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Mechanism of FACT removal from transcribed genes by anticancer drugs curaxins

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Science Advances  07 Nov 2018:
Vol. 4, no. 11, eaav2131
DOI: 10.1126/sciadv.aav2131
  • Fig. 1 FACT subunit SSRP1 is preferentially enriched on highly transcribed genes, and curaxins remove SSRP1 from the gene bodies.

    (A) Integrated genome views of the SSRP1 distributions at selected regions of chromosome 6 of the human genome. (B) Distribution of SSRP1 peaks in control and CBL0137-treated cells in relation to genome annotation features. (C) SSRP1 protein is preferentially enriched on highly transcribed genes. Average SSRP1 occupancy near the transcription start sites (TSSs), in the transcribed regions, and in transcription end sites (TESs) of the genes in HT1080 cells (incubated in the absence or presence of 3 μM curaxin CBL0137) was determined using ChIP-seq. The genes are grouped on the basis of the RNA-seq data. The corresponding heatmaps are shown in fig. S4. (D and E) Curaxins preferentially remove SSRP1 from actively transcribed genes. Density scatter plots representing average SSRP1 densities over gene bodies against the level of transcription of the corresponding genes, quantified by the average level of nascent transcripts determined in HT1080 cells by NET-seq (native elongating transcript sequencing). Analysis of all (fig. S5) or top 500 most actively transcribed genes (D and E) in the absence (D) or presence (E) of the curaxin. For the top 500 most active genes, the Pearson correlation coefficient is markedly decreased (from 0.590 to 0.003) upon treatment with curaxin.

  • Fig. 2 FACT and curaxins strongly and synergistically affect the nucleosomal structure: Analysis by spFRET.

    (A) Experimental approach. The mononucleosomes contained the single pair of Cy3 and Cy5 dyes on the nucleosomal DNA (the positions of Cy3 and Cy5 are shown by green and red circles, respectively). spFRET from nucleosomes was measured in the absence or presence of curaxin CBL0137, FACT, and/or competitor DNA. (B and C) Typical frequency distributions of FRET efficiencies (EPR). Analysis by spFRET microscopy. Sample sizes and other numerical parameters are shown in table S1. (B) Only minor changes in nucleosome structure are detected in the presence of either CBL0137 or FACT. (C) FACT and CBL0137 together induce marked and partially reversible uncoiling of the nucleosomal DNA. The uncoiling is partially reversed by subsequent addition of an excess of competitor DNA, resulting in removal of FACT from the complex. (D) Possible changes in the nucleosome structure in the presence of FACT and curaxins: nucleosome unfolding or uncoiling of nucleosomal DNA from the histone octamer.

  • Fig. 3 Nucleosomes are reversibly destabilized in the presence of curaxins and FACT.

    (A) Experimental approach. DNA-labeled nucleosomes were incubated in the presence of FACT, unlabeled competitor DNA, CBL0137 (B), or CBL0100 (C) and analyzed by native PAGE. Note that in the presence of both FACT and curaxins, nucleosomes are unstable (as reflected in release of histone-free DNA); this effect is reversed in the presence of DNA competitor that partially removes FACT from the nucleosomes (see Fig. 2).

  • Fig. 4 Curaxins and competitor nucleosomes synergistically inhibit FACT-dependent transcription in vitro.

    (A) Experimental approach. The assembled Pol II EC-119 was ligated to the 603 DNA or nucleosome (43, 58). The RNA was pulse labeled in the presence of a subset of NTPs and [α-32P]GTP, and Pol II was stalled at position −83 (43, 45, 58). Then, transcription was resumed by adding all unlabeled NTPs, FACT (to 0.2 μM), unlabeled competitor nucleosomes, and CBL0100 (B) or CBL0137 (C). Note that without competitor nucleosomes, curaxins moderately stimulate transcription through chromatin but do not inhibit FACT action. UTP, uridine 5′-triphosphate.

  • Fig. 5 Mechanism of curaxin action in cancer cells: N-trapping.

    In the cells without curaxin treatment (−Curaxins), FACT loosely interacts with the transiently exposed DNA binding surface of the H2A/H2B dimer and thus facilitates transcription through the nucleosome barrier. In the presence of curaxins (+Curaxins), FACT causes genome-wide nucleosome unfolding and is tightly trapped on the unfolded nucleosomes (n-trapping). Because the nontranscribed chromatin is present in vast excess, FACT is trapped primarily within inactive chromatin regions. Thus, curaxin-induced n-trapping reduces density of FACT on transcribed regions, resulting in loss of FACT from the transcribed genes and likely affects chromatin dynamics and/or transcript elongation process.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/11/eaav2131/DC1

    Fig. S1. Analysis of colocalization of FACT and transcribing Pol II using immunofluorescence.

    Fig. S2. Redistribution of FACT in HT1080 cells from nucleoplasm to chromatin.

    Fig. S3. Redistribution of FACT in nucleus of HT1080-treated cells from nucleoplasm to chromatin.

    Fig. S4. Heatmaps of SSRP1 occupancy in the vicinity of TSSs and TES of the genes in HT1080 cells.

    Fig. S5. Analysis of the average SSRP1 densities over gene bodies against the levels of transcription of the corresponding genes.

    Fig. S6. Curaxins preferentially remove SSRP1 from gene bodies of highly transcribed genes.

    Fig. S7. Analysis of gel-purified nucleosomes by native PAGE.

    Fig. S8. Typical frequency distributions of FRET efficiencies of the N13/91 nucleosomes.

    Fig. S9. The catalytic activity of Pol II is minimally affected by curaxins.

    Fig. S10. Trapping of FACT on immobilized competitor nucleosomes after Pol II transcription in the presence of CBL0137.

    Table S1. Statistical data for spFRET analysis.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Analysis of colocalization of FACT and transcribing Pol II using immunofluorescence.
    • Fig. S2. Redistribution of FACT in HT1080 cells from nucleoplasm to chromatin.
    • Fig. S3. Redistribution of FACT in nucleus of HT1080-treated cells from nucleoplasm to chromatin.
    • Fig. S4. Heatmaps of SSRP1 occupancy in the vicinity of TSSs and TES of the genes in HT1080 cells.
    • Fig. S5. Analysis of the average SSRP1 densities over gene bodies against the levels of transcription of the corresponding genes.
    • Fig. S6. Curaxins preferentially remove SSRP1 from gene bodies of highly transcribed genes.
    • Fig. S7. Analysis of gel-purified nucleosomes by native PAGE.
    • Fig. S8. Typical frequency distributions of FRET efficiencies of the N13/91 nucleosomes.
    • Fig. S9. The catalytic activity of Pol II is minimally affected by curaxins.
    • Fig. S10. Trapping of FACT on immobilized competitor nucleosomes after Pol II transcription in the presence of CBL0137.
    • Table S1. Statistical data for spFRET analysis.

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