Optimization of Bcl11a Knockdown By miRNA Scaffold Embedded Shrnas Leading to Enhanced Induction of Fetal Hemoglobin in Erythroid Cells for the Treatment of Beta-Hemoglobinopathies

Abstract Repression of the BCL11A protein could represent a therapeutic target for beta-hemoglobinopathies, as its knock-down has been shown to induce the expression of the fetal HBG (y-globin) gene ultimately leading to increased levels of the fetal hemoglobin tetramer (HbF, a2y2). In mice, Bcl11a is a key repressor of the murine HBG homolog Hbb-y. RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via pol III promoters has been used to modulate gene expression in a variety of mammalian cell types. However, we found a negative impact of Bcl11a knockdown on hematopoietic stem cells (HSCs), limiting the repopulation efficiency and long-term engraftment after genetic modification, which is a major impediment for its translation into human therapeutic applications. To achieve lineage-specific targeting of mRNAs in an attempt to reduce HSC toxicity, expression of shRNAs via pol II promoters is required, necessitating embedding the shRNA in mammalian microRNA (shRNAmir) sequences for expression and processing. To achieve optimal knockdown of the Bcl11a transcription factor in erythroid progenitor and precursor cells, we first compared the efficiency of mRNA modulation via pol III (U6-promoter) vs pol II (SFFV-promoter) based lentiviral vectors. We demonstrate a 100-1000 fold lower Hbb-y induction using shRNAmir vs pol III mediated shRNA vector backbones due to reduced Bcl11a knockdown efficiency. In order to understand the molecular basis for these differences, small RNA sequence analysis was performed on murine erythroleukemia cells (MEL) cells transduced by multiple shRNA–shRNAmir pairs. We show that shRNAs expressed via a U6 promoter yield guide strand sequences which differ by a 2-4 nt shift compared to pol II driven (shRNAmir) mature guide strand sequences. RNA sequencing demonstrated that the stretch of uridines making up part of the pol III termination signal is transcribed and included at the 3’ end of the shRNA. This results in the generation of mature guide strand sequences with an alternative seed sequence compared to the predicted sequence and compared to miRNA embedded shRNAs. The difference in the seed sequences between the two expression systems strongly influences the efficacy of target gene knockdown, leading to reduced knockdown in pol II based vectors. We engineered a 4bp shift into guide strands of shRNAmirs that resulted in a faithfully processed shRNA sequence (a mature guide strand sequence identical to U6-driven sh-RNAs) and improved knock-down efficiency of Bcl11a at the protein level in most cases. The improved knockdown of Bcl11a was associated with a 100-300-fold enhancement of Hbb-y induction in MEL cells. Based on these results, we propose a modified strategy for the prospective design of shRNAmirs derived from shRNA screens in pol III vector backbones to achieve lineage-specific regulation of target genes. Targeted expression of shRNAmiRs to the erythroid compartment driven by a b-globin promoter/LCR element circumvented the detrimental effect on HSC engraftment, while still mediating efficient BCL11A knockdown, leading to high y-globin induction and formation of substantial amounts of fetal hemoglobin in human CD34-derived erythroid cells in vitro. Disclosures No relevant conflicts of interest to declare.

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3'HS1 CTCF binding site in human β-globin locus regulates fetal hemoglobin expression

10.1101/2021.05.18.444713 ◽

2021 ◽

Author(s):

Pamela Himadewi ◽

Xue Qing David Wang ◽

Fan Feng ◽

Haley Gore ◽

Yushuai Liu ◽

...

Keyword(s):

Binding Site ◽

Progenitor Cells ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Ctcf Binding ◽

Ctcf Binding Site ◽

Distal Enhancer ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Ctcf Site

Mutations in the adult β-globin gene can lead to a variety of hemoglobinopathies, including sickle cell disease and β-thalassemia. An increase in fetal hemoglobin expression throughout adulthood, a condition named Hereditary Persistence of Fetal Hemoglobin (HPFH), has been found to ameliorate hemoglobinopathies. Deletional HPFH occurs through the excision of a significant portion of the 3 prime end of the β-globin locus, including a CTCF binding site termed 3'HS1. Here, we show that the deletion of this CTCF site alone induces fetal hemoglobin expression in both adult CD34+ hematopoietic stem and progenitor cells and HUDEP-2 erythroid progenitor cells. This induction is driven by the ectopic access of a previously postulated distal enhancer located in the OR52A1 gene downstream of the locus, which can also be insulated by the inversion of the 3'HS1 CTCF site. This suggests that genetic editing of this binding site can have therapeutic implications to treat hemoglobinopathies.

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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 ◽

10.1182/blood.v81.5.1384.1384 ◽

1993 ◽

Vol 81 (5) ◽

pp. 1384-1392 ◽

Cited By ~ 64

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.

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Roles for integrin very late activation antigen-4 in stroma-dependent erythropoiesis

Blood ◽

10.1182/blood.v83.10.2844.2844 ◽

1994 ◽

Vol 83 (10) ◽

pp. 2844-2850 ◽

Cited By ~ 73

Author(s):

N Yanai ◽

C Sekine ◽

H Yagita ◽

M Obinata

Keyword(s):

Progenitor Cells ◽

Adhesion Molecules ◽

Stromal Cells ◽

Ligand Molecule ◽

Erythroid Cells ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Erythroid Progenitor Cells ◽

Stem And Progenitor Cells ◽

Activation Antigen

Abstract Adhesion molecules are required for development of hematopoietic stem and progenitor cells in the respective hematopoietic microenvironments. We previously showed that development of the erythroid progenitor cells is dependent on their direct adhesion to the stroma cells established from the erythropoietic organs. In this stroma-dependent erythropoiesis, we examined the role of adhesion molecules in erythropoiesis by blocking antibodies. The development of the erythroid cells on stroma cells was inhibited by anti-very late activation antigen-4 (VLA-4 integrin) antibody, but not by anti-VLA-5 antibody, although the erythroid cells express both VLA-4 and VLA-5. Whereas high levels of expression of vascular cell adhesion molecule-1 (VCAM-1) and fibronectin, ligands for VLA-4, were detected in the stroma cells, the adhesion and development of the erythroid progenitor cells were partly inhibited by the blocking antibody against VCAM-1. VLA-5 and fibronectin could mediate adhesion of the erythroid progenitor cells to the stromal cells, but the adhesion itself may not be sufficient for the stroma-supported erythropoiesis. The stromal cells may support erythroid development by the adhesion through a new ligand molecule(s) for VLA-4 in addition to VCAM-1, and such collaborative interaction may provide adequate signaling for the erythroid progenitor cells in the erythropoietic microenvironment.

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LSD1 Plays an Important Role in GATA Switch during Erythroid Differentiation

Blood ◽

10.1182/blood.v122.21.4846.4846 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4846-4846

Author(s):

Yue Jin ◽

Yidi Guo ◽

Dongxue Liang ◽

Yue Li ◽

Zhe Li ◽

...

Keyword(s):

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Es Cells ◽

Regulatory Element ◽

Erythroid Differentiation ◽

Erythroid Cell ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Mouse Es Cells ◽

Murine Erythroleukemia

Abstract GATA factors play important role in hematopoiesis. In particular, GATA2 is critical for maintenance of hematopoietic stem and progenitor cells (HS/PCs) and GATA1 is required for erythropoiesis. GATA1 and GATA2 are expressed in reciprocal patterns during erythroid differentiation. It was shown that GATA1 occupied the -2.8Kb regulatory element and mediated repression of the GATA2 promoter in terminally differentiating erythroid cells. However, the detailed molecular mechanisms that control the enhancer/promoter activities of the GATA2 gene remain to be elucidated. In this report, we found that LSD1 and TAL1 co-localize at GATA2 1S promoter through ChIP and double-ChIP assays in murine erythroleukemia (MEL) cells. To further test whether LSD1 and its mediated H3K4 demethylation is important for repression of the GATA2 gene during erythroid differentiation, we silenced LSD1 expression in both MEL cells and mouse ES cells using retrovirus mediated shRNA knockdown and induced them to differentiate into erythroid cells with DMSO and EPO, respectively. GATA2 expression was elevated while the level of GATA1 was repressed by RT-qPCR. Furthermore, consistent with the GATA witch hypothesis, ChIP analysis revealed that the levels of H3K4me2 were increased at the GATA2 1S promoter. In addition, knock-down of LSD1 in MEL cells results in inhibition of erythroid cell differenciation and attenuation of MEL cell proliferation and survival. Thus, our data reveal that LSD1 involved in control of terminal erythroid differentiation by regulating GATA switch. The LSD1 histone demethylase complex may be recruited to the GATA2 1S promoter by interacting with TAL1. The H3K4 demethylation activity of LSD1 leads to downregulation of the active H3K4m2 mark at the GATA2 promoter that alters chromatin structure and represses transcription of the GATA2 genes. Disclosures: No relevant conflicts of interest to declare.

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Lack of Beta-1 Integrin Limits Stress Erythropoiesis and Splenomegaly in Beta-Thalassemia

Blood ◽

10.1182/blood.v126.23.2196.2196 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2196-2196

Author(s):

Roberta Chessa ◽

Ritama Gupta ◽

Bart J Crielaard ◽

Carla Casu ◽

Rick Feldman ◽

...

Keyword(s):

Bone Marrow ◽

Red Cells ◽

Poly I:C ◽

Beta Thalassemia ◽

Spleen Weight ◽

Erythroid Cells ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Stress Erythropoiesis ◽

The Impact

Abstract After blood loss, the production of red cells must be increased by stress erythropoiesis. This phenomenon is associated with increased proliferation and reduced differentiation of the erythroblasts, leading to a net increase in the number of progenitor erythroid cells and red cells (erythron). In normal conditions, after expansion of the pool of erythroblasts, these cells eventually differentiate to erythrocytes and the anemia resolves. However, in diseases such as β−thalassemia, production of healthy mature erythrocytes is impaired, resulting in anemia. Over time, the expansion, rather than the differentiation, of the erythron further exacerbates the ineffective erythropoiesis (IE), reducing the ability of the erythroid progenitors to generate erythrocytes. Interrupting the interaction between macrophages and erythroblasts (MEI) in thalassemia models is efficacious in reducing IE and alleviating the disease phenotype. We speculate that these molecules are also responsible for the homing of erythroid progenitor cells to extramedullary organs, such as the spleen and liver. Our studies in erythroblasts indicate that integrin beta−1 (Itgβ1) and also intracellular molecules such as focal adhesion kinase (Fak1), Talin−1 and Sharpin might play a role in stress erythropoiesis. Furthermore, there is increased interaction between Itgb1 and Fak1 in erythroblasts co−cultured with macrophages as demonstrated by immunocytochemistry and in vitro proximity ligation assays. In addition, targeting either Itgβ1 or Fak1 prevents expansion of erythroid cells when cultured in the presence of macrophages. Strikingly, using Itgβ1 together with Ter119 as selection parameters in flow cytometry, a distinct subset of erythroblasts, not discernable using CD44 or CD71, was observable, which we found to be part of the mixed orthochromatic erythroblast/reticulocyte population as determined with CD44 expression. Enucleation of erythroblasts was accompanied by a marked loss of Itgβ1 expression, indicating that Itgβ1 may be involved in erythroblast enucleation and differentiation. We crossed Hbbth3/+ mice with animals in which Itgβ1 or Fak1 were floxed and carrying an inducible Cre−recombinase (Mx1−Cre). From these animals, we investigated three different models; two obtained from breeding (Hbbth3/+−Itgβ1fl/fl−Mx1−Cre and Hbbth3/+−Fak1fl/fl−Mx1−Cre) and one by bone marrow transplant (BMT) of hematopoietic stem cells (HSCs) of Hbbth3/+−Itgβ1fl/fl −Mx1−Cre animals into wt mice to generate thalassemic animals that expressed the floxed Itgβ1 only in hematopoietic cells. After serial administration of Poly(I)−Poly(C) [poly(I:C)] the animals were analyzed for their erythropoiesis in the bone marrow and spleen. All the animals treated with poly(I:C) showed populations of Itgβ1 or Fak1 negative cells in the bone marrow and spleen. This indicated that all the HSCs were successfully depleted of the Itgβ1 or Fak1 gene. Interestingly, the spleen weight of all the treated animals was reduced, on average, 50% compared to untreated thalassemic mice. Similar results were seen also in Hbbth3/+−Itgβ1fl/fl−Mx1−Cre animals generated through BMT. Therefore, Itgβ1 and Fak1 might contribute to the pathophysiology of thalassemia and their removal might result in reduced stress erythropoiesis, erythroid proliferation and, as a consequence, amelioration of splenomegaly. Iron analysis and quantification of Erythroferrone (ERFE) are in progress to evaluate the impact of depleting Itgβ1 and Fak1 on these mechanisms. We are now in the process of identifying compounds that target MEI and, in particular, Itgβ1. Such molecules might be utilized for development of new treatments for thalassemia or additional disorders of aberrant erythropoiesis. Disclosures Feldman: Bayer ealthCare Phamaceuticals Inc.: Employment. Rivella:isis Pharmaceuticals: Consultancy; Merganser Biotech: Other: Stock options; Novartis Pharmaceuticals: Consultancy; Medgenics Pharmaceuticals: Consultancy; Bayer Healthcare: Consultancy, Research Funding.

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Interrogating Post-Transcriptional Mechanisms of Fetal Hemoglobin Regulation

Blood ◽

10.1182/blood-2021-151488 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3079-3079

Author(s):

Aoi Wakabayashi ◽

Maryanne Kihiu ◽

Malini Sharma ◽

Mathieu Quesnel-Vallieres ◽

Osheiza Abdulmalik ◽

...

Keyword(s):

Transcription Factors ◽

Rna Binding ◽

Rna Binding Proteins ◽

Therapeutic Potential ◽

Therapeutic Targets ◽

Fetal Hemoglobin ◽

Binding Motif ◽

Adult Type ◽

Erythroid Progenitor ◽

Hematopoietic Stem

Abstract Elevated levels of fetal hemoglobin (HbF) significantly ameliorate clinical outcomes for patients with beta-hemoglobinopathies, such as sickle cell disease (SCD). The only FDA-approved drug for treating SCD through inducing HbF is hydroxyurea, however the mechanism of action is unknown with variable effectiveness among patients. Thus, there remains a strong interest to identify more robust means of upregulating HbF, such as specific inhibition of HbF repressors. BCL11A and LRF are well-characterized transcription factors that independently repress the fetal type b-globin like genes HBG1 and HBG2 but their therapeutic potential is limited by challenging druggability and critical developmental function. However, upstream regulation of these factors, such as post-transcriptional mechanisms, are not well studied and may house novel therapeutic targets. To this end, we employed a CRISPR/Cas9 based screening approach to interrogate a library of RNA binding proteins (RBP) in the context of HbF regulation. Using HUDEP2 cells, a human adult-type erythroid progenitor cell line, we screened 341 human RBPs and identified four candidate RBPs, none of which have previously been implicated in HbF regulation. Of these candidates, RNA Binding Motif 12 (RBM12) showed the greatest level of HbF induction following in vitro depletion. Depletion of RBM12 protein in HUDEP2 cells and human CD34 + hematopoietic stem and progenitor cells (HSPC) via CRISPR/Cas9 editing raised HbF production 2-4 fold as assessed by HbF flow cytometry, HBG1/2 mRNA, and protein (γ-globin). Cell viability and maturation of RBM12 perturbed cells were largely intact. Additionally, RBM12 depletion in CD34 + HSPCs derived from SCD patients resulted in reduced percentage of sickled cells under hypoxic conditions. Unexpectedly, reduction of RBM12 had minimal effect on BCL11A and LRF expression suggesting that RBM12 may regulate HbF through a pathway that is indirectly related or independent of these transcription factors. RBM12 is an RBP that is widely expressed across diverse cell types and contains multiple RNA recognition motifs (RRM). While it has been implicated in various cancers and neurological disorders, its functions are not well studied. As an RBP, RBM12 can carry out several roles of post-transcriptional regulation, such as pre-mRNA splicing, mRNA transport, stabilization, and translation. As these activities are executed in different cellular compartments, we set out to narrow down RBM12 function by assessing its subcellular localization. Immunofluorescence staining revealed strong nuclear presence of RBM12, suggesting that it functions via mRNA biogenesis and/or processing. RNASeq and LC-MS/MS analysis of RBM12 KO CD34 + HSPCs revealed modest changes in the transcriptome and proteome. In order to gain mechanistic insight into RBM12 in the context of HbF regulation, we performed cDNA rescue experiments in RBM12-deficient HUDEP2 clones. Overexpression of full length RBM12 restored HbF repression. Notably, four out of the five RRMs were dispensable for HbF silencing, but RRM1 was essential for this activity. Interestingly, an extended form of RRM1 was also sufficient for HbF silencing. Mechanistic studies of this RRM1 module are underway and will be discussed. In sum, the identification of RBM12 as a regulator of HbF production represents a previously undescribed post-transcriptional layer of hemoglobin gene regulation. In pursuing this path, we hope to gain a deeper understanding of this understudied RBP in the context of HbF regulation which might in turn lead to the identification of potential therapeutic targets for the treatment of SCD and other hemoglobinopathies. Disclosures Blobel: Pfizer: Consultancy; Fulcrum Therapeutics, Inc.: Consultancy.

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LSD1 Inhibitors Induce Fetal Hemoglobin in Primary Human Erythroid Cells

Blood ◽

10.1182/blood-2018-99-117675 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1066-1066 ◽

Cited By ~ 1

Author(s):

Cuong LE ◽

Greggory Myers ◽

Alawi Habara ◽

David H.K. Chui ◽

Martin H. Steinberg ◽

...

Keyword(s):

Conflicts Of Interest ◽

Monoamine Oxidase Inhibitor ◽

Fetal Hemoglobin ◽

Erythroid Cell ◽

Therapeutic Effects ◽

Phase 2 ◽

Erythroid Cells ◽

Erythroid Progenitor ◽

Cd34 Cells ◽

Phase Phase

Abstract Significant amount of clinical literatures have supported that high level of fetal hemoglobin (HbF) improves the disease pathophysiology of β-globinopathies [sickle cell disease (SCD) and β-thalassemia]. Thus, we certainly can treat β-globinopathies by increasing the HbF level in adult erythroid cells. We originally reported that the lysine-specific histone demethylase 1 (LSD1) plays an important role in the regulation of the fetal γ-globin genes. Inhibition of LSD1 by using RNAi and monoamine oxidase inhibitor tranylcypromine (TCP) in primary human erythroid progenitor cells induces HbF to therapeutic levels. Furthermore, LSD1 inhibitor RN-1 treatment of SCD mice results in increased HbF synthesis and leads to effective improvement of many aspects of the disease pathology normally associated with SCD. Most recently, we examined thein vivo effects of some additional, publically available small molecule chemical inhibitors of LSD1 (including GSK-LSD1, LSD1-C12, LSD1-C76, OG-L002, and S2101) on HbF synthesis and erythroid physiology in SCD mice. There was a statistically significant increase in the percentage of HbF positive cells after 4 weeks of treatment with GSK-LSD1 or OG-L002 in SCD mice. Here, we report the effects of these two inhibitors in primary human erythroid cell derived from peripheral blood CD34+ cells. We isolated CD34+ cells using MACS column and cultured them using the two-phase-culture system. After seven days in expansion phase (phase 1) and three days in differentiation phase (phase 2), cells were treated with different doses of GSK-LSD1 and OG-L002 LSD1 inhibitors along with controls (DMSO, hydroxyurea and TCP) for 3 or 5 days in phase 2 culture. Flow cytometric assays showed that the percentage of HbF positive cells were significantly high when CD34+ cells treated with OG-L002 LSD1 (~50% at 0.1 µM) or GSK-LSD1 (~30% at 0.1 µM) as compared to control DMSO (~20%) after 5 days. These results suggest that GSK-LSD1 and OG-L002 could be two new promising HbF inducers based on LSD1 inhibition. These findings provide additional evidence to support that LSD1 comprises a useful molecular target for possible therapeutic intervention in treating SCD. Further study will be necessary to address the potential therapeutic effects of the compounds in SCD patients. Disclosures No relevant conflicts of interest to declare.

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Sustained fetal hemoglobin induction in vivo is achieved by BCL11A interference and coexpressed truncated erythropoietin receptor

Science Translational Medicine ◽

10.1126/scitranslmed.abb0411 ◽

2021 ◽

Vol 13 (591) ◽

pp. eabb0411

Author(s):

Naoya Uchida ◽

Francesca Ferrara ◽

Claire M. Drysdale ◽

Morgan Yapundich ◽

Jackson Gamer ◽

...

Keyword(s):

Gene Therapy ◽

High Efficiency ◽

Rhesus Macaques ◽

Fetal Hemoglobin ◽

B Cell Lymphoma ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Hbf Induction ◽

Xenograft Mice ◽

In Erythroid Cells

Hematopoietic stem cell gene therapy for hemoglobin disorders, including sickle cell disease, requires high-efficiency lentiviral gene transfer and robust therapeutic globin expression in erythroid cells. Erythropoietin is a key cytokine for erythroid proliferation and differentiation (erythropoiesis), and truncated human erythropoietin receptors (thEpoR) have been reported in familial polycythemia. We reasoned that coexpression of thEpoR could enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous nonhuman primate transplantation models. We generated thEpoR by deleting 40 amino acids from the carboxyl terminus, allowing for erythropoietin-dependent enhanced erythropoiesis of gene-modified cells. We then designed lentiviral vectors encoding both thEpoR and B cell lymphoma/leukemia 11A (BCL11A)–targeting microRNA-adapted short hairpin RNA (shmiR BCL11A) driven by an erythroid-specific promoter. thEpoR expression enhanced erythropoiesis among gene-modified cells in vitro. We then transplanted lentiviral vector gene-modified CD34+ cells with erythroid-specific expression of both thEpoR and shmiR BCL11A and compared to cells modified with shmiR BCL11A only. We found that thEpoR enhanced shmiR BCL11A–based fetal hemoglobin (HbF) induction in both xenograft mice and rhesus macaques, whereas HbF induction with shmiR BCL11A only was robust, yet transient. thEpoR/shmiR BCL11A coexpression allowed for sustained HbF induction at 20 to 25% in rhesus macaques for 4 to 8 months. In summary, we developed erythroid-specific thEpoR/shmiR BCL11A–expressing vectors, enhancing HbF induction in xenograft mice and rhesus macaques. The sustained HbF induction achieved by addition of thEpoR and shmiR BCL11A may represent a viable gene therapy strategy for hemoglobin disorders.

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Structure-Function Analysis of the Human TFIIB-Related Factor II Protein Reveals an Essential Role for the C-Terminal Domain in RNA Polymerase III Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.25.21.9406-9418.2005 ◽

2005 ◽

Vol 25 (21) ◽

pp. 9406-9418 ◽

Cited By ~ 17

Author(s):

Ashish Saxena ◽

Beicong Ma ◽

Laura Schramm ◽

Nouria Hernandez

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Function Analysis ◽

Rna Polymerase Iii ◽

Related Factor ◽

Pol Ii ◽

Terminal Domain ◽

U6 Promoter ◽

Pol Iii ◽

Type 3

ABSTRACT The transcription factors TFIIB, Brf1, and Brf2 share related N-terminal zinc ribbon and core domains. TFIIB bridges RNA polymerase II (Pol II) with the promoter-bound preinitiation complex, whereas Brf1 and Brf2 are involved, as part of activities also containing TBP and Bdp1 and referred to here as Brf1-TFIIIB and Brf2-TFIIIB, in the recruitment of Pol III. Brf1-TFIIIB recruits Pol III to type 1 and 2 promoters and Brf2-TFIIIB to type 3 promoters such as the human U6 promoter. Brf1 and Brf2 both have a C-terminal extension absent in TFIIB, but their C-terminal extensions are unrelated. In yeast Brf1, the C-terminal extension interacts with the TBP/TATA box complex and contributes to the recruitment of Bdp1. Here we have tested truncated Brf2, as well as Brf2/TFIIB chimeric proteins for U6 transcription and for assembly of U6 preinitiation complexes. Our results characterize functions of various human Brf2 domains and reveal that the C-terminal domain is required for efficient association of the protein with U6 promoter-bound TBP and SNAPc, a type 3 promoter-specific transcription factor, and for efficient recruitment of Bdp1. This in turn suggests that the C-terminal extensions in Brf1 and Brf2 are crucial to specific recruitment of Pol III over Pol II.

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Regulation of HDAC1 Histone Deacetylase Activity During Hematopoesis

Blood ◽

10.1182/blood.v116.21.3869.3869 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3869-3869

Author(s):

Tao Yang ◽

Yi Lou ◽

Wei Jian ◽

Jorg Bungert ◽

Constance Tom Noguchi ◽

...

Keyword(s):

Histone Deacetylase ◽

Cell Lines ◽

Protein Level ◽

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Nurd Complex ◽

Erythroid Progenitor ◽

Knock Down

Abstract Abstract 3869 Histone deacetylases (HDACs) play important roles in transcriptional regulation in eukaryotic cells. HDAC1 has been implicated in diverse cellular processes, such as developmental programming, gene expression and cell cycle progression, which are often linked to epigenetic repression. However, emerging evidence also suggests that histone deacetylase activity may be required for transcriptional activation. HDAC1 and its closely related protein HDAC2 are often present in repressor complexes, such as Sin3, NuRD and CoREST complexes. HDAC1 can undergo post transcription modifications, such as phosphorylation, sumoylation and acetylation. Acetylated HDAC1 lost deacetylase activity. Importantly, acetylated HDAC1 also inhibit the deacetylase activity of HDAC2, hence to down regulate the overall deacetylase activity of HDAC1/2 containing complexes. It is shown that NuRD corepressor complexes are important in regulating GATA-1 function during erythroid differentiation. However, it is not clear how histone deacetylase activity affects NuRD complex activity and influence hematopoiesis. In this study, we investigate the role of HDAC1 during erythroid differentiation. We tested HDAC1 level and activity in G1E-ER4 cells. G1E is a GATA-1 null erythroid progenitor cells. G1E-ER4 cells were engineered to stably express estrogen inducible GATA-1. Addition of estrogen leads to rapid induction of erythroid differentiation. HDAC1 deacetylase activity decreased upon treatment of estrogen. However, the HDAC1 protein level remains unchanged, suggesting that HDAC1 deacetylase activity, but not its protein level, is regulated. Accordingly, we found that acetylated HDAC1 level increased. Consistent with this observation, acetylated HDAC1 also increase upon Epo induction in human CD34+ cells. These results suggest that HDAC1 acetylation regulates the deacetylase activity during erythroid differentiation. To further test the role of HDAC1 in erythroid differentiation, we generated stable HDAC1 and HDAC2 knock down cell lines from MEL cells. The results show that HDAC1 and HDAC2 knock down inhibit differentiation and promote proliferation. To test the role of acetylated HDAC1 in differentiation, stable cell lines that over express HDAC1 and mutants mimicking acetylated or unacetylated HDAC1 were established. The cells that over express acetylated HDAC1 promote differentiation and cells that overexpress non acetylatable HDAC1 inhibit differentiation. We further studied whether HDAC1 modulates erythroid differentiation through regulating the activity of key erythroid transcription factor GATA-1. It is suggested that GATA-1 mediates gene activation through its association with coactivator complexes. However, recent studies indicated that GATA-1 associates with HDAC1/2 containing corepressor complexes (NuRD) throughout differentiation of erythroid cells. We investigated GATA-1 associated deacetylase activity during erythroid differentiation. We found that the deacetylase activity of the complex decreased and further diminished at during differentiation, coordinately with the increase of acetylated form of HDAC1 in both Mel cells and G1E-ER4 cells. We further demonstrated the role of HDAC1 in GATA-1 mediated gene transcription in reporter assays. These studies indicate that HDAC1 plays an important role in regulating GATA-1 activity and the deacetylase activity of the GATA-1 associated NuRD complex is also regulated. This complex may play differential roles in undifferentiated and differentiated erythroid cells. Thus, our results suggest a novel but rather general regulatory mechanism of histone deacetylase containing protein complexes. Disclosures: No relevant conflicts of interest to declare.

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