Figure 2.
The reduced DEK is responsible for chromatin relaxation and replication stress relief in HSCs. (A) Heatmap shows the expression of 270 chromatin remodelers between DMSO and Gem/HU-treated HSCs. The list on the right presents the most dysregulated genes (ranked by P value). (B) qRT-PCR analysis and the bar graph shows the expression of DEK gene in HSCs (CD48−CD150+LSK), LSK, HPCs, and Lin+ cells under indicated conditions (n = 5 samples per group). The indicated cell populations, derived from WT C57BL/6J mice, were sorted and cultured with DMSO, Gem (0.5 µM), or HU (100 µM) for 48 h. (C) Representative flow plots of DEK staining in HSCs under the condition referring to (B). The bar graph shows the MFI of DEK in HSCs (n = 4 samples per group). (D) Schematic for the generation of tissue-specific DEK-overexpressing mice. (E) Representative western blot shows the expression of DEK, Flag, and tubulin in BM Lin− cells from DekCon and DekTg mice. (F) qRT-PCR analysis and the bar graph shows the expression of DEK in HSCs from DekCon and DekTg mice (n = 5 mice per group). (G) Representative heatmaps and line plots show H3K27ac CUT&Tag peaks ± 5 kb from TSS in HSCs. HSCs (CD48−CD150+LSK) from DekCon or DekTg mice were sorted and cultured with DMSO or HU (100 µM) for 48 h, followed by H3K27ac CUT&Tag-seq. (H) Statistics and quantification of H3K27ac CUT&Tag signal in HSCs (n = 2 samples per group). (I) Representative heatmaps and line plots show ATAC peaks ± 5 kb from TSS in HSCs under indicated conditions. (J) Statistics and quantification of ATAC signal in HSCs (n = 2 samples per group). (K) Distribution of ATAC and H3K27ac CUT&Tag peaks across the indicated gene loci. (L) Experimental schematic. HSCs (CD48−CD150+LSK) from DekCon or DekTg mice were sorted and cultured with HU (100 µM) for 24 h and released from HU for 24 h or cultured with DMSO for 48 h, followed by flow cytometry analysis. (M and N) Representative flow plots of p-ATR and p-CHK1 staining in HSCs. The bar graphs show the MFI of p-ATR and p-CHK1 in HSCs (n = 5 samples per group). The data presented in panels B–N are representative of two independent experiments. The results shown in panels H and J are illustrated as violin plots, and panels B, C, F, M, and N are shown as mean ± SD. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test for panels B and C and a two-tailed unpaired Student’s t test for panels F–N. P values are presented as follows: **P < 0.01, ***P < 0.001; n.s., not significant. Source data are available for this figure: SourceData F2. Refer to the image caption for details.

The reduced DEK is responsible for chromatin relaxation and replication stress relief in HSCs. (A) Heatmap shows the expression of 270 chromatin remodelers between DMSO and Gem/HU-treated HSCs. The list on the right presents the most dysregulated genes (ranked by P value). (B) qRT-PCR analysis and the bar graph shows the expression of DEK gene in HSCs (CD48−CD150+LSK), LSK, HPCs, and Lin+ cells under indicated conditions (n = 5 samples per group). The indicated cell populations, derived from WT C57BL/6J mice, were sorted and cultured with DMSO, Gem (0.5 µM), or HU (100 µM) for 48 h. (C) Representative flow plots of DEK staining in HSCs under the condition referring to (B). The bar graph shows the MFI of DEK in HSCs (n = 4 samples per group). (D) Schematic for the generation of tissue-specific DEK-overexpressing mice. (E) Representative western blot shows the expression of DEK, Flag, and tubulin in BM Lin− cells from DekCon and DekTg mice. (F) qRT-PCR analysis and the bar graph shows the expression of DEK in HSCs from DekCon and DekTg mice (n = 5 mice per group). (G) Representative heatmaps and line plots show H3K27ac CUT&Tag peaks ± 5 kb from TSS in HSCs. HSCs (CD48−CD150+LSK) from DekCon or DekTg mice were sorted and cultured with DMSO or HU (100 µM) for 48 h, followed by H3K27ac CUT&Tag-seq. (H) Statistics and quantification of H3K27ac CUT&Tag signal in HSCs (n = 2 samples per group). (I) Representative heatmaps and line plots show ATAC peaks ± 5 kb from TSS in HSCs under indicated conditions. (J) Statistics and quantification of ATAC signal in HSCs (n = 2 samples per group). (K) Distribution of ATAC and H3K27ac CUT&Tag peaks across the indicated gene loci. (L) Experimental schematic. HSCs (CD48−CD150+LSK) from DekCon or DekTg mice were sorted and cultured with HU (100 µM) for 24 h and released from HU for 24 h or cultured with DMSO for 48 h, followed by flow cytometry analysis. (M and N) Representative flow plots of p-ATR and p-CHK1 staining in HSCs. The bar graphs show the MFI of p-ATR and p-CHK1 in HSCs (n = 5 samples per group). The data presented in panels B–N are representative of two independent experiments. The results shown in panels H and J are illustrated as violin plots, and panels B, C, F, M, and N are shown as mean ± SD. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test for panels B and C and a two-tailed unpaired Student’s t test for panels F–N. P values are presented as follows: **P < 0.01, ***P < 0.001; n.s., not significant. Source data are available for this figure: SourceData F2.

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