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PAF1 regulation of promoter-proximal pause release via enhancer activation

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Science  22 Sep 2017:
Vol. 357, Issue 6357, pp. 1294-1298
DOI: 10.1126/science.aan3269
  • Fig. 1 PAF1 regulates transcriptional activity of enhancers.

    (A) Representative genome browser track examples of Pol II, PAF1, H3K27ac, H3K4me1, and H3K4me3 ChIP-seq in HCT116 cells. The y axis represents normalized read density in reads per million (rpm). Gray and peach boxes indicate the promoter and putative enhancer regions, respectively. (B) Heat maps of the occupancy of Pol II, PAF1, H3K27ac, H3K4me1, and H3K4me3 at active promoters and enhancers. Color-scaled intensities are in units of rpm. (C) Box plots showing the ratio of PAF1 to Pol II occupancy at promoters and enhancers. P < 2.2 × 10–16. (D) Metaplots of H3K27ac occupancy in cells transduced with nontargeting (NONT) or PAF1 shRNA (shPAF1) at enhancers. (E) Genome browser track examples at the loci of activated enhancers after PAF1 depletion. (F and G) Metagene analysis of Pol II occupancy at intergenic enhancers with increased H3K27ac (F) and with no significant change of H3K27ac (G) by PAF1 knockdown. (H and I) Empirical cumulative distribution function (ECDF) plots measuring eRNA levels at intergenic enhancers with increased H3K27ac (H) and with no significant change of H3K27ac (I) by PAF1 knockdown. P values were calculated by the Kolmogorov-Smirnov test.

  • Fig. 2 Enhancer activation correlates with transcription up-regulation and pause release of nearby genes.

    (A and B) Box plots showing log2 fold change (FC) of poly(A)-depleted transcripts of genes nearby activated (A) and stable (B) enhancers by PAF1 depletion. (C and D) Box plots showing log2 fold change of poly(A)-enriched transcripts of genes nearby activated (C) and stable (D) enhancers by PAF1 depletion. (E and F) Box plots showing log2 fold change of poly(A)-depleted (E) and poly(A)-enriched (F) transcripts of genes nearby activated and stable enhancers using fewer groups. (G and H) Metagene analysis of Pol II occupancy for genes within 80 kb of activated (G) and stable (H) enhancers. The y axis represents reads per base per gene. TSS, transcription start site; TTS, transcription termination site.

  • Fig. 3 Enhancer knockout mitigates the effect of PAF1 depletion on the release of paused Pol II.

    (A and B) Genome browser track examples of Pol II ChIP-seq, H3K27ac ChIP-seq, and GRO-seq at the loci of IER5 (A) and SERPINE2 (B). Pink boxes are enhancer regions targeted by a pair of single guide RNAs (sgRNAs) for deletion. (C and D) Genome browser track examples of Pol II ChIP-seq in wild-type (WT) HCT116 cells, two clones of IER5 enhancer knockouts, and two clones of SERPINE2 enhancer knockouts transduced with NONT or shPAF1 at the loci of IER5 (C) and SERPINE2 (D). (E) Analysis of 4C-seq with the SERPINE2 promoter as the viewpoint (black arrows) in NONT and shPAF1 cells. The red box indicates the enhancer region activated by PAF1 depletion. Purple arrows indicate increased contact between the promoter and enhancer after PAF1 depletion. The median line and 20th and 80th percentiles of a sliding 5-kb window indicate the main trend. The color scale represents enrichment relative to the maximum median value at a resolution of 12 kb. (F and G) Gene expression changes in IER5 enhancer knockouts (F) and SERPINE2 enhancer knockouts (G) relative to WT, measured by RNA-seq. (H and I) Gene expression changes when comparing IER5 and SERPINE2 enhancer knockout cells for genes on chromosome 1 (H) and chromosome 2 (I). The x axis indicates the chromosome position. Gray boxes indicate 5 Mb surrounding the deleted enhancers.

  • Fig. 4 PAF1 is required for the accumulation of paused Pol II driven by the heat shock response.

    (A) Experimental design. HCT116 cells were transduced with NONT or shPAF1 for around 3.5 days and then cross-linked for ChIP-seq with or without 90 min of heat shock (HS). (B) Metagene plot of H3K27ac occupancy in cells before or after HS for the top 1000 HS-repressed enhancers. The y axis represents reads per base per gene. (C) Metagene plot of Pol II occupancy in cells before or after HS for genes within 80 kb of the top 1000 HS-repressed enhancers. (D to G) Genome browser track examples of H3K27ac occupancy at enhancers [(D) and (F)] and Pol II occupancy at nearby genes [(E) and (G)] in cells with or without HS in NONT and PAF1-depeleted cells. (H) Metagene plot of H3K27ac occupancy on HS-repressed enhancers in cells with or without PAF1 depletion during HS. (I) Empirical cumulative distribution function plot of the promoter-release ratio (PRR) distribution in cells with or without HS in NONT and PAF1-depleted cells.

Supplementary Materials

  • PAF1 regulation of promoter-proximal pause release via enhancer activation

    Fei Xavier Chen, Peng Xie, Clayton K. Collings, Kaixiang Cao, Yuki Aoi, Stacy A. Marshall, Emily J. Rendleman, Michal Ugarenko, Patrick A. Ozark, Anda Zhang, Ramin Shiekhattar, Edwin R. Smith, Michael Q. Zhang, Ali Shilatifard

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S10
    • References

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