Research ArticleGENETICS

Polycomb repressive complex 1 modifies transcription of active genes

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Science Advances  02 Aug 2017:
Vol. 3, no. 8, e1700944
DOI: 10.1126/sciadv.1700944
  • Fig. 1 PRC1 modifies transcription of active genes in BG3 cells.

    (A) Effects of Ph depletion (iPh) on transcript accumulation (total RNA-seq) and transcription (3′ NT-seq). The left dot plot shows the log2 fold change in RNA levels from expressed genes upon Ph depletion versus the log2 RNA level in the mock-treated control cells using total RNA-seq. Red points are genes in which the change in total RNA levels caused by Ph depletion is significant at a false discovery rate of ≤5% (q ≤ 0.05). The right dot plot shows the same by 3′ NT-seq. Orange indicates where the change is statistically significant (q ≤ 0.05). (B) Both dot plots show the fold change in RNA accumulation (total RNA) versus that in transcription (3′ NT-seq) upon Ph depletion (iPh). The two graphs are identical except that the left shows which changes are significant by total RNA-seq (red dots) and the right shows those significant by 3′ NT-seq (orange dots). (C) The left dot plot shows the fold change in RNA upon Ph depletion (iPh) versus the fold change upon Sce depletion (iSce) by total RNA-seq. Red dots show the changes that are statistically significant (q ≤ 0.05) upon Ph depletion. The right dot plot shows the same by 3′ NT-seq. Orange dots show changes that are statistically significant (q ≤ 0.05) upon Ph depletion. (D) Genome browser view of the bithorax complex shows occupancy by H3K27me3 [modENCODE ChIP-chip; Gene Expression Omnibus (GEO) GSE20780] and Sce (modENCODE ChIP-chip; GEO GSE20817) in BG3 cells; Pc, Ph, and Rpb3 ChIP-seq enrichment in control cells (this study); Rpb3 ChIP-seq in cells depleted for Sce (Rpb3 iSce) and Ph (Rpb3 iPh); and 3′ NT-seq data for mock control cells and cells depleted for Sce or Ph. The ChIP-chip enrichment scale is MAT score, and the ChIP-seq data scale is log2 enrichment. The RNA-seq data scales are log2 nucleotide coverage (density). (E) Enrichment of genes that change in transcript accumulation or transcription upon Ph depletion for those that have high levels of Ph. Increases (UP) and decreases (DOWN) in RNA levels were defined as those with q ≤ 0.05. P values were calculated using Fisher’s exact test.

  • Fig. 2 PRC1 subunit depletion alters the binding of other subunits to active genes, silenced genes, and regulatory sequences in BG3 cells.

    (A) Metagene analysis of Ph and Pc ChIP-seq enrichment for four gene categories, using 16 bins from −20 to +140% of annotated gene length. Ph ChIP-seq was conducted in mock control cells (blue line), cells depleted for Sce (iSce; red line), and cells depleted for Pc (iPc; purple line). Pc ChIP-seq was conducted for mock control cells (blue line), cells depleted for Sce (red line), and cells depleted for Ph (iPh; green line). Statistical tests of the differences in enrichment between the mock control and PRC1 subunit–depleted cells are in table S2. (B) Effects of PRC1 subunit depletion on the binding of other PRC1 subunits to active promoters (promoters, 7389), extragenic enhancers (enhancers, 523), PREs (195), and extragenic PREs positioned outside of regions that become transcribed upon Ph or Sce depletion (exPREs; 90). The left top panel shows the violin plot distributions of Ph enrichment at active promoters, extragenic enhancers, PREs, extragenic PREs (exPREs), and 6892 random 500–base pair (bp) regions as a control. The red line indicates no enrichment (log2 enrichment = 0). The top right panel shows the same for Pc. The left panel in the second row shows the Ph enrichment at PREs and extragenic PREs in mock control cells and cells depleted for Sce (iSce) and Pc (iPc). The blue line indicates the median enrichment level in mock control cells. The right panel in the second row shows the Pc enrichment at PREs and extragenic PREs in mock control cells and cells depleted for Sce and Ph. The left panel in the third row shows the Ph enrichment at active promoters in mock control, Sce-depleted, and Pc-depleted cells. The right panel in the third row shows the enrichment for Pc at active promoters in mock, Sce-depleted, and Ph-depleted cells. The left panel in the fourth row shows the Ph occupancy of extragenic enhancers in mock control, Sce-depleted, and Pc-depleted cells, and the right panel shows Pc enrichment at enhancers in mock, Sce-depleted, and Ph-depleted cells. All effects on Ph binding to regulatory sequences are also illustrated by dot plots in fig. S2, and all effects on Pc binding are shown in fig. S3. (C) Example Western blot showing levels of Ph, Pc, and H2Aub in whole-cell extracts of mock control, Sce-depleted (iSce), Ph-depleted (iPh), and Pc-depleted (iPc) cells. The top asterisk (*) indicates Ph protein produced by the ph-p gene, and the lower asterisk indicates di-ubiquitinylated H2A (H2Aub2) (10). Ph and Pc depletions often enhance the levels of these variants, but the degree of enhancement varies between experiments. (D) Table summarizing the effects of depleting Sce, Ph, and Pc on Sce, Ph, Pc, and H2Aub levels by Western blot and ChIP-seq at various regulatory sequences and genes. Thick blue arrows indicate large decreases, thin blue arrows indicate weak to moderate decreases, thick red arrows indicate large increases, and thin red arrows indicate weak to moderate increases. Tildes (~) indicate no obvious change, and gray boxes indicate that the levels were not measured.

  • Fig. 3 PRC1 subunits alter transcription (3′ NT-seq) and Pol II (Rpb3) occupancy of active genes.

    (A) Metagene analysis of Rpb3 ChIP-seq (left) and 3′ NT-seq (right) for three active gene categories for mock control (blue lines), Sce-depleted (iSce; red lines), Ph-depleted (iPh; green lines), and Pc-depleted (iPc; purple lines) cells. Statistical tests of the enrichment differences between mock control and PRC1 subunit–depleted cells are in table S2. (B) Top: Ratio of 3′ NT-seq metagene density in Sce- and Ph-depleted cells to the 3′ NT-seq density in mock control cells for Ph+ H3K27me3− genes [upper right panel in (A)]. The horizontal and vertical blue lines intersect at the average position of the poly(A) sites, determined by performing a metagene analysis with total RNA-seq data (not depicted). Middle: Median ratio of the 3′ NT-seq density in the 500 nucleotides (nt) upstream of AATAAA sequences to the density in the 500 nt downstream of the AATAAA sequences for all active genes in mock control, Sce-depleted, and Ph-depleted cells. Bottom: Ratio of the 3′ NT-seq data in the last 500 nt of exons to the first 500 nt of the following intron in mock control, Sce-depleted, and Ph-depleted cells.

  • Fig. 4 PRC1 subunits alter phosphorylation of the Rpb1 CTD heptad repeats.

    (A) Genome browser view of the headcase gene (transcribed from left to right) as an example showing a typical ChIP-seq log2 enrichment pattern for the Ph, Rpb3, Ser5P, Ser7P, Ser2P, Thr4P, and Spt5 antibodies. The log2 densities for 3′ NT-seq and total RNA-seq are shown for comparison. (B) Metagene analysis of the Ser5P, Ser2P, and Thr4P enrichment for Ph+ H3K27me3− genes in mock control (blue lines), Sce-depleted (iSce; red lines), Ph-depleted (iPh; green lines), and Pc-depleted (iPc; purple) cells. Left: Mean enrichment for each modification. Right: Ratio of each modification relative to Rpb3 (see Fig. 3). Metagene patterns at other gene classes and Ser7P data are shown in fig. S4. Wilcoxon signed-rank test P values of the differences in peak enrichment between the mock control and PRC1 subunit–depleted cells are in table S2.

  • Fig. 5 PRC1 globally alters phosphorylation of Rpb1 heptad repeats at active gene promoters.

    The violin plots in the left panel show the distribution of total Pol II (Rpb3) occupancy at 7398 active promoters determined from ChIP-seq data (described in Figs. 3 and 4) in mock control, Sce-depleted (iSce), Ph-depleted (iPh), and Pc-depleted (iPc) cells. The blue line indicates the median enrichment level in mock control cells. The top panels show the same for the Ser5P and Ser2P modifications. The same data are illustrated by dot plots in fig. S5. The bottom panels show the ratios of Ser5P and Ser2P to Rpb3 in mock control, Sce-depleted, Ph-depleted, and Pc-depleted cells.

  • Fig. 6 PRC1 influences the association of the Spt5 pausing-elongation factor with active genes and promoters.

    (A) Metagene analysis of Spt5 ChIP-seq enrichment for Ph+ H3K27me3− and Ph− H3K27me3− active genes in mock control (blue lines), Sce-depleted (iSce; red lines), and Ph-depleted (iPh; green lines) cells. Left: Mean enrichment. Right: Ratio of Spt5 to Rpb3 across the genes. The P values from Wilcoxon signed-rank tests are provided in table S2. (B) Occupancy of 7398 active promoters by Rpb3 and Spt5 in mock control, Sce-depleted, and Ph-depleted cells, shown in violin plot distributions. Blue lines indicate median values in mock control cells. Dot plots in fig. S5 illustrate the same results. Right: Distribution of the Spt5-to-Rpb3 ratio across all promoters.

  • Fig. 7 PRC1 facilitates association of Spt5 and Pol II with enhancers and PREs and influences Pol II phosphorylation.

    (A) Distribution of Rpb3 and Spt5 density across genes and regulatory sequences. The left two panels show the occupancy of Rpb3 and Spt5 at promoters (pink violin distributions), in gene bodies (purple), at gene ends (blue), at extragenic enhancers (red), at PREs and extragenic PREs (orange), and at random sites (tan). The right panel shows the distributions of the Spt5-to-Rpb3 ratio at the same elements. (B) Effects of Sce and Ph depletion on Spt5 and Ser5P levels at enhancers and extragenic PREs. The left panel shows the distribution of Rpb3 at extragenic enhancers (red) and PREs (orange) in mock control, Sce-depleted (iSce), and Ph-depleted (iPh) cells. The top left panel shows the same for Spt5, and the bottom left panel shows the same for Ser5P. Dot plots in fig. S6 show the same data. The two right panels show the ratios of Spt5 to Rpb3 (top) and Ser5P to Rpb3 (bottom).

  • Fig. 8 Nipped-B and cohesin control global Pc chromosome–binding dynamics in vivo.

    (A) FRAP recovery curves for Pc-EGFP in wild-type (blue diamonds), heterozygous Rad21 (verthandi) mutant (vtdex15, Rad21−/+, red boxes), and heterozygous Nipped-B mutant (Nipped-B407/+, Nipped-B−/+, orange triangles) third-instar larval salivary gland nuclei. At least 30 nuclei were averaged for each curve. Error bars are the SD of the mean. The inset shows the half-lives calculated from the recovery curves for the stable (slow; blue) and less stable (fast; red) binding forms of Pc. Error bars are SDs. The asterisk indicates a statistically significant difference from the control. (B) FRAP recovery curves for the SA-EGFP–labeled cohesin subunit in wild-type (SA-EGFP; blue diamonds) and heterozygous Pc mutant (Pc4/+, Pc−/+, red boxes) salivary glands. At least 30 nuclei were averaged for each curve, error bars are SDs of the mean, and the inset shows the chromosomal half-lives calculated for the stable (slow; blue) and less stable (fast; red) binding from the recovery curves.

  • Fig. 9 PRC1 influences Pol II modifications and Spt5 occupancy at active genes.

    TFIIH in the preinitiation complex (upper left) facilitates DNA unwinding to form the transcription bubble and phosphorylates Ser5 residues (Ser5P) in the heptad repeats in the CTD of the Rpb1 subunit of Pol II. Binding of Spt5 and NELF (negative elongation factor) pausing factors causes promoter-proximal pausing (upper right). The cohesin complex recruits PRC1 (green box), and the Sce subunit of PRC1 facilitates Ser5 phosphorylation and Spt5 binding by undefined mechanisms. Ph suppresses Ser5P at the promoter. P-TEFb in the SEC (super elongation complex) recruited by activator proteins phosphorylates Ser2 residues in the Pol II heptad repeats and Spt5 to start transcription elongation (bottom). Ph suppresses Spt5 association with elongating Pol II. The effects of PRC1 on Pol II and Spt5 coincide with changes, largely increases, in transcription of active genes.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/8/e1700944/DC1

    fig. S1. Comparison of effects of Sce, Pc, and Ph depletion on total RNA accumulation in BG3 cells.

    fig. S2. Effects of Sce and Pc depletion on Ph occupancy of PREs, active promoters, and extragenic enhancers measured by ChIP-seq.

    fig. S3. Effects of Sce and Ph depletion on Pc occupancy of PREs, active promoters, and extragenic enhancers measured by ChIP-seq.

    fig. S4. PRC1 modifies Rpb1 phosphorylation at active genes.

    fig. S5. Effects of Sce, Ph, and Pc depletion on Rpb3, Ser5P, Ser2P, and Spt5 occupancy of active promoters measured by ChIP-seq.

    fig. S6. Effects of Sce and Ph depletion on Rpb3, Ser5P Rpb1, and Spt5 levels at extragenic enhancers and extragenic PREs measured by ChIP-seq.

    table S1. RNA-seq quantification and gene ontology.

    table S2. Statistical analysis of ChIP-seq data.

    table S3. Ribosomal RNA depletion oligonucleotides.

    table S4. ChIP-seq replicates and inputs used for normalization.

    file S1. R scripts.

    file S2. Whole-gene bed file for 3′ NT-seq quantification.

    file S3. Active gene exons greater than 500 nt in length.

    file S4. Potential active gene poly(A) signals.

    file S5. Extended length active gene bed file for genes with high Ph and low H3K27me3 for metagene analysis.

    file S6. Extended length active gene bed file for genes with low Ph and low H3K27me3 for metagene analysis.

    file S7. Extended length active gene bed file for genes with high Ph and high H3K27me3 for metagene analysis.

    file S8. Extended length active gene bed file for PcG domain genes for metagene analysis.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Comparison of effects of Sce, Pc, and Ph depletion on total RNA accumulation in BG3 cells.
    • fig. S2. Effects of Sce and Pc depletion on Ph occupancy of PREs, active promoters, and extragenic enhancers measured by ChIP-seq.
    • fig. S3. Effects of Sce and Ph depletion on Pc occupancy of PREs, active promoters, and extragenic enhancers measured by ChIP-seq.
    • fig. S4. PRC1 modifies Rpb1 phosphorylation at active genes.
    • fig. S5. Effects of Sce, Ph, and Pc depletion on Rpb3, Ser5P, Ser2P, and Spt5 occupancy of active promoters measured by ChIP-seq.
    • fig. S6. Effects of Sce and Ph depletion on Rpb3, Ser5P Rpb1, and Spt5 levels at extragenic enhancers and extragenic PREs measured by ChIP-seq.

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    Other Supplementary Material for this manuscript includes the following:

    • table S1 (Microsoft Excel format). RNA-seq quantification and gene ontology.
    • table S2 (Microsoft Excel format). Statistical analysis of ChIP-seq data.
    • table S3 (Microsoft Excel format). Ribosomal RNA depletion oligonucleotides.
    • table S4 (Microsoft Excel format). ChIP-seq replicates and inputs used for normalization.
    • file S1 (.txt format). R scripts.
    • file S2 (.txt format). Whole-gene bed file for 3′ NT-seq quantification.
    • file S3 (.txt format). Active gene exons greater than 500 nt in length.
    • file S4 (.txt format). Potential active gene poly(A) signals.
    • file S5 (.txt format). Extended length active gene bed file for genes with high Ph and low H3K27me3 for metagene analysis.
    • file S6 (.txt format). Extended length active gene bed file for genes with low Ph and low H3K27me3 for metagene analysis.
    • file S7 (.txt format). Extended length active gene bed file for genes with high Ph and high H3K27me3 for metagene analysis.
    • file S8 (.txt format). Extended length active gene bed file for PcG domain genes for metagene analysis.

    Files in this Data Supplement:

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