HBO1 activates genes involved in HSC quiescence and self-renewal. (A-B) H3K14ac levels in LSK and progenitor cells in Hbo1^fl/+;CreER and Hbo1^fl/fl;CreER mice. Mice (n = 3 per genotype) were treated 3× with tamoxifen (150 mg/kg BW per dose, doses 48 hours apart). Bone marrow was collected 7 days after the first tamoxifen treatment. LSK and progenitor cells were sorted by FACS and then fixed and stained with anti-H3K14ac antibodies detected with fluorescently coupled secondary antibodies. (A) Representative FACS plots of H3K14ac levels in LSKs and progenitor cells. Gray peak, control LSK and progenitor cells (Hbo1^fl/+;CreER). Red peak, LSK and progenitor cells lacking HBO1 (Hbo1^fl/fl;CreER). (B) Mean fluorescence intensity of H3K14ac levels in control and Hbo1^fl/fl;CreER LSK and progenitor cells. Data are displayed as mean ± SEM and were analyzed using a Student t test. (C-G) RNA-seq results of LSK and progenitor cells. Hbo1^fl/+;CreER and Hbo1^fl/fl;CreER mice (n = 4 animals per genotype) were treated 3x with tamoxifen (150 mg/kg BW per dose, doses 24 hours apart). Hbo1^fl/+;Mx1Cre and Hbo1^fl/fl;Mx1Cre mice (n = 4 animals per genotype) were injected 3x with poly(I:C) (2.5 mg/kg BW per dose, doses 48 hours apart). To achieve a similar stage of HSC^DN and progenitor reduction after Mx1Cre- and CreER-mediated Hbo1 deletion (compare Figure 2 and supplemental Figure 3), bone marrow was collected 3 days after the first tamoxifen treatment and 7 days after the first poly(I:C) injection. LSK and progenitor cells were FACS sorted. Barcoded RNA sequencing libraries were generated from RNA (50 ng) of sorted LSK cells or progenitor cells and sequenced on the Illumina HiSeq 2000 platform. Between 19 million and 43 million 80 bp paired-end reads for each sample were obtained. After individual assessment, which revealed similar results (supplemental Figure 12), sequencing data were pooled from Hbo1Mx1Cre and Hbo1CreER mice within cell type. (C) Venn diagram showing the numbers of up- (red) or downregulated genes (blue) in LSK and progenitor cells with significance corrected for multiple testing (FDR < 0.05). (D) Radar charts showing differentially expressed genes (FDR < 0.05) binned by expression levels as up- (red) or downregulated (blue) in LSK and progenitor cells. (E) Hematopoietic cell lineage signature genes (path:mmu 04640) were downregulated in Hbo1-deleted LSKs when compared with control cells (P = .0003). The horizontal axis shows t statistics for all genes in LSK cells. Black bars mark positions of genes annotated in the hematopoietic cell lineage signature. Worm shows relative enrichment of the hematopoietic cell lineage signature genes relative to uniform ordering of genes in LSK data set. (F-G) Genes encoding transcription factors and receptors crucial for HSC functions significantly reduced in Hbo1-deleted LSK cells. Data are displayed as mean ± SEM and were analyzed as stated under RNA-seq in the "Material and methods" section. FDR, false discovery rate; RPKM, reads per kilobase of transcript per million mapped reads.