5'-flanking sequences mediate butyrate stimulation of embryonic globin gene expression in adult erythroid cells

1991 ◽  
Vol 11 (9) ◽  
pp. 4690-4697
Author(s):  
J G Glauber ◽  
N J Wandersee ◽  
J A Little ◽  
G D Ginder
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
Globin Gene ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Adult Chicken ◽  
Beta Globin Gene ◽  
Flanking Sequences ◽  
Murine Erythroleukemia ◽  
Globin Gene Expression

A stable transfection assay was used to test the mechanism by which embryonic globin gene transcription is stimulated in adult erythroid cells exposed to butyric acid and its analogs. To test the appropriate expression and inducibility of chicken globin genes in murine erythroleukemia (MEL) cells, an adult chicken beta-globin gene construct was stably transfected. The chicken beta-globin gene was found to be coregulated with the endogenous adult mouse alpha-globin gene following induction of erythroid differentiation of the transfected MEL cells by incubation with either 2% dimethyl sulfoxide (DMSO) or 1 mM sodium butyrate (NaB). In contrast, a stably transfected embryonic chicken beta-type globin gene, rho, was downregulated during DMSO-induced MEL cell differentiation. However, incubation with NaB, which induces MEL cell differentiation, or alpha-amino butyrate, which does not induce differentiation of MEL cells, resulted in markedly increased levels of transcription from the stably transfected rho gene. Analysis of histone modification showed that induction of rho gene expression was not correlated with increased bulk histone acetylation. A region of 5'-flanking sequence extending from -569 to -725 bp upstream of the rho gene cap site was found to be required for both downregulation of rho gene expression during DMSO-induced differentiation and upregulation by treatment with NaB or alpha-amino butyrate. These data are support for a novel mechanism by which butyrate compounds can alter cellular gene expression through specific DNA sequences. The results reported here are also evidence that 5'-flanking sequences are involved in the suppression of embryonic globin gene expression in terminally differentiated adult erythroid cells.

scholarly journals 5'-flanking sequences mediate butyrate stimulation of embryonic globin gene expression in adult erythroid cells.

1991 ◽  
Vol 11 (9) ◽  
pp. 4690-4697 ◽  
Author(s):  
J G Glauber ◽  
N J Wandersee ◽  
J A Little ◽  
G D Ginder
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
Globin Gene ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Adult Chicken ◽  
Beta Globin Gene ◽  
Flanking Sequences ◽  
Murine Erythroleukemia ◽  
Globin Gene Expression

A stable transfection assay was used to test the mechanism by which embryonic globin gene transcription is stimulated in adult erythroid cells exposed to butyric acid and its analogs. To test the appropriate expression and inducibility of chicken globin genes in murine erythroleukemia (MEL) cells, an adult chicken beta-globin gene construct was stably transfected. The chicken beta-globin gene was found to be coregulated with the endogenous adult mouse alpha-globin gene following induction of erythroid differentiation of the transfected MEL cells by incubation with either 2% dimethyl sulfoxide (DMSO) or 1 mM sodium butyrate (NaB). In contrast, a stably transfected embryonic chicken beta-type globin gene, rho, was downregulated during DMSO-induced MEL cell differentiation. However, incubation with NaB, which induces MEL cell differentiation, or alpha-amino butyrate, which does not induce differentiation of MEL cells, resulted in markedly increased levels of transcription from the stably transfected rho gene. Analysis of histone modification showed that induction of rho gene expression was not correlated with increased bulk histone acetylation. A region of 5'-flanking sequence extending from -569 to -725 bp upstream of the rho gene cap site was found to be required for both downregulation of rho gene expression during DMSO-induced differentiation and upregulation by treatment with NaB or alpha-amino butyrate. These data are support for a novel mechanism by which butyrate compounds can alter cellular gene expression through specific DNA sequences. The results reported here are also evidence that 5'-flanking sequences are involved in the suppression of embryonic globin gene expression in terminally differentiated adult erythroid cells.

scholarly journals A human beta-globin gene fused to the human beta-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1384-1392 ◽  
Author(s):  
I Plavec ◽  
T Papayannopoulou ◽  
C Maury ◽  
F Meyer
Keyword(s):  
Bone Marrow Cells ◽  
Globin Gene ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
In Erythroid Cells

Abstract Retroviral-mediated gene transfer of human beta-globin provides a model system for the development of somatic gene therapy for hemoglobinopathies. Previous work has shown that mice receiving a transplant of bone marrow cells infected with a retroviral vector containing the human beta-globin gene can express human beta-globin specifically in erythroid cells; however, the level of expression of the transduced globin gene was low (1% to 2% per gene copy as compared with that of the endogenous mouse beta-globin gene). We report here the construction of a recombinant retrovirus vector encoding a human beta- globin gene fused to the 4 major regulatory elements of the human beta- globin locus control region (LCR). The LCR cassette increases the level of expression of the globin gene in murine erythroleukemia cells by 10- fold. To study the level of expression in vivo, mouse bone marrow cells were infected with virus-producing cells and the transduced cells were injected into lethally irradiated recipients. In the majority of provirus-containing mice (up to 75%), expression of human beta-globin in peripheral blood was detected at least 3 to 6 months after transplantation. Twelve animals representative of the level of expression of the transduced gene in blood (0.04% to 3.2% of the endogenous mouse beta-globin RNA) were selected for further analysis. A range of 0.4% to 12% of circulating erythrocytes stained positive for human beta-globin protein. Based on these values, the level of expression of the transduced gene per cell was estimated to be 10% to 39% of the endogenous mouse beta-globin gene. These data demonstrate that fusion of the LCR to the beta-globin gene in a retroviral vector increases the level of beta-globin expression in murine erythroleukemia cells and suggest that high-level expression can be obtained in erythroid cells in vivo after transduction into hematopoietic stem cells.

scholarly journals Effect of in vitro DNA methylation on beta-globin gene expression.

1988 ◽  
Vol 85 (13) ◽  
pp. 4638-4642 ◽  
Author(s):  
J. Yisraeli ◽  
D. Frank ◽  
A. Razin ◽  
H. Cedar
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Globin Gene ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Globin Gene Expression

scholarly journals Fetal hemoglobin in sickle cell anemia: relation to regulatory sequences cis to the beta-globin gene. Multicenter Study of Hydroxyurea

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1604-1611 ◽  
Author(s):  
ZH Lu ◽  
MH Steinberg
Keyword(s):  
Gene Expression ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Regulatory Sequences ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Globin Gene Expression

Very different fetal hemoglobin levels among adult sickle cell anemia patients suggest genetic modulation of gamma-globin gene expression. In sickle cell anemia, different fetal hemoglobin levels are associated with distinct beta-globin gene haplotypes. Haplotype may be a marker for linked DNA that modulates gamma-globin gene expression. From 295 individuals with sickle cell anemia, we chose for detailed studies 53 patients who had the highest or the lowest fetal hemoglobin levels and 7 patients whose fetal hemoglobin levels were atypical of their haplotype. In these individuals, we examined portions of the beta- globin gene locus control region hypersensitive sites two and three, an (AT)x(T)y repeat 5′ to the beta-globin gene, a 4-bp deletion 5 to the A gamma T gene, promoters of both gamma-globin genes, 5′ flanking region of the G gamma-globin gene, and A gamma-globin gene IVS-II. Of the regions we studied all polymorphisms were always haplotype-linked and no additional mutations were present. This suggested that variations in these areas are uncommon mechanisms of fetal hemoglobin modulation in sickle cell anemia. Whereas unexamined cis-acting sequences may regulate gamma-globin gene transcription, trans-acting factors may play a more important role.

Specific Activation of Human beta-Globin Gene Expression by the Transcription Factor Ikaros.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3641-3641
Author(s):  
Andrew C. Perkins ◽  
Peter Papathanasiou ◽  
Christopher C. Goodnow ◽  
Janelle R. Keys
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Fetal Liver ◽  
Globin Gene ◽  
Regulatory Control ◽  
Haematopoietic Stem Cell ◽  
Beta Globin ◽  
Relative Expression ◽  
Beta Globin Gene ◽  
Globin Gene Expression

Abstract The zinc finger transcription factor Ikaros is recognized as a key regulator of lymphocyte differentiation. Recently generated dominant negative mutants have hinted at a broader role in haematopoietic stem cell generation. Most recently, a mouse strain, IkarosPlastic, with a point mutation in Ikaros that disrupts DNA binding but preserves efficient assembly of Ikaros protein complexes, is embryonically lethal due to severe defects in erythrocyte differentiation (Papathanasiou P, et al,. Immunity, 2003). (1). These mice display normal murine globin gene expression in the fetal liver. However in humans the globin locus is under alternative regulatory control, particularly with respect to the fetal-to-adult globin switch. Thus, to determine if Ikaros plays a role in human globin switching we crossed the IkarosPlastic mice with mice transgenic for a YAC containing the entire human b-globin locus, which show human fetal to adult globin gene switching from E12 to E17. Embryos were harvested from E12.5 to E15.5 and globin expression was determined in the fetal liver by real-time PCR (relative to actin). At all time points human gamma-globin gene expression was not significantly altered by the presence of the IkarosPlastic mutatation (relative expression Ikaroswt/wt 1±0.11, IkarosPlastic/Plastic 0.82±0.12). In contrast, human beta-globin gene expression was significantly down-regulated in IkarosPlastic fetal livers (relative expression Ikaroswt/wt 1±0.14, IkarosPlastic/Plastic 0.18±0.07). Interestingly, neither murine a- or b-globin gene expression was significantly different to wild type mice, which suggests that the transcription factor Ikaros plays a specific role in the transcriptional activation of the human b-globin gene during development. The mechanism by which this occurs remains to be elucidated, however it is intriguing to consider that Ikaros may act as a potentiator of transcription for erythroid specific transcription factors such as EKLF. Experiments to address this will be presented.

“Definitive” Erythropoiesis Has Distinct Developmental Origins and Globin Expression Patterns in the Mammalian Embryo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2539-2539
Author(s):  
Kathleen E. McGrath ◽  
Jenna M Frame ◽  
George Fromm ◽  
Anne D Koniski ◽  
Paul D Kingsley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Globin Gene ◽  
Yolk Sac ◽  
Globin Genes ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Low Levels ◽  
Globin Gene Expression

Abstract Abstract 2539 Poster Board II-516 A transient wave of primitive erythropoiesis begins at embryonic day 7.5 (E7.5) in the mouse as yolk sac-derived primitive erythroid progenitors (EryP-CFC) generate precursors that mature in the circulation and expand in numbers until E12.5. A second wave of erythroid progenitors (BFU-E) originates in the yolk sac beginning at E8.25 that generate definitive erythroid cells in vitro. These BFU-E colonize the newly forming liver beginning at E10.5, prior to the initial appearance there of adult-repopulating hematopoietic stem cells (HSCs) between E11.5-12.5. This wave of definitive erythroid yolk sac progenitors is proposed to be the source of new blood cells required by the growing embryo after the expansion of primitive erythroid cells has ceased and before HSC-derived hematopoiesis can fulfill the erythropoietic needs of the embryo. We utilized multispectral imaging flow cytometry both to distinguish erythroid lineages and to define specific stages of erythroid precursor maturation in the mouse embryo. Consistent with this model, we found that small numbers of definitive erythrocytes first enter the embryonic circulation beginning at E11.5. All maturational stages of erythroid precursors were observed in the E11.5 liver, consistent with these first definitive erythrocytes having rapidly completed their maturation in the liver. The expression of βH1 and εy-beta globin genes is thought to be limited to primitive erythroid cells. Surprisingly, examination of globin gene expression by in situ hybridization revealed high levels of βH1-, but not εy-globin, transcripts in the parenchyma of E11.5-12.5 livers. RT-PCR analysis of globin mRNAs confirmed the expression of βH1- and adult β1-, but not εy-globin, in E11.5 liver-derived definitive (ckit+, Ter119lo) proerythroblasts sorted by flow cytometry to remove contaminating primitive (ckit-, Ter119+) erythroid cells. A similar pattern of globin gene expression was found in individual definitive erythroid colonies derived from E9.5 yolk sac and from early fetal liver. In vitro differentiation of definitive erythroid progenitors from E9.5 yolk sac revealed a maturational “switch” from βH1- and β1-globins to predominantly β1-globin. βH1-globin transcripts were not observed in proerythroblasts from bone marrow or E16.5 liver or in erythroid colonies from later fetal liver. ChIP analysis revealed that hyperacetylated domains encompass all beta globin genes in primitive erythroid cells but only the adult β1- and β2-globin genes in E16.5 liver proerythroblasts. Consistent with their unique gene expression, E11.5 liver proerythroblasts have hyperacetylated domains encompassing the βh1-, β1- and β2-, but not εy-globin genes. We also examined human globin transgene expression in mice carrying a single copy of the human beta globin locus. Because of the overlapping presence and changing proportion of primitive and definitive erythroid cells during development, we analyzed sorted cell populations whose identities were confirmed by murine globin gene expression. We confirmed that primitive erythroid cells express higher levels of γ- than ε-globin and little β-globin. E11.5 proerythroblasts and cultured E9.5 progenitors express γ- and β-, but not ε-globin. E16.5 liver proerythroblasts express β- and low levels of γ-globin, while adult marrow proerythroblasts express only β-globin transcripts. In summary, two forms of definitive erythropoiesis emerge in the murine embryo, each with distinct globin expression patterns and chromatin modifications of the β-globin locus. While both lineages predominantly express adult globins, the first, yolk sac-derived lineage uniquely expresses low levels of the embryonic βH1-globin gene as well as the human γ-globin transgene. The second definitive erythroid lineage, found in the later fetal liver and postnatal marrow, expresses only adult murine globins as well as low levels of the human γ-globin transgene only in the fetus. Our studies reveal a surprising complexity to the ontogeny of erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

Butyrate Downregulates the Expression of a Number of Repressors of Fetal Hemoglobin In Definitive Erythroid Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2080-2080 ◽  
Author(s):  
Radha Raghupathy ◽  
Shay Karkashon ◽  
Melissa J Fazzari ◽  
Jane A Little
Keyword(s):  
Gene Expression ◽  
Fatty Acids ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Short Chain Fatty Acids ◽  
Short Chain ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Rt Pcr ◽  
Globin Gene Expression

Abstract Abstract 2080 Elevated fetal hemoglobin (HbF) is ameliorative for beta-globin gene disorders. Butyrate, a short chain fatty acid, is a potent inducer of fetal hemoglobin with limited clinical applicability. We wanted to examine non-globin gene targets of butyrate that are regulated in definitive erythroid cells prior to the induction of embryonic/fetal beta-type globin genes. Mechanistic insights may improve clinical utility for short chain fatty acids by identifying novel molecular therapeutic targets. Induced embryonic/fetal globin gene expression is detectable in murine fetal liver-derived definitive erythroid cells (FL EryD) from wildtype and human beta-globin YAC transgenic mice after 19 hours in culture with butyrate & erythropoietin (EPO), but not in EPO alone. Differential regulation of non-globin gene targets in wildtype FL EryD was studied on a Mouse Gene 1.0ST Affymetrix Array after culture in EPO only or butyrate & EPO at 6 hours (when no embryonic globin gene expression is detectable, n=3). Data from biological replicates were normalized by robust multichip average and analyzed with expression console software. As shown in Table 1, several confirmed and putative repressors of embryonic/fetal beta-type globin gene expression, including SOX6, Bcl11A, and Ikaros 1 (but not cMyb) were significantly down regulated by Butyrate at 6 hours (n=3); this was confirmed by RT-PCR. The histone deacetylase inhibitor trichostatin A (TSA), which also induces embryonic globin gene expression in murine FL EryD, has a directionally similar effect (Table 1). Down regulation of some fetal/embryonic globin gene repressors, relative to identically handled EPO-only treated samples, was detectable by RT-PCR as early as 60 to 120 minutes after butyrate induction. These repressors included Bcl11A (60min: 0.66±0.005, p<.001, n=2; 120min: 0.4±0.24, p<.01, n=4), Sox6 (60min: 0.55± 0.18, p=0.08, n=2; 120min: 0.63± 0.06, p<.001, n=4) and Ikaros1 (60min: 0.63±0.45, p=0.36, n=2; 120min: 0.42±0.15, p<.001, n=4). The proximate molecular mechanisms through which butyrate act, while unknown in detail, have been posited to include ‘stress’ signaling via p38 and/or direct activation of gamma-globin gene expression through inhibited histone deacetylation. We found no evidence for butyrate-mediated enhancement of p38 phosphorylation in FL EryD at 0–120 minutes in culture. However, bulk histone acetylation measured by western for histone 3 (H3), was >1.5 fold greater with butyrate induction at 60–90 minutes relative to baseline, while less than baseline in EPO-only treated FL EryD (n=2). Cumulatively, these data suggest that the down regulation by butyrate of major molecular repressors of embryonic/fetal globin gene expression, likely mediated directly or indirectly through epigenetic modifications, is a key underlying mechanism for the induction of fetal hemoglobin in definitive erythroid cells by short chain fatty acids. Disclosures: No relevant conflicts of interest to declare.

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