ATF4 regulates ribosome biogenesis by directly activating the transcription of Rps19bp1. (A) Venn diagram depicting the overlapping downregulated/transcriptionally repressed genes identified from 4 different data sets (ie, ATAC-seq, H3K4me3 CUT&Tag, RNA-seq, and scRNA-seq) after Atf4 deletion. (B) qRT-PCR analysis of the indicated genes in MEP cells sorted from the fl/fl and Δ/Δ mice (n = 3 samples). (C) Lollipop plot displaying the top 10 predicted ATF4-targeting genes within the CMP population using pySCENIC. (D) Expression of Rps19bp1 projected onto the UMAP plot of fl/fl and Δ/Δ CMP cells, based on scRNA-seq data. Color intensity indicates the expression levels. (E) IGV software was used to visually present the important genomic regions of Rps19bp1; ATAC-seq and H3K4me3 CUT&Tag peaks, predicted cis-regulated elements, and luciferase reporter clone regions are shown. (F) Schematic diagrams of the pGL3-Rps19bp1-promoter-luciferase reporter constructs (Pro-1 contains the CTCF-bound region; Pro-2 does not contain the CTCF-bound region); relative luciferase activity was determined by sequential normalization to Renilla luciferase and pGL3-vector activity (n = 3 samples). (G) qChIP of ATF4 with primers covering the promoters of Rps19bp1 (n = 3 samples). (H) Ribosomes were separated from MEL cells transduced with control or Atf4 shRNA mix (shRNA-1 and shRNA-2) and then analyzed by western blotting using antibodies targeting the indicated proteins. (I) Protein synthesis in HSPCs based on OP-Puro incorporation in vivo (n = 4 mice in 3 independent experiments). (J) Experimental workflow showing how cKit⁺ cells were sorted from the BM of fl/fl and Δ/Δ mice and then transduced with vector-TD or Flag-Rps19bp1-TD for the plating colony and SUnSET assays. (K) TD⁺ cKit⁺ cells of the fl/fl and Δ/Δ mice were treated with puromycin and then analyzed by western blotting using antibodies against the indicated proteins. (L) Ribosomes were separated from MEL cells transduced with control or Atf4 shRNA mix and vector-TD or Flag-Rps19bp1-TD then analyzed by western blotting using antibodies against the indicated proteins. (M-N) BFU-E (M) or CFU-E (N) colony assays of 2 × 10⁴ transduced TD⁺ cKit⁺ cells from fl/fl or Δ/Δ mice cultured in MethoCult SF M3436 or M3334 methylcellulose-based medium with EPO cytokine for 10 days (M) or 48 hours (N), respectively (n = 3 wells in 3 independent experiments). Representative images (left) and colony numbers (right) are shown. (O) Experimental workflow of the transplantation assay in which 10⁵ cKit⁺ cells (sorted from the BM of the fl/fl or Δ/Δ mice and transduced with vector-TD or Flag-Rps19bp1-TD, Flag-Rps19bp1-Mut-TD) and 10⁶ BM cells from donor mice (β-actin-GFP) were infected into irradiated CD45.1 recipient mice. (P) Percentage of GFP^–Ter119⁺ erythrocytes in PB erythrocytes of recipient mice at the indicated time points after BM transplantation (n = 3-5 mice). Data represent the mean ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; unpaired 2-tailed Student t test for Figure 6B,F-G,I; 1-way analysis of variance followed by an unpaired 2-tailed Student t test for figure 6M-N,P. EPO, erythropoietin; OP-Puro, O-propargyl-puromycin; qChIP, quantitative chromatin immunoprecipitation; shRNA, short hairpin RNA; SUnSET, surface sensing of translation.