scholarly journals Erythroid Krüppel-Like Factor Is Essential for β-Globin Gene Expression Even in Absence of Gene Competition, But Is Not Sufficient to Induce the Switch From γ-Globin to β-Globin Gene Expression

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2259-2263
Author(s):  
Louis-Georges Guy ◽  
Qi Mei ◽  
Andrew C. Perkins ◽  
Stuart H. Orkin ◽  
Lee Wall
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transgenic Mice ◽  
Fetal Development ◽  
Adult Stage ◽  
Globin Gene ◽  
Globin Genes ◽  
Developmental Switch ◽  
Determining Factor ◽  
Globin Gene Expression

Different genes in the β-like globin locus are expressed at specific times during development. This is controlled, in part, by competition between the genes for activation by the locus control region. In mice, gene inactivation of the erythroid Krüppel-like factor (EKLF) transcription factor results in a lethal anemia due to a specific and substantial decrease in expression of the fetal/adult-stage–specific β-globin gene. In transgenic mice carrying the complete human β-globin locus, EKLF ablation not only impairs human β-globin–gene expression but also results in increased expression of the human γ-globin genes during the fetal/adult stages. Hence, it may appear that EKLF is a determining factor for the developmental switch from γ-globin to β-globin transcription. However, we show here that the function of EKLF for β-globin–gene expression is necessary even in absence of gene competition. Moreover, EKLF is not developmental specific and is present and functional before the switch from γ-globin to β-globin–gene expression occurs. Thus, EKLF is not the primary factor that controls the switch. We suggest that autonomous repression of γ-globin transcription that occurs during late fetal development is likely to be the initiating event that induces the switch.

scholarly journals Erythroid Krüppel-Like Factor Is Essential for β-Globin Gene Expression Even in Absence of Gene Competition, But Is Not Sufficient to Induce the Switch From γ-Globin to β-Globin Gene Expression

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2259-2263 ◽  
Author(s):  
Louis-Georges Guy ◽  
Qi Mei ◽  
Andrew C. Perkins ◽  
Stuart H. Orkin ◽  
Lee Wall
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transgenic Mice ◽  
Fetal Development ◽  
Adult Stage ◽  
Globin Gene ◽  
Globin Genes ◽  
Developmental Switch ◽  
Determining Factor ◽  
Globin Gene Expression

Abstract Different genes in the β-like globin locus are expressed at specific times during development. This is controlled, in part, by competition between the genes for activation by the locus control region. In mice, gene inactivation of the erythroid Krüppel-like factor (EKLF) transcription factor results in a lethal anemia due to a specific and substantial decrease in expression of the fetal/adult-stage–specific β-globin gene. In transgenic mice carrying the complete human β-globin locus, EKLF ablation not only impairs human β-globin–gene expression but also results in increased expression of the human γ-globin genes during the fetal/adult stages. Hence, it may appear that EKLF is a determining factor for the developmental switch from γ-globin to β-globin transcription. However, we show here that the function of EKLF for β-globin–gene expression is necessary even in absence of gene competition. Moreover, EKLF is not developmental specific and is present and functional before the switch from γ-globin to β-globin–gene expression occurs. Thus, EKLF is not the primary factor that controls the switch. We suggest that autonomous repression of γ-globin transcription that occurs during late fetal development is likely to be the initiating event that induces the switch.

Comparison of Expression of Human Globin Genes Transferred Into Mouse Erythroleukemia Cells and in Transgenic Mice

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3416-3421 ◽  
Author(s):  
E. Skarpidi ◽  
G. Vassilopoulos ◽  
G. Stamatoyannopoulos ◽  
Q. Li
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Globin Gene ◽  
Mrna Levels ◽  
Globin Genes ◽  
Position Effects ◽  
Erythroleukemia Cells ◽  
Cell Clones ◽  
Globin Gene Expression ◽  
Mouse Erythroleukemia

To examine whether transfer of γ globin genes into mouse erythroleukemia cells can be used for the analysis of regulatory elements of γ globin gene promoter, Aγ gene constructs carrying promoter truncations that have been previously analyzed in transgenic mice were used for production of stably transfected mouse erythroleukemia (MEL) cell clones and pools. We found that constructs, which contain a microlocus control region (μLCR) that efficiently protects globin gene expression from the effects of the position of integration in transgenic mice, display position-dependent globin gene expression in MEL cell clones. Aγ globin gene expression among MEL cell clones carrying the μLCR(−201)Aγ and μLCR(−382)Aγ gene constructs ranged 15.5-fold and 17.6-fold, respectively, and there was no correlation between theAγ mRNA levels and the copies of the transgene (r= .28, P = .18). There was significant variation in per copy Aγ globin gene expression among MEL cell pools composed of 10 clones, but not among pools composed of 50 clones, indicating that position effects are averaged in pools composed by large numbers of clones. The overall pattern of Aγ globin gene expression in MEL cell pools resembled that observed in transgenic mice indicating that MEL cell transfections can be used in the study ofcis elements controlling γ globin gene expression. MEL cell transfections, however, are not appropriate for investigation of cis elements, which either sensitize or protect the globin transgenes from position effects. © 1998 by The American Society of Hematology.

scholarly journals Investigations of a Human Embryonic Globin Gene Silencing Element Using YAC Transgenic Mice

2006 ◽  
Vol 231 (3) ◽  
pp. 328-334 ◽  
Author(s):  
Patrick A. Navas ◽  
Qiliang Li ◽  
Kenneth R. Peterson ◽  
George Stamatoyannopoulos
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Yeast Artificial Chromosome ◽  
Globin Gene ◽  
Gene Promoter ◽  
Developmental Expression ◽  
Mrna Levels ◽  
Globin Genes ◽  
Embryonic Cells ◽  
Globin Gene Expression

A silencing element has been previously located upstream of the human ε-globin gene promoter using transient assays and transgenic mice carrying plasmid constructs in which the element has been deleted or its transcriptional motifs have been mutated. To investigate whether this element functions in the context of the whole β-globin locus, we analyzed ε-globin gene expression in transgenic mice carrying a deletion of the silencing element in the context of a 213-kilobase human β-globin yeast artificial chromosome (β-YAC). ε-Globin gene expression was measured during embryonic and fetal development and in adult mice. ε-mRNA levels in embryonic cells in Day 12 blood were as high as those measured in wild-type β-YAC controls, indicating that the deletion does not affect ε gene promoter function. ε-Globin gene expression was confined to the embryonic cells, indicating that deletion of this silencing element did not affect ε-globin developmental expression in the context of the β-YAC. These results suggest that in the context of the whole β-globin locus, other proximal and upstream ε gene promoter elements as well as competition by the downstream globin genes contribute to the silencing of the ε-globin gene in the cells of definitive erythropoiesis.

Comparison of Expression of Human Globin Genes Transferred Into Mouse Erythroleukemia Cells and in Transgenic Mice

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3416-3421 ◽  
Author(s):  
E. Skarpidi ◽  
G. Vassilopoulos ◽  
G. Stamatoyannopoulos ◽  
Q. Li
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Globin Gene ◽  
Mrna Levels ◽  
Globin Genes ◽  
Position Effects ◽  
Erythroleukemia Cells ◽  
Cell Clones ◽  
Globin Gene Expression ◽  
Mouse Erythroleukemia

Abstract To examine whether transfer of γ globin genes into mouse erythroleukemia cells can be used for the analysis of regulatory elements of γ globin gene promoter, Aγ gene constructs carrying promoter truncations that have been previously analyzed in transgenic mice were used for production of stably transfected mouse erythroleukemia (MEL) cell clones and pools. We found that constructs, which contain a microlocus control region (μLCR) that efficiently protects globin gene expression from the effects of the position of integration in transgenic mice, display position-dependent globin gene expression in MEL cell clones. Aγ globin gene expression among MEL cell clones carrying the μLCR(−201)Aγ and μLCR(−382)Aγ gene constructs ranged 15.5-fold and 17.6-fold, respectively, and there was no correlation between theAγ mRNA levels and the copies of the transgene (r= .28, P = .18). There was significant variation in per copy Aγ globin gene expression among MEL cell pools composed of 10 clones, but not among pools composed of 50 clones, indicating that position effects are averaged in pools composed by large numbers of clones. The overall pattern of Aγ globin gene expression in MEL cell pools resembled that observed in transgenic mice indicating that MEL cell transfections can be used in the study ofcis elements controlling γ globin gene expression. MEL cell transfections, however, are not appropriate for investigation of cis elements, which either sensitize or protect the globin transgenes from position effects. © 1998 by The American Society of Hematology.

scholarly journals Developmental Specificity of the Interaction between the Locus Control Region and Embryonic or Fetal Globin Genes in Transgenic Mice with an HS3 Core Deletion

1998 ◽  
Vol 18 (7) ◽  
pp. 4188-4196 ◽  
Author(s):  
Patrick A. Navas ◽  
Kenneth R. Peterson ◽  
Qiliang Li ◽  
Eva Skarpidi ◽  
Alex Rohde ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Gene Transcription ◽  
Globin Gene ◽  
Globin Genes ◽  
Embryonic Cells ◽  
Core Element ◽  
The Core ◽  
Core Elements ◽  
Globin Gene Expression

ABSTRACT The human β-globin locus control region (LCR) consists of five erythroid-lineage-specific DNase I-hypersensitive sites (HSs) and is required for activation of the β-globin locus chromatin domain and globin gene expression. Each DNase I-HS of the LCR consists of a highly conserved core element and flanking sequences. To analyze the functional role of the core elements of the HSs, we deleted a 234-bp fragment encompassing the core of HS3 (HS3c) from a β-globin locus residing on a 248-kb β-locus yeast artificial chromosome and analyzed its function in F2 progeny of transgenic mice. Human ɛ-globin gene expression was absent at day 10 and severely reduced in the day 12 embryonic erythropoiesis of mice lacking HS3c. In contrast, γ-globin gene expression was normal in embryonic erythropoiesis but it was absent in definitive erythropoiesis in the fetal liver. These results indicate that the core element of HS3 is necessary for ɛ-globin gene transcription in embryonic cells and for γ-globin gene transcription in definitive cells. Normal γ-globin gene expression in embryonic cells and the absence of γ-globin gene expression in definitive cells show that different HSs interact with γ-globin gene promoters in these two stages of development. Such results provide direct evidence for developmental stage specificity of the interactions between the core elements of HSs and the promoters of the globin genes.

In Vivo and In Vitro Analysis of an Aγ-Globin Gene Silencer in Human β-Yac Transgenic Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1220-1220
Author(s):  
Susanna Harju ◽  
Kenneth R. Peterson
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Fetal Liver ◽  
Globin Gene ◽  
Yolk Sac ◽  
Luciferase Reporter ◽  
Globin Genes ◽  
Mobility Shift ◽  
Gene Switching ◽  
Globin Gene Expression

Abstract Autonomous silencing of gene expression is one mechanism operative in the control of human β-like globin gene switching and is best exemplified by the ε-globin gene. Experiments using variously truncated Aγ-globin genes linked to LCR sequences suggested that a region of the Aγ-globin gene between −730 to −378 relative to the mRNA CAP site may function as an adult stage-specific silencer element. A 5.4 Kb marked Aγ-globin gene (Aγm) inserted between LCR 5′HS1 and the ε-globin gene in a β-YAC (Aγm 5′ ε β-YAC) was silenced in transgenic mice during adult definitive erythropoiesis, even in the absence of an adult β-globin gene. In contrast, when a marked β-globin gene (βm) was inserted in this same location in another β-YAC (βm 5′ ε β-YAC), the βm-globin gene was expressed throughout ontogeny. From these data we concluded that: 1) any gene located near the LCR will be strongly expressed throughout ontogeny, unless some gene-specific silencing mechanism exists, 2) competition between the γ- and β-globin genes for interaction with the LCR is not the exclusive mechanism controlling γ- to β-globin gene switching, and 3) that the Aγm-globin gene was autonomously silenced. A -730 to -378 deletion of the Aγm-globin gene was introduced into the Aγm 5′ ε β-YAC via homologous recombination to produce a Δ1s Aγm 5′ ε β-YAC. This YAC was microinjected and six founders were obtained. Four transgenic lines were established carrying at least one full-length β-globin locus and two were established that lacked the adult β-globin gene. All founders containing an intact β-globin gene expressed the Δ1s Aγm-globin during adult erythropoiesis (45% – 122% relative to human β-globin expression). In one line examined in detail, the Δ1s Aγm-globin gene was expressed in the embryonic yolk sac, fetal liver, and adult blood. ε-globin gene expression was not detected in the embryonic yolk sac and expression of the normally located γ-globin genes was not observed at any developmental stage. β-globin gene expression was observed in the fetal liver and adult blood, although its expression was decreased. To further delineate the function of the Δ1s fragment, transient transfection assays to test silencer function and protein-DNA interaction assays were performed. Silencer activity of the352 bp Δ1s fragment was examined using a series of pGL2 luciferase reporter plasmids that were synthesized to include the Δ1s fragment; these were electroporated into various cells. Electrophoretic mobility shift assays (EMSAs) and DNAse I footprinting were employed to begin assessment of protein binding within the Δ1s fragment. A 50 bp DNA fragment spanning −713 to −664 of the Δ1s element was used in EMSAs; DNA binding activity was observed in K562 nuclear extracts. These preliminary data suggest that the −730 to −378 γ-globin gene silencer binds a repressor protein complex.

scholarly journals Mi2β-mediated silencing of the fetal γ-globin gene in adult erythroid cells

Blood ◽  
2013 ◽  
Vol 121 (17) ◽  
pp. 3493-3501 ◽  
Author(s):  
Maria Amaya ◽  
Megha Desai ◽  
Merlin Nithya Gnanapragasam ◽  
Shou Zhen Wang ◽  
Sheng Zu Zhu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Major Part ◽  
Globin Gene ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Key Points ◽  
Globin Gene Expression ◽  
Partial Depletion

Key Points Mi2β exerts a major part of its silencing effect on embryonic and fetal globin genes by positively regulating the BCL11A and KLF1 genes. Partial depletion of Mi2β induces increased γ-globin gene expression in primary human erythroid cells without impairing differentiation.

Developmental regulation of human gamma-globin genes in transgenic mice

1993 ◽  
Vol 13 (12) ◽  
pp. 7636-7644 ◽  
Author(s):  
G Stamatoyannopoulos ◽  
B Josephson ◽  
J W Zhang ◽  
Q Li
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Gene Silencing ◽  
Copy Number ◽  
Adult Stage ◽  
Gene Promoter ◽  
Globin Genes ◽  
Gamma Globin ◽  
Stage Of Development ◽  
Upstream Promoter

We report results showing that several gamma gene promoter elements participate in the developmental control of gamma-globin genes. Four gamma gene constructs with 5' truncated at -141, -201, -382, and -730 of the A gamma gene promoter linked to a micro locus control region (microLCR) cassette were used for production of transgenic mice and analysis of gamma gene expression during development. Mice carrying a microLCR -141 A gamma construct displayed downregulation of gamma gene expression in the adult stage of development, indicating that the proximal promoter contains elements participating in gamma gene silencing. Mice carrying a microLCR -201 A gamma or a microLCR -382 A gamma construct displayed high gamma gene expression in the fetal stage of development and complete loss of gamma gene downregulation in the adult stage, suggesting that the -141 to -201 gamma gene sequence contains elements which upregulate gamma gene expression and are dominant over the negative element 3' to -141. Extension of the promoter to -730 resulted in reappearance of gamma gene downregulation, suggesting that the -382 to -730 sequences contain an adult-stage-specific silencer. gamma gene expression in the microLCR -201 A gamma and the microLCR -382 A gamma transgenic mice was copy number dependent. All the microLCR -730 A gamma transgenic mice expressed gamma mRNA; however, gamma gene expression was copy number independent, indicating that levels of gamma gene expression were modulated by the surrounding chromatin. Our results suggest that multiple elements participate in gamma gene silencing. The findings in the microLCR-201 A gamma and microLCR -382 A gamma transgenic mice are interpreted to indicate that the LCR interacts not only with the minimal gamma gene promoter but also with sequences of the upstream promoter. We postulate that gamma gene downregulation is achieved when the interaction between LCR and the upstream promoter is disturbed by the silencer located in the -382 to -730 region. We propose that gamma gene silencing is achieved by the combined effect of negative elements located 3' to -141, the negative element located between -382 and -730, and the competition by the beta gene promoter during the adult stage of development.

scholarly journals Regulatory factors specific for adult and embryonic globin genes may govern their expression in erythroleukemia cells

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 705-712 ◽  
Author(s):  
NP Anagnou ◽  
TY Yuan ◽  
E Lim ◽  
J Helder ◽  
S Wieder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Chromosome ◽  
Globin Gene ◽  
Globin Genes ◽  
Chromosome 16 ◽  
Regulatory Factors ◽  
Erythroleukemia Cells ◽  
Erythroleukemia Cell ◽  
Globin Gene Expression ◽  
Mouse Erythroleukemia

Abstract In order to test if trans-acting regulatory factors specific for globin genes of the adult and embryonic stages of development exist in erythroid cells, transcriptionally active embryonic and adult globin genes on the same chromosome were transferred by cell fusion from the human leukemia cell K562 into phenotypically adult mouse erythroleukemia cells. Restriction-fragment-length polymorphisms of the K562 zeta (embryonic) globin genes were used to establish that all three copies of human chromosome 16 present in the K562 cell showed the same pattern of human globin gene expression after transfer to the mouse erythroleukemia cell. Adult (alpha) but not embryonic (zeta) human globin mRNA was detected in all nine of the independently derived mouse erythroleukemia hybrid cells, each of which contained human chromosome 16. Restriction endonuclease studies of the K562 alpha- and zeta-globin genes after transfer into the mouse erythroleukemia cell showed no evidence of rearrangements or deletions that could explain this loss of zeta-globin gene expression. These data suggest that regulation of globin gene expression in these erythroleukemia cells involves trans-acting regulatory factors specific for the adult and embryonic stages of development.

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