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.

scholarly journals EHMT1 and EHMT2 inhibition induces fetal hemoglobin expression

Blood ◽  
2015 ◽  
Vol 126 (16) ◽  
pp. 1930-1939 ◽  
Author(s):  
Aline Renneville ◽  
Peter Van Galen ◽  
Matthew C. Canver ◽  
Marie McConkey ◽  
John M. Krill-Burger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Locus ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Cells ◽  
Adult Human ◽  
Key Points ◽  
Globin Gene Expression

Key Points EHMT1/2 inhibition increases human γ-globin and HbF expression, as well as mouse embryonic β-globin gene expression. EHMT1/2 inhibition decreases H3K9Me2 and increases H3K9Ac at the γ-globin gene locus in adult human erythroid cells.

Differences in Fetal Hemoglobin Induction in Sickle Cell Disease and beta-Thalassemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 555-555 ◽  
Author(s):  
Hassana Fathallah ◽  
Ali Taher ◽  
Ali Bazarbachi ◽  
George F. Atweh
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Mrna Levels ◽  
Globin Genes ◽  
Thalassemia Intermedia ◽  
Cell Disease ◽  
Globin Mrna ◽  
Globin Gene Expression

Abstract A number of therapeutic agents including hydroxyurea, butyrate and decitabine have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in β-thalassemia. As a first step towards understanding the molecular basis of the different clinical responses to these agents, we have studied the mechanisms of induction of fetal hemoglobin (HbF) by butyrate in BFU-E derived cells from 5 patients with SCD and 9 patients with β-thalassemia intermedia. Exposure to butyrate resulted in a dose-dependent augmentation of γ-globin mRNA levels in erythroid cells from patients with SCD. In contrast, induction of γ-globin expression in erythroid cells from patients with β-thalassemia intermedia was only seen at a high concentration of butyrate. The increase in γ-globin mRNA levels in patients with SCD and β-thalassemia intermedia was associated with opening of the DNA structure as manifested by decreased DNA methylation at the γ-globin promoters. Interestingly, butyrate exposure had markedly different effects on the expression of the β- and α-globin genes in the two categories of patients. Butyrate decreased the level of β-globin mRNA in 4 out of 5 patients with SCD (P = 0.04), while in β-thalassemia the levels of β-globin mRNA did not change in 7 patients and decreased in 2 patients after butyrate exposure (P = 0.12). Thus in patients with SCD, the effects of the induction of the γ-globin gene on the γ/(β+γ) mRNA ratios were further enhanced by the butyrate-mediated decreased expression of the β-globin gene. As a result, γ/(β+γ) mRNA ratios increased in all patients with SCD, with a mean increase of 31% (P = 0.002). In contrast, butyrate increased γ/(β+γ) mRNA ratios only in 4 out of 9 patients with β-thalassemia, with a more modest mean increase of 12% (P = 0.004). Interestingly, the decreased β-globin expression in patients with SCD was associated with closing of the DNA configuration as manifested by hypermethylation of DNA at the promoter of the β-globin gene while methylation of the same promoter did not change following butyrate exposure in patients with β-thalassemia intermedia. More surprisingly, the expression of the α-globin genes increased following butyrate exposure in 4 out of 9 patients with β-thalassemia, while the levels of α-globin mRNA decreased in 4 out of 5 patients with SCD. As a result, the favorable effects of the butyrate-induced increase in γ-globin gene expression on the α: non-α mRNA imbalance in patients with β-thalassemia intermedia were partly neutralized by the corresponding increase in α-globin gene expression. These differences may explain, at least in part, the more favorable effects of inducers of HbF in SCD than in β-thalassemia. Further studies are necessary to fully understand the molecular bases of the different responses to agents that induce HbF in patients with these disorders.

Context-Specific Roles for Erythroid Krüppel-Like Factor (EKLF) in Co-Ordinate High Level Expression of the Murine α- and β-Globin Genes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 365-365 ◽  
Author(s):  
Valerie M. Jansen ◽  
Shaji Ramachandran ◽  
Aurelie Desgardin ◽  
Jin He ◽  
Vishwas Parekh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Gene Promoters ◽  
Globin Genes ◽  
Context Specific ◽  
High Level ◽  
Kinetics Of ◽  
Globin Gene Expression

Abstract Binding of EKLF to the proximal promoter CACC motif is essential for high-level tissue-specific β-globin gene expression. More recent studies have demonstrated that EKLF regulates expression of other erythroid-specific genes, suggesting a broad role for EKLF in co-ordinating gene transcription in differentiating erythroblasts. Given these observations, we hypothesized that EKLF may play a role in synchronizing α- and β-globin gene expression. Supporting this model, studies of fetal erythroblasts derived from EKLF-null embryos revealed a 3-fold reduction in murine α-globin gene expression in fetal erythroblasts when compared to wild type littermate controls. A similar reduction in primary α-globin RNA transcripts was observed in these studies. To further examine the molecular consequences of EKLF function at the α- and β-globin genes in vivo, we utilized an erythroid cell line derived from EKLF null fetal liver cells. We have demonstrated previously that introduction into these cells of the wildtype EKLF cDNA, fused in frame with a mutant estrogen response element results in tamoxifen-dependent rescue of β-globin gene expression. Consistent with our observations in primary erythroblasts, α-globin gene expression is present in the absence of functional EKLF. However, with tamoxifen induction, we observed a 3–5 fold increase in α-globin gene transcription. Interestingly, the kinetics of the changes in transcription of the α- and β-gene transcripts were similar. Enhancement in α-gene transcription was associated with EKLF binding at the α- and β-globin promoters as determined by a quantitative chromatin immunoprecipitation (ChIP) assay. Interestingly, maximal EKLF binding and α-gene transcription was observed within 2 hours of tamoxifen induction. We hypothesized that the role of EKLF may differ function at the promoters, given that a basal level of α-globin gene expression occurs in absence of EKLF binding. Supporting this hypothesis, we observed sequential recruitment of p45NF-E2, RNA polymerase II (Pol II) and the co-activator CBP to the β-promoter with tamoxifen induction. No change in GATA-1 binding was observed. In contrast, p45NF-E2 does not bind to the α-promoter and the kinetics of GATA-1 and PolII association is unchanged after tamoxifen induction. Taken together, our results demonstrate that EKLF regulates the co-ordinate high-level transcription of the α- and β-globin genes, binding in a kinetically identical manner to the gene promoters. However, the effects of EKLF on transacting factor recruitment (and chromatin modification) differ between the promoters, consistent with the idea that EKLF acts in a context-specific manner to modulate gene transcription.

O-Glcnacylation Regulates γ-Globin Transcription

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1020-1020
Author(s):  
Kenneth R Peterson ◽  
Zhen Zhang ◽  
Ee Phie Tan ◽  
Anish Potnis ◽  
Nathan Bushue ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sodium Butyrate ◽  
Yeast Artificial Chromosome ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Globin Gene Expression ◽  
Dynamic Cycling

Abstract Patients with sickle cell disease (SCD), caused by mutation of the adult β-globin gene, are phenotypically normal if they carry compensatory mutations that result in continued expression of the fetal γ-globin genes, a condition termed hereditary persistence of fetal hemoglobin (HPFH). Thus, a logical clinical goal for treatment of SCD is to up-regulate γ-globin synthesis using compounds that are specific for increasing fetal hemoglobin (HbF) without pleiotropic effects on cellular homeostasis. Developmental regulation of the γ-globin genes is complex and normal silencing during the adult stage of erythropoiesis likely results from a combination of the loss of transcriptional activators and the gain of transcriptional repressor complexes. One mode of γ-globin silencing occurs at the GATA binding sites located at -566 or -567 relative to the Aγ-globin or Gγ-globin CAP sites respectively, and is mediated through the DNA binding moiety of GATA-1 and its recruitment of co-repressor partners, FOG-1 and Mi-2 (NuRD complex). Modifications of repressor complexes can regulate gene transcription; one such modification is O-GlcNAcylation. The O-GlcNAc post-translational modification is the attachment of a single N-acetyl-glucosamine moiety to either a serine or threonine residue on nuclear and cytoplasmic proteins. O-GlcNAc is added to proteins by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA) in response to changes in extracellular signals and nutrients. A dynamic balance in protein levels also exists between these two enzymes; an increase or decrease of one results in a like compensatory change in the other. Thus, the rate of O-GlcNAc addition and removal is a dynamic cycling event that is exquisitely controlled for a given target molecule, which may offer a point of intervention in the turning off or on of gene expression. O-GlcNAcylation is involved in the regulation of many cellular processes such as stress response, cell cycle progression, and transcription. Potentially, O-GlcNAc plays a pivotal role in regulating transcription of the human γ-globin genes. We induced human erythroleukemia cell line K562 with sodium butyrate to differentiate toward the erythroid lineage and observed the expected increase of γ-globin gene expression. A robust increase of γ-globin gene expression was measured after pharmacological inhibition of OGA using Thiamet-G (TMG). Using chromatin immunoprecipitation (ChIP), we demonstrated that OGT and OGA are recruited to the -566 region of the Aγ-globin promoter, the same region occupied by the GATA-1-FOG-1-Mi-2 (NuRD) repressor complex. However, OGT recruitment to this region was decreased when O-GlcNAc levels were artificially elevated by OGA inhibition with TMG. When γ-globin expression was not induced, Mi-2 was modified with O-GlcNAc and interacted with both OGT and OGA. After induction, O-GlcNAcylation of Mi-2 was reduced and Mi2 no longer interacted with OGT. Stable K562 cells were generated in which OGA was knocked down using shRNA. Following induction of these cells with sodium butyrate, γ-globin gene expression was higher compared to control cells. These data suggest that the dynamic cycling of O-GlcNAc on the Mi-2 (NuRD) moiety contributes towards regulation of γ-globin transcription. Concurrent ChIP experiments in human β-globin locus yeast artificial chromosome (β-YAC) transgenic mice demonstrated that GATA-1, Mi2 and OGT were recruited to the -566 Aγ-globin GATA silencer site in day E18 fetal liver when γ-globin is repressed, but not in day E12 fetal liver when γ-globin is expressed. These data demonstrate that O-GlcNAc cycling is a novel mechanism regulating γ-globin gene expression and will provide new avenues to explore in how alterations in gene regulation lead to the onset, progression, and severity of hematological disease. Disclosures: No relevant conflicts of interest to declare.

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.

Cooperative Silencing of Chicken ρ- and βH- Globin Gene Expression by Chicken β-Globin Regulatory Elements: Involvement of RNAi Mediated Repression.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3627-3627
Author(s):  
Elliot M. Epner ◽  
Jin Wang ◽  
Jing Huang
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
Globin Gene ◽  
K562 Cells ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Globin Genes ◽  
Phenotypic Analysis ◽  
Pol Ii ◽  
Globin Gene Expression

Abstract The chicken β-globin locus represents a well characterized, model system where the relationship between chromatin structure, transcription and DNA replication can be studied. The locus contains several regulatory elements including an intergenic enhancer as well as upstream regulatory elements that may function either alone or in combination with the intergenic enhancer as an LCR. The availability of the recombination proficient chicken B cell line DT40 has allowed the introduction of mutations into the endogenous chicken β-globin locus and phenotypic analysis after microcell mediated chromosome transfer into human erythroleukemia (K562) cells. Using this system, we have introduced deletions in the chicken β-globin intergenic enhancer as well as 5′ HS 1,2, and 3. Expression of the embryonic ρ and fetal βH chicken globin genes were repressed by the intergenic enhancer, 5′ HS1, or 5′HS2. No ρ or βH globin gene expression was detected in K562 cells containing control chicken chromosomes, while ρ and βH mRNA were activated when the intergenic enhancer, 5′ HS1, or 5′HS2 were deleted. Chromatin immunoprecipitation (ChIP) experiments that assayed RNA polmerase II (pol II), GATA-1 and NF-E2 p45/ p18 binding at regulatory elements and gene promoters in targeted cell lines supported this hypothesis and suggested a potential role for 5′HS3 in gene activation. However, targeted deletion of 5′ HS3, unlike the other chicken β-globin regulatory elements, showed no transcriptional phenotype. Our results demonstrate the intergenic enhancer, 5′HS1, and 5′ HS2 function through a common silencing mechanism involving pol II, GATA-1, and NF-E2/P18. The recent demonstration of the involvement of Pol II in the synthesis of miRNA’s prompted us to investigate the role of miRNA’s in gene silencing in this system. A small miRNA was identified at the intergenic enhancer region. ChIP assays showed the binding of two components of the RISC (Dicer and Ago2) at the chicken globin regulatory elements. These results are consistent with the involvement of RISC and miRNA’s in gene silencing in this system.

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 MiR-144 Regulates Hemoglobin Expression in Human Erythroid Cell Line

2020 ◽  
Vol 17 (11) ◽  
pp. 1221-1229
Author(s):  
Tipparat PENGLONG ◽  
Apisara SAENSUWANNA ◽  
Jitpanu KOCHAROENWAT ◽  
Wittawat BOORINTARAGOT ◽  
Suppanut FUPONGSIRIPHAN ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Target Gene ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Functional Studies ◽  
Genes Expression ◽  
Globin Gene Expression

The regulation of globin gene expression is significantly important to understand the pathogenesis of globin gene disorders. Recent findings have shown that microRNAs (miRNAs, miRs) play an important role in the regulation of globin gene expression. The miR-144 is an erythroid lineage-specific miRNA, in which its expression mediates NRF2 gene silencing and inhibits fetal hemoglobin expression. However, roles of miR-144 to other globin genes expression especially in ɑ-globin cluster remain unknown. This study, thus, examined the functional studies of miR-144 to globin gene expression in K562 human erythroid cell line. The results revealed that ɑ-globin and z-globin gene expression were silenced by the overexpressed miR-144 and that correlated with the reduced expression of KLF1- the suspected target gene. By contrast, transfection with miR-144 inhibitor reversed the silencing effect of miR-144. On the other hand, miR-144 had no effect to β-globin gene expression. Our results sustain the findings of the previous studies that the overexpression of miR-144 correlates with the repressing of NRF2 and 𝛄-globin gene expression. Taken together, our results suggest that miR-144 plays a key role in globin gene expression by silencing 𝛄-globin through NRF2 target mRNA and repressing adult ɑ-globin and embryonic z-globin gene expression possibly by targeting KLF1 gene.

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.

Induction of Fetal Hemoglobin by Transcriptional Co-Activators MTF-1 and TSPYL1

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 353-353 ◽  
Author(s):  
Kenneth R Peterson ◽  
Flavia C Costa ◽  
Halyna Fedosyuk ◽  
Renee Neades ◽  
Johana Bravo de los Rios ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Protein Complex ◽  
Globin Gene ◽  
Mrna Level ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Zinc Metabolism ◽  
Globin Promoter ◽  
Globin Gene Expression

Abstract Abstract 353 Sickle cell disease (SCD) impacts one of 400 African-Americans born each year. Augmentation of fetal hemoglobin (HbF) levels is widely accepted as the most effective method for treating SCD, but hydroxyurea (HU) is currently the only approved drug that increases HbF. Thus, there is a need for the development of new therapies for this disease, including the identification of transcriptional activators that specifically up-regulate γ-globin (HbF). Developmental regulation of human β-like globin gene switching is controlled by several parameters, including cis- and trans-acting transcriptional determinants. Understanding the mechanisms underlying control of globin gene expression, particularly those involved in activation of γ-globin expression (HbF) is important for developing new treatments for SCD. Metal-responsive transcription factor-1 (MTF-1) is a key regulator of zinc metabolism in higher eukaryotes that controls the metal-inducible expression of metallothioneins and a number of other genes directly involved in the intracellular sequestration and efflux transport of zinc. Previous studies demonstrated that MTF-1 plays an essential role in liver development and that MTF-1-deficient mice display an anemic phenotype, suggesting a role for MTF-1 in hematopoiesis. In our study, when murine MTF-1 was expression was enforced, we observed a 5-fold increase in γ-globin expression in K562 cells. We also demonstrated increased γ-globin expression in adult blood from MTF-1 human β-globin locus yeast artificial chromosome (β-YAC) bi-transgenic (bigenic) mouse lines at the mRNA level by quantitative real-time RT-PCR (qPCR) and at the protein level by FACS analysis. Lastly, γ-globin gene expression was induced 12-fold in bone marrow cells (BMCs) derived from these bigenic mice compared to BMCs derived from β-YAC-only mice, and 3-fold after 6 hours of zinc treatment in β-YAC-only BMCs. Corroborative studies including zinc-deficient and zinc replete diets in β-YAC mice and erythroid-specific MTF-1 loss-of-function in loxP-flanked-MTF-1 LCR-β-globin promoter-Cre β-YAC mice further support a role for MTF-1 in g-globin gene expression. Chromatin immunoprecipitation (ChIP) analysis did not show recruitment of MTF-1 to any γ-globin gene-proximal metal response elements (MREs), the DNA motif that MTF-1 binds to control zinc metabolism genes. However, GATA-2 co-immunoprecipitated with MTF-1 in MTF-1 β-YAC BMCs, but not in β-YAC-only BMCs, suggesting that reactivation of γ-globin expression by MTF-1 might be mediated by a MTF-1-GATA-2 protein complex. ChIP experiments indicated that MTF-1 and GATA-2 co-occupy the same sites in the γ-globin promoter. Two of the stronger co-recruitment regions contain not only GATA sites, but also non-canonical MREs that vary by 1 or 2 bp from the canonical 7 bp MRE core. Interestingly, GATA-2 was induced 2-fold in adult blood of MTF-1 β-YAC mice, and also 3.5-fold in MTF-1 β-YAC BMCs treated with zinc for 6 hours. Our data suggest that activation of γ-globin by MTF-1 is mediated by protein-protein interaction with GATA-2 and that this multi-protein complex is targeted to GATA sites located in the γ-globin gene-promoters via binding of the GATA-2 protein. In a previous study we identified testis-specific protein Y-like 1 (TSPYL1) as a candidate gene involved in activation of γ-globin (de Andrade et al., 2006, Blood Cells, Mol. & Dis. 37:82). TSPYL1 mRNA level was increased 2–5 fold in deletional hereditary persistence of fetal hemoglobin (HPFH-2) subjects and decreased in a carrier of the Sicilian δβ-thalassemia trait. TSPYL1 is a transcription factor that is a member of the nucleosome assembly protein (NAP) family. TSPYL1 is not a DNA-binding protein; thus it exerts its effect through protein-protein interactions. When we enforced expression of human TSPYL1 in K562 cells an 11-fold induction of γ-globin expression was obtained. A reduction of γ-globin expression was observed following TSPYL1 knockdown in K562 cells. qPCR analysis of blood from TSPYL1 β-YAC bigenic mice showed that γ-globin expression was increased 4–12-fold. Taken together, our data strongly support the evidence that MTF-1 and TSPYL1 reactivate γ-globin expression in adult erythropoiesis. These two proteins represent potential new targets in strategies to reactivate γ-globin in hemoglobinopathies where higher levels of HbF would have beneficial effects. Disclosures: No relevant conflicts of interest to declare.

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