Fig. 7. PU.1 depletion in yHSCs leads to “old-like” HSCs.
a–d UMAP plots for CD45.2pos GFPpos LSK cells, isolated 2 months post-transplantation with yHSCs infected with shRen, shPU#1 or shPU#2 lentiviruses. Six clusters (0–6) were identified by graph-based clustering (a). HSC-like cells (red) were annotated based on AUC gene set activity for an LT-HSC-specific gene set (b) and the corresponding shRNAs are color-coded (c, d). e Volcano plot of differential gene expression in the HSC-like population comparing shRen vs. shPU1 LT-HSCs. f–h UMAP plots showing the activity of indicated gene sets (color-coded based on the AUC values). i Scatter plots showing the aHSC and shPU1.up AUC values of shRen (left) or shPU#1/#2 cells (right) plotted against each other. j GSEA analysis of shPU1 vs. shRen transduced HSCs using a gene set consisting of the Top100 genes downregulated by shPU1 in BM-HPC#5 cells. k GST pulldowns with cell extracts from 293T cells incubated with either GST or GST-PU-1. Bound TAZ was detected by immunoblot. The experiment was independently repeated three times. l Luciferase assay with a reporter construct containing three PU-Boxes. Cells were transfected with increasing amounts of a TAZ S89A construct, either in the absence (−PU.1) or presence (+PU.1) of a PU.1 construct (n = 3 independent experiments, one-way ANOVA with Tukey HSD post hoc test). RLUs = relative light units. m Heatmap of PU.1 binding at all (n = 3163) PU.1 peaks in Cut&Run. IgG serves as negative control. n Homer motif analysis, of the most significantly enriched motifs in all PU.1 peaks. o Distribution of PU.1 peaks in the genome. p Exemplary browser track of PU.1 and IgG Cut&Run signals in the promoter region of Exosc9. q Venn diagram of gene promoters bound by PU.1 (green) and genes that are downregulated after shTAZ in HSCs (red). r Top5 gene ontology terms overrepresented in all PU.1 peaks (n = 3523, green) or TAZ-dependent regulated PU.1 peaks (n = 60, red). FDR = false discovery rate. s Schematic illustrating the changes in TAZ and PU.1 expression and their effect on PU.1 transcriptional activity. t Proposed model of how increased TAZ expression in oHSCs maintains PU.1 activity despite reduced PU.1 expression. Data in k, l are presented as mean values +/− SEM. Source data are provided as a Source data file.