Figure 5.
DEK haploinsufficiency promotes chromatin relaxation, replication stress relief, and function recovery of Fancd2-deficient HSCs. (A) A schematic for the generation of Fancd2-deficient allele. (B) qRT-PCR analysis. The left bar graph shows the expression of Fancd2 in LSK cells from Fancd2+/+ and Fancd2−/− mice (n = 4 mice per group). The right bar graph shows DEK expression in HSCs (n = 4 samples per group). HSCs (CD48−CD150+LSK) were treated with DMSO or HU (100 µM) for 24 h (n = 4 sample per group). (C) qRT-PCR analysis and the bar graph shows DEK expression in HSCs from indicated mice (n = 4 mice per group). (D) Representative heatmaps and line plots show ATAC peaks ± 5 kb from TSS in HSCs with indicated genotypes. (E) Statistics and quantification of ATAC signal in HSCs (n = 2 samples per group). (F) Representative flow plots of γH2AX staining in HSCs with indicated genotypes. The right bar graph shows the MFI of γH2AX (n = 5 samples per group). (G) Representative flow plots of p-ATR staining in HSCs with indicated genotypes. The bar graphs show the MFI of p-ATR (left) and p-CHK1 (right) in HSCs (n = 5 samples per group). (H) Representative flow plots show the EdU+ in HSCs with indicated genotypes. The right bar graph shows the frequency of EdU+ cells in HSCs with indicated genotypes (n = 5 samples per group). The freshly sorted HSCs (CD48−CD150+LSK) were cultured in vitro for 24 h, followed by adding EdU (10 µM) into the medium and culture for 2 h, then flow cytometry analysis of EdU incorporation was conducted. (I) Representative flow plots of LSK cells in BM from mice with indicated genotypes. (J) The bar graphs show the count of LSK (left) and HSCs (CD48−CD150+LSK) (right) in BM (n = 6 mice per group). (K) Experimental schematic for the competitive transplantation assay. (L) The chart shows the donor chimerism in PB of the recipient mice at indicated time points after transplantation (n = 6 recipient mice per group). (M) The bar graphs show the donor chimerism in LSK (left) and HSCs (right) of recipient mice at 16 wk after transplantation (n = 6 recipient mice per group). The data presented in panels B–M are representative of two independent experiments. The results shown in panel E are illustrated as violin plots, and other panels are shown as mean ± SD. Statistical analyses were performed using a two-tailed unpaired Student’s t test for panels B and E and one-way ANOVA followed by Tukey’s post hoc test for panels C and F–M. P values are presented as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. Refer to the image caption for details.

DEK haploinsufficiency promotes chromatin relaxation, replication stress relief, and function recovery of Fancd2-deficient HSCs. (A) A schematic for the generation of Fancd2-deficient allele. (B) qRT-PCR analysis. The left bar graph shows the expression of Fancd2 in LSK cells from Fancd2+/+ and Fancd2−/− mice (n = 4 mice per group). The right bar graph shows DEK expression in HSCs (n = 4 samples per group). HSCs (CD48−CD150+LSK) were treated with DMSO or HU (100 µM) for 24 h (n = 4 sample per group). (C) qRT-PCR analysis and the bar graph shows DEK expression in HSCs from indicated mice (n = 4 mice per group). (D) Representative heatmaps and line plots show ATAC peaks ± 5 kb from TSS in HSCs with indicated genotypes. (E) Statistics and quantification of ATAC signal in HSCs (n = 2 samples per group). (F) Representative flow plots of γH2AX staining in HSCs with indicated genotypes. The right bar graph shows the MFI of γH2AX (n = 5 samples per group). (G) Representative flow plots of p-ATR staining in HSCs with indicated genotypes. The bar graphs show the MFI of p-ATR (left) and p-CHK1 (right) in HSCs (n = 5 samples per group). (H) Representative flow plots show the EdU+ in HSCs with indicated genotypes. The right bar graph shows the frequency of EdU+ cells in HSCs with indicated genotypes (n = 5 samples per group). The freshly sorted HSCs (CD48−CD150+LSK) were cultured in vitro for 24 h, followed by adding EdU (10 µM) into the medium and culture for 2 h, then flow cytometry analysis of EdU incorporation was conducted. (I) Representative flow plots of LSK cells in BM from mice with indicated genotypes. (J) The bar graphs show the count of LSK (left) and HSCs (CD48−CD150+LSK) (right) in BM (n = 6 mice per group). (K) Experimental schematic for the competitive transplantation assay. (L) The chart shows the donor chimerism in PB of the recipient mice at indicated time points after transplantation (n = 6 recipient mice per group). (M) The bar graphs show the donor chimerism in LSK (left) and HSCs (right) of recipient mice at 16 wk after transplantation (n = 6 recipient mice per group). The data presented in panels B–M are representative of two independent experiments. The results shown in panel E are illustrated as violin plots, and other panels are shown as mean ± SD. Statistical analyses were performed using a two-tailed unpaired Student’s t test for panels B and E and one-way ANOVA followed by Tukey’s post hoc test for panels C and F–M. P values are presented as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.

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