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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 shields the TER94 promoter from eve PRE activity.A: map of the eve-TER94 transgene. The 3rd exon of the TER94 protein coding region is fused to that of GFP, while the eve coding region is replaced by that of lacZ. Otherwise, the transgene consists of the entire genomic region from −6.4 kb to +11.3 kb relative to the eve TSS, and includes all of the eve enhancers, plus TER94 enhancers located within its first two introns. The locations of the major eve PRE (“eve PRE”), 3′ insulator (“Homie”), early embryonic stripe enhancers (numbered), and late embryonic enhancers (labeled) are shown as colored boxes. B: embryonic expression, at the indicated stages, of GFP RNA, driven by the TER94 promoter, from the transgene shown in A (top row “intact t'gene”), or the same transgene modified by deletion of either the insulator alone (2nd row “ΔHomie”), the PRE alone (bottom row “ΔPRE”), or both (3rd row “ΔHomie ΔPRE”), each inserted at attP landing site 95E5, visualized by whole-mount in situ hybridization. Note that the normal, ubiquitous expression (mimicking TER94) is changed to resemble the eve pattern (shown for comparison at the bottom) by deletion of Homie, while further deletion of the PRE restores TER94-like expression. Deletion of the PRE alone does not noticeably affect the embryonic pattern.
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pgen-1003883-g001: Homie shields the TER94 promoter from eve PRE activity.A: map of the eve-TER94 transgene. The 3rd exon of the TER94 protein coding region is fused to that of GFP, while the eve coding region is replaced by that of lacZ. Otherwise, the transgene consists of the entire genomic region from −6.4 kb to +11.3 kb relative to the eve TSS, and includes all of the eve enhancers, plus TER94 enhancers located within its first two introns. The locations of the major eve PRE (“eve PRE”), 3′ insulator (“Homie”), early embryonic stripe enhancers (numbered), and late embryonic enhancers (labeled) are shown as colored boxes. B: embryonic expression, at the indicated stages, of GFP RNA, driven by the TER94 promoter, from the transgene shown in A (top row “intact t'gene”), or the same transgene modified by deletion of either the insulator alone (2nd row “ΔHomie”), the PRE alone (bottom row “ΔPRE”), or both (3rd row “ΔHomie ΔPRE”), each inserted at attP landing site 95E5, visualized by whole-mount in situ hybridization. Note that the normal, ubiquitous expression (mimicking TER94) is changed to resemble the eve pattern (shown for comparison at the bottom) by deletion of Homie, while further deletion of the PRE restores TER94-like expression. Deletion of the PRE alone does not noticeably affect the embryonic pattern.

Mentions: In order to analyze the function of the eve 3′ insulator Homie, we employed a pseudo-locus that contains all the regulatory DNA necessary for normal expression of both eve[56]–[59] and the 3′ adjacent gene TER94[60]–[62]. This transgene extends from −6.4 to +11.3 kb relative to the eve TSS, from the 5′-most enhancer of eve to the 3rd exon of TER94. In addition to all of the eve enhancers, this region contains a characterized PRE [51] located just upstream (on the eve side) of Homie [55]. On the other side of Homie is the TER94 promoter and TSS, which are sufficient for ubiquitous expression, augmented by enhancers in the TER94 introns (data not shown). The eve coding region was replaced with lacZ coding DNA, and the 3rd exon of TER94 was fused with the EGFP coding region (Figure 1A). In this study, we make repeated use of a version of recombinase-mediated cassette exchange (RMCE) [63] that allows modified transgenes to be inserted in either orientation at pre-defined chromosomal landing sites. All aspects of transgene expression were consistent for both orientations and at multiple landing sites, with a few minor exceptions (as noted below).


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 shields the TER94 promoter from eve PRE activity.A: map of the eve-TER94 transgene. The 3rd exon of the TER94 protein coding region is fused to that of GFP, while the eve coding region is replaced by that of lacZ. Otherwise, the transgene consists of the entire genomic region from −6.4 kb to +11.3 kb relative to the eve TSS, and includes all of the eve enhancers, plus TER94 enhancers located within its first two introns. The locations of the major eve PRE (“eve PRE”), 3′ insulator (“Homie”), early embryonic stripe enhancers (numbered), and late embryonic enhancers (labeled) are shown as colored boxes. B: embryonic expression, at the indicated stages, of GFP RNA, driven by the TER94 promoter, from the transgene shown in A (top row “intact t'gene”), or the same transgene modified by deletion of either the insulator alone (2nd row “ΔHomie”), the PRE alone (bottom row “ΔPRE”), or both (3rd row “ΔHomie ΔPRE”), each inserted at attP landing site 95E5, visualized by whole-mount in situ hybridization. Note that the normal, ubiquitous expression (mimicking TER94) is changed to resemble the eve pattern (shown for comparison at the bottom) by deletion of Homie, while further deletion of the PRE restores TER94-like expression. Deletion of the PRE alone does not noticeably affect the embryonic pattern.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814318&req=5

pgen-1003883-g001: Homie shields the TER94 promoter from eve PRE activity.A: map of the eve-TER94 transgene. The 3rd exon of the TER94 protein coding region is fused to that of GFP, while the eve coding region is replaced by that of lacZ. Otherwise, the transgene consists of the entire genomic region from −6.4 kb to +11.3 kb relative to the eve TSS, and includes all of the eve enhancers, plus TER94 enhancers located within its first two introns. The locations of the major eve PRE (“eve PRE”), 3′ insulator (“Homie”), early embryonic stripe enhancers (numbered), and late embryonic enhancers (labeled) are shown as colored boxes. B: embryonic expression, at the indicated stages, of GFP RNA, driven by the TER94 promoter, from the transgene shown in A (top row “intact t'gene”), or the same transgene modified by deletion of either the insulator alone (2nd row “ΔHomie”), the PRE alone (bottom row “ΔPRE”), or both (3rd row “ΔHomie ΔPRE”), each inserted at attP landing site 95E5, visualized by whole-mount in situ hybridization. Note that the normal, ubiquitous expression (mimicking TER94) is changed to resemble the eve pattern (shown for comparison at the bottom) by deletion of Homie, while further deletion of the PRE restores TER94-like expression. Deletion of the PRE alone does not noticeably affect the embryonic pattern.
Mentions: In order to analyze the function of the eve 3′ insulator Homie, we employed a pseudo-locus that contains all the regulatory DNA necessary for normal expression of both eve[56]–[59] and the 3′ adjacent gene TER94[60]–[62]. This transgene extends from −6.4 to +11.3 kb relative to the eve TSS, from the 5′-most enhancer of eve to the 3rd exon of TER94. In addition to all of the eve enhancers, this region contains a characterized PRE [51] located just upstream (on the eve side) of Homie [55]. On the other side of Homie is the TER94 promoter and TSS, which are sufficient for ubiquitous expression, augmented by enhancers in the TER94 introns (data not shown). The eve coding region was replaced with lacZ coding DNA, and the 3rd exon of TER94 was fused with the EGFP coding region (Figure 1A). In this study, we make repeated use of a version of recombinase-mediated cassette exchange (RMCE) [63] that allows modified transgenes to be inserted in either orientation at pre-defined chromosomal landing sites. All aspects of transgene expression were consistent for both orientations and at multiple landing sites, with a few minor exceptions (as noted below).

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