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Mapping polycomb response elements at the Drosophilla melanogaster giant locus.

Abed JA, Cheng CL, Crowell CR, Madigan LL, Onwuegbuchu E, Desai S, Benes J, Jones RS - G3 (Bethesda) (2013)

Bottom Line: Although the sequence requirements for PREs are not well-defined, the presence of Pho, a PRE-binding PcG protein, is a very good PRE indicator.PRE-containing fragments, which coincide with localized presence of Pho in chromatin immunoprecipitations, were shown to maintain restricted expression of a lacZ reporter gene in embryos and to cause pairing-sensitive silencing of the mini-white gene in eyes.Our results also reinforce previous observations that although PRE maintenance and pairing-sensitive silencing activities are closely linked, the sequence requirements for these functions are not identical.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Dedman Life Sciences Building, Southern Methodist University, Dallas, Texas 75275-0376.

ABSTRACT
Polycomb-group (PcG) proteins are highly conserved epigenetic transcriptional regulators. They are capable of either maintaining the transcriptional silence of target genes through many cell cycles or enabling a dynamic regulation of gene expression in stem cells. In Drosophila melanogaster, recruitment of PcG proteins to targets requires the presence of at least one polycomb response element (PRE). Although the sequence requirements for PREs are not well-defined, the presence of Pho, a PRE-binding PcG protein, is a very good PRE indicator. In this study, we identify two PRE-containing regions at the PcG target gene, giant, one at the promoter, and another approximately 6 kb upstream. PRE-containing fragments, which coincide with localized presence of Pho in chromatin immunoprecipitations, were shown to maintain restricted expression of a lacZ reporter gene in embryos and to cause pairing-sensitive silencing of the mini-white gene in eyes. Our results also reinforce previous observations that although PRE maintenance and pairing-sensitive silencing activities are closely linked, the sequence requirements for these functions are not identical.

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The giant inserts tested for their abilities to maintain en-like expression pattern of β-galactosidase. (A) A schematic of gt upstream regulatory region showing fragments gt1–gt5 that were cloned into the SD10 vector and tested for PRE activity The locations of previously mapped gt enhancers gt_(-1), gt_(-3), and gt_(-6) are indicated in brackets. Pho-positive regions are indicated by arrows and correspond to PCR amplified regions 4 and 9 (Figure 1). (B) A schematic representation of the en-lacZ reporter construct SD10. Inserts are flanked by FRT sites. (C) Stage 14 embryos from transgenic lines stained for β-galactosidase expression. Lateral views of embryos are shown, anterior to the left, dorsal up. Transgenic lines tested are indicated to the left of the embryos. Expression patterns are representative of those produced by multiple lines of each construct. However, lines that failed to maintain the en-like pattern exhibited varying degrees of ectopic expression. Embryos containing intact transgenes are on the left. ΔInsert lines (right) are FLP recombinase–mediated deletion derivatives of the same lines shown on the left.
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fig2: The giant inserts tested for their abilities to maintain en-like expression pattern of β-galactosidase. (A) A schematic of gt upstream regulatory region showing fragments gt1–gt5 that were cloned into the SD10 vector and tested for PRE activity The locations of previously mapped gt enhancers gt_(-1), gt_(-3), and gt_(-6) are indicated in brackets. Pho-positive regions are indicated by arrows and correspond to PCR amplified regions 4 and 9 (Figure 1). (B) A schematic representation of the en-lacZ reporter construct SD10. Inserts are flanked by FRT sites. (C) Stage 14 embryos from transgenic lines stained for β-galactosidase expression. Lateral views of embryos are shown, anterior to the left, dorsal up. Transgenic lines tested are indicated to the left of the embryos. Expression patterns are representative of those produced by multiple lines of each construct. However, lines that failed to maintain the en-like pattern exhibited varying degrees of ectopic expression. Embryos containing intact transgenes are on the left. ΔInsert lines (right) are FLP recombinase–mediated deletion derivatives of the same lines shown on the left.

Mentions: The gt fragments were inserted between the en upstream regulatory region and en promoter to produce SD10-gt constructs (Figure 2B). Each insert was flanked by FRT sites to allow precise excision of gt insert by FLP recombinase in vivo. By comparing reporter expression from the intact transgenes and insert-deleted derivatives, it is possible to distinguish regulatory effects of the gt insert from potential position effect of the genomic location of the transgene.


Mapping polycomb response elements at the Drosophilla melanogaster giant locus.

Abed JA, Cheng CL, Crowell CR, Madigan LL, Onwuegbuchu E, Desai S, Benes J, Jones RS - G3 (Bethesda) (2013)

The giant inserts tested for their abilities to maintain en-like expression pattern of β-galactosidase. (A) A schematic of gt upstream regulatory region showing fragments gt1–gt5 that were cloned into the SD10 vector and tested for PRE activity The locations of previously mapped gt enhancers gt_(-1), gt_(-3), and gt_(-6) are indicated in brackets. Pho-positive regions are indicated by arrows and correspond to PCR amplified regions 4 and 9 (Figure 1). (B) A schematic representation of the en-lacZ reporter construct SD10. Inserts are flanked by FRT sites. (C) Stage 14 embryos from transgenic lines stained for β-galactosidase expression. Lateral views of embryos are shown, anterior to the left, dorsal up. Transgenic lines tested are indicated to the left of the embryos. Expression patterns are representative of those produced by multiple lines of each construct. However, lines that failed to maintain the en-like pattern exhibited varying degrees of ectopic expression. Embryos containing intact transgenes are on the left. ΔInsert lines (right) are FLP recombinase–mediated deletion derivatives of the same lines shown on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852391&req=5

fig2: The giant inserts tested for their abilities to maintain en-like expression pattern of β-galactosidase. (A) A schematic of gt upstream regulatory region showing fragments gt1–gt5 that were cloned into the SD10 vector and tested for PRE activity The locations of previously mapped gt enhancers gt_(-1), gt_(-3), and gt_(-6) are indicated in brackets. Pho-positive regions are indicated by arrows and correspond to PCR amplified regions 4 and 9 (Figure 1). (B) A schematic representation of the en-lacZ reporter construct SD10. Inserts are flanked by FRT sites. (C) Stage 14 embryos from transgenic lines stained for β-galactosidase expression. Lateral views of embryos are shown, anterior to the left, dorsal up. Transgenic lines tested are indicated to the left of the embryos. Expression patterns are representative of those produced by multiple lines of each construct. However, lines that failed to maintain the en-like pattern exhibited varying degrees of ectopic expression. Embryos containing intact transgenes are on the left. ΔInsert lines (right) are FLP recombinase–mediated deletion derivatives of the same lines shown on the left.
Mentions: The gt fragments were inserted between the en upstream regulatory region and en promoter to produce SD10-gt constructs (Figure 2B). Each insert was flanked by FRT sites to allow precise excision of gt insert by FLP recombinase in vivo. By comparing reporter expression from the intact transgenes and insert-deleted derivatives, it is possible to distinguish regulatory effects of the gt insert from potential position effect of the genomic location of the transgene.

Bottom Line: Although the sequence requirements for PREs are not well-defined, the presence of Pho, a PRE-binding PcG protein, is a very good PRE indicator.PRE-containing fragments, which coincide with localized presence of Pho in chromatin immunoprecipitations, were shown to maintain restricted expression of a lacZ reporter gene in embryos and to cause pairing-sensitive silencing of the mini-white gene in eyes.Our results also reinforce previous observations that although PRE maintenance and pairing-sensitive silencing activities are closely linked, the sequence requirements for these functions are not identical.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Dedman Life Sciences Building, Southern Methodist University, Dallas, Texas 75275-0376.

ABSTRACT
Polycomb-group (PcG) proteins are highly conserved epigenetic transcriptional regulators. They are capable of either maintaining the transcriptional silence of target genes through many cell cycles or enabling a dynamic regulation of gene expression in stem cells. In Drosophila melanogaster, recruitment of PcG proteins to targets requires the presence of at least one polycomb response element (PRE). Although the sequence requirements for PREs are not well-defined, the presence of Pho, a PRE-binding PcG protein, is a very good PRE indicator. In this study, we identify two PRE-containing regions at the PcG target gene, giant, one at the promoter, and another approximately 6 kb upstream. PRE-containing fragments, which coincide with localized presence of Pho in chromatin immunoprecipitations, were shown to maintain restricted expression of a lacZ reporter gene in embryos and to cause pairing-sensitive silencing of the mini-white gene in eyes. Our results also reinforce previous observations that although PRE maintenance and pairing-sensitive silencing activities are closely linked, the sequence requirements for these functions are not identical.

Show MeSH
Related in: MedlinePlus