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The Drosophila eve insulator Homie promotes eve expression and protects the adjacent gene from repression by polycomb spreading.

Fujioka M, Sun G, Jaynes JB - PLoS Genet. (2013)

Bottom Line: Ubiquitous TER94 expression is "replaced" by expression in an eve pattern when Homie is deleted, and this effect is reversed when the PRE is also removed.The full activity of the eve promoter is also dependent on Homie, and other insulators can promote normal eve enhancer-promoter communication.Our data suggest that this is not due to preventing promoter competition, but is likely the result of the insulator organizing a chromosomal conformation favorable to normal enhancer-promoter interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and the Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Insulators can block the action of enhancers on promoters and the spreading of repressive chromatin, as well as facilitating specific enhancer-promoter interactions. However, recent studies have called into question whether the activities ascribed to insulators in model transgene assays actually reflect their functions in the genome. The Drosophila even skipped (eve) gene is a Polycomb (Pc) domain with a Pc-group response element (PRE) at one end, flanked by an insulator, an arrangement also seen in other genes. Here, we show that this insulator has three major functions. It blocks the spreading of the eve Pc domain, preventing repression of the adjacent gene, TER94. It prevents activation of TER94 by eve regulatory DNA. It also facilitates normal eve expression. When Homie is deleted in the context of a large transgene that mimics both eve and TER94 regulation, TER94 is repressed. This repression depends on the eve PRE. Ubiquitous TER94 expression is "replaced" by expression in an eve pattern when Homie is deleted, and this effect is reversed when the PRE is also removed. Repression of TER94 is attributable to spreading of the eve Pc domain into the TER94 locus, accompanied by an increase in histone H3 trimethylation at lysine 27. Other PREs can functionally replace the eve PRE, and other insulators can block PRE-dependent repression in this context. The full activity of the eve promoter is also dependent on Homie, and other insulators can promote normal eve enhancer-promoter communication. Our data suggest that this is not due to preventing promoter competition, but is likely the result of the insulator organizing a chromosomal conformation favorable to normal enhancer-promoter interactions. Thus, insulator activities in a native context include enhancer blocking and enhancer-promoter facilitation, as well as preventing the spread of repressive chromatin.

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Homie facilitates eve 3′ enhancer action on the eve promoter through a mechanism that does not involve the TER94 promoter.Expression of lacZ RNA driven by the eve promoter from the transgene diagrammed at the top (described in Figure 1A) and its derivatives was monitored by in situ hybridization. A: Representative embryos are shown at either stage 5 (left column) or stage 13 (middle and right columns, which show two different orientations and focal planes at higher magnification). Note that when Homie is deleted (“ΔHomie”), stripes 1, 4, 5, and 6 are weakened relative to stripes 2, 3, and 7 (left column), while all aspects of expression at later stages, in the mesoderm, CNS, and APR, are also weakened (middle and right columns). These weakened expression elements are all driven by enhancers located between the eve and TER94 promoters. These effects are also seen when both Homie and the PRE are deleted (“ΔHomie ΔPRE”), but not when the PRE alone is deleted (“ΔPRE”). B: Representative embryos are shown at 3 embryonic stages (stages 5, 11, and 13, in the left, middle, and right columns, respectively). In ΔHomie ΔTER94, the entire TER94-GFP gene including the TER94 promoter was removed. Note that the weakened activity of 3′ enhancers seen for ΔHomie is also seen for ΔHomie ΔTER94, indicating that competition with the TER94 promoter is not causing the reduced eve promoter activity.
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pgen-1003883-g005: Homie facilitates eve 3′ enhancer action on the eve promoter through a mechanism that does not involve the TER94 promoter.Expression of lacZ RNA driven by the eve promoter from the transgene diagrammed at the top (described in Figure 1A) and its derivatives was monitored by in situ hybridization. A: Representative embryos are shown at either stage 5 (left column) or stage 13 (middle and right columns, which show two different orientations and focal planes at higher magnification). Note that when Homie is deleted (“ΔHomie”), stripes 1, 4, 5, and 6 are weakened relative to stripes 2, 3, and 7 (left column), while all aspects of expression at later stages, in the mesoderm, CNS, and APR, are also weakened (middle and right columns). These weakened expression elements are all driven by enhancers located between the eve and TER94 promoters. These effects are also seen when both Homie and the PRE are deleted (“ΔHomie ΔPRE”), but not when the PRE alone is deleted (“ΔPRE”). B: Representative embryos are shown at 3 embryonic stages (stages 5, 11, and 13, in the left, middle, and right columns, respectively). In ΔHomie ΔTER94, the entire TER94-GFP gene including the TER94 promoter was removed. Note that the weakened activity of 3′ enhancers seen for ΔHomie is also seen for ΔHomie ΔTER94, indicating that competition with the TER94 promoter is not causing the reduced eve promoter activity.

Mentions: We then tested whether Homie affects eve promoter activity. To do this, we monitored transgenic lacZ expression, which is driven by the eve promoter (Figure 5, Figure S5A,B). When Homie is removed, there is a reduction in expression driven by enhancers located 3′ of the eve coding region. Interestingly, these are the eve enhancers located between Homie and the eve TSS. Comparing “ΔHomie” with the intact transgene at stage 5 (Figure 5 left column), we see that stripes 1, 4, 5, and 6 are weakened relative to stripes 2, 3, and 7. A similar reduction of expression is seen at later stages, where mesodermal, CNS, and APR expression are weakened by deletion of Homie (Figure 5 middle and right columns). This effect is seen at all transgene landing sites tested (Figure 5A, Figure S5A,B), although it varies in strength with the direction of transgene insertion (data not shown). Despite these differences, we consistently see significant disruptions of normal eve expression when Homie is removed.


The Drosophila eve insulator Homie promotes eve expression and protects the adjacent gene from repression by polycomb spreading.

Fujioka M, Sun G, Jaynes JB - PLoS Genet. (2013)

Homie facilitates eve 3′ enhancer action on the eve promoter through a mechanism that does not involve the TER94 promoter.Expression of lacZ RNA driven by the eve promoter from the transgene diagrammed at the top (described in Figure 1A) and its derivatives was monitored by in situ hybridization. A: Representative embryos are shown at either stage 5 (left column) or stage 13 (middle and right columns, which show two different orientations and focal planes at higher magnification). Note that when Homie is deleted (“ΔHomie”), stripes 1, 4, 5, and 6 are weakened relative to stripes 2, 3, and 7 (left column), while all aspects of expression at later stages, in the mesoderm, CNS, and APR, are also weakened (middle and right columns). These weakened expression elements are all driven by enhancers located between the eve and TER94 promoters. These effects are also seen when both Homie and the PRE are deleted (“ΔHomie ΔPRE”), but not when the PRE alone is deleted (“ΔPRE”). B: Representative embryos are shown at 3 embryonic stages (stages 5, 11, and 13, in the left, middle, and right columns, respectively). In ΔHomie ΔTER94, the entire TER94-GFP gene including the TER94 promoter was removed. Note that the weakened activity of 3′ enhancers seen for ΔHomie is also seen for ΔHomie ΔTER94, indicating that competition with the TER94 promoter is not causing the reduced eve promoter activity.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814318&req=5

pgen-1003883-g005: Homie facilitates eve 3′ enhancer action on the eve promoter through a mechanism that does not involve the TER94 promoter.Expression of lacZ RNA driven by the eve promoter from the transgene diagrammed at the top (described in Figure 1A) and its derivatives was monitored by in situ hybridization. A: Representative embryos are shown at either stage 5 (left column) or stage 13 (middle and right columns, which show two different orientations and focal planes at higher magnification). Note that when Homie is deleted (“ΔHomie”), stripes 1, 4, 5, and 6 are weakened relative to stripes 2, 3, and 7 (left column), while all aspects of expression at later stages, in the mesoderm, CNS, and APR, are also weakened (middle and right columns). These weakened expression elements are all driven by enhancers located between the eve and TER94 promoters. These effects are also seen when both Homie and the PRE are deleted (“ΔHomie ΔPRE”), but not when the PRE alone is deleted (“ΔPRE”). B: Representative embryos are shown at 3 embryonic stages (stages 5, 11, and 13, in the left, middle, and right columns, respectively). In ΔHomie ΔTER94, the entire TER94-GFP gene including the TER94 promoter was removed. Note that the weakened activity of 3′ enhancers seen for ΔHomie is also seen for ΔHomie ΔTER94, indicating that competition with the TER94 promoter is not causing the reduced eve promoter activity.
Mentions: We then tested whether Homie affects eve promoter activity. To do this, we monitored transgenic lacZ expression, which is driven by the eve promoter (Figure 5, Figure S5A,B). When Homie is removed, there is a reduction in expression driven by enhancers located 3′ of the eve coding region. Interestingly, these are the eve enhancers located between Homie and the eve TSS. Comparing “ΔHomie” with the intact transgene at stage 5 (Figure 5 left column), we see that stripes 1, 4, 5, and 6 are weakened relative to stripes 2, 3, and 7. A similar reduction of expression is seen at later stages, where mesodermal, CNS, and APR expression are weakened by deletion of Homie (Figure 5 middle and right columns). This effect is seen at all transgene landing sites tested (Figure 5A, Figure S5A,B), although it varies in strength with the direction of transgene insertion (data not shown). Despite these differences, we consistently see significant disruptions of normal eve expression when Homie is removed.

Bottom Line: Ubiquitous TER94 expression is "replaced" by expression in an eve pattern when Homie is deleted, and this effect is reversed when the PRE is also removed.The full activity of the eve promoter is also dependent on Homie, and other insulators can promote normal eve enhancer-promoter communication.Our data suggest that this is not due to preventing promoter competition, but is likely the result of the insulator organizing a chromosomal conformation favorable to normal enhancer-promoter interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and the Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Insulators can block the action of enhancers on promoters and the spreading of repressive chromatin, as well as facilitating specific enhancer-promoter interactions. However, recent studies have called into question whether the activities ascribed to insulators in model transgene assays actually reflect their functions in the genome. The Drosophila even skipped (eve) gene is a Polycomb (Pc) domain with a Pc-group response element (PRE) at one end, flanked by an insulator, an arrangement also seen in other genes. Here, we show that this insulator has three major functions. It blocks the spreading of the eve Pc domain, preventing repression of the adjacent gene, TER94. It prevents activation of TER94 by eve regulatory DNA. It also facilitates normal eve expression. When Homie is deleted in the context of a large transgene that mimics both eve and TER94 regulation, TER94 is repressed. This repression depends on the eve PRE. Ubiquitous TER94 expression is "replaced" by expression in an eve pattern when Homie is deleted, and this effect is reversed when the PRE is also removed. Repression of TER94 is attributable to spreading of the eve Pc domain into the TER94 locus, accompanied by an increase in histone H3 trimethylation at lysine 27. Other PREs can functionally replace the eve PRE, and other insulators can block PRE-dependent repression in this context. The full activity of the eve promoter is also dependent on Homie, and other insulators can promote normal eve enhancer-promoter communication. Our data suggest that this is not due to preventing promoter competition, but is likely the result of the insulator organizing a chromosomal conformation favorable to normal enhancer-promoter interactions. Thus, insulator activities in a native context include enhancer blocking and enhancer-promoter facilitation, as well as preventing the spread of repressive chromatin.

Show MeSH
Related in: MedlinePlus