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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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Related in: MedlinePlus

Other Drosophila insulators block eve PRE action, and other PREs are blocked by Homie.A: fluorescence from transgenic TER94-GFP in the context of the transgene diagrammed in Figure 1A at the 95E5 landing site, with Homie replaced by either λ phage DNA (“ΔHomie”) or the indicated insulator. Diagrams below each panel show the arrangement of regulatory elements affecting GFP expression: yellow-filled boxes are PREs, either from eve (unlabeled), en, or the bxd region of Ubx, as indicated; red-filled boxes are insulators, either Homie (unlabeled), or as labeled. Note that each of these insulators restore TER94 promoter activity, although scs does so when inserted in one orientation (“scs(inv)”) but not the other (“scs”) relative to the TER94-GFP promoter. Note also that restoration of GFP expression is somewhat weaker for scs' and scs (even in the “inverted” orientation) than for gypsy and Fab-7, indicating that they only partially block eve PRE action. B: same as in A, except that the transgenes were inserted in opposite orientation at the same landing site relative to that in A, with either the gypsy insulator or the Fab-8 insulator. Note that eve PRE action is blocked in both cases, causing strong fluorescence. C: fluorescence from transgenic TER94-GFP in the context of the transgene diagrammed in Figure 1A, with Homie deleted, and with either the eve PRE deleted (“ΔHomie ΔPRE”), or replaced by the bxd PRE (“+ bxd PRE”) or the en PRE (“+ en PRE”). Note that each of these PREs repress TER94-GFP to a similar degree as does the eve PRE in the absence of an insulator (compare to “ΔHomie” panels in A and B, where the eve PRE is present). D: fluorescence from transgenic TER94-GFP in the context of the transgene diagrammed in Figure 1A, with the eve PRE either removed (“ΔPRE”), or replaced by either the bxd PRE (“+ bxd PRE”) or the en PRE (“+ en PRE”). Note that neither of these PREs is able to repress TER94-GFP when Homie is present, showing that Homie blocks the repressive action of these heterologous PREs.
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pgen-1003883-g003: Other Drosophila insulators block eve PRE action, and other PREs are blocked by Homie.A: fluorescence from transgenic TER94-GFP in the context of the transgene diagrammed in Figure 1A at the 95E5 landing site, with Homie replaced by either λ phage DNA (“ΔHomie”) or the indicated insulator. Diagrams below each panel show the arrangement of regulatory elements affecting GFP expression: yellow-filled boxes are PREs, either from eve (unlabeled), en, or the bxd region of Ubx, as indicated; red-filled boxes are insulators, either Homie (unlabeled), or as labeled. Note that each of these insulators restore TER94 promoter activity, although scs does so when inserted in one orientation (“scs(inv)”) but not the other (“scs”) relative to the TER94-GFP promoter. Note also that restoration of GFP expression is somewhat weaker for scs' and scs (even in the “inverted” orientation) than for gypsy and Fab-7, indicating that they only partially block eve PRE action. B: same as in A, except that the transgenes were inserted in opposite orientation at the same landing site relative to that in A, with either the gypsy insulator or the Fab-8 insulator. Note that eve PRE action is blocked in both cases, causing strong fluorescence. C: fluorescence from transgenic TER94-GFP in the context of the transgene diagrammed in Figure 1A, with Homie deleted, and with either the eve PRE deleted (“ΔHomie ΔPRE”), or replaced by the bxd PRE (“+ bxd PRE”) or the en PRE (“+ en PRE”). Note that each of these PREs repress TER94-GFP to a similar degree as does the eve PRE in the absence of an insulator (compare to “ΔHomie” panels in A and B, where the eve PRE is present). D: fluorescence from transgenic TER94-GFP in the context of the transgene diagrammed in Figure 1A, with the eve PRE either removed (“ΔPRE”), or replaced by either the bxd PRE (“+ bxd PRE”) or the en PRE (“+ en PRE”). Note that neither of these PREs is able to repress TER94-GFP when Homie is present, showing that Homie blocks the repressive action of these heterologous PREs.

Mentions: Are the functions of Homie seen in our assays unique, or are they shared among insulators? In order to test this, we replaced Homie with other known insulators. As a negative control, a >500 bp stretch of λ phage DNA was tested. It had no effect on repression of the TER94 promoter by the eve PRE (Figure 3A “ΔHomie”). In contrast, other characterized Drosophila insulators can substitute for Homie to block repression. gypsy (Figure 3A, B), Fab-7, scs' (Figure 3A), and Fab-8 (Figure 3B) each prevented TER94 promoter repression. Although in one orientation, scs did not work (Figure 3A, “+ scs”), it did work in the opposite orientation (Figure 3A, “+ scs(inv)”). Fab-8 and gypsy showed a minor directionality in their effectiveness (not shown). Restoration of GFP expression is somewhat weaker for scs' and scs(inv) than for gypsy and Fab-7, indicating that they only partially block eve PRE action. Despite differences in efficiency, blocking of PRE action in this context is a shared property of insulators.


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)

Other Drosophila insulators block eve PRE action, and other PREs are blocked by Homie.A: fluorescence from transgenic TER94-GFP in the context of the transgene diagrammed in Figure 1A at the 95E5 landing site, with Homie replaced by either λ phage DNA (“ΔHomie”) or the indicated insulator. Diagrams below each panel show the arrangement of regulatory elements affecting GFP expression: yellow-filled boxes are PREs, either from eve (unlabeled), en, or the bxd region of Ubx, as indicated; red-filled boxes are insulators, either Homie (unlabeled), or as labeled. Note that each of these insulators restore TER94 promoter activity, although scs does so when inserted in one orientation (“scs(inv)”) but not the other (“scs”) relative to the TER94-GFP promoter. Note also that restoration of GFP expression is somewhat weaker for scs' and scs (even in the “inverted” orientation) than for gypsy and Fab-7, indicating that they only partially block eve PRE action. B: same as in A, except that the transgenes were inserted in opposite orientation at the same landing site relative to that in A, with either the gypsy insulator or the Fab-8 insulator. Note that eve PRE action is blocked in both cases, causing strong fluorescence. C: fluorescence from transgenic TER94-GFP in the context of the transgene diagrammed in Figure 1A, with Homie deleted, and with either the eve PRE deleted (“ΔHomie ΔPRE”), or replaced by the bxd PRE (“+ bxd PRE”) or the en PRE (“+ en PRE”). Note that each of these PREs repress TER94-GFP to a similar degree as does the eve PRE in the absence of an insulator (compare to “ΔHomie” panels in A and B, where the eve PRE is present). D: fluorescence from transgenic TER94-GFP in the context of the transgene diagrammed in Figure 1A, with the eve PRE either removed (“ΔPRE”), or replaced by either the bxd PRE (“+ bxd PRE”) or the en PRE (“+ en PRE”). Note that neither of these PREs is able to repress TER94-GFP when Homie is present, showing that Homie blocks the repressive action of these heterologous PREs.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003883-g003: Other Drosophila insulators block eve PRE action, and other PREs are blocked by Homie.A: fluorescence from transgenic TER94-GFP in the context of the transgene diagrammed in Figure 1A at the 95E5 landing site, with Homie replaced by either λ phage DNA (“ΔHomie”) or the indicated insulator. Diagrams below each panel show the arrangement of regulatory elements affecting GFP expression: yellow-filled boxes are PREs, either from eve (unlabeled), en, or the bxd region of Ubx, as indicated; red-filled boxes are insulators, either Homie (unlabeled), or as labeled. Note that each of these insulators restore TER94 promoter activity, although scs does so when inserted in one orientation (“scs(inv)”) but not the other (“scs”) relative to the TER94-GFP promoter. Note also that restoration of GFP expression is somewhat weaker for scs' and scs (even in the “inverted” orientation) than for gypsy and Fab-7, indicating that they only partially block eve PRE action. B: same as in A, except that the transgenes were inserted in opposite orientation at the same landing site relative to that in A, with either the gypsy insulator or the Fab-8 insulator. Note that eve PRE action is blocked in both cases, causing strong fluorescence. C: fluorescence from transgenic TER94-GFP in the context of the transgene diagrammed in Figure 1A, with Homie deleted, and with either the eve PRE deleted (“ΔHomie ΔPRE”), or replaced by the bxd PRE (“+ bxd PRE”) or the en PRE (“+ en PRE”). Note that each of these PREs repress TER94-GFP to a similar degree as does the eve PRE in the absence of an insulator (compare to “ΔHomie” panels in A and B, where the eve PRE is present). D: fluorescence from transgenic TER94-GFP in the context of the transgene diagrammed in Figure 1A, with the eve PRE either removed (“ΔPRE”), or replaced by either the bxd PRE (“+ bxd PRE”) or the en PRE (“+ en PRE”). Note that neither of these PREs is able to repress TER94-GFP when Homie is present, showing that Homie blocks the repressive action of these heterologous PREs.
Mentions: Are the functions of Homie seen in our assays unique, or are they shared among insulators? In order to test this, we replaced Homie with other known insulators. As a negative control, a >500 bp stretch of λ phage DNA was tested. It had no effect on repression of the TER94 promoter by the eve PRE (Figure 3A “ΔHomie”). In contrast, other characterized Drosophila insulators can substitute for Homie to block repression. gypsy (Figure 3A, B), Fab-7, scs' (Figure 3A), and Fab-8 (Figure 3B) each prevented TER94 promoter repression. Although in one orientation, scs did not work (Figure 3A, “+ scs”), it did work in the opposite orientation (Figure 3A, “+ scs(inv)”). Fab-8 and gypsy showed a minor directionality in their effectiveness (not shown). Restoration of GFP expression is somewhat weaker for scs' and scs(inv) than for gypsy and Fab-7, indicating that they only partially block eve PRE action. Despite differences in efficiency, blocking of PRE action in this context is a shared property of insulators.

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