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. 2020 Aug 3;27(9):836–845. doi: 10.1038/s41594-020-0466-9

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© The Author(s), under exclusive licence to Springer Nature America, Inc. 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a, Glutathione S-transferase (GST; control) and GST-SSX1 (78 aa) purified recombinant proteins incubated with mammalian mononucleosomes (purified by MNase digestion) were captured using glutathione resin and visualized using Colloidal Blue stain. b, Quantitative MS analysis of maltose binding protein (MBP) pull-down experiments using the MBP-SSX 78-aa protein and endogenous mammalian nucleosomes purified using MNase digestion from 293T cells. The log₂(fold change (FC)) is calculated relative to the input sample. Red, enriched; blue, depleted. Raw data are provided in Supplementary Table 2. c, IF analysis of V5-tagged SS18 and SS18-SSX relative to RING1B and SUZ12 in 293T cells. Arrowheads indicate Barr bodies. Scale bars, 5 μm. d, SSX1 protein sequence alignment across species and compared to related PRDM7 and PRDM9 proteins. Highly conserved basic (blue) and acidic (red) regions are indicated. e, Pull-down experiments with biotinylated SSX peptides (scrambled (aa 155–188), SSX 34 aa (aa 155–188), SSX 24 aa (aa 164–188) and SSX 23 aa (aa 165–188) incubated with mammalian mononucleosomes and visualized with Colloidal Blue. f, Pull-down experiments of N-terminally biotinylated SSX peptides, including scrambled control, WT and mutant variants (single alanine substitutions as well as regional substitutions; that is, basic-A, basic region RLRERK → AAAAAA; acidic-A, acidic region DPEEDDE → AAAAAAA), incubated with mammalian mononucleosomes and visualized with Colloidal Blue. g, ChIP-seq density heatmaps reflecting the chromatin occupancy of V5-SS18-SSX1, V5-SS18, V5-SS18-SSX (24 aa) and V5-SS18-SSX (34 aa) over all V5 peaks (38,014 total peaks). h, Heatmap reflecting the top 5% upregulated and downregulated genes (Z-score) by RNA-seq for each condition. i, Proliferation of SYO1 SS cells infected with either control hairpin (shCt) or shSSX with overexpression of empty vector control, SS18-SSX 78 aa or SS18-SSX 34 aa variants. Error bars represent mean ± s.d. of n = 3 independent experimental replicates; **P < 0.01 determined from a two-tailed t-test. Data for i are available as Source data. Uncropped gel images for a, e and f are presented in Supplementary Fig. 1.

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