Figure S2.
Chromatin compaction induced by H1C overexpression causes replication stress in HSCs. (A) Experimental schematic. Freshly enriched BM HSPCs (Lin−c-Kit+) from WT C57BL/6J mice (CD45.2) were transduced with the indicated retrovirus. Then EGFP+HSCs (CD48−CD150+LSK) were sorted for further analysis (qRT-PCR, cell cycle analysis, and intracellular protein analysis). Approximately 500 EGFP+HSCs were transplanted into lethally irradiated recipient mice (CD45.1). (B) qRT-PCR analysis and the bar graph shows the expression of H1C in HSCs (n = 3 samples per group). (C) Representative line plots show ATAC peaks ± 5 kb from TSS in HSCs under indicated conditions. (D) Representative flow plots of cell cycle distribution in EGFP+HSCs by Ki-67 + DAPI staining. The bar graph on the right shows the percentage of the cell cycle distribution (n = 5 samples per group). (E) The bar graphs show the MFI of p-ATR (left), p-CHK1 (middle), and γH2AX (right) in EGFP+HSCs using flow cytometry staining and analysis (n = 5 samples per group). (F) Representative flow plots of LSK analysis in BM of the recipient mice at 16 wk after transplantation. The right bar graphs show the cell count of EGFP+LSK and EGFP+HSCs (EGFP+CD48−CD150+LSK) in BM from the recipient mice (n = 6 mice per group). (G) A proposed model shows the effect of chromatin hyper-compaction on HSCs. (H) Representative heatmaps and line plots show ATAC peaks ± 5 kb from TSS in HPCs under indicated conditions. HPCs (Lin−c-Kit+) from DekCon or DekTg mice were sorted and cultured with DMSO or HU (100 µM) for 48 h, followed by ATAC-seq. (I) Statistics and quantification of ATAC signal in HPCs (n = 2 samples per group). (J) Representative heatmaps and line plots show DEK CUT&Tag peaks ± 5 kb from TSS in HSCs (CD48−CD150+LSK) from WT C57BL/6J, DekTg, and Dekfl/fl,Tie2-Cre (Dek-cKO) mice. (K) Statistics and quantification of DEK CUT&Tag signal in HSCs (n = 2 samples per group). (L) Pathway analysis of the genes with significantly changed DEK CUT&Tag peaks (greater than twofold change, P < 0.01) in both comparison: DekTg versus WT and Dek-cKO versus WT. (M) The violin plots show distribution of DEK CUT&Tag signal at genomic regions (±5 kb from TSS) of IEGs in HSCs. The list IEGs is presented in Table S1. (N and O) The violin plots show distribution of H3K27ac CUT&Tag and ATAC signal at genomic regions (±5 kb from TSS) of IEGs in HSCs. The data in Dekfl/fl and Dek-cKO groups were analyzed using a published data (GSE166434). The data presented in panels B and D–K are representative of two independent experiments. The results shown in panels I, K, and M–O are illustrated as violin plots, and panels B and D–F are shown as mean ± SD. Statistical analyses were performed using a two-tailed unpaired Student’s t test. P values are presented as follows: *P < 0.05, ***P < 0.001. Refer to the image caption for details.

Chromatin compaction induced by H1C overexpression causes replication stress in HSCs. (A) Experimental schematic. Freshly enriched BM HSPCs (Lin−c-Kit+) from WT C57BL/6J mice (CD45.2) were transduced with the indicated retrovirus. Then EGFP+HSCs (CD48−CD150+LSK) were sorted for further analysis (qRT-PCR, cell cycle analysis, and intracellular protein analysis). Approximately 500 EGFP+HSCs were transplanted into lethally irradiated recipient mice (CD45.1). (B) qRT-PCR analysis and the bar graph shows the expression of H1C in HSCs (n = 3 samples per group). (C) Representative line plots show ATAC peaks ± 5 kb from TSS in HSCs under indicated conditions. (D) Representative flow plots of cell cycle distribution in EGFP+HSCs by Ki-67 + DAPI staining. The bar graph on the right shows the percentage of the cell cycle distribution (n = 5 samples per group). (E) The bar graphs show the MFI of p-ATR (left), p-CHK1 (middle), and γH2AX (right) in EGFP+HSCs using flow cytometry staining and analysis (n = 5 samples per group). (F) Representative flow plots of LSK analysis in BM of the recipient mice at 16 wk after transplantation. The right bar graphs show the cell count of EGFP+LSK and EGFP+HSCs (EGFP+CD48−CD150+LSK) in BM from the recipient mice (n = 6 mice per group). (G) A proposed model shows the effect of chromatin hyper-compaction on HSCs. (H) Representative heatmaps and line plots show ATAC peaks ± 5 kb from TSS in HPCs under indicated conditions. HPCs (Lin−c-Kit+) from DekCon or DekTg mice were sorted and cultured with DMSO or HU (100 µM) for 48 h, followed by ATAC-seq. (I) Statistics and quantification of ATAC signal in HPCs (n = 2 samples per group). (J) Representative heatmaps and line plots show DEK CUT&Tag peaks ± 5 kb from TSS in HSCs (CD48−CD150+LSK) from WT C57BL/6J, DekTg, and Dekfl/fl,Tie2-Cre (Dek-cKO) mice. (K) Statistics and quantification of DEK CUT&Tag signal in HSCs (n = 2 samples per group). (L) Pathway analysis of the genes with significantly changed DEK CUT&Tag peaks (greater than twofold change, P < 0.01) in both comparison: DekTg versus WT and Dek-cKO versus WT. (M) The violin plots show distribution of DEK CUT&Tag signal at genomic regions (±5 kb from TSS) of IEGs in HSCs. The list IEGs is presented in Table S1. (N and O) The violin plots show distribution of H3K27ac CUT&Tag and ATAC signal at genomic regions (±5 kb from TSS) of IEGs in HSCs. The data in Dekfl/fl and Dek-cKO groups were analyzed using a published data (GSE166434). The data presented in panels B and D–K are representative of two independent experiments. The results shown in panels I, K, and M–O are illustrated as violin plots, and panels B and D–F are shown as mean ± SD. Statistical analyses were performed using a two-tailed unpaired Student’s t test. P values are presented as follows: *P < 0.05, ***P < 0.001.

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