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. 2023 Sep 21;30(11):1640–1652. doi: 10.1038/s41594-023-01096-3

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© The Author(s) 2023

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. 1 — a, MBD-mediated targeting to methylated CpG. Here, KDM2B is redirected to methylated CpG via the MBD. b, Schematic of MBD-Luc and MBD-KDM2B fusions containing a V5 tag. MBD-KDM2B contains the histone demethylase domain (JmjC) and a mutated CxxC. c, Left: immunofluorescence in HS-SY-II cells of the MBD constructs (V5, magenta) with BCOR (green) and H2AK119ub1 (cyan). Arrowheads point to the MBD foci. Scale bars, 5 μm. Right: percentage of BCOR or H2AK119ub1 foci overlapping a V5 focus in n = 3 (MBD-Luc) or n = 4 (MBD-KDM2B) biological replicates. Data represent the mean and the s.e.m. d, Left: immunofluorescence for V5 (magenta) and SS18-SSX1 (HA, cyan). Right: percentage of HA (SS18-SSX1) foci overlapping a V5 focus. Data represent the mean ± s.e.m. in n = 5 biological replicates. e, Left: immunofluorescence of MBD-KDM2B (V5, magenta) in the presence of different sgRNAs (eGFP background fluorescence) with SS18-SSX1 (HA, cyan) in HS-SY-II-Cas9 cells. Right: percentage of HA (SS18-SSX1) foci overlapping a V5 focus in n = 2 (sgBCOR, sgEZH2), n = 3 (sgPCGF1, sgEED) or n = 4 (sgCTRL) biological replicates. Data represent the mean ± s.e.m. P values determined by unpaired one-tailed t-test between groups (**P = 0.003; ***P = 0.0005). f, Heatmaps of H2AK119ub1 calibrated ChIP (purple) and SS18-SSX scaled HA CUT&RUN signals (blue) in HS-SY-II-Cas9 cells expressing empty sgRNA as control (sgEV) or targeting PCGF1 (sgPCGF1). Both heatmaps represent signals over H2AK119ub1 peaks (n = 11,099) called using H2AK119ub1 CUT&RUN in HS-SY-II (Extended Data Fig. 1h). Rows correspond to ±10-kb regions across the midpoint of each enriched region, ranked by increasing signal. g, H2AK119ub1 and SS18-SSX corresponding score distributions. h, Gene tracks for H2AK119ub1 and SS18-SSX at the SHH, IGF2, FGF4-FGF3 and WNT7B loci. i, k-means clustering of H2AK119ub1 and SS18-SSX log₂ ratio of sgPCGF1 over sgEV. j, Salt extraction assay displaying SS18-SSX1 levels by western blot in HS-SY-II-Cas9 cells expressing an empty vector (EV) or sgRNAs against PCGF1 or PCGF3. k, Percentage of total SS18-SSX per salt extraction fractions. Data represent the mean ± s.e.m. of n = 3 biological replicates. P values determined by paired one-tailed t-test between groups (*P = 0.03).

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