Fig 2: Additional PU.1 knockdown experiments.(A) Validation of Pu.1 shRNA by Western blotting. Experiment was performed in MLP-GFP transduced B-ALL cells following puromycin selection. (B–C) Effect of PU.1 knockdown on TRIM33 occupancy at Atp1b3. ChIP-qPCR analysis at the indicated locations of the Atp1b3 locus in B-ALL. Labels refer to kilobase distance relative to Atp1b3 TSS. Plotted is the average of three biological replicates. Error bars denote S.E.M. (D) Effect of PU.1 knockdown on Bim expression. RNA was prepared from B-ALL cells transduced with indicated shRNAs and used for reverse transcription. Plotted is the average of three biological replicates. Results were normalized to Gapdh.DOI: http://dx.doi.org/10.7554/eLife.06377.014

Fig 3: Irf8-/- CNS tissues contain significantly less microglial processes than Irf8+/+ tissues. (A) Dorsal columns at the Th1 spinal level were immunolabeled for CD11b and PU.1, and inverted confocal fluorescence images of 12 µm-thick transverse sections are shown in monochrome. Only cross-sectional areas of the dorsal columns are shown. The location of each microglial nucleus positive for PU.1 is indicated by a circle to demonstrate similar distributions of microglia between the Irf8+/+ and Irf8-/- dorsal columns, which contrasts to lower densities of CD11b-positive microglial processes in Irf8-/- mice than in Irf8+/+ mice. (B) Similar difference was observed in the coronal sections of the corpus callosum. (C) The percentages of CD11b-positive areas in the whole cross-sectional area of the dorsal column were measured and compared quantitatively between Irf8-/- (closed bar) and Irf8+/+ mice (open bar). Three 3-month-old animals were used in each genotype. **P < 0.01. Scale bar: 100 µm.

Fig 4: IRF8 supports PU.1’s chromatin occupancy and transcriptional output(A) Scatterplot of IRF8 CERES versus PU.1 CERES in leukemia cell lines.(B) GSEA analysis of IRF8-KO (left) and MEF2D-KO (right) RNA-seq data in MOLM-13 cells. PU.1 signature scatterplot of MEF2D CERES versus IRF8 CERES in leukemia cell lines, where the PU.1 signature is defined as the top 500 downregulated genes on PU.1 depletion. Normalized enrichment score (NES) and false discovery rate (FDR) q value are shown.(C and D) Density plot (C) and meta-profiles (D) of PU.1, H3K27ac and H3K4me1 ChIP-seq signals at IRF8 binding sites in MOLM13-dIRF8 cells (Cao et al., 2021) on 4 h treatment of DMSO or dTAG that rapidly eliminates IRF8 protein. Peaks are ranked by IRF8 ChIP-seq tag counts (n = 3).(E) Immunoblotting of IRF8, PU.1 and GAPDH in whole-cell lysates of MOLM13-dIRF8 cells treated with 500nM dTAG-47 for 4h.(F and G) Gene tracks of ChIP-seq signal from Figures 4C and 4D at MEF2D (E) or CD180 (F) locus.

Fig 5: (A–B) Ranked ordering of PU.1 and H3K27 acetylation peaks identified using ChIP-seq analysis in B-ALL.Peaks were ranked based on sequence tag counts. Positions of Bim-117 and Atp1b3-35 are indicated.DOI: http://dx.doi.org/10.7554/eLife.06377.012