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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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Model for LCR 5′HS3 gene activation specificity. These illustrations emphasize the interaction of 5′HS3 with a specific globin gene at each developmental stage. Panels A–C represent the interaction of the intact wt LCR with the ε- and γ-globin genes during primitive erythropoiesis (panels A and B) and fetal definitive erythropoiesis (panel C). Panels D–F represent the interaction of the 5′HS3 mutant LCRs with the ε- and γ-globin genes during primitive erythropoiesis (panels D and E) and fetal definitive erythropoiesis (panel F). Panel D shows the effect of the Δ5′HS3 on ε- and γ-globin during primitive erythropoiesis; panels E and F show the effect of the 5′ΔHS3c on these two genes during primitive erythropoiesis and fetal definitive erythropoiesis, respectively. For each panel, the developmental stage and globin gene are indicated at the top. The intact 5′HS3 is shown as a black oval (panels A–C), the complete 5′HS3 deletion (Δ5′HS3) is indicated by a missing oval (panel D) and the Δ5′HS3c is displayed as a hatched oval (panels E and F). The ε- and γ-globin genes are shown as rectangles; the darker color shade for each gene represents the promoter. (A) In the embryonic yolk sac, LCR 5′HS3 is essential for activation of ε-globin gene expression. (B) LCR 5′HS3 is not required for interaction with the γ-globin genes; another LCR 5′HS may be necessary. (C) In the fetal liver, LCR 5′HS3 is essential for activation of γ-globin gene expression. For the complete 5′HS3 and 5′ΔHS3c deletions [(D) and (E), respectively], γ-globin is expressed normally in yolk sac because the 5′HS3-mutated LCRs cannot interact with the ε-globin gene. However, LCR 5′HS3 is required for γ-globin expression during fetal definitive hematopoiesis (F), and in the absence of the 5′HS3 core region, γ-globin levels are markedly reduced. Altogether, this model suggests that HS site-specificity for gene activation is an important determinant of correct developmental expression of the β-like globin genes.
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gks900-F9: Model for LCR 5′HS3 gene activation specificity. These illustrations emphasize the interaction of 5′HS3 with a specific globin gene at each developmental stage. Panels A–C represent the interaction of the intact wt LCR with the ε- and γ-globin genes during primitive erythropoiesis (panels A and B) and fetal definitive erythropoiesis (panel C). Panels D–F represent the interaction of the 5′HS3 mutant LCRs with the ε- and γ-globin genes during primitive erythropoiesis (panels D and E) and fetal definitive erythropoiesis (panel F). Panel D shows the effect of the Δ5′HS3 on ε- and γ-globin during primitive erythropoiesis; panels E and F show the effect of the 5′ΔHS3c on these two genes during primitive erythropoiesis and fetal definitive erythropoiesis, respectively. For each panel, the developmental stage and globin gene are indicated at the top. The intact 5′HS3 is shown as a black oval (panels A–C), the complete 5′HS3 deletion (Δ5′HS3) is indicated by a missing oval (panel D) and the Δ5′HS3c is displayed as a hatched oval (panels E and F). The ε- and γ-globin genes are shown as rectangles; the darker color shade for each gene represents the promoter. (A) In the embryonic yolk sac, LCR 5′HS3 is essential for activation of ε-globin gene expression. (B) LCR 5′HS3 is not required for interaction with the γ-globin genes; another LCR 5′HS may be necessary. (C) In the fetal liver, LCR 5′HS3 is essential for activation of γ-globin gene expression. For the complete 5′HS3 and 5′ΔHS3c deletions [(D) and (E), respectively], γ-globin is expressed normally in yolk sac because the 5′HS3-mutated LCRs cannot interact with the ε-globin gene. However, LCR 5′HS3 is required for γ-globin expression during fetal definitive hematopoiesis (F), and in the absence of the 5′HS3 core region, γ-globin levels are markedly reduced. Altogether, this model suggests that HS site-specificity for gene activation is an important determinant of correct developmental expression of the β-like globin genes.

Mentions: The second hypothesis states that the conformation of the LCR in the ACH is the most important determinant of LCR–globin gene interaction. If this hypothesis is true, than in the embryonic stage, the LCR would be expected to adopt a three-dimensional conformation that favors interaction with the first gene in the complex, the ε-globin gene. Consistent with this hypothesis, following the first switch from ε-globin gene expression in the primitive yolk sac to γ-globin gene expression in the fetal liver, the LCR would be predicted to assume an alternate conformation favorable to γ-globin activation. When 5′HS3 is deleted, an alternate conformation is assumed that decreases the chance that there will be an interaction between the LCR and the ε-globin gene. However, in 5′ΔHS3c mice, the next genes in locus, the γ-globin genes, are expressed (30). Thus, we assume that the LCR must still interact with the γ-globin genes during primitive erythropoiesis. Although γ-globin gene expression is normal during primitive erythropoiesis in these mutant mice, expression is extinguished during the fetal stage of definitive erythropoiesis, in contrast to mice carrying a normal β-YAC construct. These data suggest that a conformational change occurs in the Δ5′HS3c LCR during the switch from embryonic to definitive erythropoiesis, from one that supports γ-globin gene expression to one that does not (Figure 9). Alternately, the embryonic trans-acting environment may allow the mutant LCR to interact with and activate the γ-globin genes, but the fetal trans-acting environment may not support this interaction in the absence of the 5′HS3 core.Figure 9.


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)

Model for LCR 5′HS3 gene activation specificity. These illustrations emphasize the interaction of 5′HS3 with a specific globin gene at each developmental stage. Panels A–C represent the interaction of the intact wt LCR with the ε- and γ-globin genes during primitive erythropoiesis (panels A and B) and fetal definitive erythropoiesis (panel C). Panels D–F represent the interaction of the 5′HS3 mutant LCRs with the ε- and γ-globin genes during primitive erythropoiesis (panels D and E) and fetal definitive erythropoiesis (panel F). Panel D shows the effect of the Δ5′HS3 on ε- and γ-globin during primitive erythropoiesis; panels E and F show the effect of the 5′ΔHS3c on these two genes during primitive erythropoiesis and fetal definitive erythropoiesis, respectively. For each panel, the developmental stage and globin gene are indicated at the top. The intact 5′HS3 is shown as a black oval (panels A–C), the complete 5′HS3 deletion (Δ5′HS3) is indicated by a missing oval (panel D) and the Δ5′HS3c is displayed as a hatched oval (panels E and F). The ε- and γ-globin genes are shown as rectangles; the darker color shade for each gene represents the promoter. (A) In the embryonic yolk sac, LCR 5′HS3 is essential for activation of ε-globin gene expression. (B) LCR 5′HS3 is not required for interaction with the γ-globin genes; another LCR 5′HS may be necessary. (C) In the fetal liver, LCR 5′HS3 is essential for activation of γ-globin gene expression. For the complete 5′HS3 and 5′ΔHS3c deletions [(D) and (E), respectively], γ-globin is expressed normally in yolk sac because the 5′HS3-mutated LCRs cannot interact with the ε-globin gene. However, LCR 5′HS3 is required for γ-globin expression during fetal definitive hematopoiesis (F), and in the absence of the 5′HS3 core region, γ-globin levels are markedly reduced. Altogether, this model suggests that HS site-specificity for gene activation is an important determinant of correct developmental expression of the β-like globin genes.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks900-F9: Model for LCR 5′HS3 gene activation specificity. These illustrations emphasize the interaction of 5′HS3 with a specific globin gene at each developmental stage. Panels A–C represent the interaction of the intact wt LCR with the ε- and γ-globin genes during primitive erythropoiesis (panels A and B) and fetal definitive erythropoiesis (panel C). Panels D–F represent the interaction of the 5′HS3 mutant LCRs with the ε- and γ-globin genes during primitive erythropoiesis (panels D and E) and fetal definitive erythropoiesis (panel F). Panel D shows the effect of the Δ5′HS3 on ε- and γ-globin during primitive erythropoiesis; panels E and F show the effect of the 5′ΔHS3c on these two genes during primitive erythropoiesis and fetal definitive erythropoiesis, respectively. For each panel, the developmental stage and globin gene are indicated at the top. The intact 5′HS3 is shown as a black oval (panels A–C), the complete 5′HS3 deletion (Δ5′HS3) is indicated by a missing oval (panel D) and the Δ5′HS3c is displayed as a hatched oval (panels E and F). The ε- and γ-globin genes are shown as rectangles; the darker color shade for each gene represents the promoter. (A) In the embryonic yolk sac, LCR 5′HS3 is essential for activation of ε-globin gene expression. (B) LCR 5′HS3 is not required for interaction with the γ-globin genes; another LCR 5′HS may be necessary. (C) In the fetal liver, LCR 5′HS3 is essential for activation of γ-globin gene expression. For the complete 5′HS3 and 5′ΔHS3c deletions [(D) and (E), respectively], γ-globin is expressed normally in yolk sac because the 5′HS3-mutated LCRs cannot interact with the ε-globin gene. However, LCR 5′HS3 is required for γ-globin expression during fetal definitive hematopoiesis (F), and in the absence of the 5′HS3 core region, γ-globin levels are markedly reduced. Altogether, this model suggests that HS site-specificity for gene activation is an important determinant of correct developmental expression of the β-like globin genes.
Mentions: The second hypothesis states that the conformation of the LCR in the ACH is the most important determinant of LCR–globin gene interaction. If this hypothesis is true, than in the embryonic stage, the LCR would be expected to adopt a three-dimensional conformation that favors interaction with the first gene in the complex, the ε-globin gene. Consistent with this hypothesis, following the first switch from ε-globin gene expression in the primitive yolk sac to γ-globin gene expression in the fetal liver, the LCR would be predicted to assume an alternate conformation favorable to γ-globin activation. When 5′HS3 is deleted, an alternate conformation is assumed that decreases the chance that there will be an interaction between the LCR and the ε-globin gene. However, in 5′ΔHS3c mice, the next genes in locus, the γ-globin genes, are expressed (30). Thus, we assume that the LCR must still interact with the γ-globin genes during primitive erythropoiesis. Although γ-globin gene expression is normal during primitive erythropoiesis in these mutant mice, expression is extinguished during the fetal stage of definitive erythropoiesis, in contrast to mice carrying a normal β-YAC construct. These data suggest that a conformational change occurs in the Δ5′HS3c LCR during the switch from embryonic to definitive erythropoiesis, from one that supports γ-globin gene expression to one that does not (Figure 9). Alternately, the embryonic trans-acting environment may allow the mutant LCR to interact with and activate the γ-globin genes, but the fetal trans-acting environment may not support this interaction in the absence of the 5′HS3 core.Figure 9.

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
Related in: MedlinePlus