Targeting a Novel Epigenetic Silencing Mechanism to Efficiently Upregulate Fetal Globin Gene Expression

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 352-352
Author(s):  
Fabiana Perna ◽  
Ly P. Vu ◽  
Maria Themeli ◽  
Ruben Hoya-Arias ◽  
Xinyang Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chromatin Structure ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Ips Cells ◽  
Gamma Globin ◽  
Cd34 Cells ◽  
Hes Cells ◽  
Globin Gene Expression

Abstract Abstract 352 L3MBTL1 is a Polycomb group protein, commonly deleted in patients with myeloid disorders associated with the 20q- chromosomal abnormality. After crystallizing the MBT repeat domain, we demonstrated that L3MBTL1 compacts chromatin by binding mono- and di-methylated lysine residues in histones H1 (H1K26) and H4 (H4K20), ultimately leading to gene repression. Despite its role in affecting the chromatin structure, the role of L3MBTL1 in hematopoiesis has remained largely unknown. We recently demonstrated that lack of L3MBTL1 accelerates the erythroid differentiation of human hematopoietic stem cells and here we reveal that L3MBTL1 represses the expression of the fetal gamma globin gene. We lentivirally expressed shRNAs targeting L3MBTL1 in human cord blood (CB) CD34+ cells and in K562 erythroleukemia cells, and consistently observed upregulation of gamma globin gene expression, while beta globin gene expression decreased. Remarkably, we observed similar findings in human embryonic stem (hES) cells, where knock-down of L3MBTL1 triggered a BMP4-like spontaneous differentiation. Given the potential impact of therapeutically increasing fetal hemoglobin expression in patients with hemoglobinopathies, we targeted L3MBTL1 in induced pluripotent stem (iPS) cells derived from patients with β-thalassemia. The gene expression profile of L3MBTL1-KD normal and thalassemic iPS cells indicated clear activation of fetal hemoglobin (HbF) expression, activation of BMP4 signaling and upregulation of specific smad5 target genes (e.g. EKLF, HHEX, ID2/3). We generated and utilized a model of “stress erythropoiesis” in L3MBTL1 KO mice and observed in vivo BMP4-mediated expansion of spleen immature erythroid progenitors, as indicated by increased spleen weight and splenic BFU-E colonies in KO mice compared to controls. We also examined K562 cells, human CB CD34+ cells and hES cells, using chromatin immunoprecipitation assays, and found that L3MBTL1 directly associates with the human β-globin locus, occupying discrete regions within the human β-globin cluster. Furthermore, L3MBTL1 colocalized with H4K20me within the Locus Control Region (LCR), a primary attachment site for chromatin modifiers. We observed clearance of L3MBTL1 and its associated histone marks (H4K20me1/2) from the LCR upon treatment with hemin, erythropoietin or TGFβ, three agents that potently induce erythroid differentiation. This suggests that this polycomb repressor complex responds to cytokine signaling. In summary, we have identified a novel epigenetic regulatory mechanism to control fetal globin gene expression; the Polycomb protein L3MBTL1 regulates BMP4 signaling and the chromatin structure of globin genes. Targeting this regulatory system represents a means to efficiently increase HbF in a human model of β-thalassemia (i.e. with the use of patient-derived iPS cells) and to potentially ameliorate hematological and clinical symptoms of patients with red cell disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Fetal hemoglobin in sickle cell anemia: relation to regulatory sequences cis to the beta-globin gene. Multicenter Study of Hydroxyurea

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1604-1611 ◽  
Author(s):  
ZH Lu ◽  
MH Steinberg
Keyword(s):  
Gene Expression ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Regulatory Sequences ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Globin Gene Expression

Very different fetal hemoglobin levels among adult sickle cell anemia patients suggest genetic modulation of gamma-globin gene expression. In sickle cell anemia, different fetal hemoglobin levels are associated with distinct beta-globin gene haplotypes. Haplotype may be a marker for linked DNA that modulates gamma-globin gene expression. From 295 individuals with sickle cell anemia, we chose for detailed studies 53 patients who had the highest or the lowest fetal hemoglobin levels and 7 patients whose fetal hemoglobin levels were atypical of their haplotype. In these individuals, we examined portions of the beta- globin gene locus control region hypersensitive sites two and three, an (AT)x(T)y repeat 5′ to the beta-globin gene, a 4-bp deletion 5 to the A gamma T gene, promoters of both gamma-globin genes, 5′ flanking region of the G gamma-globin gene, and A gamma-globin gene IVS-II. Of the regions we studied all polymorphisms were always haplotype-linked and no additional mutations were present. This suggested that variations in these areas are uncommon mechanisms of fetal hemoglobin modulation in sickle cell anemia. Whereas unexamined cis-acting sequences may regulate gamma-globin gene transcription, trans-acting factors may play a more important role.

Regulation of Globin Gene Expression and Erythroid Differentiation by Sp/KLF Factors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4241-4241
Author(s):  
Jie Hong ◽  
George Stamatoyannopoulos ◽  
Chao-Zhong Song
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Expression System ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Regulatory Elements ◽  
Mrna Levels ◽  
Gene Promoters ◽  
Gamma Globin ◽  
Globin Gene Expression

Abstract Sp/Krüppel-like factor (KLF) family of proteins are characterized by the presence of three highly homologous Cys2His2 type zinc-fingers near the C-terminus that bind GC/CACCC boxes, which are one of the most common regulatory elements found in promoters of many cellular and viral genes. Currently, more than 20 members have been identified in the family. This family of factors plays important roles in cell growth, differentiation, development and homeostasis by regulating the expression of their target genes. The GC and GT/CACCC boxes in the globin gene promoters and the beta globin locus control region play an important role in the tissue- and developmental stage- specific expression of globin genes. We have carried out extensive studies to identify the KLF factors that regulate gamma globin expression and erythroid differentiation. Gene expression analysis revealed that most of the Sp/KLF factors are expressed, albeit at variable levels, in human fetal liver and adult blood cells. To determine the role of the Sp/KLF factors in gamma globin expression and erythroid differentiation, functional studies using systematic RNAi to knockdown selected Sp/KLF factors were performed. We used a lentiviral mediated siRNA expression system for specific silencing selected Sp/KLF factors. Effective knockdown of Sp/KLF factors was achieved as judged by a 70–90% decrease in their mRNA levels in the cells. Analyses of globin gene expression showed that the knockdown of some Sp/KLF factors resulted in changes in globin gene expression in K562 cells. We also observed that knockdown of specific Sp/KLF factors resulted in erythroid differentiation of K562 cells. These results suggest that specific Sp/KLF factors may play a role in regulation of globin gene expression and erythroid differentiation.

A Zinc-Finger Transcriptional Activator Designed to Interact with the Gamma-Globin Gene Promoters Enhances Fetal Hemoglobin Production in Erythroid Cells Derived From Normal and Beta-Thalassemic CD34+ Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3567-3567
Author(s):  
Andrew Wilber ◽  
Uli Tschulena ◽  
Phillip W. Hargrove ◽  
Yoon-Sang Kim ◽  
Carlos F. Barbas ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Lentiviral Vector ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Beta Thalassemia ◽  
Normal Donor ◽  
Gene Promoters ◽  
Gamma Globin ◽  
Cd34 Cells

Abstract Abstract 3567 Poster Board III-504 Fetal hemoglobin (α2γ2; HbF) is a potent genetic modifier of the severity of beta-thalassemia and sickle cell anemia. Clinical studies indicate that moderate elevation in production of HbF achieved through heritable persistence of HbF or administration of hydroxyurea, effectively reduce the severity of beta-chain defects. Accordingly, we are exploring strategies to maintain expression of the endogenous gamma-globin genes following lentiviral vector-mediated gene transfer. The artificial zinc-finger transcription factor (GG1-VP64) was designed to interact with sequences in the proximal gamma-globin gene promoters and has been shown to enhance gamma-globin expression in human erythroleukemia cells and mouse marrow cells which are transgenic for the human beta-globin locus. Here, we describe studies designed to evaluate the impact of expression of GG1-VP64 on gamma-globin expression by maturing adult erythroblasts derived from CD34+ cells of normal and thalassemic donors. We utilized an in vitro culture model of human erythropoiesis in which late stage erythroblasts are derived from human CD34+ hematopoietic cells. In this system, cytokine-mobilized peripheral blood or steady state bone marrow CD34+ cells from adults yielded erythroblasts containing 2% or less HbF. The lentiviral vector encodes for bicistronic expression of the GG1-VP64 transactivator and GFP under transcriptional control of the beta-spectrin or ankyrin-1 promoter which give low but progressive increase in expression during erythroid development. Three normal donor CD34+ cells were transduced 48 hours after initiation of culture by overnight exposure to the GG1-VP64 vector or GFP control vector. Approximately 50-60% of the cells were successfully transduced with the control and GG1-VP64 vectors as monitored by flow cytometry analysis for GFP expression. Control vector transduction had no effect on cell proliferation or differentiation monitored by consistent increases in cell numbers and the appearance of CD71 (transferrin receptor) and CD235 (glycophorin A) on most cells (>98% and >80%, respectively) whereas GG1-VP64 gene transfer reduced cell proliferation slightly without affecting erythroid differentiation. Erythroblasts derived from GFP transduced cells expressed low levels of HbF (1.7+/−0.6%) whereas those derived from cells transduced with GG1-VP64 demonstrated induction of HbF ranging from 12-21% with an average vector copy number of 0.8 to 1.0. When cells from a normal donor were sorted into GFP- and GFP+ populations, significant levels of HbF were present only in the GFP+ fraction. We next tested the GG1-VP64 transactivator in three independent studies using bone marrow CD34+ cells from two patients with beta-thalassemia major. Gene transfer was effective as reflected by 74+/−6% (control) and 47+/− 2% (GG1-VP64) GFP marking in bulk cultures. Again, GG1-VP64 gene transfer in beta-thalassemia CD34+ cells reduced cell growth somewhat but did not perturb erythroid differentiation as monitored by the appearance of transferrin receptor (>98%) and Glycophorin A (>80%) as well as cell morphology. Erythroblasts derived from GFP transduced cells expressed levels of HbF in the range of 26+/−5% whereas those derived from cells transduced with GG1-VP64 demonstrated a 2-fold induction of HbF to 52+/−9% with an average vector copy number of 0.5-0.9. Our data show that lentiviral-mediated, enforced expression of GG1-VP64 under the control of erythroid-specific promoters induced significant amounts of HbF in normal and thalassemic erythroblasts derived from adult CD34+ cells without altering their capacity for erythroid maturation following transduction. These observations demonstrate the potential for sequence specific enhancement of HbF in patients with beta-thalassemia or sickle cell anemia. Disclosures: No relevant conflicts of interest to declare.

Novel Hybrids of Hydroxyurea and Thalidomide Based Pharmacophores Induce Fetal Hemoglobin and Block Monocyte Activation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2673-2673
Author(s):  
Carolina Lanaro ◽  
Carla Franco-Penteado ◽  
Jean Leandro Santos ◽  
Marlene Wade ◽  
Shobha Yerigenahally ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mononuclear Cells ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Cytokine Release ◽  
Gamma Globin ◽  
Tnf Α ◽  
Activated Monocytes ◽  
Humidified Air ◽  
Globin Gene Expression

Abstract Abstract 2673 Introduction: Fetal hemoglobin induction is an effective strategy for the treatment of sickle cell disease (SCD). In addition, targeting systemic inflammation, which consists of activated and injured vascular endothelium, elevated pro-inflammatory cytokines, and activated WBC, promises to reduce vaso-occlusion and disease severity. We therefore aimed to develop novel compounds that combine potent HbF-inducing and anti-inflammatory activities with the goal to improve treatment outcomes. In principle, our rational drug design used molecular hybridization to synthesize three compounds (Lapdesf 1, 2, 3) that link Hydroxyurea's nitric oxide-donating nitrate ester subunit and thalidomide's phthalimide ring with three different intermolecular spacers. Hydroxyurea is to date the only FDA approved drug for the treatment of adult patients with SCD that induces HbF and reduces clinical complications. The release of nitric oxide by HU is an important mechanism of its HbF-inducing properties. On the other hand, thalidomide and its recently developed IMiD derivatives potently inhibit cytokine release from activated monocytes and suppress adhesion molecule expression on vascular endothelium. These properties are critically linked to the phthalimide ring in the thalidomide molecule. The aim of this study was to evaluate the effects of three novel hybrid compounds Lapdesf 1 (2-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)phenyl]ethyl nitrate), Lapdesf 2 (4-[1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl]-N-hydroxybenzenesulfonamide), and Lapdesf 3 (3-[1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl]benzyl nitrate) on γ-globin gene expression and cytokine release from activated monocytes. Methods: 1. Gamma-globin gene expression. Human K562 cells were maintained in DMEM with 10% FBS, Pen/Strep, in humidified air (5% CO2, 37°C). Cells (1×107cells/100mL) were incubated with compounds at different concentrations (5, 30, 60, and 100μM) for 24, 48, 72 and 96h. Gamma-globin gene expression was analyzed by qRT-PCR and quantified using the Gnorm program. Results were expressed as arbitrary units. 2. Monocyte stimulation. Animals: Adult knockout-transgenic sickle cell mice were anesthetized with Ketamine/Xylazine and blood collected by intracardiac puncture. Mononuclear cells were purified from peripheral blood using Ficoll gradient separation. Mononuclear cells were placed in plastic dishes with DMEM with 10% calf serum and incubated for 2 h in humidified air (5% CO2, at 37°C). Purity of monocytes was > 90% as determined by May-Giemsa staining. Lapdesf 1, 2, and 3 were added to the monocytes cultures at different concentrations (100μM, 300μM and 600μM) 30 minutes before LPS (1 μg/ml). After 20 h incubation supernatants were collected and stored at −80°C. Cytokines were determined by ELISA. Results: 1. Gamma-globin gene expression. Lapdesf 1 and 2 were equipotent and demonstrated inverse dose-response relationships achieving the highest levels of γ-globin induction at 5 and 30 μM; in contrast, Lapdesf 2 achieved maximal γ-globin induction as early as 24h after treatment, whereas Lapdesf 1 required 72h of incubation (Lapdesf 2 [24h; 5 μM]: 1.48±0.06 AU; control 0.78±0.1 AU; P<0.05; Lapdesf 1 [72h; 5 μM] 1.78±0.5 AU; control 0.64±0.26; P<0.05). Testing of Lapdesf 3 is currently underway. 2. Monocyte stimulation. Monocytes from sickle mice produced significantly higher levels of TNF-α (1.9 fold), IL-6 (3.48 fold), IL-8 (1.95 fold) and IL-1β (3.42 fold) after LPS stimulation compared to hemizygous controls (P<0.05). Lapdesf 1 and 3 (300 μM) significantly suppressed cytokine production compared to vehicle in LPS-stimulated sickle monocytes and were more potent inhibitors than dexamethasone (Lapdesf 1; Lapdesf 3; Dex: TNF-α : −14.71 fold; −4.64 fold; −4.44 fold; n ≥5; P<0.001; IL-6: −35.68 fold; −12.46 fold, −6.13 fold; n ≥5; P<0.001; IL-8: −7.83 fold; −3.10 fold; −2.55 fold; n ≥5; P<0.001; IL-1β: −14.34 fold; −5.26 fold; −8.9 fold; n ≥5; P<0.01). Lapdesf 2 (300 μM) failed to block cytokine release. Conclusions: These results provide proof-of-concept that our hybrid compounds utilizing critical hydroxyurea and thalidomide based pharmacophores are capable of potent HbF stimulation and anti-inflammatory activities. Testing of our lead compound Lapdesf 1in a preclinical mouse model of SCD is currently underway to evaluate its efficacy in the treatment of this hemoglobinopathy. Disclosures: No relevant conflicts of interest to declare.

High Throughput Screen To Identify Small Molecules That Differentially Regulate Expression of the Globin Genes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3632-3632
Author(s):  
Benjamin L. Ebert ◽  
Raymond Mak ◽  
Jennifer L. Pretz ◽  
David Peck ◽  
Stephen Haggerty ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Beta Globin ◽  
Diversity Oriented Synthesis ◽  
Gamma Globin ◽  
Globin Gene Expression

Abstract Several lines of evidence indicate that the pharmacological activation of fetal hemoglobin is an effective therapy for sickle cell anemia and beta thalassemia, but novel treatments for these diseases are needed. We developed and validated a high throughput assay to detect differential regulation of the globin genes and utilized this assay in a small molecule screen to identify novel compounds that increase the relative expression of gamma globin. In our assay, transcripts for the alpha, beta, delta, epsilon, gamma, theta, and zeta globin genes are amplified by multiplexed ligation-mediated PCR. Labeled amplicons are captured on different fluorescent microspheres using molecular barcodes, and the relative abundance of labeled amplicons is detected by high speed flow cytometry. To recapitulate the activity of compounds in the bone marrow of patients as accurately as possible, the screen was performed using primary human erythroid progenitor cells cultured in vitro. The assay was adapted to 384-well format with robotic liquid handling. In validation studies, the assay detected the expected increases in globin gene expression during erythroid differentiation, increased gamma globin expression in umbilical cord blood progenitor cells, and increased gamma globin expression in cells treated with known inducers of fetal hemoglobin including hydroxyurea and sodium butyrate. We screened a library of 1040 known bioactive compounds, 75% of which are FDA approved drugs, and a library of 600 compounds produced by diversity oriented synthesis that have been shown to inhibit histone deacetylase (HDAC) activity. In the screen, we rediscovered previously identified globin gene regulators, further validating our globin assay. For example, corticosteroids, known activators of fetal hemoglobin, increased the relative expression of gamma globin. Thyroid hormone specifically increased expression of delta globin, consistent with clinical observations that hemoglobin A2 levels are increased in hyperthyroidism and decreased in hypothyroidism. We identified ten novel compounds from the diversity oriented synthesis library that powerfully induce expression of the gamma globin gene relative to beta globin. Moreover, HDAC inhibition reversed the ontogeny of globin gene expression, coordinately increasing expression of fetal and embryonic relative to the adult globin genes. Relative to beta globin gene expression, gamma and epsilon globin were induced while delta globin was unaffected by HDAC inhibitors; relative to alpha globin expression, zeta globin was increased and theta globin was unaffected. The identification of compounds that differentially regulate globin gene expression may provide lead compounds for the development of novel therapies for sickle cell disease and beta thalassemia and may help elucidate the molecular events underlying switching of the globin genes during normal development.

Gamma-Globin Gene Expression in Adult Human Erythroblasts Is Associated with Concurrent Changes in the Nuclear Protein Levels of at Least Seven Transcription Factors.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1866-1866
Author(s):  
Orapan Sripichai ◽  
Y. Terry Lee ◽  
Toshihiko Tanno ◽  
Seung-Jae Noh ◽  
Colleen Byrnes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Transcription Factors ◽  
Nuclear Protein ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Binding Motif ◽  
Gamma Globin ◽  
Protein Levels ◽  
Globin Gene Expression

Abstract Alterations of globin-gene cluster expression mediated through cytokine signal transduction have been previously established. In adult erythroblasts, ex vivo combinatorial signaling from cytokines such as erythropoietin (EPO), stem cell factor (SCF), and transforming growth factor-beta (TGF-B) causes a robust reactivation of fetal hemoglobin. To determine if cytokine-activated expression of fetal hemoglobin significantly alters the transcript and protein abundance of nuclear transcription factors, profiling studies of transcription factor expression were performed. Primary CD34+ cells were cultured as donor-matched pairs using cytokine combinations that produced low vs. high levels of gamma-globin mRNA and fetal hemoglobin (Bhanu et al. Blood, 2005). To identify candidate genes, total RNA from 15 separate healthy volunteer donors was collected and combined into 5 pools. The pooled RNA was labeled and hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips. Informatics strategies were aimed toward identifying differential expression of those transcription factors with binding motif on any of the 10 human globin gene promoter regions (553 transcription factors; 115 binding motif families), versus all transcription factors demonstrating robust expression with changes of at least 3-fold. Combining both strategies, 11 candidate transcription factors were identified (BCL11A, CBFB, EGR-1, ELK-1, HHEX, ID2, MAFF, MNDA, SOX-6, TCF3, and THRB). RNA-based descriptions of gene activity do not necessarily reflect changes in protein levels; therefore, Western analyses were performed. GATA-1 was utilized as a control, since no significant change in GATA-1 mRNA was detected by expression array profiling. Nuclear protein extracts were obtained from 3 additional donors’ CD34+ cells cultured under conditions of low vs. high gamma-globin mRNA synthesis (1.6E+06 ± 3.8E+05 and 1.5E+07 ± 6.3E+06 copies/ng total RNA, respectively). Western blot analysis revealed reproducible and robust differences in protein expression levels for 7 of the 11 candidate transcription factors (EGR-1, ELK-1, HHEX, ID2, MAFF, MNDA, and SOX-6). Two other candidates were expressed below the protein detection limit. GATA-1 and the remaining 2 candidates demonstrated no change in the nuclear protein levels. Among the group with confirmed changes in gene expression, ELK-1 and EGR-1 are downstream targets of the MAP kinase signal transduction cascade. MAFF, ID2, and SOX-6 are known regulators of globin gene expression. The differential expression of HHEX and MNDA warrants further investigation. These data demonstrate that cytokine signal transduction causes changes in the intranuclear levels of at least 7 transcription factors concurrently with activation of gamma-globin gene expression.

Depletion of L3MBTL1, a PcG Gene Deleted in Patients with 20q-Associated with Hematologic Malignancies, Accelerates Erythroid Differentiation of Hematopoietic Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3740-3740
Author(s):  
Fabiana Perna ◽  
Nadia Gurvich ◽  
Ruben Hoya-Arias ◽  
Francesca Voza ◽  
Vladimir Jankovic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Polycythaemia Vera ◽  
Globin Genes ◽  
Hematopoietic Stem ◽  
Knock Down ◽  
Cd34 Cells ◽  
Globin Gene Expression

Abstract L3MBTL1 is a polycomb gene located in 20q12, within the common deleted region identified in patients with 20q deletion associated with polycythaemia vera (PV), myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). L3MBTL1 is expressed within CD34+ haematopoietic progenitor cells from which myeloid malignancies arise and its Drosophila homologue encodes a tumour suppressor protein. L3MBTL1 represents then a candidate target gene in 20q deletion patients. To gain insight into the role of L3MBTL1 in hematopoiesis we knocked down the level of L3MBTL1 mRNA through lentiviral expression of short hairpin RNAs in CD34+ hematopoietic stem/progenitor cells isolated from human cord blood (CB) cells. We achieved an approximately 80% drop in endogenous L3MBTL1 mRNA level, as determined using quantitative RT-PCR. Transduced and sorted CD34+ GFP+ CB cells were plated in liquid cultures and induced to cytokine-driven differentiation. The effect of L3MBTL1-knock down was assessed by colony assays and by fluorescence-activated cell sorting (FACS), using lineage–specific cell surface markers. We demonstrate that the knock down (KD) of L3MBTL1 remarkably accelerates the differentiation of hematopoietic CD34+ cells into erythrocytes. The relative percentage of mature erythroid precursors cells, defined as CD71+ and Glycophorin A+ cells, consistently increased in the L3MBTL1-KD population, as the CD34+ cells reached this advanced stage of erythroid differentiation in fewer days than control cells. As confirmation, Giemsa staining after cytospin preparations of L3MBTL1-KD cells showed more mature morphology compared to the control cells, and benzidine staining revealed many more Hb containing-positive cells in the L3MBTL1-depleted cell population compared to the control. Monitoring of globin gene expression demonstrated that L3MBT-Knock down is involved in the regulation of only a subset of these genes, primarily the expression of the epsilon and zeta globin genes, the embryonic globin genes belonging to the beta and alpha globin gene clusters respectively. In addition to these effects a significant slowing of proliferation was seen, which likely reflects the increased differentiation of these cells. We have previously identified a role for L3MBT in binding histones H1 and H4 that contain monomethylated and dimethylated lysine residues H1k2b and H4k20 (Kalakonda et al. 2008) and compacting chromatin (Trojer et al. Cell 2007). Precisely how this feature of L3MBTL1 function in regulating the erythroid differentiation of hematopoietic cells is unknown. We however link lack of L3MBTL1 with the possible pathogenesis of PV associated with 20q deletion. We will present additional data that attempt to define the kinetics of alpha-like and beta-like globin gene expression during the erythroid maturation of L3MBTL1-KD cells in culture and to investigate a supposed synergism with the known molecular pathways of normal and malignant erythroid differentiation.

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

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-5
Author(s):  
Donald Lavelle ◽  
Vinzon Ibanez ◽  
Kestis Vaitkus ◽  
Yogenthiran Saunthararajah ◽  
Robert E. Molokie
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cell Line ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Cd34 Cells ◽  
Murine Erythroleukemia ◽  
Globin Gene Expression ◽  
In Cells

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

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