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. 2020 Oct 27;11:5414. doi: 10.1038/s41467-020-18955-y

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© The Author(s) 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. 6 — a Correlation between differentiation-ssociated and H3.3-G34W-associated gene expression changes. For the differentiation log-fold changes (LFC) were calculated between average of profiles from each differentiation timepoint group (very early: 0.5 to 2 h; early: 4 to 554 16 h, mid: 24 to 168 h; and late: 336 to 504 h) and the profiles of mesenchymal stem cells (MSCs) sampled prior to the start of the experiment. Each hexagon reflects the density of the underlying data points. Genes with most pronounced changes in both data sets are marked. b Principal component analysis of RNA-Seq data from osteogenic differentiation of MSC (greyscale rectangles) and expression profiles of GCTB samples, H3.3 WT (blue triangles) and H3.3 MUT (red circles), only PC2 and PC3 are shown, while PC1 capturing batch effects between two experiments is not shown. The sampled differentiation timepoints were grouped as in a. For H3.3 WT and H3.3 MUT each data point represents one patient. The isochores and background shading give a surface fit-based estimate of differentiation time for each point of the (PC2, PC3) plane. c Alkaline phosphatase staining of nt-SC, H3.3 WT, and H3.3 MUT cells during osteogenic differentiation. d Quantification of alkaline phosphatase (ALP) activity relative to the viability as a surrogate for cell count over the time course of 15 days of in vitro osteogenic differentiation. Each data point represents one patient. Middle bars represent means, error bars show standard deviations within each group. H3.3 WT in blue, n = 8 and H3.3 MUT in red, n = 7 biologically independent cell lines from different patients. P-values are from unpaired (for within-group comparisons) and paired (for between-group comparisons) two-tailed t-tests. e. Model for GCTB tumorigenesis: The H3F3A mutation encoding H3.3-G34W occurs in osteoblastic precursor cells and leads to alterations in osteogenic differentiation. Epigenetic differences described between H3.3 WT and H3.3 MUT stromal cells are the result of measuring cells at different differentiation stages and direct effects of the mutated histone.