Molecular basis for H3K36me3 recognition by the Tudor domain of PHF1

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1 Supplementary information Molecular basis for H3K36me3 recognition by the Tudor domain of PHF1 Catherine A. Musselman 1, Nikita Avvakumov 2, Reiko Watanabe 3, Christopher G. Abraham 4, Marie-Eve Lalonde 2, Zehui Hong 5, Christopher Allen 6, Siddhartha Roy 1, James K. Nuñez 1, Jac Nickoloff 6, Caroline A. Kulesza 4, Akira Yasui 3, Jacques Côté 2, and Tatiana G. Kutateladze 1 1 Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado, USA. 2 Laval University Cancer Research Center, Hôtel-Dieu de Québec (CHUQ), Quebec City, Québec, Canada. 3 Division of Dynamic Proteome in Cancer and Aging, Institute of Development, Aging and Cancer, Tohoku University, Aobaku, Sendai, Japan. 4 Department of Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USA. 5 Department of Genetics and Developmental Biology, Medical School of Southeast University, Nanjing, China. 6 Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado, USA. Correspondence should be addressed to T.G.K. (tatiana.kutateladze@ucdenver.edu)

2 Supplementary Figure 1 The PHF1 Tudor domain recognizes H3K36me3. (a) Pulldown assay of GST-fusion PHF1 Tudor with the indicated singly modified biotinylated histone peptides, analyzed by Western with an antibody against GST. (b) Representative ITC curves for PHF1 Tudor binding to trimethylated, dimethylated or monomethylated H3K36 peptides, shown from left to right. (c) Differences in NMR resonance perturbations that occurred in the Tudor domain upon binding to H4K20me3 (top left), H3K4me3 (top right), H3K9me3 (bottom left) and H3K27me3 (bottom right) as compared to binding to H3K36me3. Resonances that were perturbed notably less than H3K36me3 are labeled and highlighted in orange.

3 Supplementary Figure 2 Binding affinities of the PHF1 Tudor domain. (a) A representative ITC curve used to determine the binding affinity to H3K27me3. (b) Representative NMR curves used to determine the binding affinity of the wild type Tudor domain to methylated and unmodified histone peptides and the W41A mutant to H3K36me3. (c) Alignment of the H3K36, H4K20, H3K4, H3K9 and H3K27 sequences. The methylated Lys residue is shown in red, and adjacent basic, hydrophobic and polar residues are shown in blue, yellow and green, respectively.

4 Supplementary Figure 3 Substitution of Y47 or W41 disrupts binding to H3K36me3 but not Tudor domain structure. 1 H, 15 N HSQC overlays of W41A (top) and Y47A (bottom) mutants of the PHF1 Tudor domain in the presence of increasing amounts of H3K36me3 peptide, color coded by the protein:peptide molar ratio. The dispersion of resonances indicates that the mutant proteins are folded.

5 Supplementary Figure 4 Comparison of the H3K36me3 binding mechanisms. The PHF1 Tudor domain (left), the BRPF1 PWWP domain (middle) and the EAF3 chromo-barrel domain (right) are shown as solid surfaces and the methylated H3K36 peptides are shown as a stick model.

6 Supplementary Figure 5 Binding of PHF1 to H3K36me3 inhibits PRC2 activity. (a) Fluorography of the gel on which were loaded HMT assays with PHF1-PRC2 on native wild type chromatin (SON) and chromatin lacking H3K36-specific HKMT Set2 (Δset2 SON). The lower labeled band is from a truncation product of histone H3 often seen in yeast histones 1. (b) Comassie stain of the HMT assay gel showing wild type and Δset2 histone bands in the absence or presence of Flag-PHF1. Molecular weight (MW) marker is shown.

7 Supplementary Figure 6 Kinetic analysis of GFP-PHF1 accumulation and retention at laser irradiated sites. (a) Localization of GFP-PHF1 wild type (top), GFP-PHF1 W41A (middle), and GFP-PHF1 Y47A (bottom), at the DSB sites in U2OS cells at indicated time after irradiation. (b) Inhibition of the GFP-PHF1 accumulation at DNA DSBs by the PARP1 and ATM/ATR kinase inhibitors. Kinetic analysis of intensities of GFP-PHF1 wild type, GFP-PHF1 W41A and GFPPHF1 Y47A at laser irradiated sites upon treatment with PARP1 or ATM/ATR kinase inhibitors. (c, d) Loss of GFP-XRCC1 accumulation at DNA single-strand breaks (produced with a low dose of irradiation) (c) as well as loss of poly(adp ribose) (PAR) as detected by Western analysis (d) upon treatment with PARP1 inhibitor (AZD2281).

8 Supplementary Movie 1 H3K36me-dependent recruitment of PHF1 to the sites of DSBs. Accumulation and dissociation of GFP-PHF1 wild type (a), GFP-PHF1 W41A (b) and GFP- PHF1 Y47A (c) at laser-irradiated DSB sites within six minutes in U2OS cells. Supplementary Movie 2 Inhibition of the GFP-PHF1 accumulation at DNA DSBs by inhibitors. Accumulation and dissociation of GFP-PHF1 wild type (top panel), GFP-PHF1 W41A (middle panel) and GFP-PHF1 Y47A (bottom panel) at laser-irradiated DSB sites within six minutes in U2OS cells either untreated (left panel), treated with the PARP1 inhibitor AZD2281 (middle panel) or an ATM/ATR kinase inhibitor (right panel). References: 1. Santos-Rosa, H. et al. Histone H3 tail clipping regulates gene expression. Nature structural & molecular biology 16, (2009).