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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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Related in: MedlinePlus

Pho binds to two regions within the giant cis-regulatory region that colocalizes with peaks of E(z) and Pc distribution. (A) Schematic representation showing the giant genomic region and flanking genes. Regions amplified by PCR in ChIP assays (1–13) are shown. Region 1 and 13 are, respectively, within the CG32797 and tko genes and serve as negative controls. (B) ChIP analysis of Oregon-R embryos 2 h 50 min to 3 h 20 min after egg lay shows two Pho peaks, one close to the gt promoter (region 4) and the other ∼6 kb upstream (region 9). E(z) and Pc, subunits of PRC2 and PRC1, respectively, colocalize with Pho but are more broadly distributed. Preliminary ChIP assays using 33 primer sets spanning this 19-kb region did not show the presence of Pho at additional sites (data not shown). ChIP was performed as indicated with anti-Pho antibody (upper right panel), anti-E(z) antibody (lower left panel), anti-Pc antibody (lower right panel), and rabbit preimmune antiserum for mock (upper left panel). ChIP signals are presented as a percent of input chromatin. Error bars represent SD.
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fig1: Pho binds to two regions within the giant cis-regulatory region that colocalizes with peaks of E(z) and Pc distribution. (A) Schematic representation showing the giant genomic region and flanking genes. Regions amplified by PCR in ChIP assays (1–13) are shown. Region 1 and 13 are, respectively, within the CG32797 and tko genes and serve as negative controls. (B) ChIP analysis of Oregon-R embryos 2 h 50 min to 3 h 20 min after egg lay shows two Pho peaks, one close to the gt promoter (region 4) and the other ∼6 kb upstream (region 9). E(z) and Pc, subunits of PRC2 and PRC1, respectively, colocalize with Pho but are more broadly distributed. Preliminary ChIP assays using 33 primer sets spanning this 19-kb region did not show the presence of Pho at additional sites (data not shown). ChIP was performed as indicated with anti-Pho antibody (upper right panel), anti-E(z) antibody (lower left panel), anti-Pc antibody (lower right panel), and rabbit preimmune antiserum for mock (upper left panel). ChIP signals are presented as a percent of input chromatin. Error bars represent SD.

Mentions: To date, all PREs that have been functionally tested bind Pho (Oktaba et al. 2008; Kassis and Brown 2013). Therefore, the presence of Pho at a given genomic region is a good indicator of the location of a biologically active PRE. To identify gt PREs, we began by precisely mapping the distribution of Pho across a 19-kb region that encompasses the gt locus and extends into flanking genes CG32797 and technical knockout (tko) (Figure 1A). ChIP assays were performed on Oregon-R blastoderm stage embryos using anti-Pho antibodies. Pho was detected at two regions within the gt upstream regulatory region of gt (Figure 1B). The first is near the promoter at the PCR-amplified region 4 (+9 to −168). The second is approximately 6 kb upstream at region 9 (−6108 to −6320) (Figure 1). These results are consistent with a genome-wide ChIP-on-chip study that reported the presence of Pho at the gt locus in embryos (Oktaba et al. 2008). ChIP assays with anti-E(z) and anti-Pc antibodies show colocalization of these PRC2 and PRC1 subunits with Pho, but with broader distributions (Figure 1B). Even though the presence of Pho at these regions is highly suggestive that they contain PREs, in vivo tests, such as PRE maintenance or pairing-sensitive silencing assays, are required to confirm these predictions.


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)

Pho binds to two regions within the giant cis-regulatory region that colocalizes with peaks of E(z) and Pc distribution. (A) Schematic representation showing the giant genomic region and flanking genes. Regions amplified by PCR in ChIP assays (1–13) are shown. Region 1 and 13 are, respectively, within the CG32797 and tko genes and serve as negative controls. (B) ChIP analysis of Oregon-R embryos 2 h 50 min to 3 h 20 min after egg lay shows two Pho peaks, one close to the gt promoter (region 4) and the other ∼6 kb upstream (region 9). E(z) and Pc, subunits of PRC2 and PRC1, respectively, colocalize with Pho but are more broadly distributed. Preliminary ChIP assays using 33 primer sets spanning this 19-kb region did not show the presence of Pho at additional sites (data not shown). ChIP was performed as indicated with anti-Pho antibody (upper right panel), anti-E(z) antibody (lower left panel), anti-Pc antibody (lower right panel), and rabbit preimmune antiserum for mock (upper left panel). ChIP signals are presented as a percent of input chromatin. Error bars represent SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Pho binds to two regions within the giant cis-regulatory region that colocalizes with peaks of E(z) and Pc distribution. (A) Schematic representation showing the giant genomic region and flanking genes. Regions amplified by PCR in ChIP assays (1–13) are shown. Region 1 and 13 are, respectively, within the CG32797 and tko genes and serve as negative controls. (B) ChIP analysis of Oregon-R embryos 2 h 50 min to 3 h 20 min after egg lay shows two Pho peaks, one close to the gt promoter (region 4) and the other ∼6 kb upstream (region 9). E(z) and Pc, subunits of PRC2 and PRC1, respectively, colocalize with Pho but are more broadly distributed. Preliminary ChIP assays using 33 primer sets spanning this 19-kb region did not show the presence of Pho at additional sites (data not shown). ChIP was performed as indicated with anti-Pho antibody (upper right panel), anti-E(z) antibody (lower left panel), anti-Pc antibody (lower right panel), and rabbit preimmune antiserum for mock (upper left panel). ChIP signals are presented as a percent of input chromatin. Error bars represent SD.
Mentions: To date, all PREs that have been functionally tested bind Pho (Oktaba et al. 2008; Kassis and Brown 2013). Therefore, the presence of Pho at a given genomic region is a good indicator of the location of a biologically active PRE. To identify gt PREs, we began by precisely mapping the distribution of Pho across a 19-kb region that encompasses the gt locus and extends into flanking genes CG32797 and technical knockout (tko) (Figure 1A). ChIP assays were performed on Oregon-R blastoderm stage embryos using anti-Pho antibodies. Pho was detected at two regions within the gt upstream regulatory region of gt (Figure 1B). The first is near the promoter at the PCR-amplified region 4 (+9 to −168). The second is approximately 6 kb upstream at region 9 (−6108 to −6320) (Figure 1). These results are consistent with a genome-wide ChIP-on-chip study that reported the presence of Pho at the gt locus in embryos (Oktaba et al. 2008). ChIP assays with anti-E(z) and anti-Pc antibodies show colocalization of these PRC2 and PRC1 subunits with Pho, but with broader distributions (Figure 1B). Even though the presence of Pho at these regions is highly suggestive that they contain PREs, in vivo tests, such as PRE maintenance or pairing-sensitive silencing assays, are required to confirm these predictions.

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