion ◽  
Beta Thalassemia ◽  
Ribonuclease Protection Assay ◽  
Beta Globin ◽  
Gamma Delta ◽  
Beta Globin Gene ◽  
Thalassemia Minor

We have previously described an English family with gamma delta beta- thalassemia in which a large deletion stops 25 kilobases (kb) upstream from the beta-globin gene locus, and yet the beta-globin gene is inactive in vivo. Affected family members had a beta-thalassemia minor phenotype with a normal hemoglobin A2 level. Gene mapping showed that these subjects were heterozygous for a chromosome bearing a large deletion that began in the G gamma-globin gene, extended through the epsilon-globin gene, and continued upstream for at least 75 kb. The A gamma-, delta-, and beta-globin gene loci on this chromosome were intact. To examine the possibility that an additional defect was present in the beta-globin gene, we cloned, sequenced, and examined the expression of the beta-globin gene from the affected chromosome. No mutation was found in the beta-globin gene sequence from 990 base-pairs 5′ to the cap site to 350 basepairs 3′ to the polyadenylation signal. The gene was subcloned into an expression vector and introduced into HeLa cells. Analysis of RNA derived from these cells, using a ribonuclease protection assay, revealed qualitatively and quantitatively normal transcription. Thus a structurally and functionally normal beta-globin gene is inactive in the presence of a large deletion more than 25 kb upstream. The loss of beta-globin gene function may be due to disturbance of chromatin conformation caused by the deletion or may be the result of loss of upstream sequences that are necessary for beta-globin gene expression in vivo.

scholarly journals Current Perspective of Beta Thalassemia and Its Treatment Strategies

2019 ◽  
Author(s):  
Shaukat Ali ◽  
Shumaila Mumtaz ◽  
Hafiz Abdullah Shakir ◽  
Hafiz Muhammad Tahir ◽  
Tafail Akbar Mughal
Keyword(s):  
Genetic Testing ◽  
Blood Transfusion ◽  
Dna Analysis ◽  
Complete Blood Count ◽  
Globin Gene ◽  
Thalassemia Major ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Hematopoietic Stem ◽  
Thalassemia Minor

Thalassemia is genetic blood disease cause by absence or decrease of one or more of the globin chain synthesis. Beta thalassemia is characterized by one or more mutations in beta globin gene. Absence or reduced amount the of beta globin chains cause ineffective erythropoiesis which leads to anemia. Beta thalassemia has been further divided into three main forms: Thalassemia minor/silent carrier, major and intermedia. More severe form is thalassemia major in which patients depend upon blood transfusion for survival and high level of iron occur as a consequence of consistent blood transfusion. Over loaded iron invokes the synthesis of reactive oxygen species that are toxic in redundancy and triggering the impairment to vascular, endocrine and hepatic system. Thalassemia can be diagnosed and detected through various laboratory tests such as blood smear, prenatal testing (genetic testing of amniotic fluid), DNA analysis (genetic testing) and complete blood count. Treatment of thalassemia intermedia is symptomatic but it can also be managed by splenectomy and folic supplementation. While thalassemia major can be treated by transplantation of bone marrow, regular transfusion of blood and iron chelation treatment, stimulation of fetal hemoglobin production, hematopoietic stem cell transplantation and gene therapy.

scholarly journals Functional Investigation of the Argonaute Proteins in Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-32
Author(s):  
Gordon G. L. Wong ◽  
Gabriela Krivdova ◽  
Olga I. Gan ◽  
Jessica L. McLeod ◽  
John E. Dick ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Erythroid Differentiation ◽  
Lineage Commitment ◽  
Myeloid Differentiation ◽  
Hematopoietic Stem ◽  
Erythroid Precursor ◽  
Lineage Differentiation ◽  
Erythroid Precursors

Micro RNA (miRNA)-mediated gene silencing, largely mediated by the Argonaute (AGO) family proteins, is a post-transcriptional gene expression control mechanism that has been shown to regulate hematopoietic stem and progenitor cells (HSPCs) quiescence, self-renewal, proliferation, and differentiation. Interestingly, only the function of AGO2 in hematopoiesis has been investigated. O'Carroll et al. (2007) showed that AGO2 knockout in mice bone marrow cells interferes with B220low CD43- IgM-pre-B cells and peripheral B cell differentiation and impairs Ter119high, CD71high erythroid precursors maturation. However, the functional significance of other AGO proteins in the regulation of stemness and lineage commitment remains unclear. AGO submembers, AGO1-4 in humans, are traditionally believed to act redundantly in their function. However, our previous proteomic analysis from sorted populations of the human hematopoietic hierarchy shows each sub-member is differentially expressed during HSPCs development, suggesting each sub-member may have a specialized function in hematopoiesis. Here, we conducted CRISPR-Cas9 mediated knockout of AGO1-4 in human cord blood derived long-term (LT-) and short-term hematopoietic stem cells (ST-HSCs) and investigated the impact of the loss of function of individual AGOs in vitro and in vivo in xenograft assays. From the in vitro experiment, we cultured CRISPR-edited LT- or ST-HSCs in a single cell manner on 96-well plates pre-cultured with murine MS5 stroma cells in erythro-myeloid differentiation condition. The colony-forming capacity and lineage commitment of each individual HSC is assessed on day 17 of the culture. Initial data showed that AGO1, AGO2 and AGO3 knockout decreased the colony formation efficacy of both LT- and ST-HSCs, suggesting AGO1, AGO2 and AGO3 are involved in LT- and ST-HSCs proliferation or survival. As for lineage output, AGO1 knockout increases CD56+ natural killer cell commitment in LT-HSCs and erythroid differentiation in ST-HSCs; AGO2 knockout increases erythroid differentiation in both LT- and ST-HSCs and decreases myeloid differentiation in ST-HSCs; while AGO4 knockout seems to decrease erythroid output. For the in vivo experiment, we xenotransplanted AGO1 and AGO2 knockout LT-HSCs in irradiated immunodeficient NSG mice and assessed the change in LT-HSCs engraftment level and lineage differentiation profile at 12- and 24-week time points. We found that AGO2 knockout increased CD45+ engraftment at both 12- and 24-weeks. Aligning with our in vitro data, AGO2 knockout increases GlyA+ erythroid cells at 12- and 24-weeks. The increase in GlyA+ erythroid cells is a consequence of the 2-fold increase in GlyA+ CD71+ erythroid precursor cells, recapitulating previous findings that AGO2 knockout in mice impairs CD71high erythroid precursor maturation leading to the accumulation of undifferentiated CD71+ erythroid precursors (O'Carroll et al., 2007). Accumulation of early progenitors of the erythroid lineage, including the common myeloid progenitors (CMPs) and myelo-erythroid progenitor (MEPs) were observed, as well as their progeny including CD33+ myeloid and CD41+ megakaryocytes. For the myeloid lineage, AGO2 knockout shifts myeloid differentiation toward CD66b+ granulocytes from CD14+ monocytes. For lymphoid, AGO2 knockout decreases CD19+ CD10- CD20+ mature B-lymphoid cells, which again aligns with previous AGO2 knockout mice results. On the other hand, AGO1 knockout LT-HSCs share some similar phenotype with AGO2 knockout LT-HSCs, where AGO1 knockout increases CD71+ erythroid precursors. However, AGO1 knockout in LT-HSCs also results in unique phenotypes, with a decrease in neutrophil formation and an increase in CD4+ CD8+ T progenitor cells are observed. AGO3 and AGO4 knockout experiments are in progress. In summary, our AGO2 knockout experiments recapitulate the reported results from murine studies but also illustrate a more complete role of AGO2 in hematopoietic lineage differentiation. Moreover, AGO knockout experiments of individual submembers are revealing novel insights into their role in the regulation of stemness and lineage commitment of LT-HSCs and ST-HSCs. These data point to a unique role of different AGO isoforms in lineage commitment in human HSCs and argue against redundant functioning. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells

1989 ◽  
Vol 9 (4) ◽  
pp. 1426-1434
Author(s):  
M A Bender ◽  
R E Gelinas ◽  
A D Miller
Keyword(s):  
Dna Analysis ◽  
Integration Site ◽  
High Titer ◽  
Globin Gene ◽  
Myeloid Cell ◽  
Beta Globin ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Beta Globin Gene ◽  
Provirus Integration Site

Murine bone marrow was infected with a high-titer retrovirus vector containing the human beta-globin and neomycin phosphotransferase genes. Anemic W/Wv mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human beta-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human beta-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human beta-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide. DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active beta-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.

scholarly journals The Pu.1 Locus Is Differentially Regulated at the Level of Chromatin Structure and Noncoding Transcription by Alternate Mechanisms at Distinct Developmental Stages of Hematopoiesis

2007 ◽  
Vol 27 (21) ◽  
pp. 7425-7438 ◽  
Author(s):  
Maarten Hoogenkamp ◽  
Hanna Krysinska ◽  
Richard Ingram ◽  
Gang Huang ◽  
Rachael Barlow ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Transcription Factors ◽  
B Cells ◽  
Regulatory Element ◽  
Precursor Cells ◽  
Hematopoietic Stem ◽  
Tissue Specific ◽  
Specific Regulation

ABSTRACT The Ets family transcription factor PU.1 is crucial for the regulation of hematopoietic development. Pu.1 is activated in hematopoietic stem cells and is expressed in mast cells, B cells, granulocytes, and macrophages but is switched off in T cells. Many of the transcription factors regulating Pu.1 have been identified, but little is known about how they organize Pu.1 chromatin in development. We analyzed the Pu.1 promoter and the upstream regulatory element (URE) using in vivo footprinting and chromatin immunoprecipitation assays. In B cells, Pu.1 was bound by a set of transcription factors different from that in myeloid cells and adopted alternative chromatin architectures. In T cells, Pu.1 chromatin at the URE was open and the same transcription factor binding sites were occupied as in B cells. The transcription factor RUNX1 was bound to the URE in precursor cells, but binding was down-regulated in maturing cells. In PU.1 knockout precursor cells, the Ets factor Fli-1 compensated for the lack of PU.1, and both proteins could occupy a subset of Pu.1 cis elements in PU.1-expressing cells. In addition, we identified novel URE-derived noncoding transcripts subject to tissue-specific regulation. Our results provide important insights into how overlapping, but different, sets of transcription factors program tissue-specific chromatin structures in the hematopoietic system.

Sign in / Sign up

Export Citation Format

Share Document