PU.1 enforces quiescence and limits hematopoietic stem cell expansion during inflammatory stress

Abstract

Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress.

Figure 1.

Chronic IL-1 induces repression of cell cycle and protein synthesis genes. (A) Experimental design for RNA-seq studies (n = 4–7 pools of SLAM cells from mice treated for 20 d ± IL-1β). Pools were generated from three independent cohorts of mice. (B) Volcano plot of DEGs (P_adj ≤ 0.05) in IL-1–exposed SLAM cells (LSK/Flk2⁻/CD48⁻/CD150⁺) from A showing log₂ fold change (FC) versus −log₁₀ P value significance. See also Table S1. (C) GO category enrichment of downregulated DEGs in IL-1–exposed SLAM cells from A, expressed as −log₁₀ P value. See also Table S2. (D) IPA showing enriched upstream regulators of DEGs in IL-1–exposed SLAM cells from A. See also Table S3. (E) GSEA analysis of significantly downregulated DEGs. GSEA plots show negative enrichment of translation and Myc pathway genes in IL-1–exposed SLAM cells from A. See also Table S4. (F) Experimental design for Fluidigm qRT-PCR analyses and intracellular FACS staining of HSC^LT (LSK/Flk2⁻/CD48⁻/CD150⁺/CD34⁻/EPCR⁺) from mice treated for 20 d with or without IL-1β (left), and quantification by Fluidigm qRT-PCR array of cell cycle and protein synthesis gene expression in HSC^LT (n = 8/group). Data are expressed as log₁₀ fold expression versus −IL-1β. Box represents upper and lower quartiles with line representing median value. Whiskers represent minimum and maximum values. Data are representative of two independent experiments. (G) Intracellular flow cytometry analysis of Myc protein levels in HSC^LT (n = 10 −IL-1β; 8 +IL-1β). Data are expressed as fold change of MFI versus −IL-1β. Individual values are shown with bars representing mean values. Data are compiled from three independent experiments. (H) Intracellular flow cytometry analysis of puro incorporation in HSC^LT (n = 9 −IL-1β; 10 +IL-1β). Data are expressed as fold change of MFI versus −IL-1β. Individual values are shown with bars representing mean values. Data are compiled from three independent experiments. (I) Experimental design for cell cycle analyses of SLAM cells and HSC^LT from mice treated for 20 d with or without IL-1. (J) Representative flow cytometry plots showing cell cycle distribution in SLAM cells and HSC^LT from I. (K) Quantification of cell cycle phase distribution in SLAM cells and HSC^LT from I (n = 5/group). Data are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney U test or ANOVA with Tukey’s test in K. Error bars represent SD. See also Fig. S1.

Figure S1.

Characterization of SLAM cells and reporter mice. (A) GO category enrichment of significantly upregulated DEGs in SLAM cells from mice treated for 20 d with or without IL-1β versus 20 d −IL-1β, expressed as −log₁₀ P value. See Table S2 for complete list of GO categories. (B and C) Quantification by Fluidigm qRT-PCR array of gene expression in SLAM cells from mice treated with or without IL-1β for 20 d (n = 8/group). Data are expressed as log₁₀ fold expression versus −IL-1β. Box represents upper and lower quartiles with line representing median value. Whiskers represent minimum and maximum values. Data are representative of two independent experiments. (D) Comparison of normalized enrichment scores (NESs) for indicated gene signatures from IL-1–exposed SLAM cells and SLAM cells from CIA mice (GSE129511). (E) Representative FACS plots showing frequencies of phenotypic HSC^LT fraction within the SLAM gate from mice treated with IL-1β versus without IL-1β for either 1 or 20 d. Data are representative of multiple (more than three) experiments. (F) Geometric MFI of Myc from one of three experiments (n = 5/group). Individual values are shown with bars representing mean values. (G) Geometric MFI of puro from one of three experiments (n = 5/group). Individual values are shown with bars representing mean values. (H) Experimental design for H2B-GFP in vivo labeling experiments. Dox, doxycycline. (I) Representative sort gates showing identification of GFP^hi and GFP^lo populations within the HSC^LT gate. Myeloid progenitors (MyPro), which rapidly dilute the GFP label, are shown in gray. (J) Representative FACS plot showing GFP and YFP profiles of single-color control Lin⁻/c-Kit⁺ HSPC from Myc-GFP (G⁺Y⁻) or PU1-EYFP (G⁻Y⁺) mice relative to G⁻Y⁻ controls (left) and representative FACS plots of defined HSPC populations from Myc-GFP::PU.1-EYFP (G⁺Y⁺) mice versus G⁻Y⁻ controls (right). (K) Quantification of GFP and YFP levels in each HSPC population from mice in J (n = 2/group). Individual values are shown with bars representing mean values. Data in J and K are representative of at least two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney U test or ANOVA with Tukey's post-test in F and G. Error bars represent SD.

Figure 2.

IL-1–induced gene repression is rapid and independent of HSC^LT divisional history. (A) Experimental design for cell cycle analyses of SLAM cells and HSC^LT from mice treated for 1 d with or without IL-1β. (B) Representative flow cytometry plots showing cell cycle distribution in SLAM cells and HSC^LT from A. (C) Quantification of cell cycle phase distribution in SLAM cells and HSC^LT from A (n = 10/group). Data are compiled from three independent experiments. (D) Quantification by Fluidigm qRT-PCR array of cell cycle gene expression in HSC^LT from mice treated for 1 d with or without IL-1β (n = 8/group). Data are expressed as log₁₀ fold expression versus −IL-1β. Box represents upper and lower quartiles with line representing median value. Whiskers represent minimum and maximum values. Data are representative of two independent experiments. (E) Quantification of IL-1–repressed protein synthesis and cell cycle genes from Fluidigm qRT-PCR array in D. Data are representative of two independent experiments. (F) Experimental design for analysis of divisional history in HSC^LT using H2B-GFP mice treated for 20 d with or without IL-1β. (G) Representative FACS plots showing analysis of HSC^LT divisional history via GFP dilution from H2B-GFP mice treated for 20 d with or without IL-1β. (H) Quantification of divisional history of mice in F based on GFP dilution (n = 5–6/group). Data are compiled from two independent experiments. (I) Quantification by Fluidigm qRT-PCR array of IL-1–repressed genes in GFP^hi and GFP^lo HSC^LT from H2B-GFP mice in F (n = 8/group). Data are expressed as log₁₀ fold expression versus −IL-1β. Box represents upper and lower quartiles with line representing median value. Whiskers represent minimum and maximum values. Data are representative of two independent experiments. (J) Quantification of IL-1 target genes from Fluidigm qRT-PCR array in I. Data are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney U test or ANOVA with Tukey’s test in C, H, I, and J. Error bars represent SD. See also Fig. S1.

Figure 3.

IL-1–induced gene repression is associated with high PU.1 levels. (A) GSEA enrichment of significantly downregulated genes in publicly available datasets versus RNA-seq analysis of SLAM cells from mice treated with or without IL-1β for 20 d. Data show downregulated genes as negatively enriched in SLAM cells from mice treated for 20 d with or without IL-1β. See also Table S5. (B) Venn diagram showing intersections between gene sets in A. A partial list of common genes is depicted at the right of the diagram. See also Table S6 for complete list of genes. (C) Left: Experimental design for analysis of PU.1-EYFP::GFP-Myc mice treated with or without IL-1β for 20 d. Right: Representative FACS plots showing gating strategy to identify PU.1^lo and PU.1^hi SLAM cells based on PU.1-EYFP expression levels in these mice. (D) Representative FACS plots (left) and quantification (right) showing PU.1-EYFP expression levels in PU.1^lo and PU.1^hi SLAM cell fractions from C (n = 3 −IL-1β; 5 +IL-1β). PU.1-EYFP negative control is shown in gray. Individual values are shown with bars representing mean values. Data are representative of two independent experiments. (E) Representative FACS plots (left) and quantification (right) showing GFP-Myc expression levels in PU.1^lo and PU.1^hi SLAM cell fractions from C (n = 3 −IL-1β; 5 +IL-1β). GFP-Myc negative control is shown in gray. Individual values are shown with bars representing mean values. Data are representative of two independent experiments. (F) Quantification by Fluidigm qRT-PCR array of cell cycle and protein synthesis gene expression in PU.1^hi and PU.1^lo SLAM cells from mice treated with or without IL-1β for 20 d (n = 8/group). Data are expressed as log₁₀ fold expression versus −IL-1β. Box represents upper and lower quartiles with line representing median value. Whiskers represent minimum and maximum values. Data are representative of two independent experiments. (G) Representative FACS plots (left) and quantification (right) of cell cycle distribution in PU.1^hi SLAM cells from mice treated with or without IL-1β for 20 d (n = 3/group) using Ki-67 and DAPI. Data are compiled from two independent experiments. (H) Representative FACS plots (left) and quantification (right) showing PU.1-EYFP expression levels in HSC^LT from mice in C (n = 3 −IL-1β; 5 +IL-1β). PU.1-EYFP negative control is shown in gray in FACS plots. Individual values are shown with bars representing mean values. Data are representative of two independent experiments. (I) Representative FACS plots (left) and quantification (right) showing GFP-Myc expression levels in PU.1^lo and PU.1^hi SLAM cell fractions from mice in C. GFP-Myc negative control is shown in gray in FACS plots. Individual values are shown with bars representing mean values. Data are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney U test or ANOVA with Tukey’s test in D–G. Error bars represent SD. See also Figs. S1 and S2.

Figure S2.

Characterization of SLAM cell fractions based on PU.1 level. (A) Experimental design for analysis of Myc levels in PU.1^hi SLAM cells. (B) Representative FACS plot (left) and quantification (right) of Myc levels in PU.1^hi SLAM HSCs from PU.1-EYFP mice treated with or without IL-1β for 20 d (n = 4/group). Individual values are shown with bars representing means. Data are compiled from two independent experiments. (C–E) Quantification by Fluidigm qRT-PCR array of IL-1 target gene expression (C), lineage gene expression (D), and HSC gene expression (E) in PU.1^lo and PU.1^hi SLAM cells from PU.1-EYFP mice treated with or without IL-1β for 20 d (n = 8/group). Data are expressed as log₁₀ fold expression versus −IL-1β. Box represents upper and lower quartiles with line representing median value. Whiskers represent minimum and maximum values. Data are representative of two independent experiments. (F) Representative FACS plots (left) and quantification (right) of cell cycle distribution in PU.1^lo SLAM cells from mice treated with or without IL-1β for 20 d (n = 3/group) using Ki-67 and DAPI. Data are compiled from two independent experiments. (G) Experimental design (left) and quantification (right) of cell cycle distribution in PU.1^lo and PU.1^hi SLAM cells from mice treated with or without IL-1β for 1 d (n = 4/group). Data are compiled from two independent experiments. (H) Quantification by Fluidigm qRT-PCR array of cell cycle and protein synthesis genes in PU.1^lo and PU.1^hi SLAM cells from PU.1-EYFP mice treated with or without IL-1β for 1 d (n = 8/group). Data are expressed as log₁₀ fold expression versus −IL-1β. Box represents upper and lower quartiles with line representing median value. Whiskers represent minimum and maximum values. Data are representative of two independent experiments. (I) Quantification of fold change (f.c.) in geometric MFI of HSC^LT from PU.1-EYFP mice treated with or without IL-1β for 1 d (n = 5/group). Individual values are shown with bars representing mean values. Data are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney U test or ANOVA with Tukey’s test in C–E, and H. Error bars represent SD.

Figure 4.

Direct IL-1 stimulation in vitro induces PU.1 and restricts HSC^LT cell cycle entry. (A) Representative FACS plots (left) and quantification (right) of cell cycle distribution in HSC^LT cultured for 24 h with or without IL-1β (n = 6/group). Data are compiled from three independent experiments. (B) Experimental design for single-cell tracking studies of PU.1-EYFP HSC^LT cultured with or without IL-1β. Time to first cell division was tracked via microscopy. (C) Graph showing kinetics of first cell division in HSC^LT from B (n = 194 −IL-1β; 139 IL-1β). Data are compiled from three independent experiments. (D) PU.1-EYFP levels in HSC^LT before first cell division (n = 137 −IL-1β; 192 +IL-1β). Data are compiled from three independent experiments. Box shows upper and lower quartiles with line showing median value, and whiskers upper and lower 10th percentile and individual dots represent outliers. (E) Hierarchical clustering analysis of PU.1-EYFP expression over time from the start of observation until the first division in PU.1-EYFP HSC^LT cultured with or without IL-1 (Euclidean distance with Ward linkage; n = 557 −IL-1β; 489 +IL-1β). Data are compiled from three independent experiments. (F) Quantification of PU.1-EYFP level (as arbitrary units [AU]; left) and time to division (right) of HSC^LT cultured with or without IL-1β in E. Individual values (representing means from cells cultured either with or without IL-1β in an experiment) are shown with bars representing mean values (n = 6). Data are compiled from three independent experiments. (G) Distribution of HSC^LT from E in different clusters based on culture with or without IL-1β. Data are compiled from three independent experiments. (H) Experimental design for Fluidigm qRT-PCR array analysis of HSC^LT cultured with or without IL-1β for 12 h. (I) Quantification by Fluidigm qRT-PCR array of cell cycle and protein synthesis gene expression in HSC^LT from F (n = 8/group). Data are expressed as log₁₀ fold expression versus −IL-1β. Box represents upper and lower quartiles with line representing median value. Whiskers represent minimum and maximum values. Data are representative of two independent experiments. (J) Experimental design for cell cycle analysis of HSC^LT cultured with or without IL-1β and with or without 100 nM Oma for 24 h. (K) Representative FACS plots (left) and quantification (right) of cell cycle distribution in HSC^LT from H (n = 3/group). Data are from one experiment. (L) Proportion of HSC^LT with >2N DAPI signal from H. Individual values are shown with means (horizontal line). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney U test or one-way ANOVA with Tukey’s test in F, G, K, and L. Error bars represent SD. See also Fig. S3.

Figure S3.

Impact of PU.1 expression and Oma on HSC^LT. (A) Single-cell tracking studies of PU-ERT HSC^LT cultured with or without 4-OHT. Quantification (left) of time to first cell division (n = 57 −4-OHT; 86 +4-OHT) and graph showing kinetics of first cell division in HSC^LT (right). Data are representative of two independent experiments. Box shows upper and lower quartiles with line showing median value, and whiskers upper and lower 10th percentile and individual dots represent outliers. (B) Representative FACS plots (left) and quantification (right) of FSC^hi HSCs based on FSC/DAPI in HSC^LT treated in vitro with or without IL-1β or with or without Oma for 24 h. Individual values are shown with lines representing mean values. Data are representative of two independent experiments. *, P < 0.05; **, P < 0.01 by Mann-Whitney U test or ANOVA with Tukey’s test in B. Error bars represent SD.

Figure 5.

PU.1 directly binds cell cycle and protein synthesis genes repressed by IL-1. (A) Experimental design for ChIP-seq experiment (n = 2/group). (B) Heatmap showing PU.1 ChIP-seq peak intensities versus WCE control at TSS ± 1 kb. (C) Transcription factor binding site motif enrichment at ChIP-seq peak sites. (D) Pie chart comparing IL-1–downregulated genes identified in SLAM cells by RNA-seq analysis in Fig. 1 and presence of PU.1 peaks at or near these genes (TSS ± 20 kb). (E) Pie chart showing PU.1 peak locations in IL-1–downregulated genes. (F) Transcription factor binding site motif enrichment at PU.1 ChIP-seq peak sites in IL-1–downregulated genes (TSS ± 20 kb). (G) GO category enrichment of IL-1–downregulated DEGs containing PU.1 peaks in C. Representative genes from the indicated categories are shown to the right. Data are expressed as −log₁₀ P value. (H) UCSC genome browser rendering of a PU.1 peak location in Myc gene body. Tracks show PU.1 ChIP-Seq and WCE control, with corresponding peak locations and intensities in thioglycollate-elicited primary mouse macrophage (ThioMac) and BM-derived macrophage (BMDM) PU.1 ChIP-seq datasets from GSE21512. (I) Myc and PU.1 motif enrichment (motif score) at TSS ± 1 kb in IL-1–downregulated, –upregulated, or unchanged genes containing PU.1 peaks. Box shows upper and lower quartiles with line showing median value, and whiskers upper and lower 10th percentile and individual dots represent outliers. ***, P ≤ 0.001 based on Wilcoxon rank sum test. (J) Comparison of IL-1–downregulated genes in SLAM HSCs with genes containing PU.1 peaks within TSS ± 20 kb in PU-ER cells with or without 4-OHT. Based on PU.1 ChIP-seq datasets in GSE21512. (K) GO category enrichment of IL-1–downregulated DEGs containing PU.1 peaks in PU-ER cell ChIP-seq dataset at 0 h +4-OHT versus combined 1–48 h +4-OHT. Top three GO categories are shown. Data are expressed as −log₁₀ P value. See also Fig. S4.

Figure S4.

PU.1 binding to IL-1–upregulated genes and analysis of PU-ER cells. (A) Pie chart comparing IL-1–upregulated genes identified in SLAM cells by RNA-seq analysis in Fig. 1 D and presence of PU.1 peaks at or near these genes (TSS ± 20 kb) in ChIP-seq data. See also Table S4. (B) Pie chart showing proportion of PU.1 peak locations in all genes versus IL-1–upregulated genes. (C) Transcription factor binding site motif enrichment at PU.1 ChIP-seq peak sites located at TSS ± 20 kb in IL-1–downregulated genes. (D) GO category enrichment of IL-1–upregulated DEGs containing PU.1 peaks. Representative genes in the indicated categories are shown to the right. Data are expressed as −log₁₀ P value. See also Table S5. (E) UCSC genome browser rendering of PU.1 peak location in Itgam gene body. Tracks show PU.1 ChIP-seq, WCE control, and peak locations and intensities in thioglycollate-elicited primary mouse macrophage (ThioMac) and BM-derived macrophage (BMDM) PU.1 ChIP-seq datasets from GSE21512. (F) Luciferase reporter assay measuring MYC promoter activity in 293T cells with or without PU.1 (n = 3/group). Data are representative of two independent experiments. (G) Quantification of Myc protein expression in PU-ER cells after 24 h culture with or without 4-OHT (n = 6/group). Data are representative of two independent experiments. Individual values are shown with bars representing mean values. (H) Cell cycle activity in PU-ER cells after 24 h culture with or without 4-OHT (n = 6/group). Data are representative of two independent experiments. (I) Comparison of PU.1 peak overlaps between PU.1 ChIP-seq data in A and datasets from GSE21512. (J) Comparison of IL-1–upregulated genes in SLAM HSCs with genes containing PU.1 peaks within TSS ± 20 kb in PU-ER cells with or without 4-OHT. Based on PU.1 ChIP-seq datasets in GSE21512. (K) GO category enrichment of IL-1–downregulated DEGs containing PU.1 peaks in PU-ER cell ChIP-seq dataset at 0 h with 4-OHT versus combined 1–48 h with 4-OHT. The top three GO categories are shown. Data are expressed as −log₁₀ P value. *, P < 0.05; **, P < 0.01 by Mann-Whitney U test or ANOVA with Tukey’s test in H. Error bars represent SD.

Figure 6.

Aberrant cell cycle and protein synthesis gene expression in PU.1-deficient HSC^LT. (A) Venn diagram comparing genes downregulated by IL-1 in SLAM cells (Fig. 1) and genes upregulated in PU.1^KI/KI SLAM cells. (B) Experimental design of Fluidigm qRT-PCR array analyses of HSC^LT from PU.1^+/+ and PU.1^KI/KI mice treated with or without IL-1β for 20 d. (C) Heatmap with hierarchical clustering analysis (Pearson correlation with average linkage) of gene expression data from HSC^LT in B. (D) Quantification by Fluidigm qRT-PCR array of Spi1 gene expression in HSC^LT from B. Data are expressed as log₁₀ fold expression versus −IL-1β. Box represents upper and lower quartiles with line representing median value. Whiskers represent minimum and maximum values. Data are representative of two independent experiments. (E) Quantification of cell cycle and protein synthesis gene expression by Fluidigm qRT-PCR array analysis in D. (F) Quantification of cell cycle gene expression by Fluidigm qRT-PCR array analysis in D. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by ANOVA with Tukey’s test. See also Fig. S5.

Figure S5.

PU.1 conditional knockout analysis and culture of PU.1^KI/KI HSC^LT. (A) Quantification by Fluidigm qRT-PCR array of cell cycle and protein synthesis gene expression in HSC^LT from mice treated with or without IL-1β for 1 d (n = 8/group). Data are expressed as log₁₀ fold expression versus −IL-1β. Box represents upper and lower quartiles with line representing median value. Whiskers represent minimum and maximum values. Data are from one experiment. (B) Study design for Cre induction with 4-OHT and analysis of HSC^LT from SCL-CreERT PU.1^+/+ and PU.1^Δ/Δ mice treated with or without IL-1β for 20 d (n = 2–3/group). (C) Intracellular flow cytometry analysis of puro incorporation in HSC^LT from mice in E. Puro was injected i.p. 1 h before BM harvest. Data are expressed as fold change of MFI versus −IL-1β. Individual values are shown with bars representing mean values. Data are representative of two independent experiments. (D) Quantification of cell cycle phase distribution in HSC^LT from PU.1^+/+ and PU.1^Δ/Δ mice in B based on Ki-67 and DAPI. Data are representative of two independent experiments. (E) Experimental design for analysis of HSC^LT from PU.1^+/+ and PU.1^KI/KI mice treated with or without IL-1β for 1 d. (F) Representative FACS plots (left) and quantification (right) of cell cycle phase distribution in HSC^LT from mice in H based on Ki-67 and DAPI (n = 7–8/group). Data are compiled from two experiments. (G) Myc levels from PU.1^+/+ and PU.1^KI/KI mice treated with IL-1β for 1 d (n = 4–5/group). Individual values are shown with bars representing mean values. Data are compiled from two experiments. (H) Experimental design for competitive in vitro assays on Boy/J and PU.1^KI/KI HSC^LT cultured in a 1:1 ratio with or without IL-1β for 12 d. (I) Quantification of total cells (left) and immature Sca-1⁺/cKit⁺ progenitors at the indicated time points (n = 3/group). (J) Quantification of frequency of total (left) and immature (right) CD45.2⁺ cells derived from PU.1^KI/KI HSC^LT cultured with or without IL-1β at the indicated time points. Percentages >50 (color coded green in the graphs) are indicative of a competitive advantage for PU.1^KI/KI cells. Data are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney U test in G or ANOVA with Tukey’s test. Error bars represent SD.

Figure 7.

Chronic IL-1 induces aberrant cell cycle activity and expansion of PU.1-deficient HSC^LT. (A) Experimental design for analyses of HSC^LT from PU.1^+/+ and PU.1^KI/KI mice treated for 20 d with or without IL-1β. (B) Intracellular flow cytometry analysis of Myc protein levels in HSC^LT from PU.1^+/+ and PU.1^KI/KI mice treated with or without IL-1β for 20 d (n = 4–9/group) Data are expressed as fold change of MFI versus −IL-1β. Individual values are shown with bars representing mean values. Data are compiled from two independent experiments. (C) Intracellular flow cytometry analysis of puro incorporation in HSC^LT from PU.1^+/+ and PU.1^KI/KI mice treated with or without IL-1β for 20 d (n = 4 PU.1^+/+; 5 PU.1^KI/KI). Data are expressed as fold change of MFI versus −IL-1β. Individual values are shown with bars representing mean values. Data are from one experiment. (D) Quantification of cell cycle phase distribution in HSC^LT from mice in A. Data are compiled from two independent experiments. (E) Quantification of BM HSC^LT from mice in A. Individual values are shown with bars representing mean values. Data are compiled from three independent experiments. (F) Representative FACS plots showing SLAM cells in the spleens of PU.1^+/+ and PU.1^KI/KI mice treated for 20 d with or without IL-1β. (G) Quantification of SLAM cells in the spleens of mice in A. Individual values are shown with bars representing mean values. Data are compiled from two independent experiments. (H) Cartoon showing key features of WT and PU.1-deficient HSC^LT. WT HSC^LT (left) engage a cell cycle and protein synthesis repression gene program that limits protein synthesis, cell cycle activity, and HSC^LT pool size following challenge with IL-1. On the other hand, PU.1-deficient HSC^LT overexpress cell cycle and protein synthesis genes, priming them for increased protein synthesis and cell cycle activity that is associated with aberrant expansion of the HSC^LT pool following IL-1 challenge. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by ANOVA with Tukey’s test. Error bars represent SD. See also Fig. S5.