Loss of H3K14ac in the absence of HBO1 affects most genes, including important hematopoietic genes. (A-E) Levels of acetylated lysine 14 on histone H3 (H3K14ac) in Hbo1^fl/fl;Mx1-Cre vs Hbo1^fl/+;Mx1-Cre control LSK cells was assessed by CUT&Tag sequencing. This experiment was repeated twice. (A) H3K14ac sequencing read coverage plot of gene bodies plus or minus 5 kb in Hbo1^fl/fl;Mx1-Cre vs Hbo1^fl/+;Mx1-Cre control LSK cells. (B) Barcode plot depicting the correlation between the H3K14ac levels changes in the gene bodies in Hbo1^fl/fl;Mx1-Cre vs Hbo1^fl/+;Mx1-Cre control LSK cells (depicted by the light blue/gray/pink box) and the top 5% highest (red) and bottom 5% lowest (blue) expressed genes (depicted by the vertical lines). (C) H3K14ac levels in genes regulating hematopoietic stem cell function in Hbo1^fl/fl;Mx1-Cre vs Hbo1^fl/+;Mx1-Cre control LSK cells. A correlation between mRNA levels of genes depicted in panel F and the H3K14 acetylation changes at these genes. The downregulated gene expression changes shown graphically have a proportional lack of H3K14 acetylation, whereas the 3 upregulated genes are comparatively less affected. (D) Sequencing read depth plots over the Mpl gene of Hbo1^fl/fl;Mx1-Cre vs Hbo1^fl/+;Mx1-Cre control LSK cells corrected for D. melanogaster spike-in. One animal per genotype shown, illustrating the higher level of H3K14ac around the transcription start site and the coverage of the gene body in the control samples and the near absence of reads in the Hbo1-deleted samples. Black tracts represent control samples and gray tracts represent Hbo1-deleted samples. Supportive data shown in supplemental Figures 13 and 14 including replicate tracts of the Mpl locus and tracts of the Tek locus. (E) Consequences of loss of HBO1 on hematopoiesis. Insufficient expansion and premature differentiation of HSCs results in insufficient numbers of mature cell types. (F) Working model of HBO1 function in adult hematopoiesis. HBO1 is a major regulator for adult hematopoiesis through promoting the expression of multiple genes encoding transcription factors and receptors crucial for HSC quiescence and self-renewal. Important transcriptional and signaling regulators that are required for HSC functions are listed in a simplified schematic of adult hematopoiesis. Genes in blue are downregulated (fold-change in brackets) and genes in red are upregulated (fold-change in brackets) in Hbo1-deleted LSK cells compared with control cells. The specific functions indicated are based on published reports for Gata2,^63-66,Mpl,^60,61Mpl, Stat5, Tek,^62,Hoxa9,^68,69Pbx1,^70,Meis1,^71-73,Gfi1b,^75,Egr1⁷⁶ (see text for "Discussion"), as well as Tal1/Scl,^77,Erg,^78,79Msi2⁸⁰; Runx1;^81,Spi1,^51,52Gata1,^82,Cebpa,⁵³ and Cebpb.⁸³