Fig 2: Rad21 deficiency leads to changes in chromatin accessibility in LSK cells in response to inflammation. (A–K) LSK cells from 24-mo-old mice were infected with an shRNA against Rad21 or a control shRNA (shLuci) and cultured for 2 d followed by an exposure to LPS/Pam3 (LPS: 200 ng/ml; Pam3: 1 µg/ml) or PBS control for 14 h. At that time point, LSK cells were subjected to ATAC-seq. (A) Venn diagrams showing the number of ATAC-seq peaks (P value cutoff = 1e-3) in PBS-treated LSK cells. There was a reduction in the overall number of peaks in LSK cells that were infected with an shRNA against Rad21 versus control shRNA (shLuci)–infected LSK cells. (B) The histogram depicts the reduction in ATAC-seq peaks in the indicated genomic regions of shRad21 versus shLuci-infected LSK cells that were treated with PBS or LPS/Pam3. For the calculation, only common peaks found in both of the two replicates have been used. (C) Distribution of the ATAC-seq peaks on promoters and enhancers (±3 kb) in LSK cells infected with the indicated shRNAs. (D) Hierarchical clustering of the Pearson’s correlation of the ATAC-seq reads in all the samples analyzed. (E) Signaling pathway enrichment (x axis) of the genes associated with peaks depleted in shRad21-LPS/Pam3 compared with shLuci-LPS/Pam3–treated cells. (F) Upstream regulators associated with the genes depicted in E. X axis shows the activation Z-score of the target genes, and color code shows enrichment P value (-log10). (G–I) Genomic views of the Ccl gene cluster (G), Tlr4 (H), and Il6 (I). The yellow highlight marks the genomic regions where ATAC-seq identified increases in chromatin accessibility in response to LPS/Pam3 treatment, but diminished increases in chromatin accessibility in Rad21 knockdown versus control shRNA infected cells. (J and K) Motif discovery results on the peaks enriched in shLuci-LPS/Pam3 compared with shLuci-PBS–treated cells (J) or depleted in shRad21-LPS/Pam3 compared with shLuci-LPS/Pam3–treated cells (K).

Fig 3: Rad21 mediates differentiation of HSPCs in response to inflammation through NF-κB–dependent signaling. (A) Analysis of DEGs in two RNA-seq experiments: (1) on GFP+CD48−Sca-1+ cells purified from cultured LSK cells, 3 d after isolation and lentiviral infection with an shRNA targeting Rad21 or a control shRNA (luciferase = luci) and (2) on freshly isolated GFP+ LSK cells from mice that were transplanted with lentivirally infected LSK cells (shRNA-Rad21 or shRNA-luci), 4 mo after transplantation. KEGG pathway analysis shows the 14 most down-regulated pathways in Rad21 knockdown cells versus control cells. The analysis did not identify significantly (q < 0.1) up-regulated pathways. Green asterisks highlight NF-κB–related pathways. DEGs in these pathways from the in vivo analysis are depicted in B. (B) The heat map shows DEGs based on GO-terms related to the KEGG pathways highlighted in A (in vivo) in freshly isolated Rad21 knockdown LSK cells (shRad21) versus control (shLuci) LSK cells reisolated 4 mo after LSK cell transplantation. The heat map shows row-scaled and variance-stabilized expression counts for each gene. Red asterisks highlight inflammatory receptor genes. (C) The seven top transcription factor complexes, regulating DEGs in the experiment on culture-activated LSK cells (Fig. S2 A), were identified with the help of the GeneTrail web service. All significant DEGs were sorted according to their fold change and used as input for GeneTrail with default parameters. An adjusted P value cutoff of 0.05 was applied to select the most significant enriched/depleted transcription factor complexes, regulating these DEGs. Seven depleted transcription factor complexes (q < 0.05) were identified in shRad21-LSK cells. (D and E) 5,000 GFP+LSK cells were isolated from the recipient mice of Rad21 knockdown or control LSK cells at 4 mo after transplantation and cultured for 3 d. The percentage of CD11b+ cells in the cultures was determined by FACS. (D) Representative FACS plots showing percentages of CD11b+ cells in the indicated groups. (E) The histogram shows the percentage of CD11b+ cells in the LSK cell cultures in the indicated groups (in total, n = 8 mice per group were analyzed in n = 2 independent experiments). Statistical significance was assessed with two-way ANOVA followed by Tukey’s multiple comparison test on logit-transformed data. (F) LSK cells from 16-mo-old NF-κB1−/− or wild-type mice were infected with viruses carrying shRNAs against Rad21 or luciferase and cultured with or without LPS/Pam3 for 3 d. The graphs depict the increase in differentiation of LSK cells into myeloid cells (CD11b+) in LPS/Pam3-treated cultures relative to PBS-treated cultures of LSK cells from wild-type mice (left) and LSK cells from NF-κB1−/− mice (right) that were infected with the indicated shRNAs. The dots represent individual mice (in total, n = 6 wild-type mice and n = 4 NF-κB1−/− mice were analyzed in n = 2 independent experiments). Statistical significance was assessed with paired t test after log transformation. All data represent mean ± SD; *, P < 0.05; ****, P < 0.0001; ns, not significant.

Fig 4: mRNA expression signatures of MPPs upon in vivo LPS stimulation. (A–C) NanoString analysis was performed on MPPs (CD34+LSK) from young (2 mo old) and old (24 mo old) mice, 24 h after injection of LPS (1.5 mg/kg body weight) or control, PBS injection (n = 12 young mice in three pools of 4 mice per pool, and n = 3 old mice as individual samples; the same experiment as depicted in Fig. 6, D–I). The heat maps show the clustering of the indicated groups according to changes in the mRNA expression of inflammatory genes (A), differentiation factors (B), and cohesin genes and cohesin regulator genes (C).

Fig 5: Age-dependent impairment in expansion potential of HSCs in response to LPS exposure associates with a failure to down-regulate inflammation signaling and cohesin expression in the resolution phase of inflammation. (A) HSCs (CD150+CD34-LSK) were isolated from young (2–3 mo old) and old (24 mo old) wild-type mice and treated with PBS or LPS (200 ng/ml) for 2 h. mRNA expression of Il6 relative to Actb was analyzed at the indicated time points after LPS stimulation (n = 3–4 mice per group). Statistical significance was assessed with Welch’s t test after log transformation. (B–I) In vivo LPS stimulation was performed in young (2–3 mo old) and old (24 mo old) wild-type mice at a dose of 1.5 mg/kg body weight. Mice were sacrificed at 24 h after LPS injection. (B and C) The charts depict the fold change in the percentages of HSCs (B) and MPPs (C) in BM of LPS-stimulated mice relative to control (PBS injected) mice of the indicated age groups. Multiple pairwise comparisons of fold changes of HSCs (CD150+CD41-CD34-LSK; B) and MPPs (CD34+ LSK; C) were performed. Wilcoxon test was used to test difference of all pairwise ratios for each cell type (in total, n = 16 young mice and n = 4 old mice were analyzed in four independent experiments). (D–I) NanoString analysis was performed on HSCs from young (2–3 mo old) and old (24 mo old) mice, 24 h after LPS injection along with controls. The heat maps show the clustering of the indicated groups according to changes in the mRNA expression of inflammatory genes (D), differentiation factors (F), and cohesin genes and cohesin regulator genes (H). (E, G, and I) Histograms show the relative mRNA expression of representative genes encoding for cytokines (E), differentiation inducing factors (G), or cohesin components (I). The depicted representative examples were selected from the NanoString analysis in D, F, and H. The data are shown as linear fold change relative to the nontreated young control (n = 12 young mice in three pools of 4 mice per pool, and n = 3 old mice as individual samples). 12 housekeeping genes were used for the normalization of the expression value. Statistical significance was assessed with Welch’s t test after log transformation. Data represent mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.