Fig 2: Baf155 CKO EMPs Have Reduced Chromatin Accessibility at the Myeloid and EryD Gene Loci(A) A Venn diagram of the numbers of ATAC-seq peaks found in Baf155 CKO and WT EMPs.(B) ATAC-seq signals over 10-kb regions centered on the differentially accessible regions (DARs) with reduced signals in Baf155 CKO EMPs compared with the WT (left) and the presence of the Spi1/Pu.1 motif in the DARs (right).(C) Heatmaps of HOMER known TF motif fold enrichment in the DARs and unaffected accessible regions. Gray cells indicate no enrichment found (p > 0.05).(D) Enriched Gene Ontology (GO) terms and their binomial p values from analyzing the DARs with reduced signals in Baf155 CKO EMPs (white) and the unaffected peaks (red) using GREAT.(E) Epigenome browser views of representative myeloid gene loci.(F) Epigenome browser views of representative erythroid gene loci.See also Figure S4.

Fig 3: Relative levels of Dectin-1, TLR-2 and TLR-4 (A) mRNA and (B) protein expression in THP-1 cells at 48 h following transduction with AdPU.1, as determined by RT-qPCR and western blot analyses, respectively (*P<0.05 vs. Mock group; #P<0.05 vs. Ad group). Relative levels of Dectin-1, TLR-2 and TLR-4 (C) mRNA and (D) protein expression in THP-1 cells at 48 h following transfection with siPU.1, as determined by RT-qPCR and western blot analyses, respectively (*P<0.05 vs. Mock group; #P<0.05 vs. siNC group). Dectin-1, dendritic cell-associated C-type lectin receptor-1; TLR, toll-like receptor; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Ad-PU.1, recombinant adenoviral-PU.1 vector; siPU.1, small-interfering RNA targeting PU.1; mock, untransfected/transduced cells; Ad, adenovirus-transduced cells; siNC, negative control siRNA.

Fig 4: SPI1 is the master regulator of LSC signature genes and controls HAVCR2 and c-MYC expression.(A) Nearly 70% of the genes highly expressed in the HAVCR2high subgroup—the LSC signature genes—are potential SPI1 target genes (purple);(B) Pseudotemporal ordering of single cells based on Spi1 or Myc expression; (C) FACS analysis shows the correlation of HAVCR2 cell surface expression and intracellular SPI1 and c-MYC levels in the LSC-enriched (Lin-CD3+KITmid; red) and blast (Lin-CD3+KIT-; blue) subpopulations. Gray, isotype control; (D–E) ChIP-qPCR analysis identifies SPI1 binding regions in the HAVCR2 promoter (left) and Tcra/d enhancer A(EA) region (right), using Blast-SPI1 cells; (F) ChIP analysis identifies a SPI1 binding site in the endogenous Myc enhancer;(G) q-PCR shows the fold changes in Havcr2, Itgax, Lmo2 and Myc expression between Blast-SPI1 cells (red) and Blast-EGFP cells (blue); (H) Western blotting shows the SPI1, HAVCR2 and c-Myc protein levels in WT thymus, Blast-EGFP and Blast-SPI1 cells. The fold changes relative to expression in the WT thymus are shown above each lane; (I) q-PCR analysis shows the fold changes in HAVCR2, ITGAX, LMO2 and MYC expression in control shRNA (blue) and shSPI1 knockdown human T-ALL KE-37 cells (red); (D–I) All experiments were performed at least three independent times, and the data in D, E, F, G, and I are the means ± S.Ds; *p=0.05; **p=0.01; ***p=0.001.

Fig 5: Spi1 expression is maintained by ß-catenin-SPI1-HAVCR2 regulatory circuit.(A) q-PCR analysis of SPI1 and SPI1-regulated HAVCR2 and c-MYC expressions after the overexpression of active ß-catenin in the Jurkat T-ALL cell line (red bars). The data are normalized to that of empty plasmid controls (blue bars); (B–E) Upper panels: quantitative intracellular FACS analyses of P-GSK-3ß, non-phospho-ß-catenin, P-p65 and SPI1 levels in the HAVCR2high, HAVCR2low and blast subgroups; lower panels: representative intracellular FACS analysis of P-GSK-3ß, non-phospho-ß-catenin, P-p65 and SPI1 levels in the HAVCR2high, HAVCR2low and blast subgroups. Gray line, isotype control;(F) FACS analysis shows cells in the HAVCR2high subgroup at the ETP/DN1 stage, which are absent in WT and dKO mice; (G) Representative FACS plots show the number of cells in the HAVCR2high subgroup in the different drug treatment groups. The data in A, B, C, D and E are the means ± S.Ds of 3 independent tests; *p=0.05; **p=0.01; ***p=0.001.