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.

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.

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

Role of gene order in developmental control of human gamma- and beta-globin gene expression

1993 ◽  
Vol 13 (8) ◽  
pp. 4836-4843
Author(s):  
K R Peterson ◽  
G Stamatoyannopoulos
Keyword(s):  
Gene Expression ◽  
Gene Order ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Gene Promoters ◽  
Globin Genes ◽  
Beta Globin ◽  
Gamma Globin ◽  
Globin Gene Expression ◽  
Beta Gene

To determine the effect of gene order on globin gene developmental regulation, we produced transgenic mice containing two tandemly arranged gamma- or beta-globin or gamma beta- and beta gamma-globin genes linked to a 2.5-kb cassette containing sequences of the locus control region (LCR). Analysis of constructs containing two identical gamma or beta genes assessed the effect of gene order on globin gene expression, while analysis of constructs containing tandemly arranged gamma and beta genes assessed any additional effects of the trans-acting environment. When two gamma genes were tandemly linked to the LCR, expression from the proximal gamma gene was three- to fourfold higher than expression from the distal gamma gene, and the ratio of proximal to distal gene expression remained unchanged throughout development. Similarly, when two beta genes were tandemly linked to the LCR, the proximal beta gene was predominantly expressed throughout development. These results indicate that proximity to LCR increases gene expression, perhaps by influencing the frequency of interaction between the LCR and globin gene promoters. An arrangement where the gamma gene was proximal and the beta gene distal to the LCR resulted in predominant gamma-gene expression in the embryo. When the order was reversed and the gamma gene was placed distally to the LCR, gamma-gene expression in the embryo was still up to threefold higher than expression of the LCR-proximal beta gene. These findings suggest that the embryonic trans-acting environment interacts preferentially with the gamma genes irrespective of their order or proximity to the LCR. We conclude that promoter competition rather than gene order plays the major role in globin gene switching.

The PcG Protein L3MBTL1 Transcriptionally Represses Human Embryonic and Fetal Globin Genes: a Novel Prospect for HbF Activation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1014-1014
Author(s):  
Fabiana Perna ◽  
Ruben Hoya-Arias ◽  
Ly Phuong Vu ◽  
Fan Liu ◽  
Francesca Voza ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Globin Gene ◽  
Transcriptional Repressor ◽  
Mrna Levels ◽  
Globin Genes ◽  
Beta Globin ◽  
Hematopoietic Stem ◽  
Globin Gene Expression

Abstract Abstract 1014 L3MBTL1 is the human homolog of the Drosophila Polycomb Group tumor suppressor gene, lethal(3)malignant brain tumor. We demonstrated that human L3MBTL1 functions as a transcriptional repressor and after crystallizing the MBT repeat domain determined that L3MBTL1 compacts chromatin by binding mono- and di-methylated lysine residues in histones H1 (H1K26) and H4 (H4K20). Despite the known role of L3MBTL1 in affecting chromatin structure, the function of L3MBTL1 in human hematopoiesis has remained largely unknown. We recently demonstrated that L3MBTL1 enforces cell fate decision toward the erythroid lineage and that knockdown of L3MBTL1 accelerates the erythroid differentiation of human hematopoietic stem/progenitor cells, suggesting that its deletion contributes to the pathogenesis of 20q- erythroid malignancies. Consistently with its role in erythropoiesis, here we reveal that L3MBTL1 is a novel transcriptional repressor of fetal globin genes and it may work in concert with BCL11A and EKLF to control globin gene expression. By utilizing RNA interference to reduce L3MBTL1 expression, we have found that knockdown of L3MBTL1 in human cord blood hematopoietic stem/progenitor cells consistently upregulates the expression of the epsilon, gamma, and zeta globin genes, but not the beta globin gene. Similar effects were seen following knockdown of L3MBTL1 in the human erythroleukemia cell line K562, and knockdown of L3MBTL1 in human embryonic stem cells (ESCs) led to the inappropriate expression of fetal and embryonic globin genes (which increases more than 50-fold after the L3MBTL1-KD). These data suggest a role for L3MBTL1 in regulating the globin switch. To investigate the mechanism by which L3MBTL1 silences embryonic and fetal globin gene expression, we used chromatin immunoprecipitation (ChIP) assays to show that L3MBTL1 directly associates with the human β-globin locus. L3MBTL1 occupies several discrete regions within the human β-globin cluster and colocalizes with H4K20me within the Locus Control Region (LCR), a primary attachment site for chromatin modifiers. As confirmation, we found that treatment of K562 cells with hemin, which broadly increases H3K9 acetylation over the β-globin locus and activates the transcription of globin genes, leads to decreases in expression of the repressive H4K20me2 methylmark and L3MBTL1 to the beta-globin cluster. Given the recent identification of the repressor of gamma globin gene expression, BCL11A, we investigated a potential relationship between L3MBTL1 and BCL11A. We found that knockdown of L3MBTL1 led to downregulation of BCL11A mRNA. Accordingly, we have also found that overexpression of L3MBTL1 is associated with an upregulation of BCL11A mRNA, suggesting that L3MBTL1 and BCL11A may function cooperatively to silence globin gene expression. Knockdown of L3MBTL1 also upregulated EKLF mRNA levels which could relate to the decreased BCL11A expression. In summary our data demonstrate that knock-down of L3MBTL1 upregulates embryonic and fetal globin genes in cell contexts where they are usually silenced, indicating the functional importance of this Polycomb protein for repressing the globin gene locus. The clearance of L3MBTL1 and its associated histone mark (H4K20me2) during treatments that induce potent transcriptional activation of globin genes suggest that repression induced by L3MBTL1 is dynamic and may be involved in the fetal-to-adult globin switch. L3MBTL1 therefore emerges as a novel transcriptional repressor of fetal globin genes whose expression may be coordinated with that of BCL11A and EKLF. Understanding the role of L3MBTL1 and the H4K20 methylmark in globin gene switching offers the prospect of the targeted activation of HbF in erythroid cells of patients with hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Role of gene order in developmental control of human gamma- and beta-globin gene expression.

1993 ◽  
Vol 13 (8) ◽  
pp. 4836-4843 ◽  
Author(s):  
K R Peterson ◽  
G Stamatoyannopoulos
Keyword(s):  
Gene Expression ◽  
Gene Order ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Gene Promoters ◽  
Globin Genes ◽  
Beta Globin ◽  
Gamma Globin ◽  
Globin Gene Expression ◽  
Beta Gene

To determine the effect of gene order on globin gene developmental regulation, we produced transgenic mice containing two tandemly arranged gamma- or beta-globin or gamma beta- and beta gamma-globin genes linked to a 2.5-kb cassette containing sequences of the locus control region (LCR). Analysis of constructs containing two identical gamma or beta genes assessed the effect of gene order on globin gene expression, while analysis of constructs containing tandemly arranged gamma and beta genes assessed any additional effects of the trans-acting environment. When two gamma genes were tandemly linked to the LCR, expression from the proximal gamma gene was three- to fourfold higher than expression from the distal gamma gene, and the ratio of proximal to distal gene expression remained unchanged throughout development. Similarly, when two beta genes were tandemly linked to the LCR, the proximal beta gene was predominantly expressed throughout development. These results indicate that proximity to LCR increases gene expression, perhaps by influencing the frequency of interaction between the LCR and globin gene promoters. An arrangement where the gamma gene was proximal and the beta gene distal to the LCR resulted in predominant gamma-gene expression in the embryo. When the order was reversed and the gamma gene was placed distally to the LCR, gamma-gene expression in the embryo was still up to threefold higher than expression of the LCR-proximal beta gene. These findings suggest that the embryonic trans-acting environment interacts preferentially with the gamma genes irrespective of their order or proximity to the LCR. We conclude that promoter competition rather than gene order plays the major role in globin gene switching.

Hydroxyurea Cytotoxicity and Fetal Hemoglobin Induction.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3607-3607
Author(s):  
Heather M. Rogers ◽  
Xiaobing Yu ◽  
Constance Tom Noguchi
Keyword(s):  
Transcription Factor ◽  
Progenitor Cells ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Cell Toxicity ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
High Concentrations ◽  
Globin Gene Expression

Abstract An important treatment strategy for sickle cell anemia is to increase fetal hemoglobin (HbF) in circulating erythrocytes. We use hydroxyurea (HU) as a model compound to induce HbF in adult human erythroid progenitor cells to examine the relationship between cell toxicity and globin gene expression. HU inhibits ribonucleotide reductase and its use is limited by hematopoietic toxicity at high dose. Cultures of primary human hematopoietic progenitor cells were stimulated with erythropoietin (EPO) and the effect of increasing doses of HU (from 1 to 200 mM) was determined on cell proliferation and differentiation, globin production, and erythroid transcription factors expression. At the lowest concentration (1 mM) we observed a minimal increase in cell proliferation with little change in % benzidine positive cells after 12 days of culture with EPO. As HU concentration increased, proliferation and % benzidine positive cells decreased, with concentrations of 100 and 200 mM being highly toxic, reducing cell number by 10 fold or more. Analysis of globin gene expression indicates that low concentrations of HU increase both g-globin and b-globin, resulting in only a modest increase in the g/(g+b) ratio compared with control. The g/(g+b) ratio increases with increasing HU concentration reaching a value of 0.25 or greater for concentrations of 50 mM or more, and approaching 1.0 at 200 mM, a consequence of the suppression of b-globin expression. This concentration of HU also inhibited g-globin expression, so that although the g/(g+b) ratio is quite high, it is at a cost in overall globin production and cell toxicity. Hemoglobin expression is determined primarily at the transcription level. We examined expression of GATA-1, GATA-2, SCL/Tal-1 and EKLF as regulatory proteins critical to erythropoiesis. We found that HU affects expression of select transcription factors associated with erythroid differentiation. EPO induction of GATA-1, a zinc-finger transcription factor required for survival and differentiation of erythroid progenitor cells, is delayed with HU, and the peak level of GATA-1 decreases at mid- and high concentrations, falling by 10 fold or more at 100 mM or greater. At the lowest concentration (1 mM) GATA-1 increases higher than the control. HU also delays EPO induction of SCL/Tal-1, a basic-helix-loop-helix transcription factor that positively regulates erythroid differentiation and is required for the production of mature erythrocytes, and EKLF, a zinc-finger transcription factor necessary for induction of b-globin in adult erythroid cells that acts by direct binding to the b-globin promoter. At the lowest concentration (1 mM), the delay in EPO induction of SCL/Tal-1 and EKLF is followed by a marked increase leading to peak levels greater than the control. At mid- and high concentrations, overall levels of SCL/Tal-1 and EKLF are reduced. GATA-2, a member of the GATA-family that plays a critical role in proliferation and survival of early erythroid progenitor cells, is down-regulated with EPO stimulation and is not markedly affected by HU. Therefore, HU concentration is crucial in optimizing the production of HbF. At low levels, HU increases both b- and g-globin resulting in small increases in g/(g+b) ratio, while at high concentrations the maximal increases in g/(g+b) ratio are concomitant with cytotoxicity. These data explain in part the importance of the maximum tolerated dose to achieve maximum increase in %HbF in hydroxyurea therapy.μμμγβγγβγγβμμβγγγβμμμβγγγβγγβββ

Inhibition of G9a Methyltransferase in Adult Human Erythroblasts Stimulates Fetal Hemoglobin Production By Facilitating Looping Between LCR and γ-Globin Gene

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 332-332
Author(s):  
Ivan Krivega ◽  
Colleen Byrnes ◽  
Jaira F. de Vasconcellos ◽  
Y. Terry Lee ◽  
Megha Kaushal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone Modification ◽  
Progenitor Cells ◽  
Protein Complex ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Gene Promoters ◽  
Globin Genes ◽  
Adult Human ◽  
Globin Gene Expression

Abstract Globin gene expression undergoes developmental switching from embryonic (ε) through fetal (γ) to adult (δ and β) genes. Inherited mutations or deletions at the β-gene cause beta-thalassemia. One of the most propitious strategies of treatment for the disease is forced switching from mutated β-gene to unaffected fetal γ-gene expression in adult erythroid cells. Expression of globin genes is regulated by the upstream LCR enhancer. The LCR enhancer loops to globin gene promoters utilizing the LDB1/GATA-1/TAL1/LMO2 protein complex. Additionally histone-modifying enzymes play a significant role in regulation of globin gene expression. G9a methyltransferase, responsible for establishing H3K9me2 histone modification, is involved in repressing fetal and activating adult globin gene expression in mouse erythroid cells. Moreover, inhibition of G9a methyltransferase activity by the synthetic chemical compound UNC0638 activates γ- and represses β-gene expression in adult human hematopoietic precursor CD34(+) cells. Using ex vivo differentiation of primary CD34(+) adult human cells as a model system, we investigated the effect of UNC0638 on switching from β- to γ-globin gene expression, LDB1 complex occupancy and LCR/β-gene promoter looping patterns in adult erythroblast cells. Human peripheral blood CD34(+) progenitor cells from three healthy adult donors were differentiated for 21 days in a three phase serum-free media system. Based upon dose titration studies, 1µM UNC0638 was added to the medium during the most proliferative phase of culture (days 7-14) and compared to control cells grown without UNC0638. Under these conditions, a highly significant 5-fold increase in γ-globin gene expression was observed. UNC0638 treatment also caused a pronounced (3-fold) reduction in β-globin gene expression without substantial change in α-globin. At the end of the culture period, HPLC analyses also demonstrated that UNC0638 treatment resulted in a considerable increase in the cellular fetal hemoglobin (HbF / HbA + HbF: control: 2.9 +/- 1.2%; UNC0638: 30.9 +/- 2.5%, p=0.003). Chromatin immunoprecipitation and chromosome conformation capture assays were utilized to determine if the increase of fetal hemoglobin along with activation of γ-gene expression and concomitant reduction of β-gene expression were associated with epigenetic modification of the β-globin locus. UNC0638 erased H3K9me2 histone modification in the β-globin locus and caused changes in LCR looping from interaction with the β- to the γ-globin gene. Mirroring differences in looping pattern, LDB1 containing protein complex occupancy was significantly increased at the γ-globin gene and decreased at δ- and β-gene promoters. These results support a model whereby G9a establishes conditions preventing activation of γ-gene by interacting with the LCR and facilitating LCR looping with δ- and β-gene promoters and subsequent strong activation of adult globin genes expression during differentiation of adult erythroid progenitor cells. In this view, G9a inhibition represents a promising approach for treatment of β-hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

Activity of RN-1, an LSD-1 Inhibitor, on Fetal Globin Expression in Sickle Mice and Sickle Erythroid Progenitors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 961-961 ◽  
Author(s):  
Shuaiying Cui ◽  
Jose Sangerman ◽  
Seyed Mehdi Nouraie ◽  
Yan Dai ◽  
Oluwakemi Owoyemi ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Hemolytic Anemia ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Clinical Severity ◽  
Globin Genes ◽  
Erythroid Progenitors ◽  
Irreversible Inhibitor ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

Abstract Sickle cell disease (SCD) is the most common monogenic disorder, afflicting millions worldwide, and causing hemolytic anemia and chronic organ damage from vaso-occlusion. Fetal hemoglobin (HbF) is an endogenous type of hemoglobin present in all humans during development, which is normally suppressed in infancy. Biochemical and clinical studies have shown that increased synthesis of HbF inhibits sickle hemoglobin (HbS) polymerization and reduces clinical severity. Concerted efforts have been made to induce the synthesis of HbF in adult erythroid cells with chemical inducers of HbF and through disruption of transcription factors in repressor complexes. As wide variability in individual responses to drug candidates have been observed in clinical trials, consistently effective HbF inducers are highly desired. We previously identified that Lysine-specific histone demethylase 1 (LSD1) is involved in the regulation of the fetal γ-globin genes, and inhibition of LSD1 using either RNAi or by the momoamine oxidase inhibitor tranylcypromine (TC) in primary human erythroid progenitor cells induces HbF to therapeutic levels. However, TC treatment has potentially problematic side effects, and at high concentrations decreases adult b-globin mRNAs and impairs erythroid maturation. We have now investigated another LSD1 inhibitor, RN-1, which is a cell-permeable TC analog that acts as a potent, irreversible inhibitor of LSD1 with a lower IC50 than TC. We investigated in vivo effects of RN-1 on γ-globin gene expression and erythroid physiology in a transgenic mouse model of SCD which expresses human α- and sickle β-globin, and has many genetic, hematologic, and pathophysiological features found in SCD patients, including irreversibly sickled RBCs, hemolytic anemia, high reticulocyte counts, hepatosplenomegaly and organ pathology. We found a robust increase in human fetal γ-globin (15-fold) and murine embryonic εY- and βH1-globin mRNAs (36 and 54-fold) and 4-fold increases in human HbF in SCD mice following repeated RN-1 treatment (at 10 μg/g body weight) within 4 weeks. Further, irreversibly sickled RBCs were significantly reduced, and RBC lifespan increased markedly in RN-1-treated SCD mice, leading to significantly decrease pathophysiologic indicators (hemolysis, splenomegaly, and organ necrosis) compared to untreated SCD mice. To begin to evaluate potential effects of RN-1 on erythroid progenitor cells from patients with SCD, peripheral blood from 5 adult SCD patients was cultured with RN-1 (0.07 to 0.25 μM) in a 2-phase progenitor assay, with mRNA analyzed on day 12 and F-reticulocytes on day 13-14 of the erythroid differentiation phase. RN-1 treated progenitors demonstrated a mean 3.4-fold higher g-globin mRNA (p=0.04) and 5% higher absolute F-reticulocytes than were observed in untreated progenitors from the same subject, with responses occurring in 5/5 subjects' assays. These preclinical studies provide additional evidence that modulating LSD-1 activity is a promising approach to inducing HbF expression as a mechanism to reduce clinical severity of SCD. Disclaimer: "Research reported in this publication was supported by the NHLBI under Award Number P50HL118006. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health" R01 DK052962 10A1 R42-HL-110727 Disclosures No relevant conflicts of interest to declare.

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.

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.

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