Research ArticleGENETICS

Structure and function of an ectopic Polycomb chromatin domain

See allHide authors and affiliations

Science Advances  09 Jan 2019:
Vol. 5, no. 1, eaau9739
DOI: 10.1126/sciadv.aau9739
  • Fig. 1 Characterization of en80.

    (A) ChIP-seq distribution of Pho, Ph, and H3K27me3 at the inv-en domain in WT (rows 1 to 3), en80 (rows 4 to 6), HAen79@attP40 enΔ110 (rows 7 to 9), and HAen79@attP3; enΔ110 (rows 10 to 12) is shown. Inv and en strong, constitutive PREs are indicated by red stars, and weak PcG peaks are indicated by green stars. The region deleted in en80 is highlighted with a crossed box. ChIP-seq signals from transgenes are within the red dashed box. Positions of the genes (navy blue) are shown at the bottom. The arrows indicate the direction of the transcription. The genomic coordinates are shown at the top (FlyBase R5). (B) The extent of genomic DNA present in the HAen79 transgene is shown (2R:7386838–7466000). (C) Zoomed-in view of the en upstream region. The ChIP-seq scale has been readjusted to highlight the weak peaks; H3K27me3 data are not included. At one of the small peaks, Pho and Ph are strongly reduced in size in HAen79@attP40 and HAen79@attP3 in comparison with en80 (red dotted box). All transgenes and en deletions are homozygous.

  • Fig. 2 H3K27me3 spreads to regions flanking the HAen79 insertion sites.

    The ChIP-seq distribution of Pho, Ph, H3K27me3, H3K27me3 significant enrichment, and H3K36me3 at attP40 in en80 (top five rows) and HAen79@attP40 en∆110 (bottom five rows) (A) and at attP3 in en80 (top five rows) and HAen79@attP3; en∆110 (bottom five rows) (B) is shown. The attP40 (A) or attP3 (B) insertion site is indicated with a red dashed line. A schematic of the transgene inserted at the sites is shown above (not to scale). Note that the scale of Pho and Ph ChIP-seq is the same as that in Fig. 1C. There are small but significant Pho and Ph peaks at the attP40 insertion site and nearby. In contrast, there are only very weak Pho or Ph site in the region of attP3. All transgenes and en deletions are homozygous.

  • Fig. 3 Active and repressed chromatin regions are segregated efficiently in different chromatin contexts.

    (A) 4C interaction map at attP40 of en80 and HAen79@attP40 en∆110. Significant 4C interactions are shown under the interaction profile. Distribution of H3K36me3 at the insertion site is shown in the bottom row. enPREs interacts significantly with flanking chromatin; schematic of interactions of attP40 flanking sequences with the enPRE bait is shown on top with black arrows, and this interaction intensity is abruptly reduced at active chromatin (indicated with red arrowheads). (B) 4C interaction map at attP3 of HAen79@attP3; en∆110. Significant 4C interactions are shown under the interaction profile. Distribution of H3K36me3 surrounding the insertion site is shown in the bottom row. enPREs interacts significantly with flanking chromatin; schematic of interactions of attP3 flanking sequences with the enPRE bait is shown on top with black arrows, and this interaction intensity is abruptly reduced at active chromatin (indicated with red arrowheads). Positions of the genes (navy blue) are shown at the bottom of (A) and (B). All transgenes and en deletions are homozygous.

  • Fig. 4 The strong, constitutive PREs are important for silencing of the HAen79 transgene.

    (A) View of adult female and male abdomen. Genotypes of the flies are indicated above the abdominal pictures. ph is present on the X chromosome of Drosophila genome. (B) Top: Schematic diagram of the transgenic lines used for this experiment. Strong PREs are highlighted with red vertical lines. Green double-headed arrows highlight Drosophila somatic chromosome pairing in interphase nucleus. Bottom: View of adult female and male abdomen of HAen79∆1.5@attP40/enPREs@attP40 (left), HAen79∆1.5@attP40/vector alone@attP40 (middle), and HAen79∆1.5@attP40/PRED@attP40 (right) is shown. (C) Top: Schematic diagram of the transgenic lines used for this experiment (females; males only have one X chromosome); strong PREs are highlighted with red vertical lines. The flies all contain a WT inv-en domain (boxed). Photo credit: S.D., NICHD, NIH.

  • Fig. 5 en strong PREs are essential at attP40.

    (A) Immunostaining with anti-HA and anti-Inv antibody in HAen79@attP40/+ (top) and HAen79∆1.5@attP40/+ (bottom) embryos, ventral view of central nervous system, and anterior in left corner. Photo credit: J.A.K., NICHD, NIH. (B) Quantification of H3K27me3 over the mini-y gene and ~1 kb upstream and downstream of the attP40 site in HAen79 and HAen79∆1.5 larval brains and discs; homozygous for the transgenes and for WT inv-en domains.

  • Fig. 6 en enhancers from HAen79 transgene can drive flanking gene expression.

    (A) Schematic diagram of the HAen79@attP40 line at the attP40 insertion site. H3K36me3 accumulation is highlighted with green color; arrows indicate the direction of transcription. (B) RNA in situ hybridization with w (top box) and y (bottom box) probe in embryos of HAen79@attP40 (homozygous) and vector only @attP40 (homozygous). (C) Schematic diagram of the HAen79@attP3 line at the attP3 insertion site (not to scale). Arrows indicate the direction of transcription. (D) RNA in situ hybridization with w (top box), y (middle box), and CG1504 (bottom box) probe in embryos of HAen79@attP3 (homozygous) and vector only @attP3 (homozygous). In each box, stages 11 or 12 embryo (top) and stage 13 embryo (bottom). All embryos are lateral views, anterior left, dorsal up. Photo credit: N.D.G., NICHD, NIH.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/1/eaau9739/DC1

    Fig. S1. Distribution of active and repressive epigenetic marks at breakpoint of WT and T(2;3)enEs/enX31.

    Fig. S2. Distribution of active and repressive marks at ectopic sites.

    Fig. S3. Expression of HA-En and HA-EnSTOP from HAen79 or HAen79STOP.

    Fig. S4. en80 flies are homozygous viable and fertile and express En correctly.

    Fig. S5. The 79-kb HA-en transgene sets up a 3D PcG domain at ectopic sites.

    Fig. S6. H3K27me3 spreads beyond the HAen79 transgene to the marker genes.

    Fig. S7. 4C interactions between the enPREs and flanking DNA in T(2;3)enEs/enX31 larvae.

    Fig. S8. H3K27me3 distribution in en80∆1.5.

    Fig. S9. RNA in situ of genes flanking the attP40 and attP3 sites.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Distribution of active and repressive epigenetic marks at breakpoint of WT and T(2;3)enEs/enX31.
    • Fig. S2. Distribution of active and repressive marks at ectopic sites.
    • Fig. S3. Expression of HA-En and HA-EnSTOP from HAen79 or HAen79STOP.
    • Fig. S4. en80 flies are homozygous viable and fertile and express En correctly.
    • Fig. S5. The 79-kb HA-en transgene sets up a 3D PcG domain at ectopic sites.
    • Fig. S6. H3K27me3 spreads beyond the HAen79 transgene to the marker genes.
    • Fig. S7. 4C interactions between the enPREs and flanking DNA in T(2;3)enEs/enX31 larvae.
    • Fig. S8. H3K27me3 distribution in en80∆1.5.
    • Fig. S9. RNA in situ of genes flanking the attP40 and attP3 sites.

    Download PDF

    Files in this Data Supplement:

Navigate This Article