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.

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.

Human Fetal Hemoglobin Expression Is Regulated by the Developmental Stage-Specific Repressor BCL11A

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 487-487 ◽  
Author(s):  
Vijay G Sankaran ◽  
Tobias F. Menne ◽  
Thomas E. Akie ◽  
Guillaume Lettre ◽  
Joel N. Hirschhorn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developmental Stage ◽  
Disease Severity ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Integrative Approach ◽  
Erythroid Cells ◽  
Nucleosome Remodeling ◽  
Globin Gene Expression

Abstract Numerous molecular approaches have been taken to elucidate the regulation of the human β-like globin genes, and particularly the “fetal” (γ- to β-) globin switch, given the role of fetal hemoglobin (HbF) levels on disease severity in the β-hemoglobin disorders. Despite these efforts, no developmental stage-specific nuclear regulators of HbF expression have been identified and validated. Recent genome-wide single nucleotide polymorphism (SNP) association studies by us and others have revealed novel loci that are significantly associated with HbF levels in normal, sickle cell, and thalassemia populations. One variant, lying within intron 2 of the chromosome 2 gene BCL11A, accounts for >10% of the variation in HbF levels. We have now tested the hypothesis that BCL11A, a zinc-finger transcription factor, serves as a stage-specific regulator of HbF expression, rather than merely a genetic marker of HbF status. We found that BCL11A is expressed as two major isoforms (termed XL and L) in human erythroid progenitors. The level of BCL11A expression is inversely correlated with the expression of the HbF gene, γ-globin, in human erythroid cell types representative of different developmental stages. Expression of BCL11A is negligible in embryonic, and high in adult, erythroid cells. Correlation of SNP genotypes with levels of BCL11A RNA in cells derived from individuals of known genotypes indicates that the “high HbF” genotype is associated with reduced BCL11A expression. To better characterize its potential role in erythropoiesis and globin gene regulation, we identified interacting protein partners of BCL11A in erythroid cells through affinity purification and protein microsequencing. We found that the BCL11A protein exists in complexes with the nucleosome remodeling and histone deacetylase (NuRD) corepressor complex, as well as the erythroid transcription factors GATA-1 and FOG-1. Taken together, the genetic, developmental, and biochemical data are most consistent with a model in which BCL11A functions as a repressor of γ-globin gene expression. To directly test this possibility, we modulated expression of BCL11A in primary human erythroid precursors expanded from adult CD34+ progenitors. Transient or persistent knockdown of BCL11A accomplished by siRNA or lentiviral shRNA delivery, respectively, led to robust induction of γ-globin gene expression. Importantly, down-regulation of BCL11A expression did not alter the differentiation state or global transcriptional profile of the cells, suggesting an effect on a limited number of targets, including the γ-globin gene. In summary, these studies establish BCL11A as a potent regulator of human globin switching. As an adult-stage repressor, BCL11A represents a primary target for therapy aimed at reactivating HbF expression in patients with β-hemoglobin disorders. Our studies illustrate the power of an integrative approach to reveal the functional connection between a common genetic variant and a trait that serves as a prominent modifier of disease severity.

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.

Correct Function of the Locus Control Region May Require Passage Through a Nonerythroid Cellular Environment

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 703-712 ◽  
Author(s):  
George Vassilopoulos ◽  
Patrick A. Navas ◽  
Evangelia Skarpidi ◽  
Kenneth R. Peterson ◽  
Chris H. Lowrey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Region ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Globin Mrna ◽  
L Cells ◽  
Correct Function ◽  
Globin Gene Expression

Abstract The function of the β-globin locus control region (LCR) has been studied both in cell lines and in transgenic mice. We have previously shown that when a 248-kb β-locus YAC was first microinjected into L-cells and then transferred into MEL cells by fusion, the YAC loci of the LxMEL hybrids displayed normal expression and developmental regulation.To test whether direct transfer of a β-globin locus (β-YAC) into MEL cells could be used for studies of the function of the LCR, a 155-kb β-YAC that encompasses the entire β-globin locus was used. This YAC was retrofitted with a PGK-neo selectable marker and with two I-PpoI sites at the vector arm-cloned insert junctions, allowing detection of the intact globin loci on a single I-PpoI fragment by pulsed field gel electrophoresis (PFGE). ThePpo-155 β-YAC was used to directly lipofect MEL 585 cells. In 7 β-YAC MEL clones with at least one intact copy of the YAC, the levels of total human globin mRNA (ie, ɛ + γ + β) per copy of integrated β-YAC varied more than 97-fold between clones. These results indicated that globin gene expression was strongly influenced by the position of integration of the β-YAC into the MEL cell genome and suggested that the LCR cannot function properly when the locus is directly transferred into an erythroid cell environment as naked β-YAC DNA. To test whether passage of the β-YAC through L-cells before transfer into MEL cells was the reason for the previously observed correct developmental regulation of human globin genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by lipofection. Three clones carried the intact 144-kb I-PpoI fragment and transcribed the human globin genes with a fetal-like pattern. Subsequent transfer of the YAC of these L(β-YAC) clones into MEL cells by fusion resulted in LxMEL hybrids that synthesized human globin mRNA. The variation in human β-globin mRNA (ie, ɛ + γ + β) levels between hybrids was 2.5-fold, indicating that globin gene expression was independent of position of integration of the transgene, as expected for normal LCR function. The correct function of the LCR when the YAC is first transferred into the L-cell environment raises the possibility that normal activation of the LCR requires interaction with the transcriptional environment of an uncommitted, nonerythroid cell. We propose that the activation of the LCR may represent a multistep process initiated by the binding of ubiquitous transcription factors early during the differentiation of hematopoietic stem cells and completed with the binding of erythroid type of factors in the committed erythroid progenitors.

Correct Function of the Locus Control Region May Require Passage Through a Nonerythroid Cellular Environment

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 703-712 ◽  
Author(s):  
George Vassilopoulos ◽  
Patrick A. Navas ◽  
Evangelia Skarpidi ◽  
Kenneth R. Peterson ◽  
Chris H. Lowrey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Region ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Globin Mrna ◽  
L Cells ◽  
Correct Function ◽  
Globin Gene Expression

The function of the β-globin locus control region (LCR) has been studied both in cell lines and in transgenic mice. We have previously shown that when a 248-kb β-locus YAC was first microinjected into L-cells and then transferred into MEL cells by fusion, the YAC loci of the LxMEL hybrids displayed normal expression and developmental regulation.To test whether direct transfer of a β-globin locus (β-YAC) into MEL cells could be used for studies of the function of the LCR, a 155-kb β-YAC that encompasses the entire β-globin locus was used. This YAC was retrofitted with a PGK-neo selectable marker and with two I-PpoI sites at the vector arm-cloned insert junctions, allowing detection of the intact globin loci on a single I-PpoI fragment by pulsed field gel electrophoresis (PFGE). ThePpo-155 β-YAC was used to directly lipofect MEL 585 cells. In 7 β-YAC MEL clones with at least one intact copy of the YAC, the levels of total human globin mRNA (ie, ɛ + γ + β) per copy of integrated β-YAC varied more than 97-fold between clones. These results indicated that globin gene expression was strongly influenced by the position of integration of the β-YAC into the MEL cell genome and suggested that the LCR cannot function properly when the locus is directly transferred into an erythroid cell environment as naked β-YAC DNA. To test whether passage of the β-YAC through L-cells before transfer into MEL cells was the reason for the previously observed correct developmental regulation of human globin genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by lipofection. Three clones carried the intact 144-kb I-PpoI fragment and transcribed the human globin genes with a fetal-like pattern. Subsequent transfer of the YAC of these L(β-YAC) clones into MEL cells by fusion resulted in LxMEL hybrids that synthesized human globin mRNA. The variation in human β-globin mRNA (ie, ɛ + γ + β) levels between hybrids was 2.5-fold, indicating that globin gene expression was independent of position of integration of the transgene, as expected for normal LCR function. The correct function of the LCR when the YAC is first transferred into the L-cell environment raises the possibility that normal activation of the LCR requires interaction with the transcriptional environment of an uncommitted, nonerythroid cell. We propose that the activation of the LCR may represent a multistep process initiated by the binding of ubiquitous transcription factors early during the differentiation of hematopoietic stem cells and completed with the binding of erythroid type of factors in the committed erythroid progenitors.

scholarly journals Identification of a Novel Enhancer/Chromatin Opening Element Associated with High-Level γ-Globin Gene Expression

2018 ◽  
Vol 38 (19) ◽  
Author(s):  
Yong Shen ◽  
MacLean A. Bassett ◽  
Aishwarya Gurumurthy ◽  
Rukiye Nar ◽  
Isaac J. Knudson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Chromosome 11 ◽  
Erythroleukemia Cell ◽  
High Level ◽  
Globin Gene Expression

ABSTRACT The organization of the five β-type globin genes on chromosome 11 reflects the timing of expression during erythroid cell development, with the embryonic ε-globin gene being located at the 5′ end, followed by the two fetal γ-globin genes, and with the adult β- and δ-globin genes being located at the 3′ end. Here, we functionally characterized a DNase I-hypersensitive site (HS) located 4 kb upstream of the Gγ-globin gene (HBG-4kb HS). This site is occupied by transcription factors USF1, USF2, EGR1, MafK, and NF-E2 in the human erythroleukemia cell line K562 and exhibits histone modifications typical for enhancers. We generated a synthetic zinc finger (ZF) DNA-binding domain targeting the HBG-4kb HS (HBG-4kb ZF). The HBG-4kb ZF interacted with the target site in vitro and in the context of cells with a high affinity and specificity. Direct delivery of the HBG-4kb ZF to K562 and primary human erythroid cells caused a reduction in γ-globin gene expression which was associated with decreased binding of transcription factors and active histone marks at and downstream of the HS. The data demonstrate that the HBG-4kb HS is important for fetal globin production and suggest that it may act by opening chromatin in a directional manner.

Endogenous Elevations of Short Chain Fatty Acids (SCFAs) Can Up-Regulate Embryonic Globin Gene Expression.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1770-1770
Author(s):  
Himanshu Bhatia ◽  
Jennifer Hallock ◽  
Lauren Sterner ◽  
Toru Miyazaki ◽  
Ann Dean ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Short Chain Fatty Acids ◽  
Yolk Sac ◽  
Globin Genes ◽  
Key Enzyme ◽  
Globin Gene Expression

Abstract Persistence of fetal hemoglobin can ameliorate adult beta (β)-globin gene disorders. Since SCFAs can affect embryonic and fetal globin gene expression, we examined their role during development. Murine globin gene expression, β-type (embryonic βH1, and epsilon-y, εY, and adult βmajor), and alpha (α)-type (embryonic zeta, ζ, >α, adult α), were compared between wildtype (wt) and transgenic mice, in which a key enzyme for SCFA metabolism, PCCA, had been knocked out (PCCA−/−, (Miyazaki et al, 2001). E10.5 PCCA−/− yolk sac (n= 9), showed increased α, βH1 and ζ gene expression, at respectively 2-, 2.6- and 1.6-fold relative to wt (n=13, p<.05), and εY gene expression, at 1.7-fold (p=0.07). The embryonic-to-adult globin gene switch was modestly delayed in yolk sacs from E12.5 PCCA−/− (n=9) vs. wt (n=4) and E 14.5 PCCA−/− (n=6) vs. wt (n=6). % embryonic β-type globin gene expression (% βH1 and εY of total β globin) was 77±6 PCCA−/− and 74±3 wt at E12.5, p=n.s., and 42±13 PCCA−/− and 21±3 wt at E14.5, p<.05; % emvbryonic α-type expression (% ζ of total α) was 32±3 PCCA−/−, 25±1wt at E12.5, p<.05 and 7±2 PCCA−/− and 4±1 wt at E14.5, p<.05). Embryonic globin gene expression in E 12.5 and 14.5 fetal livers was not different between PCCA−/− and wt embryos. Cultures of pooled E14.5 wt fetal liver cells (FLCs, n=4 separate experiments), however, suggested that embryonic globin genes can be activated in FLCs. The percent of total β-type globin gene expression that was embryonic after culture with butyrate (1mM) was 11.6±2.6%, with propionate (2.5 mM) was 3.6±0.2%, and insulin/erythropoietin or basal media was 0.03±0.03% and 0.42±0.26% respectively (p<.05 relative to SCFAs). Dose-response with propionate (n=2 seaparate experiments) suggest inadequate endogenous propionate levels for activation in PCCA −/− fetal liver, as % embryonic β-type globin gene expression rose above basal levels only at concentrations of 1 to 5 mM (2.5 mM maximal) but not at <0.6 mM. We conclude that endogenous SCFAs, at levels achievable in vivo can activate embryonic globin gene expression during development in the murine yolk-sac. However, higher levels than achievable endogenously currently are necessary to produce this effect in murine fetal livers.

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.

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