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LCR 5' hypersensitive site specificity for globin gene activation within the active chromatin hub.

Peterson KR, Fedosyuk H, Harju-Baker S - Nucleic Acids Res. (2012)

Bottom Line: To distinguish between these possibilities, human β-globin locus yeast artificial chromosome (β-YAC) lines were produced in which the ε-globin gene was replaced with a second marked β-globin gene (β(m)), coupled to an intact LCR, a 5'HS3 complete deletion (5'ΔHS3) or a 5'HS3 core deletion (5'ΔHS3c).Although the 5'HS3 core was not required for β(m)-globin expression, previous work showed that the 5'HS3 core is necessary for ε-globin expression during embryonic erythropoiesis.These data support a site specificity model of LCR HS-globin gene interaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA. kpeterson@kumc.edu

ABSTRACT
The DNaseI hypersensitive sites (HSs) of the human β-globin locus control region (LCR) may function as part of an LCR holocomplex within a larger active chromatin hub (ACH). Differential activation of the globin genes during development may be controlled in part by preferential interaction of each gene with specific individual HSs during globin gene switching, a change in conformation of the LCR holocomplex, or both. To distinguish between these possibilities, human β-globin locus yeast artificial chromosome (β-YAC) lines were produced in which the ε-globin gene was replaced with a second marked β-globin gene (β(m)), coupled to an intact LCR, a 5'HS3 complete deletion (5'ΔHS3) or a 5'HS3 core deletion (5'ΔHS3c). The 5'ΔHS3c mice expressed β(m)-globin throughout development; γ-globin was co-expressed in the embryonic yolk sac, but not in the fetal liver; and wild-type β-globin was co-expressed in adult mice. Although the 5'HS3 core was not required for β(m)-globin expression, previous work showed that the 5'HS3 core is necessary for ε-globin expression during embryonic erythropoiesis. A similar phenotype was observed in 5'HS complete deletion mice, except β(m)-globin expression was higher during primitive erythropoiesis and γ-globin expression continued into fetal definitive erythropoiesis. These data support a site specificity model of LCR HS-globin gene interaction.

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γ- to β-globin gene switching during development in wt and Δ5′HS3c Δε::βm β-YAC transgenic mice. RPA was utilized as described in the ‘Materials and Methods’ section to generate these data. y-axis, percent human globin, [γ/(γ+β) × 100]; x-axis, developmental day, days post-conception or adult. Square and solid line, γ-globin, Δ5′HS3c Δε::βm β-YAC; diamond and dashed line, β-globin, Δ5′HS3c Δε::βm β-YAC; triangle and dotted line, γ-globin, wt β-YAC; circle and dot-dashed line, β-globin, wt β-YAC.
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gks900-F4: γ- to β-globin gene switching during development in wt and Δ5′HS3c Δε::βm β-YAC transgenic mice. RPA was utilized as described in the ‘Materials and Methods’ section to generate these data. y-axis, percent human globin, [γ/(γ+β) × 100]; x-axis, developmental day, days post-conception or adult. Square and solid line, γ-globin, Δ5′HS3c Δε::βm β-YAC; diamond and dashed line, β-globin, Δ5′HS3c Δε::βm β-YAC; triangle and dotted line, γ-globin, wt β-YAC; circle and dot-dashed line, β-globin, wt β-YAC.

Mentions: The γ-globin to β-globin switch was also completed much earlier during development in Δ5′HS3c Δε::βm mice compared with wt β-YAC mice, essentially when the site of hematopoiesis moved from the yolk sac to the fetal liver between Days E11 and E12 (Figure 4). This observation supports our results above and previous data (30) demonstrating that 5′HS3 is required for γ-globin synthesis during fetal liver definitive erythropoiesis. When the 5′HS3 core is deleted, the switch to β-globin expression is completed rapidly during the transition from embryonic to definitive erythropoiesis. In contrast to published Δ5′HS3c β-YAC lines, where the γ-globin gene was highly expressed in the primitive yolk sac (30), Δ5′HS3c Δε::βm mice express lower levels of γ-globin mRNA (10–15%; Figure 5A), suggesting that the presence of the βm-globin gene competitively inhibits γ-globin gene expression. This observation agrees with our previously published experiments, in which we analyzed the effect of gene order on temporal regulation of β-like globin gene expression (46).Figure 4.


LCR 5' hypersensitive site specificity for globin gene activation within the active chromatin hub.

Peterson KR, Fedosyuk H, Harju-Baker S - Nucleic Acids Res. (2012)

γ- to β-globin gene switching during development in wt and Δ5′HS3c Δε::βm β-YAC transgenic mice. RPA was utilized as described in the ‘Materials and Methods’ section to generate these data. y-axis, percent human globin, [γ/(γ+β) × 100]; x-axis, developmental day, days post-conception or adult. Square and solid line, γ-globin, Δ5′HS3c Δε::βm β-YAC; diamond and dashed line, β-globin, Δ5′HS3c Δε::βm β-YAC; triangle and dotted line, γ-globin, wt β-YAC; circle and dot-dashed line, β-globin, wt β-YAC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526258&req=5

gks900-F4: γ- to β-globin gene switching during development in wt and Δ5′HS3c Δε::βm β-YAC transgenic mice. RPA was utilized as described in the ‘Materials and Methods’ section to generate these data. y-axis, percent human globin, [γ/(γ+β) × 100]; x-axis, developmental day, days post-conception or adult. Square and solid line, γ-globin, Δ5′HS3c Δε::βm β-YAC; diamond and dashed line, β-globin, Δ5′HS3c Δε::βm β-YAC; triangle and dotted line, γ-globin, wt β-YAC; circle and dot-dashed line, β-globin, wt β-YAC.
Mentions: The γ-globin to β-globin switch was also completed much earlier during development in Δ5′HS3c Δε::βm mice compared with wt β-YAC mice, essentially when the site of hematopoiesis moved from the yolk sac to the fetal liver between Days E11 and E12 (Figure 4). This observation supports our results above and previous data (30) demonstrating that 5′HS3 is required for γ-globin synthesis during fetal liver definitive erythropoiesis. When the 5′HS3 core is deleted, the switch to β-globin expression is completed rapidly during the transition from embryonic to definitive erythropoiesis. In contrast to published Δ5′HS3c β-YAC lines, where the γ-globin gene was highly expressed in the primitive yolk sac (30), Δ5′HS3c Δε::βm mice express lower levels of γ-globin mRNA (10–15%; Figure 5A), suggesting that the presence of the βm-globin gene competitively inhibits γ-globin gene expression. This observation agrees with our previously published experiments, in which we analyzed the effect of gene order on temporal regulation of β-like globin gene expression (46).Figure 4.

Bottom Line: To distinguish between these possibilities, human β-globin locus yeast artificial chromosome (β-YAC) lines were produced in which the ε-globin gene was replaced with a second marked β-globin gene (β(m)), coupled to an intact LCR, a 5'HS3 complete deletion (5'ΔHS3) or a 5'HS3 core deletion (5'ΔHS3c).Although the 5'HS3 core was not required for β(m)-globin expression, previous work showed that the 5'HS3 core is necessary for ε-globin expression during embryonic erythropoiesis.These data support a site specificity model of LCR HS-globin gene interaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA. kpeterson@kumc.edu

ABSTRACT
The DNaseI hypersensitive sites (HSs) of the human β-globin locus control region (LCR) may function as part of an LCR holocomplex within a larger active chromatin hub (ACH). Differential activation of the globin genes during development may be controlled in part by preferential interaction of each gene with specific individual HSs during globin gene switching, a change in conformation of the LCR holocomplex, or both. To distinguish between these possibilities, human β-globin locus yeast artificial chromosome (β-YAC) lines were produced in which the ε-globin gene was replaced with a second marked β-globin gene (β(m)), coupled to an intact LCR, a 5'HS3 complete deletion (5'ΔHS3) or a 5'HS3 core deletion (5'ΔHS3c). The 5'ΔHS3c mice expressed β(m)-globin throughout development; γ-globin was co-expressed in the embryonic yolk sac, but not in the fetal liver; and wild-type β-globin was co-expressed in adult mice. Although the 5'HS3 core was not required for β(m)-globin expression, previous work showed that the 5'HS3 core is necessary for ε-globin expression during embryonic erythropoiesis. A similar phenotype was observed in 5'HS complete deletion mice, except β(m)-globin expression was higher during primitive erythropoiesis and γ-globin expression continued into fetal definitive erythropoiesis. These data support a site specificity model of LCR HS-globin gene interaction.

Show MeSH