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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

Homie blocks PRE action in ovaries.Fluorescence and GFP RNA levels in ovarioles from fly lines carrying the indicated transgenic reporters (described in Figure 1), or no transgene (“no t'gene”). Note the strong fluorescence from TER94 promoter-driven GFP with the intact transgene (“intact t'gene”) at all stages of oogenesis (which proceeds from left to right within each string of ovarioles), and that this is lost when Homie is deleted (“ΔHomie”). Remarkably, strong GFP expression is restored when both Homie and the PRE are deleted (“ΔHomie ΔPRE”), indicating that in the absence of Homie, the PRE is responsible for repression of TER94-GFP. Strong expression is also seen when only the PRE is removed (“ΔPRE”). The graph at the bottom shows, on a log scale, the results of quantitation in triplicate (averages with standard deviations) of GFP RNA from ovaries of lines carrying the indicated transgenes (see Materials and Methods). Note that GFP RNA levels decrease more than 50-fold when Homie is deleted, and are restored by additional deletion of the PRE.
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pgen-1003883-g002: Homie blocks PRE action in ovaries.Fluorescence and GFP RNA levels in ovarioles from fly lines carrying the indicated transgenic reporters (described in Figure 1), or no transgene (“no t'gene”). Note the strong fluorescence from TER94 promoter-driven GFP with the intact transgene (“intact t'gene”) at all stages of oogenesis (which proceeds from left to right within each string of ovarioles), and that this is lost when Homie is deleted (“ΔHomie”). Remarkably, strong GFP expression is restored when both Homie and the PRE are deleted (“ΔHomie ΔPRE”), indicating that in the absence of Homie, the PRE is responsible for repression of TER94-GFP. Strong expression is also seen when only the PRE is removed (“ΔPRE”). The graph at the bottom shows, on a log scale, the results of quantitation in triplicate (averages with standard deviations) of GFP RNA from ovaries of lines carrying the indicated transgenes (see Materials and Methods). Note that GFP RNA levels decrease more than 50-fold when Homie is deleted, and are restored by additional deletion of the PRE.

Mentions: Early, ubiquitous expression of TER94 comes from maternally deposited RNA, based on its early appearance and the fact that TER94 is expressed strongly in developing oocytes [60]–[62]. This was confirmed by staining for transgene expression in the absence of a maternal contribution, which is much weaker at early stages than the maternally derived signal (Figure S2 “intact t'gene”; compare to Figure 1B, Figure S1). Since TER94-GFP RNA is deposited maternally, we examined expression in ovaries. TER94 mRNA is present in both the germline, including nurse cells, and somatic epithelial follicle cells [60]–[62]. No eve expression in ovaries has been reported. In our transgenic lines, strong TER94-GFP expression was seen at all stages of oogenesis (Figure 2 “intact t'gene”, Figure S4) in both germline and somatic epithelial cells (Figure S3 “intact t'gene”). However, the level depended to some extent on chromosomal location (compare Figure 2 and Figure S4). In each case, expression was severely repressed when Homie was deleted (Figure 2 “ΔHomie”, Figure S4). As was seen in embryos, it was restored when the PRE was also deleted (Figure 2 “ΔHomie ΔPRE”, Figure S4). These data confirm that in ovaries, Homie is required for TER94 promoter activity, due to its blocking of PRE-dependent repression.


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 blocks PRE action in ovaries.Fluorescence and GFP RNA levels in ovarioles from fly lines carrying the indicated transgenic reporters (described in Figure 1), or no transgene (“no t'gene”). Note the strong fluorescence from TER94 promoter-driven GFP with the intact transgene (“intact t'gene”) at all stages of oogenesis (which proceeds from left to right within each string of ovarioles), and that this is lost when Homie is deleted (“ΔHomie”). Remarkably, strong GFP expression is restored when both Homie and the PRE are deleted (“ΔHomie ΔPRE”), indicating that in the absence of Homie, the PRE is responsible for repression of TER94-GFP. Strong expression is also seen when only the PRE is removed (“ΔPRE”). The graph at the bottom shows, on a log scale, the results of quantitation in triplicate (averages with standard deviations) of GFP RNA from ovaries of lines carrying the indicated transgenes (see Materials and Methods). Note that GFP RNA levels decrease more than 50-fold when Homie is deleted, and are restored by additional deletion of the PRE.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003883-g002: Homie blocks PRE action in ovaries.Fluorescence and GFP RNA levels in ovarioles from fly lines carrying the indicated transgenic reporters (described in Figure 1), or no transgene (“no t'gene”). Note the strong fluorescence from TER94 promoter-driven GFP with the intact transgene (“intact t'gene”) at all stages of oogenesis (which proceeds from left to right within each string of ovarioles), and that this is lost when Homie is deleted (“ΔHomie”). Remarkably, strong GFP expression is restored when both Homie and the PRE are deleted (“ΔHomie ΔPRE”), indicating that in the absence of Homie, the PRE is responsible for repression of TER94-GFP. Strong expression is also seen when only the PRE is removed (“ΔPRE”). The graph at the bottom shows, on a log scale, the results of quantitation in triplicate (averages with standard deviations) of GFP RNA from ovaries of lines carrying the indicated transgenes (see Materials and Methods). Note that GFP RNA levels decrease more than 50-fold when Homie is deleted, and are restored by additional deletion of the PRE.
Mentions: Early, ubiquitous expression of TER94 comes from maternally deposited RNA, based on its early appearance and the fact that TER94 is expressed strongly in developing oocytes [60]–[62]. This was confirmed by staining for transgene expression in the absence of a maternal contribution, which is much weaker at early stages than the maternally derived signal (Figure S2 “intact t'gene”; compare to Figure 1B, Figure S1). Since TER94-GFP RNA is deposited maternally, we examined expression in ovaries. TER94 mRNA is present in both the germline, including nurse cells, and somatic epithelial follicle cells [60]–[62]. No eve expression in ovaries has been reported. In our transgenic lines, strong TER94-GFP expression was seen at all stages of oogenesis (Figure 2 “intact t'gene”, Figure S4) in both germline and somatic epithelial cells (Figure S3 “intact t'gene”). However, the level depended to some extent on chromosomal location (compare Figure 2 and Figure S4). In each case, expression was severely repressed when Homie was deleted (Figure 2 “ΔHomie”, Figure S4). As was seen in embryos, it was restored when the PRE was also deleted (Figure 2 “ΔHomie ΔPRE”, Figure S4). These data confirm that in ovaries, Homie is required for TER94 promoter activity, due to its blocking of PRE-dependent repression.

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