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  • 1

    SUPPLEMENTARY INFORMATION

    Mapping the chemical chromatin reactivation landscape identifies BRD4-TAF1 cross-talk

    Sara Sdelci1, Charles-Hugues Lardeau1,2, Cynthia Tallant3,4, Freya Klepsch1, Björn Klaiber1, James Bennett3,4, Philipp Rathert5,10, Michael Schuster1, Thomas Penz1, Oleg Fedorov3,4, Giulio Superti-Furga1,6, Christoph Bock1,7,8, Johannes Zuber4, Kilian V. M. Huber1,3,4, Stefan Knapp3,4,9, Susanne Müller3,4, Stefan Kubicek1,2, *

    1CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences. Lazarettgasse 14, 1090 Vienna, Austria

    2Christian Doppler Laboratory for Chemical Epigenetics and Antiinfectives, CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria. 3Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK 4Target Discovery Institute, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK 5Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria

    6Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria 7Department of Laboratory Medicine, Medical University of Vienna, 1090 Vienna, Austria 8Max Planck Institute for Informatics, 66123 Saarbrücken, Germany 9Johann Wolfgang Goethe-University, Institute for Pharmaceutical Chemistry and Buchmann Institute for Life Sciences, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany 10Present address: Institute of Biochemistry, University Stuttgart, Stuttgart, Germany

    *To whom correspondence should be addressed:

    Stefan Kubicek CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences. Lazarettgasse 14, 1090 Vienna, Austria Phone : +43-1-40160-70036, Fax: +43-1-40160-970 000, Email: skubicek@cemm.oeaw.ac.at

    Nature Chemical Biology: doi:10.1038/nchembio.2080Nature Chemical Biology: doi:10.1038/nchembio.2080

  • 2

    SUPPLEMENTARY RESULTS

    Supplementary Fig. 1 ǀ Characterization of RED3 cells

    Supplementary Fig. 2 ǀ Characterization of the RFP integration site in REDS3 cells

    Supplementary Fig. 3 ǀ Characterization of screening hits

    Supplementary Fig. 4 ǀ Characterization of the binding of CeMMEC1 and CeMMEC2 to bromodomain proteins

    Supplementary Fig. 5 ǀ Full western blots

    Supplementary Fig. 6 ǀ CeMMEC1 analogs with improved selectivity

    Supplementary Fig. 7 ǀ Interaction of BRD4 and TAF1

    Supplementary Table 1 ǀ Small molecule screening data

    Supplementary Table 2 ǀ Summary of bioactivity data of hit compounds

    Supplementary Table 3 ǀ Structure-activity relationships of CeMMEC1 analogs

    Nature Chemical Biology: doi:10.1038/nchembio.2080Nature Chemical Biology: doi:10.1038/nchembio.2080

  • 3

    Supplementary Fig. 1 ǀ Characterization of RED3 cells (a) Western Blot analysis showing c-MYC downregulation in WT-KBM7 treated with 0.5 μM (S)-JQ1 for 18 hours; an equal volume of DMSO was used as control. (b) Cell cycle profiles evaluated by PI-staining and DNA content analysis by FACS. WT-KBM7 cells were treated with 0.5 μM of (S)-JQ1 or (R)-JQ1 for 24 hours; an equal amount of DMSO was added as control. Four biological replicates have been done (mean). (c) Examples of live cell imaging pictures of REDS1, REDS2 and REDS3 cells treated with 0.5 μM of (S)-JQ1 or (R)-JQ1; an equal amount of DMSO was added as control. Scale bar 100 μm. (d) RFP and (e) zeocin expression performed by RT-PCR in REDS3 cells treated with 0.5 μM (S)-JQ1 or (R)-JQ1 for 18 hours; an equal volume of DMSO was used as control. Three biological replicates were performed for each experimental condition (meanSTD). (f) Left panel: cell cycle profiles of REDS3 untreated cells or REDS3 cells treated for 20 hours with thymidine (2 mM), RO3306 (9 μM) or nocodazole (1μM). Nuclei were stained with PI and DNA content quantified by FACS; representative cell cycle profiles from one of the two experiments done (30,000 cells were FACS analyzed in each experiment). Right panel: examples of live cell imaging pictures of REDS3 cells treated as above and with 0.5 μM of (S)-JQ1; an equal volume of DMSO was used as control. Representative pictures from one of the three replicates done. Scale bar 100 μm. (g) Quantification of RFP-positive cells by live cell imaging pictures of REDS3 cells treated with PMA (200 nM), PHA (5ug/ml), (S)-JQ1 (1 μM) or a combination of PMA or PHA with (S)- JQ1 for 24 hours. An equal amount of DMSO was added as control; three replicates were done and at least 1,500 cells were quantified. (h) BRD3, BRD4 and RFP expression assessed by RT-PCR in BRD3 or BRD4 downregulated REDS3 cells; three biological replicates were done for each experimental condition (meanSTD).

    Nature Chemical Biology: doi:10.1038/nchembio.2080Nature Chemical Biology: doi:10.1038/nchembio.2080

  • 4

    Supplementary Fig. 2 ǀ Characterization of the RFP integration site in REDS3 cells (a) Representation of the RFP locus. RFP (red arrow) is inserted in the sense direction at 12 chromosome (chr12:131,323,912- 131,324,450) between STX2 (reverse direction, cyan arrow) and RAN (sense direction, cyan arrow); primers used in b are indicated in the representation (WT genome: green lines; REDS3 genome: purple lines). The region between STX2 and RAN is

    enhancer-rich (light orange dashed line), while the region downstream of STX2 is heterochromatic (gray dashed loops). In the

    box: representation of ENCODE/Broad institute chromatin state segmentation and Chip-seq (H3K4m1, H3K4m3, H3K27ac,

    H3K27m3 and H3H36m3) data (K-562 cell line). (b) PCR of WT and REDS3 DNA using specific primers for the locus of the RFP insertion; primers amplifying the insulin promoter (Ins_P) have been used as control. (c) DAVID functional annotation analysis of WT-KBM7 (S)-JQ1-upregulated genes.

    Nature Chemical Biology: doi:10.1038/nchembio.2080Nature Chemical Biology: doi:10.1038/nchembio.2080

  • 5

    Supplementary Fig. 3 ǀ Characterization of screening hits (a) Pipeline for the selection of validated hits. Compound library: collection of 89,355 small molecules. DR: Dose Response; TC: Time Course. (b) Representation of the criteria used for the selection of the positive control concentration ((S)-JQ1 0.5 µM) applied in the screening in terms of RFP fluorescence increase (from live cell imaging pictures quantification, duplicates) and (c) its associated Z-Factor. (d) Example of live cell imaging pictures of REDS3 cells treated with 10 μM of (S)-JQ1, CeMMEC1 (1) or CeMMEC2 (2) for 24 hours. An equal volume of DMSO was used as control. (e) Quantification of RFP-positive nuclei of REDS3 cells treated with (S)-JQ1, CeMMEC1 and CeMMEC2 at 0.5, 1, 2.5, 5, 10 and 20 μM from live cell imaging pictures. An equal

    amount of DMSO was added as control; three replicates were done and at least 1,500 cells were quantified in each experiment

    (meanSTD). (f) RNA-seq analysis: Venn diagrams indicating the number of up- and down-regulated genes in WT-KBM7 cells

    treated with (S)-JQ1 1 μM (grey), CeMMEC1 10 μM (red) and CeMMEC2 10 μM (pink) for 24 hours, and the overlapping of these

    groups. (g) Scatter plot representing the correlation of gene expression variation in cells treated as in d.

    Nature Chemical Biology: doi:10.1038/nchembio.2080Nature Chemical Biology: doi:10.1038/nchembio.2080

  • 6

    Supplementary Fig. 4 ǀ Characterization of the binding of CeMMEC1 and CeMMEC2 to bromodomain proteins (a) Dose response (12 dilutions, from 20 to 0.02 μM) AlphaLISA assay for the first (BD1) and the second (BD2) bromodomain of

    BRD4 incubated with (S)-JQ1 and CeMMEC2 (2). The assay was done in duplicate (meanSTD); (b) AlphaLISA dose response

    (12 dilutions, from 20 to 0.02 μM) for full length BRD4 (GST-tagged) incubated with (S)-JQ1 or CeMMEC2. (c) BromoKdELECT assay for CeMMEC1 (1) against TAF1 (2), BRD9, CREBBP and EP300 bromodomains (11 dilutions, from 10 to 0.017 μM). (d) Fluorescence Resonance Energy Transfer (FRET) results for CeMMEC1 with TAF1 (2). (e) KinomeScan profile for CeMMEC1 (10 µM). Bubbles indicate the percentage of inhibition of the binding of the analyzed kinase to an immobilized ligand. (f) Docking of CeMMEC2 to the first bromodomain of BRD4 and (g) of CeMMEC1 to the second bromodomain of TAF1. 3D model (top panel) and 2D ligand interaction diagram (bottom panel). Red dots indicate water molecules.

    Nature Chemical Biology: doi:10.1038/nchembio.2080Nature Chemical Biology: doi:10.1038/nchembio.2080

  • 7

    Supplementary Fig. 5 ǀ Full western blots Full scans of western blots shown in Figure 3c.

    Nature Chemical Biology: doi:10.1038/nchembio.2080Nature Chemical Biology: doi:10.1038/nchembio.2080

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    Supplementary Fig. 6 ǀ CeMMEC1 analogs with improved selectivity (a) AlphaLISA data illustrating the inhibition by 10 μM CeMMEC1 analogs of binding BRD4(1), CREBBP and TAF1(2) bromodomains to an acetylated substrate. On the bottom of the figure: RFP-positive cell values obtained by live imaging pictures

    quantification (DMSO normalized; duplicates, 1,500 cells quantified each replicate). (b) Docking poses of analog CEMMEC13 (13) and (c) CEMMEC14 (14) to TAF1. (d) BromoELECT assay for analog CeMMEC13 (10 μM) against BRD4 (1), BRD9, CREBBP, EP300 and TAF1 (2).

    Nature Chemical Biology: doi:10.1038/nchembio.2080Nature Chemical Biology: doi:10.1038/nchembio.2080

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