Globin Gene Silencing in Primary Erythroid Cultures

Ferry, A E; Baliga, Surendra; Monteiro, Carlos Augusto; Pace, Betty S.

doi:10.1074/jbc.272.32.20030

Cited by 29 publications

(18 citation statements)

References 54 publications

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“…We have demonstrated in this study that IL-6 reduces steady-state HbF synthesis in fetal erythroid progenitors thus corroborating our observations made previously (6). Interestingly, IL-6 inhibited the proliferative capacity of fetal-stage progenitors contrary to what we observe routinely in cultures established with adult erythroid progenitors.…”

Section: Discussionsupporting

confidence: 53%

“…Protein tyrosine kinases of the Janus kinase (JAK) family activate STAT proteins upon binding of cytokines to their cognate receptors (1,2). The JAK-STAT pathway mediates the effect of several cytokines (3)(4)(5) including interleukin 6 (IL-6), which was previously demonstrated to inhibit ␥ gene expression (6) and to most likely exert its negative effect at the level of the bi-potential progenitor cell. Although several cis-acting elements have been identified in the globin gene promoters (7,8), the precise mechanism for cytokine-mediated globin gene regulation during development remains to be clarified.…”

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confidence: 99%

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Stat3β Inhibits γ-Globin Gene Expression in Erythroid Cells

Foley

Ofori-Acquah

Yoshimura

et al. 2002

Journal of Biological Chemistry

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We demonstrated previously ␥-globin gene inhibition in K562 cells and primary erythroid progenitors treated with interleukin-6. Although several cis-acting elements have been identified in the globin promoters, the precise mechanism for cytokine-mediated globin gene regulation remains to be elucidated. In this report we demonstrate inhibitors of Stat3 phosphorylation abrogate interleukin-6-mediated ␥ gene silencing in erythroid cells. DNA-protein binding studies established Stat3 interaction in the 5-untranslated ␥-globin promoter region. Furthermore, co-transfection experiments with Stat3␤ demonstrate ␥ promoter inhibition in a concentrationdependent manner, which was significantly reversed when the cognate Stat3-binding site in the 5-untranslated region was mutated. These studies establish a novel mechanism for ␥ gene silencing through the STAT signal transduction pathway.

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Section: Discussionsupporting

confidence: 53%

mentioning

confidence: 99%

Stat3β Inhibits γ-Globin Gene Expression in Erythroid Cells

Foley

Ofori-Acquah

Yoshimura

et al. 2002

Journal of Biological Chemistry

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“…Treatment of the human erythroleukemia cell line K562 with IL-6 induces the expression of megakaryocytic markers and the silencing of certain globin genes (10). Derived from an acute erythroblastic leukemia, K562 cells are multipotent in that they can be directed into two separate differentiation pathways (11).…”

mentioning

confidence: 99%

Interleukin-6 Regulation of the Human DNA Methyltransferase (HDNMT) Gene in Human Erythroleukemia Cells

Hodge¹,

Xiao²,

Clausen³

et al. 2001

Journal of Biological Chemistry

141

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The transfer of a methyl group to the cytosine portion of the CpG dinucleotide by dnmt-1 permits or enables the binding of methyl-specific DNA-binding proteins to the methylated CpG site (1,2,4,5). The binding of methyl-specific proteins such as MeCP1 and MeCP2 to genetic regulatory elements represses transcription by blocking the binding of other positive acting transactivation factors (6). Methylcytosine-DNA-binding proteins can attract histone deacetylases to the site, which remodel chromatin into highly repressed states (7). Thus, DNA methylation can result in permanent epigenetic alteration of genes and is important in promoting or guiding the differentiation of cells and the establishment of tissue-specific gene expression patterns (8).The inflammatory cytokine IL-6 1 is able to induce the maturation and differentiation of cells (9). Treatment of the human erythroleukemia cell line K562 with IL-6 induces the expression of megakaryocytic markers and the silencing of certain globin genes (10). Derived from an acute erythroblastic leukemia, K562 cells are multipotent in that they can be directed into two separate differentiation pathways (11). K562 cells express low levels of both erythrocytic-and megakaryocyticspecific genetic markers and can be induced to differentiate along one of these two major pathways depending upon the external stimuli applied to the cells (12,13). This ability suggests some form of epigenetic control over the differentiation process. The ETS family of transcription factors represent a large family of differentially expressed, positive and negative regulators of transcription and are involved in cell differentiation (3). Here we show that when K562 cells are induced to enter the megakaryocytic differentiation pathway by IL-6, an increase in Fli-1 expression occurs, which results in the transactivation of the human methyltransferase-1 gene expression. EXPERIMENTAL PROCEDURESCell Culture-COS-1 cells were obtained from the American Type Culture Collection (CRL-1650) and maintained in Dulbecco's modified Eagle's medium high glucose supplemented with 10% FBS, glutamine, and penicillin-streptomycin solutions. Human erythroleukemia K562 cells (ATCC CCL-243) were maintained in RPMI 1640 medium supplemented with 10% FBS, glutamine, and penicillin-streptomycin solutions. Recombinant interleukin-6 (catalog number 200-06) was purchased from Pepro-Tech Inc. (Rocky Hill, NJ). For IL-6 stimulation, K562 cells were collected by centrifugation, rinsed twice in phosphatebuffered saline, pH 7.4, then resuspended in RPMI 1640 medium supplemented with glutamine, penicillin-streptomycin, and 0.05% FBS for 48 h, then treated with IL-6.Methylation Assay-Cell nuclear pellets were freeze-thawed three times and centrifuged to remove debris. Clarified lysates were mixed with an equal volume of Chelex-100 resin (50%v/v) to remove DNA and RNA from the sample. For each replicate, 5 g of the protein lysate was added to 200 l of an assay mixture consisting of 20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 25% glycerol, 0.5% Trit...

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“…In theory, therapeutic ␥-globin reactivation could be accomplished by inhibition of repressor proteins to prevent silencing or enforced expression of trans-activators. Our group investigated proximal ␥-gene Stat3 binding site in the 5Ј-untranslated region (Ferry et al, 1997). We demonstrated ␥-globin silencing by interleukin-6, a known activator of Stat3 signaling.…”

Section: ␥-Globin Gene Regulationmentioning

confidence: 99%

Understanding mechanisms of γ‐globin gene regulation to develop strategies for pharmacological fetal hemoglobin induction

Pace

Zein

2006

Developmental Dynamics

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The developmental regulation of ␥-globin gene expression has shaped research efforts to establish therapeutic modalities for individuals affected with sickle cell disease (SCD). Fetal hemoglobin (Hb F) synthesis is high at birth, followed by a decline to adult levels by 10 months of age. The expression of ␥-globin is controlled by a developmentally regulated transcriptional program that is recapitulated during normal erythropoiesis in the adult bone marrow. It is known that naturally occurring mutations in the ␥-gene promoters cause persistent Hb F synthesis after birth, which ameliorates symptoms in SCD by inhibiting hemoglobin S polymerization and vaso-occlusion. Several pharmacological agents have been identified over the past 2 decades that reactivate ␥-gene transcription through different cellular systems. We will review the progress made in our understanding of molecular mechanisms that control ␥-globin expression and insights gained from Hb F-inducing agents that act through signal transduction pathways.

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Globin Gene Silencing in Primary Erythroid Cultures

Cited by 29 publications

References 54 publications

Stat3β Inhibits γ-Globin Gene Expression in Erythroid Cells

Stat3β Inhibits γ-Globin Gene Expression in Erythroid Cells

Interleukin-6 Regulation of the Human DNA Methyltransferase (HDNMT) Gene in Human Erythroleukemia Cells

Understanding mechanisms of γ‐globin gene regulation to develop strategies for pharmacological fetal hemoglobin induction

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