Fig. 6: SPT2 binding to H3–H4 regulates the levels of soluble and chromatin-bound H3.3 in human cells.

a, Distribution of HsSPT2 between soluble and chromatin fractions of U-2 OS cells; one representative experiment of two. b, GFP–HsSPT2 binding to chromatin in fixed or pre-extracted U-2 OS cells assessed by high-content microscopy. Graph shows the mean GFP intensity normalized to the fixed sample; n = 2, data are represented as mean of two independent experiments. Right, representative images. c, Schematics of the SNAP-tag assay for new histone H3.3 incorporation. d, Western blot showing SPT2 levels in WT, SPT2 KO-3 and KO-4 clones and in the rescued cell lines after reintroduction of untagged SPT2 WT or HBM; one representative experiment of two. e,f, Fluorescence intensity of new TMR-labeled SNAP-H3.3 in cell nuclei of the indicated U-2 OS SNAP-H3.3 cell lines after direct fixation (e) or pre-extraction (f). Data from each one of the four independent replicates is indicated with a different symbol, showing the reproducible trend of SPT2^WT rescuing new H3.3-SNAP levels to a higher degree than SPT2^HBM. Data are represented as mean ± s.d. from four independent experiments and normalized to WT. Statistical testing: one-way ANOVA with Dunnett’s post-test. NS, P > 0.05; *P < 0.05, **P < 0.01, ***P < 0.0001. One-way ANOVA P value results: KO-3 direct fixation, 0.0164; KO-4 direct fixation, 0.0510; KO-3 pre-extraction, 0.0017; KO-4 pre-extraction, 0.0139. Exact P values of Dunnett’s post-test comparisons are reported in Supplementary Table 3. g, Chromatin accessibility levels at DA genes in WT and SPT2 KO cells, measured by ATAC–seq and expressed in reads per million. The chromatin accessibility profile of a control dataset of 100 random, non-DA genes is shown in Extended Data Fig. 6m. h, Example of ATAC–seq tracks on histone genes in WT and SPT2 KO cells.

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