The Effects Of The Histone Methyltransferase Inhibitor UNC0638 Upon Gamma Globin Gene and Protein Expression Are Erythroblast Differentiation Stage Specific

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3453-3453
Author(s):  
Ki Soon Kim ◽  
Colleen Byrnes ◽  
Y. Terry Lee ◽  
Jaira F. de Vasconcellos ◽  
Megha Kaushal ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Epigenetic Modification ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Cells ◽  
Erythroid Progenitor ◽  
Gamma Globin ◽  
Adult Human ◽  
Gene And Protein Expression ◽  
Differentiation Stage

Abstract Epigenetic modification of chromatin in erythroid cells represents an active field of study aimed, in part, toward increased expression of fetal hemoglobin in patients with beta-thalassemia. The homologous methyltransferases G9a and GLP regulate globin gene transcription by catalyzing mono- and dimethylation at Lys 9 and dimethylation at Lys 27 of histone H3. Inhibition of these methyltransferases by the small molecule named UNC0638 was recently shown to increase gamma-globin gene expression in adult human hematopoietic precursor and stem cells. Here UNC0638 was explored further to include fetal hemoglobin expression among more mature erythroid cells cultured from CD34(+) cells of three healthy adult human donors in a serum-free culture medium. According to this culture model, the main erythroblast population on culture days 0-7 consists of CD36(+), CD45(+), CD71(moderate), CD235a(-) erythroid progenitor cell. On culture days 7-14, the progenitor cells differentiate in the presence of erythropoietin to become CD36(+), CD45(-), CD71(high), CD235a(+) precursor cells. During the final week in culture, the erythroblasts undergo nuclear condensation, enucleation, and loss of RNA combined with the loss of CD36 and CD71 on the plasma membrane to become mature erythrocytes. To investigate different stages of erythroblast maturation, the cells were cultured in medium containing 1µM UNC0638 for periods of seven days (culture days 0-7, 7-14, or 14-21) and compared to control cultures without UNC0638. The effects of UNC0638 were determined by flow cytometry, Q-RT-PCR and hemoglobin chromatography (HPLC). Unexpectedly, fetal hemoglobin expression was highly-dependent upon the differentiation stage of the cells in the presence of UNC0638. When cultured in UNC0638 supplemented medium on culture days 0-7 or 14-21, the cells underwent terminal maturation, but there was no significant increase in the fetal hemoglobin content of the mature cells (see abstract figure). In contrast, UNC0638 added on culture days 7-14, caused a significant increase in fetal hemoglobin (HbF; control: 3.9 ± 3.5% vs. day 7-14 UNC0638: 32.6 ± 0.95%, p=0.007). The increase in HbF was associated with a similar increase in gamma-globin mRNA (control: 1.5E+06 ± 1.7E+05 copies/ng vs. day 7-14 UNC0638: 7.5E+06 ± 1.4E+06 copies/ng, p=0.021). Additionally, terminal maturation and enucleation were partially inhibited when compared to the other conditions or controls. These data suggest that UNC0638 causes a robust increase in fetal hemoglobin as the cells undergo maturation. Fetal hemoglobin increases were more pronounced after exposure to UNC0638 during the erythropoietin-dependent transition from CD235a(-) to CD235a(+) erythroblasts. The results suggest that fetal hemoglobin regulation by G9a and GLP may be differentiation stage dependent. Disclosures: No relevant conflicts of interest to declare.

Knockdown of HNF4A Gene Increases Fetal Hemoglobin Synthesis in Hudep-2

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 968-968
Author(s):  
Ingrid Grazielle Sousa ◽  
Priscila Keiko Matsumoto Martin ◽  
Dulcinéia Martins de Albuquerque ◽  
Carolina Lanaro ◽  
Ryo Kurita ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Transcription Factor ◽  
Conflicts Of Interest ◽  
Clonal Selection ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Inherited Disease ◽  
Erythroid Progenitor ◽  
Gamma Globin ◽  
Cd34 Cells

Sickle cell anemia is a recessive inherited disease caused by a single nucleotide polymorphism in the β-globin gene the resulting substitution of glutamic acid by valine causes red blood cell sickling when deoxygenated. Some hypomethylating agents are able to induce the expression of γ-globin by inhibiting DNA methylation at the gene promoter. However, it is not completely understood how this regulation occurs and which genes are involved in this process. To understand fetal hemoglobin (HbF) regulation, CD34+ cells were treated with 1µM of decitabine on the 9th day of culture to induce HbF production. The expression levels of transcription factors and chromatin modifiers were evaluated through the PCR Array platform (Qiagen™ Germany). The transcription factor, HNF4A (Hepatocyte Nuclear Factor 4 Alpha), was highly upregulated in cells treated with decitabine, compared to the control cells, and was chosen as a candidate for CRISPR/Cas9 knockout in HUDEP-2 cells (immortalized human erythroid progenitor cells). The HNF4A gene has been reported as a transcription factor, which regulates the expression of several hepatic genes, and is able to play a role in the development of the liver, kidney, and intestines. Moreover, HNF4A is expressed in the hematopoietic tissue. To the best of our knowledge, the association between HNF4A and gamma globin gene synthesis has not been previously described. HUDEP-2 cells were cultured and then treated with 50nM decitabine. After 72 hours, HbF levels were measured with anti-HbF antibody by flow cytometry in three biological replicates. The percentage of cells positive for HbF in decitabine-treated HUDEP-2 were 12.27 ± 0.7%, N=3, while in control cells the percentage was 1.0 ± 0.06%, N=3 (p<0.0001). These results corroborate the increased expression measured in CD34+ cells. To knock out HNF4A in HUDEP-2 cells, we used the CRISPR/Cas9 system. We generated INDELs in heterozygosity for HNF4A. Briefly, HUDEP-2 cells were nucleofected with Cas9 high fidelity ribonucleoprotein (104 pmol), crRNA:tracrRNA (120 pmol) complex and 1µM of gRNA HNF4A using a CD34+ human cell kit and the E-001 program in an AMAXA Nucleofector 4D-device (Lonza). Two days after nucleofection, edited HUDEP-2 cells were submitted to clonal selection and expanded for approximately 28 days. Genomic DNA from clones was analyzed by a Sanger Sequencer, and four edited HNF4A-HUDEP-2 clones were selected with INDELs in the fourth exon of HNF4A gene. These clones were expanded in culture with controls, and the HbF levels were quantified by flow cytometry. HbF levels in these four clones were 8.2 ± 2.4%, while in the wild type HUDEP-2, HbF was 0.9 ± 0.02% (p< 0.05). The edited clones expressed significantly more HbF than the controls, although this expression was not homogeneous. Western blotting of edited HNF4A-HUDEP-2 clones demonstrated decreased HNF4A at the protein level (0.35 ± 0.9 AU N=8), compared to controls (2.02± 1.1 AU N=3, p< 0.05). Results suggest that HNF4A may play a role in the gamma globin gene transcription in HUPED-2 cells. The mechanism of how HNF4A may regulate gamma globin gene expression remains to be clarified. Disclosures No relevant conflicts of interest to declare.

A sustained and pancellular reversal of gamma-globin gene silencing in adult human erythroid precursor cells

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 387-393 ◽  
Author(s):  
Natarajan V. Bhanu ◽  
Tiffany A. Trice ◽  
Y. Terry Lee ◽  
Nicole M. Gantt ◽  
Patricia Oneal ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Synergistic Effects ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Mrna ◽  
Erythroid Precursor ◽  
Gamma Globin ◽  
Adult Human ◽  
Gene And Protein Expression ◽  
Complete Reversal

Abstract We systematically compared cytokine-mediated increases or decreases in proliferation with globin gene and protein expression in adult human erythroblasts. Despite their opposite effects on growth, stem cell factor (SCF) and transforming growth factorbeta (TGF-B) had synergistic effects with respect to fetal hemoglobin (HbF): average HbF/HbF + adult hemoglobin (HbA) ratio in erythropoietin (EPO) = 1.4 ± 1.0%; EPO + TGF-B = 10.8 ± 1.9%; EPO + SCF = 19.1 ± 6.2%; and EPO + SCF + TGF-B (EST) = 39.3 ± 6.3%. Polymerase chain reaction (PCR) revealed significant increases in gamma-globin transcripts that were balanced by reduced beta-globin transcripts. Single-cell quantitative PCR demonstrated a complete reversal of gamma-globin gene silencing with detectable gamma-globin mRNA in more than 95% of the cells. Immunostaining with HbF antibodies also showed a pancellular distribution in EST (96.2 ± 0.01% HbF positive) compared with a heterocellular distribution in EPO (42.9 ± 0.01% HbF positive). As shown here for the first time, a robust and pancellular reversal of gamma-globin gene silencing among hemoglobinized erythroblasts from adult humans may be achieved in the absence of hereditary mutation or direct genomic manipulation. (Blood. 2005;105:387-393)

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.

scholarly journals alpha-Amino-N-butyric acid stimulates fetal hemoglobin in the adult

Blood ◽  
1988 ◽  
Vol 72 (6) ◽  
pp. 1961-1967
Author(s):  
P Constantoulakis ◽  
T Papayannopoulou ◽  
G Stamatoyannopoulos
Keyword(s):  
Butyric Acid ◽  
Globin Gene ◽  
Chromatin Modification ◽  
Fetal Hemoglobin ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Gamma Globin ◽  
Intracellular Environment ◽  
Synergistic Induction

The effect of alpha-amino-N-butyric acid (alpha ABA) on fetal hemoglobin production in the adult was examined in vivo after being administered to normal and anemic baboons and in erythroid progenitor cell cultures. Infusion of alpha ABA for five days resulted in four- to fivefold increases in the level of F reticulocytes of normal or chronically anemic baboons. The induction of HbF by alpha ABA was strikingly enhanced by the administration of 5-azacytidine. The addition of alpha ABA in culture produced a concentration-related increase of HbF in baboon CFUe and e-cluster colonies. In addition to the induction of HbF, alpha ABA stimulated the growth of all classes of erythroid progenitors in vivo or in culture. The activation of gamma- globin gene expression by alpha ABA is attributed to an interaction between regulatory sites of globin chromatin modified by alpha ABA and the immature intracellular environment of the expanding erythropoiesis. The combination of chromatin modification, DNA methylation, and the immature intracellular environment of rapid erythroid regeneration may explain the synergistic induction of HbF by alpha ABA and 5-azacytidine.

scholarly journals LSD1 Inhibitors Induce Fetal Hemoglobin in Primary Human Erythroid Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1066-1066 ◽  
Author(s):  
Cuong LE ◽  
Greggory Myers ◽  
Alawi Habara ◽  
David H.K. Chui ◽  
Martin H. Steinberg ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Monoamine Oxidase Inhibitor ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Therapeutic Effects ◽  
Phase 2 ◽  
Erythroid Cells ◽  
Erythroid Progenitor ◽  
Cd34 Cells ◽  
Phase Phase

Abstract Significant amount of clinical literatures have supported that high level of fetal hemoglobin (HbF) improves the disease pathophysiology of β-globinopathies [sickle cell disease (SCD) and β-thalassemia]. Thus, we certainly can treat β-globinopathies by increasing the HbF level in adult erythroid cells. We originally reported that the lysine-specific histone demethylase 1 (LSD1) plays an important role in the regulation of the fetal γ-globin genes. Inhibition of LSD1 by using RNAi and monoamine oxidase inhibitor tranylcypromine (TCP) in primary human erythroid progenitor cells induces HbF to therapeutic levels. Furthermore, LSD1 inhibitor RN-1 treatment of SCD mice results in increased HbF synthesis and leads to effective improvement of many aspects of the disease pathology normally associated with SCD. Most recently, we examined thein vivo effects of some additional, publically available small molecule chemical inhibitors of LSD1 (including GSK-LSD1, LSD1-C12, LSD1-C76, OG-L002, and S2101) on HbF synthesis and erythroid physiology in SCD mice. There was a statistically significant increase in the percentage of HbF positive cells after 4 weeks of treatment with GSK-LSD1 or OG-L002 in SCD mice. Here, we report the effects of these two inhibitors in primary human erythroid cell derived from peripheral blood CD34+ cells. We isolated CD34+ cells using MACS column and cultured them using the two-phase-culture system. After seven days in expansion phase (phase 1) and three days in differentiation phase (phase 2), cells were treated with different doses of GSK-LSD1 and OG-L002 LSD1 inhibitors along with controls (DMSO, hydroxyurea and TCP) for 3 or 5 days in phase 2 culture. Flow cytometric assays showed that the percentage of HbF positive cells were significantly high when CD34+ cells treated with OG-L002 LSD1 (~50% at 0.1 µM) or GSK-LSD1 (~30% at 0.1 µM) as compared to control DMSO (~20%) after 5 days. These results suggest that GSK-LSD1 and OG-L002 could be two new promising HbF inducers based on LSD1 inhibition. These findings provide additional evidence to support that LSD1 comprises a useful molecular target for possible therapeutic intervention in treating SCD. Further study will be necessary to address the potential therapeutic effects of the compounds in SCD patients. Disclosures No relevant conflicts of interest to declare.

During Terminal Differentiation, HbF-Inducing Cytokines Cause Decreased Expression and Reduced Globin Locus Occupancy of BCL11A in Human Erythroblasts.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 457-457
Author(s):  
Orapan Sripichai ◽  
Christine M. Kiefer ◽  
Y. Terry Lee ◽  
Emily Riehm Meier ◽  
Colleen Byrnes ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Protein Expression ◽  
Culture Condition ◽  
Conflicts Of Interest ◽  
Association Studies ◽  
Globin Gene ◽  
Terminal Differentiation ◽  
Fetal Hemoglobin ◽  
Beta Globin ◽  
Adult Human

Abstract Abstract 457 Genetic association studies and gene regulation studies demonstrate that the transcription factor BCL11A is a regulator of fetal hemoglobin (HbF) expression in humans. Cytokine signal transduction also regulates fetal hemoglobin expression in cultured adult human erythroblasts. To further explore the potential for BCL11A in the cytokine-mediated induction of HbF during adult erythropoiesis, transcript and protein expression levels of BCL11A were measured during erythroblast differentiation. BCL11A expression was detected at all stages of erythroid differentiation with the highest level expression in proerythroblasts during the first week in culture under both low-HbF (%HbF ≤3) and high-HbF (%HbF ≥30) culture conditions. Despite a reduction in BCL11A mRNA expression, Western analyses failed to demonstrate reduced levels of BCL11A nuclear protein expression at the proerythroblast stage of differentiation. However, BCL11A protein expression in the high-HbF producing cells was reduced relative to the low-HbF cells during the later period of culture as the cells underwent terminal differentiation. During this later culture period, hemoglobinization occurred, and cells grown in the high-HbF condition revealed a pancellular distribution of HbF compared with a heterocellular distribution in the low-HbF culture condition. Chromatin immunoprecipitation further demonstrated that the addition of HbF-inducing cytokines caused a nearly complete loss of BCL11A chromatin occupancy within the beta-globin locus under the high-HbF culture condition. Specifically, the loss of chromatin occupancy was detected in a region approximately 3 kb downstream of the (A)gamma-globin gene. Further examination of this genomic region demonstrated several BCL11A binding domains located on a cluster of non-coding, intronless RNAs previously named “BGL3” that possess an expression pattern in vivo that is largely restricted to the fetal-liver. In addition to increased and pancellular expression of fetal hemoglobin in the high-HbF erythroblasts, the loss of BCL11A chromatin occupancy in that region of the beta-globin locus was associated with increased expression of BGL3 mRNA (GenBank: AY034471) measured by RT-PCR. These findings demonstrate that defined combinations of cytokines regulate the expression level and chromatin occupancy of BCL11A in adult human erythroblasts as they undergo terminal differentiation. In addition to inheritance and ontogeny, the data also support a role for BCL11A in the regulation of HbF by cytokine signal transduction. Disclosures: No relevant conflicts of interest to declare.

KLF1 Dependent Pathways In Developmental Globin Gene Switching

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5162-5162
Author(s):  
Alexander E. Felice
Keyword(s):  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Progenitors ◽  
Gamma Globin ◽  
Genome Wide ◽  
Hereditary Persistence ◽  
Informative Family ◽  
Gene Switching

Abstract Abstract 5162 We provide additional data on members of the family from Malta with Hereditary Persistence of Fetal Hemoglobin (HPFH) due to KLF1 haplo-insufficiency. The data indicated a possible role of additional loci in the pathway of globin gene control. We showed that KLF1 functions as a master regulator of erythropoiesis and developmental globin gene switching (Borg et al., Nature Genetics doi: 10. 1038/ng.630, 2010), at least partly through BCL11A. Given the phenoytpes of the HPFH heterozygotes, the truncating KLF1 p.K288X was best described as a dominant mutation with variable penetrance; most likely due to interplay with other regulatory factors that we have been seeking. Genome-wide association analysis, in the context of genome-wide expression profiles from cultured Human Erythroid Progenitors (HEPs) of critically informative family members, revealed additional loci of potential interest. The effect of the Hb F inducer Hydroxyurea on the gamma globin profiles of the KLF1- (p.K288X) HPFH HEPs was enhanced compared to the wild type, and 74 loci were differentially expressed. It is anticipated that extensive re-sequencing of these new targets may reveal the extent of the molecular pathways under control of KLF1 in erythropoiesis and globin gene switching, and in particular those that may be targeted for therapeutics in patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals High levels of human gamma-globin gene expression in adult mice carrying a transgene of deletion-type hereditary persistence of fetal hemoglobin.

1997 ◽  
Vol 17 (4) ◽  
pp. 2076-2089 ◽  
Author(s):  
M O Arcasoy ◽  
M Romana ◽  
M E Fabry ◽  
E Skarpidi ◽  
R L Nagel ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Dna Sequences ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Gamma Globin ◽  
Transgenic Lines ◽  
Normal Human ◽  
Hereditary Persistence

Persistent expression of the gamma-globin genes in adults with deletion types of hereditary persistence of fetal hemoglobin (HPFH) is thought to be mediated by enhancer-like effects of DNA sequences at the 3' breakpoints of the deletions. A transgenic mouse model of deletion-type HPFH was generated by using a DNA fragment containing both human gamma-globin genes and HPFH-2 breakpoint DNA sequences linked to the core sequences of the locus control region (LCR) of the human beta-globin gene cluster. Analysis of gamma-globin expression in six HPFH transgenic lines demonstrated persistence of gamma-globin mRNA and peptides in erythrocytes of adult HPFH transgenic mice. Analysis of the hemoglobin phenotype of adult HPFH transgenic animals by isoelectric focusing showed the presence of hybrid mouse alpha2-human gamma2 tetramers as well as human gamma4 homotetramers (hemoglobin Bart's). In contrast, correct developmental regulation of the gamma-globin genes with essentially absent gamma-globin gene expression in adult erythroid cells was observed in two control non-HPFH transgenic lines, consistent with autonomous silencing of normal human gamma-globin expression in adult transgenic mice. Interestingly, marked preferential overexpression of the LCR-distal (A)gamma-globin gene but not of the LCR-proximal (G)gamma-globin gene was observed at all developmental stages in erythroid cells of HPFH-2 transgenic mice. These findings were also associated with the formation of a DNase I-hypersensitive site in the HPFH-2 breakpoint DNA of transgenic murine erythroid cells, as occurs in normal human erythroid cells in vivo. These results indicate that breakpoint DNA sequences in deletion-type HPFH-2 can modify the developmentally regulated expression of the gamma-globin genes.

scholarly journals The breakpoint of a large deletion causing hereditary persistence of fetal hemoglobin occurs within an erythroid DNA domain remote from the beta-globin gene cluster

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2178-2186
Author(s):  
EA Feingold ◽  
BG Forget
Keyword(s):  
Globin Gene ◽  
Adult Life ◽  
Fetal Hemoglobin ◽  
Large Deletion ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Enhancer Element ◽  
Gamma Globin ◽  
Hereditary Persistence

The DNA juxtaposed to the gamma-globin genes as a result of a large deletion associated with hereditary persistence of fetal hemoglobin (HPFH) was studied to define the role it may play in maintaining active expression of these genes in adult erythroid cells. The DNA located immediately 3′ to the deletion breakpoint was found to function as an enhancer element in gene transfer experiments and to be specifically hypomethylated in normal erythroid cells of both fetal and adult origin. This DNA also contains a long open reading frame encoding a polypeptide chain 292 amino acids in length. Therefore, in this form of HPFH (HPFH-1), the continued expression of gamma-globin genes in adult life may result from the inclusion of these genes within a new chromosomal domain that is potentially transcriptionally active in adult erythroid cells. The 3′ breakpoint of another large deletion causing delta beta thalassemia rather than HPFH was also identified. This deletion (Spanish G gamma A gamma (delta beta) degrees thalassemia) is nearly identical in size and location to that of HPFH- 1, but extends an additional 8.5 to 9 kb in the 3′direction, and therefore results in loss of the sequences near the 3′ breakpoint of HPFH-1. Thus, the presence of these sequences appears to be important for the expression of the HPFH phenotype.

scholarly journals Effects of butyrate and glucocorticoids on gamma- to beta-globin gene switching in somatic cell hybrids.

1995 ◽  
Vol 15 (2) ◽  
pp. 790-795 ◽  
Author(s):  
G Zitnik ◽  
K Peterson ◽  
G Stamatoyannopoulos ◽  
T Papayannopoulou
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Cellular Systems ◽  
Erythroid Cell ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Cellular Mechanisms ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Gene Switching

Butyrate and its analogs have been shown to induce fetal hemoglobin in humans and primates and in erythroid cell cultures. To obtain insights concerning the cellular mechanisms of butyrate action, we analyzed the effects of butyrate on human globin gene expression in hybrids produced by fusing mouse erythroleukemia cells (MEL) with human fetal erythroid cells (HFE). These hybrids initially express human fetal hemoglobin but subsequently switch to adult globin expression after several weeks in culture. We found that alpha-aminobutyric acid, a butyrate analog which does not induce terminal maturation, strikingly delays the rate of the gamma- to beta-globin gene (gamma-to-beta) switch in the HFE x MEL hybrids. The effect of butyrate on globin expression is transient, with the result that the delay of globin gene switching requires the continuous presence of this compound in culture. Furthermore, butyrate fails to induce fetal hemoglobin expression in hybrids which have switched, suggesting that the effect of this compound on gamma-globin expression is due to inhibition of gamma gene silencing rather than to induction of gamma gene transcription. Since in other cellular systems, glucocorticoids antagonize the action of butyrate, the effect of dexamethasone on the gamma-to-beta switch in HFE x MEL hybrids was examined. Dexamethasone strikingly accelerated the gamma-to-beta switch, and its effect was irreversible. The effects of dexamethasone and butyrate on the gamma-to-beta switch of the HFE x MEL hybrids appear to be codominant. These results indicate that steroids can have a direct effect on globin gene switching in erythroid cells.

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