scholarly journals Human fetal globin gene expression is regulated by LYAR

2014 ◽  
Vol 42 (15) ◽  
pp. 9740-9752 ◽  
Author(s):  
Junyi Ju ◽  
Ying Wang ◽  
Ronghua Liu ◽  
Yichong Zhang ◽  
Zhen Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
K562 Cells ◽  
Binding Motif ◽  
Dependent Manner ◽  
Histone H4 ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Globin Gene Expression ◽  
Selection Of

AbstractHuman globin gene expression during development is modulated by transcription factors in a stage-dependent manner. However, the mechanisms controlling the process are still largely unknown. In this study, we found that a nuclear protein, LYAR (human homologue of mouse Ly-1 antibody reactive clone) directly interacted with the methyltransferase PRMT5 which triggers the histone H4 Arg3 symmetric dimethylation (H4R3me2s) mark. We found that PRMT5 binding on the proximal γ-promoter was LYAR-dependent. The LYAR DNA-binding motif (GGTTAT) was identified by performing CASTing (cyclic amplification and selection of targets) experiments. Results of EMSA and ChIP assays confirmed that LYAR bound to a DNA region corresponding to the 5′-untranslated region of the γ-globin gene. We also found that LYAR repressed human fetal globin gene expression in both K562 cells and primary human adult erythroid progenitor cells. Thus, these data indicate that LYAR acts as a novel transcription factor that binds the γ-globin gene, and is essential for silencing the γ-globin gene.

Inhibition of β-globin gene expression by 3′-azido-3′-deoxythymidine in human erythroid progenitor cells

1999 ◽  
Vol 44 (3) ◽  
pp. 167-177 ◽  
Author(s):  
Maria-Grazia Spiga ◽  
Douglas A Weidner ◽  
Chantal Trentesaux ◽  
Robert D LeBoeuf ◽  
Jean-Pierre Sommadossi
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Globin Gene ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Globin Gene Expression

Developmental and Differential Changes of Gene Expression in Erythroid Progenitor Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3648-3648
Author(s):  
Vladan P. Cokic ◽  
Bhaskar Bhattacharya ◽  
Raj K. Puri ◽  
Alan N. Schechter
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
G Protein ◽  
Peripheral Blood ◽  
Globin Gene ◽  
Globin Genes ◽  
Erythroid Progenitor ◽  
Gamma Globin ◽  
Erythroid Progenitor Cells ◽  
Globin Gene Expression

Abstract During erythropoiesis and human development different globin genes (α, β, γ, δ and ε) are expressed as a result of globin gene switching. We investigated globin gene expression in comparison to the expression of other genes in erythroid progenitor cells (EPC) during ontogenesis using in-house produced microarrays containing 16,659 oligonucleotides. Human primitive CD34+ cells were isolated from fetal liver (FL), cord blood (CB), adult bone marrow (BM), peripheral blood (PB) and mobilized peripheral blood (mPB), and developed into EPC in the presence of erythropoietin and other cytokines. The differentiation to EPC was confirmed by flow cytometry as 100% cells were CD71+. In microarray studies, a total of 2996 genes were highly expressed in FL, 2673 genes in CB, 2580 in mPB, 1465 in PB and 1259 in BM derived EPC. 661 of these genes were common for all type of cells. The high level of expression, beside globin genes, was observed for the following genes: transferrin receptor, proteoglycans, ALAS2, Charcot-Leyden crystal protein, nucleophosmin, eosinophil peroxidase, myeloperoxidase and ribonucleases. Most of the analyzed genes demonstrated down-regulation during ontogenesis (elastase 2, glutathione peroxidase 1, SERPINB1, nudix, mitochondrial proteins, ribosomal proteins, enthoprotin, serine proteinase inhibitor), but some showed up-regulation (hexokinase, superoxide dismutase 2, spectrin). Besides developmental changes of globin gene expression during ontogenesis, we also analyzed changes in their expression during erythropoiesis in these different tissues by quantitative PCR. Beta-globin gene expression reached the maximum levels in cells of adult blood origin: BM (176 fmol/μg) and PB (110 fmol/μg). Gamma-globin gene expression, of FL origin, had steady levels during erythroid differentiation (20 fmol/μg), whereas cord blood derived EPC demonstrated consistent up-regulation (60 fmol/μg) in contrast to cells originated from adult blood (3–15 fmol/μg at day 14th). G protein related genes and histone deacetylases were elevated in CB derived EPC, concomitant with increased gamma-globin gene expression. We also analyzed the gamma-globin induction by hydroxyurea, a well known inducer, and established which G protein-coupled receptors involved pathways are activated in PB derived EPC: dopamine receptors D1, D2 and D5, beta 2 adrenergic receptor, human DP prostanoid receptor and prostaglandin E receptor 1, as well as genes activated by cAMP/PKA, PI-3 kinase, MAP and NO/cGMP pathways. This study establishes concomitant changes in expression of globin genes and other known and/or previously unrecognized genes, which appear to be involved in erythropoiesis.

SATB1 family protein expressed during early erythroid differentiation modifies globin gene expression

Blood ◽  
2005 ◽  
Vol 105 (8) ◽  
pp. 3330-3339 ◽  
Author(s):  
Jie Wen ◽  
Suming Huang ◽  
Heather Rogers ◽  
Liliane A. Dickinson ◽  
Terumi Kohwi-Shigematsu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Erythroid Progenitor ◽  
Family Protein ◽  
Globin Promoter ◽  
Globin Gene Expression

AbstractSpecial AT-rich binding protein 1 (SATB1) nuclear protein, expressed predominantly in T cells, regulates genes through targeting chromatin remodeling during T-cell maturation. Here we show SATB1 family protein induction during early human adult erythroid progenitor cell differentiation concomitant with ϵ-globin expression. Erythroid differentiation of human erythroleukemia K562 cells by hemin simultaneously increases γ-globin and down-regulates SATB1 family protein and ϵ-globin gene expression. Chromatin immunoprecipitation using anti-SATB1 anti-body shows selective binding in vivo in the β-globin cluster to the hypersensitive site 2 (HS2) in the locus control region (LCR) and to the ϵ-globin promoter. SATB1 overexpression increases ϵ-globin and decreases γ-globin gene expression accompanied by histone hyperacetylation and hypomethylation in chromatin from the ϵ-globin promoter and HS2, and histone hypoacetylation and hypermethylation associated with the γ-globin promoter. In K562 cells SATB1 family protein forms a complex with CREB-binding protein (CBP) important in transcriptional activation. In cotransfection experiments, increase in ϵ-promoter activity by SATB1 was amplified by CBP and blocked by E1A, a CBP inhibitor. Our results suggest that SATB1 can up-regulate the ϵ-globin gene by interaction with specific sites in the β-globin cluster and imply that SATB1 family protein expressed in the erythroid progenitor cells may have a role in globin gene expression during early erythroid differentiation. (Blood. 2005;105:3330-3339)

scholarly journals Thalidomide induces γ-globin gene expression through increased reactive oxygen species–mediated p38 MAPK signaling and histone H4 acetylation in adult erythropoiesis

Blood ◽  
2007 ◽  
Vol 110 (8) ◽  
pp. 2864-2871 ◽  
Author(s):  
Wulin Aerbajinai ◽  
Jianqiong Zhu ◽  
Zhigang Gao ◽  
Kyung Chin ◽  
Griffin P. Rodgers
Keyword(s):  
Gene Expression ◽  
Signaling Pathway ◽  
P38 Mapk ◽  
Globin Gene ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Histone H4 ◽  
Histone H4 Acetylation ◽  
Dose Dependent ◽  
Globin Gene Expression

Abstract Although thalidomide has been shown to improve anemia in some patients with myelodysplastic syndromes and stimulates erythropoietin in patients with multiple myeloma, thalidomide's specific effects on γ-globin gene expression during erythroid differentiation have not been studied. Here, we investigated the effects of thalidomide on γ-globin gene expression and the involved signaling pathway using an ex vivo culture system of primary human CD34+ cells. We found that thalidomide induced γ-globin mRNA expression in a dose-dependent manner, but had no effect on β-globin expression. We also demonstrated that intracellular reactive oxygen species (ROS) levels were increased by treatment with thalidomide for 48 hours (from day 3 to day 5). Western blot analysis demonstrated that thalidomide activated the p38 mitogen-activated protein kinase (MAPK) signaling pathway in a time- and dose-dependent manner and increased histone H4 acetylation. Pretreatment of cells with the antioxidant enzyme catalase and the intracellular hydroxyl scavenger dimethylthiourea (DMTU) abrogated the thalidomide-induced p38 MAPK activation and histone H4 acetylation. Moreover, pretreatment with catalase and DMTU diminished thalidomide-induced γ-globin gene expression. These data indicate that thalidomide induces increased expression of the γ-globin gene via ROS-dependent activation of the p38 MAPK signaling pathway and histone H4 acetylation.

scholarly journals Restoration of the CCAAT Box or Insertion of the CACCC Motif Activate δ-Globin Gene Expression

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 421-427 ◽  
Author(s):  
Delia C. Tang ◽  
David Ebb ◽  
Ross C. Hardison ◽  
Griffin P. Rodgers
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Globin Gene ◽  
Treatment Strategies ◽  
K562 Cells ◽  
Promoter Sequence ◽  
Flanking Sequence ◽  
Globin Promoter ◽  
Ccaat Box ◽  
Globin Gene Expression

Abstract Hemoglobin A2 (HbA2 ), which contains δ-globin as its non–α-globin, represents a minor fraction of the Hb found in normal adults. It has been shown recently that HbA2 is as potent as HbF in inhibiting intracellular deoxy-HbS polymerization, and its expression is therefore relevant to sickle cell disease treatment strategies. To elucidate the mechanisms responsible for the low-level expression of the δ-globin gene in adult erythroid cells, we first compared promoter sequences and found that the δ-globin gene differs from the β-globin gene in the absence of an erythroid Krüppel-like factor (EKLF ) binding site, the alteration of the CCAAT box to CCAAC, and the presence of a GATA-1 binding site. Second, serial deletions of the human δ-globin promoter sequence fused to a luciferase (LUC) reporter gene were transfected into K562 cells. We identified both positive and negative regulatory regions in the 5′ flanking sequence. Furthermore, a plasmid containing a single base pair (bp) mutation in the CCAAC box of the δ promoter, restoring the CCAAT box, caused a 5.6-fold and 2.4-fold (P < .05) increase of LUC activity in transfected K562 cells and MEL cells, respectively, in comparison to the wild-type δ promoter. A set of substitutions that create an EKLF binding site centered at −85 bp increased the expression by 26.8-fold and 6.5-fold (P < .05) in K562 and MEL cells, respectively. These results clearly demonstrate that the restoration of either an EKLF binding site or the CCAAT box can increase δ-globin gene expression, with potential future clinical benefit.

scholarly journals Differing responses of globin and glycophorin gene expression to hemin in the human leukemia cell line K562

Blood ◽  
1982 ◽  
Vol 59 (4) ◽  
pp. 738-746 ◽  
Author(s):  
BL Tonkonow ◽  
R Hoffman ◽  
D Burger ◽  
JT Elder ◽  
EM Mazur ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Globin Gene ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
Globin Mrna ◽  
Human Leukemia Cell Line ◽  
Globin Gene Expression

Abstract The human leukemia cell line, K562, produces embryonic and fetal hemoglobins and glycophorin A, proteins normally associated only with erythroid cells. Hemoglobin accumulation is enhanced by exposure of the cells to 0.05 mM hemin. We have examined K562 cells before and after exposure to hemin to determine whether expression of these erythroid proteins was shared by all cells or confined to specific subpopulations. Globin gene expression was examined by quantitation of globin mRNA sequences, using a 3H-globin cDNA molecular hybridization probe. Constitutive cells produced globin mRNA, the content of which was increased 3–4-fold by hemin. Cell-to-cell distribution of globin mRNA was determined by in situ hybridization of 3H-globin cDNA to constitutive and hemin-treated K562 cells. Virtually all cells in the culture exhibited grain counts above background, indicating globin gene expression by all cells, rather than a confined subpopulation. Virtually all hemin-treated cells had 3–5-fold higher grain counts, indicating uniformly increased globin gene expression. The glycophorin content of K562 cells was estimated by fluorescence-activated cell sorting (FACS) of cells labeled with fluorescein-labeled antiglycophorin antiserum. The vast majority of constitutive cells contained glycophorin, but exhibited to apparent increase in glycophorin accumulation after hemin exposure. Thus, glycophorin and globin genes exhibited differential responses to hemin. These differences could reflect normal differences in the patterns of specialized gene expression in stem cells. Alternatively, different aberrations of gene expression could be occurring in response to the determinants of the neoplastic properties of K562.

Globin Genes Induction by Nitric Oxide of Stromal Cell Origin.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4102-4102
Author(s):  
Vladan P. Cokic ◽  
Bojana B. Beleslin-Cokic ◽  
Constance Tom Noguchi ◽  
Alan N. Schechter
Keyword(s):  
Progenitor Cells ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Globin Genes ◽  
Globin Mrna ◽  
Erythroid Progenitor ◽  
Gamma Globin ◽  
Erythroid Progenitor Cells ◽  
Globin Gene Expression

Abstract We have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). Here we report that co-culture of human bone marrow endothelial cells with erythroid progenitor cells induced gamma-globin mRNA expression (1.8 fold), and was further elevated (2.4 fold) in the presence of hydroxyurea (40 μM). Based on these results, NOS-dependent stimulation of NO levels by bradykinin and lipopolysaccharide has been observed in endothelial (up to 0.3 μM of NO) and macrophage cells (up to 6 μM of NO), respectively. Bradykinin slightly increased gamma-globin mRNA levels in erythroid progenitor cells, but failed to increase gamma-globin mRNA levels in endothelial/erythroid cell co-cultures indicating that stimulation of endothelial cell production of NO alone is not sufficient to induce gamma-globin expression. In contrast, lipopolysaccharide and interferon-gamma mutually increased gamma-globin gene expression (2 fold) in macrophage/erythroid cell co-cultures. In addition, hydroxyurea (5–100 μM) induced NOS-dependent production of NO in human (up to 0.7 μM) and mouse macrophages (up to 1.2 μM). Co-culture studies of macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to 3 fold) in the presence of hydroxyurea (20–100 μM). These results demonstrate a mechanism by which hydroxyurea may induce globin genes and affect changes in the phenotype of hematopoietic cells via the common paracrine effect of bone marrow stromal cells.

Cucurbitacin D Upregulates Fetal Hemoglobin Expression in K562 Cells and Human Erythroid Progenitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3833-3833
Author(s):  
Hongtao Xing ◽  
Siwei Zhang ◽  
H. Phillip Koeffler ◽  
Ming Chiu Fung
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Negative Control ◽  
Optimal Dosage ◽  
Erythroid Progenitors ◽  
Cucurbitacin D ◽  
Globin Gene Expression ◽  
Sodium Phenylbutyrate

Abstract The search for novel therapeutic candidates causing reactivation of fetal hemoglobin (a2g2; HbF) to reduce the imbalance of globin gene expression is important in order to develop effective approach for the clinical management of sickle cell anemia and b-thalassemia. For the first time, we have identified cucurbitacin D (CuD), a naturally occurring oxygenated tetracyclic triterpenoid, as a molecular entity inducing g-globin gene expression and HbF synthesis in K562 cells and human erythroid progenitors from either peripheral blood or bone marrow. The upregulation of HbF induced by CuD was dose- and time- dependent. CuD was compared to hydroxyurea (HU), 5-azacytidine, amifostine, recombinant human erythropoietin (rhEPO), and sodium phenylbutyrate. At their optimal dosage, CuD (12.5 ng/mL) and HU (25.0 μg/mL) induced nearly 70% K562 cells to express total hemoglobin after 6 days culture, which was higher than the induction by Amifostine (30%), 5-azacytidine (36%), rhEPO (16%), sodium phenylbutyrate (23%) at their optimal concentrations and negative control (11%). Fetal hemoglobin ELISA showed that CuD (12.5 ng/mL) and 5-azacytidine (400 ng/mL) induced higher levels of fetal hemoglobin in K562 cells (15.4 ng/μL and 29.3 ng/μL, respectively), compared to HU (10.3 ng/μL), amifostine (7.8 ng/μL), rhEPO (10.9 ng/μL), sodium phenylbutyrate (9.9 ng/μL) at their optimal concentrations and negative control (5.3 ng/μL). CuD induced a significantly higher fetal cell percentage than HU in K562 cells (65% vs 37% maximum) and primary erythroid progenitors (36% vs 21% maximum) based on the immunofluorescence imaging and flow cytometry analysis. Real-time PCR results showed that the amount of γ-globin mRNA increased from 2.5-fold in CuD-optimal-treated cells (12.5 ng/mL, 48 hours) compared with 1.5-fold in HU-optimal-treated cells (25.0 μg/mL, 48 hours). Growth inhibition assay (MTT) demonstrated that CuD at its optimal γ-globin inducing dosage (12.5 ng/mL) inhibited proliferation of K562 by less than 10% of untreated control cells; while hydroxyurea at its optimal dosage (25.0 μg/mL) inhibited 80% of cell division. The in vitro therapeutic index (calculated by dividing the dose inhibiting 50% cell growth (IC50) by dose inducing 50% maximal HbF production (ED50)) of CuD was 40-fold greater than HU. Taken together, the results suggest that CuD has the potential to be a therapeutic agent for treatment of sickle cell anemia and b-thalassemia.

Talen-Mediated Knock Outs Of Cis and Trans Elements Potentially Involved In Globin Gene Switching

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 436-436
Author(s):  
Patrick A Navas ◽  
Yongqi Yan ◽  
Minerva E Sanchez ◽  
Ericka M Johnson ◽  
George Stamatoyannopoulos
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Globin Gene ◽  
K562 Cells ◽  
Globin Mrna ◽  
Core Elements ◽  
Gene Knockouts ◽  
Gene Switching ◽  
Cis And Trans ◽  
Globin Gene Expression

Abstract Transcription activator-like effector nucleases (TALEN) are engineered proteins used for precise genome editing by generating specific DNA double strand that are repaired by homologous recombination and by non-homologous end joining. TALENs can be used to study gene regulation by deleting putative regulatory elements in the context of the native chromosome and measuring mRNA synthesis. We designed TALENs to delete individual DNAse I-hypersensitive sites (HS) of the β-globin locus control region (LCR) followed by an assessment of globin gene expression and assessment of epigenetic effects in K562 erythroleukemia cells. The β-globin LCR is composed of five HSs and functions as a powerful regulatory element responsible for appropriate levels of the five β-like globin genes during development. Introduction of plasmid DNA encoding a pair of TALENs and targeting individually the flanking region of the HS2, HS3 and HS4 core elements along with a donor 100 base single-stranded oligonucleotide resulted in the successful deletions of each of the three core elements in K562 cells. Individual K562 cells were seeded to produce clones and the mutations were screened by PCR to identify both heterozygous and homozygous clones. The TALEN-mediated 288 bp HS2 core deletion resulted 32 heterozygous (48.5%) and 6 homozygous clones (9.1%) in a total of 66 clones screened. K562 carries three copies of chromosome 11 emphasizing the robustness of TALEN technology to target each of the alleles. In the 199 bp HS3 core deletion, from 113 clones we identified 28 heterozygous (24.8%) and 3 (2.7%) homozygous clones. Lastly, the 301 bp HS4 core deletion yielded 9 homozygous (5.9%) and 12 heterozygous (7.9%) clones from 151 clones screened. Total RNA was isolated from wild-type K562 cells, and from both the heterozygous and homozygous mutant clones and subjected to RNase Protection analysis to quantitate the levels of globin mRNA. Deletion if the HS3 core in K562 cells in a ∼30% reduction in ε-globin mRNA and 2-fold reduction in γ-globin mRNA. A more dramatic effect on globin expression is observed in the HS2 core deletion, as ε- and γ-globin expression is reduced by 2- and 5-fold, respectively. These results suggest that HS2 contributes the majority of the LCR enhancer function in K562 cells. The HS4 core deletion resulted in a modest ∼20% reduction in both ε- and γ-globin expression. TALENs were designed to knockout trans-acting factors implicated to be involved in globin gene regulation and/or globin switching. TALENs bracketing the gene promoters and the first exon of 25 genes encoding either a transcription factor or histone-modifying enzyme were synthesized and post-transfection PCR screens of the transfected pool of K562 cells resulted in the successful identification of 17 gene knockouts. The 17 target genes are PRMT5, LDB1, EIF2AK3, BCL11A, HBSIL, MYB, SOX6, NFE4, NR2F2, NR2C1, NR2C2, CHTOP, NFE2, DNMT3A, RBBP4, MTA2 and MBD2. Single cell clones have been generated by limited dilution of transfected K562 pools and thus far we have identified heterozygous and homozygous clones of 8 of 17 gene knockouts, importantly all clones were identified without selection. The frequency of identifying the knockout clones, represented by the number of clones screened/ number of heterozygous clones/ number of homozygous clones, are as follows: HBS1L (63/3/0), SOX6 (68/13/2), NFE4 (56/13/7), LBD1 (300/2/0), MBD2 (301/0/1), CHTOP (288/66/6), NFE2 (712/44/5) and NR2C1 (96/40/11). The remaining nine gene knockouts and globin gene expression data will be presented at the meetings. These studies highlight a powerful TALEN-mutagenesis platform for target deletions of both cis- and trans-elements to study globin gene switching. TALENs can be synthesized in several days and the screening of the individual clones for the desired knockouts is completed within two weeks. This highly efficient mutagenesis platform will further our understanding of the molecular basis of globin switching. Disclosures: No relevant conflicts of interest to declare.

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