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

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.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4690 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4690-4697 ◽

Cited By ~ 74

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.

Download Full-text

The role of trans-acting factors and DNA-bending in the silencing of human beta-globin gene expression

Nucleic Acids Research ◽

10.1093/nar/28.14.2823 ◽

2000 ◽

Vol 28 (14) ◽

pp. 2823-2830 ◽

Cited By ~ 6

Author(s):

L. R. Drew

Keyword(s):

Gene Expression ◽

Globin Gene ◽

Dna Bending ◽

Beta Globin ◽

Beta Globin Gene ◽

Globin Gene Expression

Download Full-text

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.85.13.4638 ◽

1988 ◽

Vol 85 (13) ◽

pp. 4638-4642 ◽

Cited By ~ 39

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

Download Full-text

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

Blood ◽

10.1182/blood.v87.4.1604.bloodjournal8741604 ◽

1996 ◽

Vol 87 (4) ◽

pp. 1604-1611 ◽

Cited By ~ 36

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.

Download Full-text

Induction of HLA class I and beta globin gene expression by IFN alpha and IFN gamma in K562 cells: A proposed mechanism for differential regulation

Human Immunology ◽

10.1016/0198-8859(96)85508-x ◽

1996 ◽

Vol 47 (1-2) ◽

pp. 149

Author(s):

Chatterjee Moitreyee ◽

Agrawal Suraksha ◽

S. Agarwal Shyam

Keyword(s):

Gene Expression ◽

Globin Gene ◽

Hla Class I ◽

K562 Cells ◽

Differential Regulation ◽

Beta Globin ◽

Ifn Gamma ◽

Beta Globin Gene ◽

Ifn Alpha ◽

Globin Gene Expression

Download Full-text

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

Blood ◽

10.1182/blood.v114.22.2539.2539 ◽

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.

Download Full-text