The histone acetyltransferase KAT6B is required for hematopoietic stem cell development and function

Abstract

The histone lysine acetyltransferase KAT6B (MYST4, MORF, QKF) is the target of recurrent chromosomal translocations causing hematological malignancies with poor prognosis. Using Kat6b germline deletion and overexpression in mice, we determined the role of KAT6B in the hematopoietic system. We found that KAT6B sustained the fetal hematopoietic stem cell pool but did not affect viability or differentiation. KAT6B was essential for normal levels of histone H3 lysine 9 (H3K9) acetylation but not for a previously proposed target, H3K23. Compound heterozygosity of Kat6b and the closely related gene, Kat6a, abolished hematopoietic reconstitution after transplantation. KAT6B and KAT6A cooperatively promoted transcription of genes regulating hematopoiesis, including the Hoxa cluster, Pbx1, Meis1, Gata family, Erg, and Flt3. In conclusion, we identified the hematopoietic processes requiring Kat6b and showed that KAT6B and KAT6A synergistically promoted HSC development, function, and transcription. Our findings are pertinent to current clinical trials testing KAT6A/B inhibitors as cancer therapeutics.

Keywords: KAT6A; KAT6B; chromatin; hematopoiesis; histone acetyltransferase; stem cells; transplantation.

Figure 1

Deletion of Kat6b alleles results in a gene-dose-dependent reduction in the percentage of E14.5 fetal liver hematopoietic stem cells (A) Representative flow cytometry plot of LSK cells in the E14.5 fetal liver. (B and C) LSK cell percentage in Kat6b^+/+C57BL/6, Kat6b^+/− and Kat6b^−/− (B), or Kat6b^{+/+FVBxBALB/c} and Tg(Kat6b) (C) overexpressing E14.5 fetal livers. (D) Representative flow cytometry plot. (E and F) Percentage of HSCs in Kat6b^+/− and Kat6b^−/− mice compared with controls (E) and Tg(Kat6b) mice compared with controls (F). (G) Representative flow cytometry plot of Ki67 vs. DAPI in HSCs. (H and I) Cell cycle quantitation for HSCs from Kat6b^+/− and Kat6b^−/− mice compared with controls (H) and Tg(Kat6b) mice compared with controls (I). n = 6–10 (A), 8–10 (B), 6–10 (E), 8–10 (F), 3–5 (H), and 4–5 (I) mice per genotype. Data are presented as mean ± standard error of the mean (SEM). Each circle represents an individual mouse. Data were analyzed by one-way ANOVA with Tukey post hoc correction (B, E), Student’s t test (C, F) or two-way ANOVA with Tukey (H) or Sidak post hoc correction (I).

Figure 2

Loss and gain of Kat6b reduces and enhances hematopoietic progenitor colony formation, respectively, but neither affects differentiation in vitro (A and B) Number (A) and average size (B) of colonies formed in vitro from Kat6b^+/+C57BL/6, Kat6b^+/−, and Kat6b^−/− animals. (C) Proportion colony types formed from Kat6b^+/+C57BL/6, Kat6b^+/−, and Kat6b^−/− mice. (D and E) Number (D) and average size (E) of colonies formed in vitro from Kat6b^{+/+FVBxBALB/c} and Tg(Kat6b)animals. (F) Proportion colony types formed from Kat6b^{+/+FVBxBALB/c} and Tg(Kat6b) mice. (G) Representative images of colonies. Scale bar, 100 μm. N = 4–5 mice per genotype. Data are presented as mean ± SEM, analyzed by one-way ANOVA (A and B), unpaired Student’s t test (D and E) or two-way ANOVA with Tukey (C) or Sidak (F) post hoc correction. Data in (B) and (E) based on area assessments of 50 colonies per sample, per genotype. B: blast colony; G: granulocyte colony; GM: granulocyte/macrophage colony; M: megakaryocyte colony; E: erythroid colony; ML: mixed lineage colony.

Figure 3

Deletion of Kat6b causes a reduction in multilineage contribution of fetal liver cells in competitive transplantation assays (A) Experimental design. (B–D) Gating strategy (B) and percentage contribution of CD45.2⁺ donor cells from Kat6b^+/+C57BL/6, Kat6b^+/−, or Kat6b^−/− donor fetuses, to peripheral white blood cells at 4 weeks (C) and 20 weeks (D) post-transplantation. (E–G) Gating strategy (E) and percentage contribution (F and G) of CD45.2⁺ donor cells from Kat6b^+/+C57BL/6, Kat6b^+/−, or Kat6b^−/− fetal livers at 20 weeks post-transplantation. N = 4–7 fetal liver donors per genotype. Data are presented as mean ± SEM, analyzed by two-way ANOVA with Tukey (C, D, F, and G) post hoc correction. Each circle represents the average of three transplant recipient mice that received cells from a single donor. Granulo: granulocyte, Mono: monocyte, HSC: hematopoietic stem cell, MPP: multipotent progenitor cell, HPC-1: restricted hematopoietic progenitor type 1, HPC-2: restricted hematopoietic progenitor type 2, CMP: common myeloid progenitor GMP: granulocyte, macrophage progenitor, MEP: megakaryocyte, erythrocyte progenitor, CLP: common lymphoid progenitor.

Figure 4

Sorted Kat6b^+/− and Kat6b^−/− HSCs show impaired short and long-term multilineage reconstitution (A) Experimental design. (B–D) Percentage contribution of CD45.2⁺Kat6b^+/+C57BL/6, Kat6b^+/−, or Kat6b^−/− fetal liver donor cells to peripheral white blood cells (B) and bone marrow cells (C and D) at 15 weeks post-transplantation. (E) Percentage contribution of CD45.2⁺Kat6b^{+/+FVBxBALB/c} and Tg(Kat6b) cells to peripheral white blood cells at 4 weeks post-transplantation. (F–H) Percentage contribution of CD45.2⁺Kat6b^+/+C57BL/6, Kat6b^+/−, or Kat6b^−/− cells from primary recipients to peripheral white blood cells (F) and bone marrow cells (G, H) in the secondary recipient mice at 15 weeks post-transplantation. N = 4–7 mice per genotype in primary transplants; N = 2–7 primary recipients per genotype in secondary transplants. Data are presented as mean ± SEM and analyzed using Mann-Whitney (B, C, D, F, G, and H) or Welch t tests (E). Each circle represents the average of transplant recipients that received cells from a single fetal liver donor (B–E) or a single secondary transplant recipient (F and G). Abbreviation as in Figure 3.

Figure 5

Deletion of Kat6b alleles has a gene-dose-dependent effect on histone H3 lysine 9 acetylation (A) Gating strategy. (B and C) Median fluorescence intensity (MFI) of H3K9ac levels in Kat6b^−/−, Kat6b^+/−, and Kat6b^{+/+ C57BL/6} LSK cells and HSCs by intracellular flow cytometry. (C) Western immunoblot of H3K9ac and pan H3 in histones derived from Kat6b^−/−, Kat6b^+/−, and Kat6b^+/+ C57BL/6 E14.5 fetal liver LSK cells. Quantitation shown beside the immunoblot. (D) MFI of H3K9ac levels in Kat6b^{+/+FVBxBALB/c} and Tg(Kat6b) E14.5 fetal liver LSK cells and HSCs by intracellular flow cytometry. (E) Western immunoblot of H3K9ac relative to pan H3 in histones from Kat6b^{+/+FVBxBALB/c} and Tg(Kat6b) fetal liver LSK cells. (F) MFI of H3K23ac levels in Kat6a^+/− and Kat6a^{+/+ C57BL/6} E14.5 fetal liver LSK cells and HSCs. (G) Western immunoblot of H3K23ac relative to pan H3 in histones from Kat6a^−/−and Kat6a^{+/+ C57BL/6} fetal liver LSK cells. (H–K) MFI of H3K9ac (H) and H3K23ac (J) levels in Kat6b^+/−Kat6a^+/− and wild-type LSK cells and HSCs by intracellular flow cytometry (H and J) and LSK cells by western immunoblotting (I and K). N = 3–8 mice per genotype. Western blots: each lane represents histones from sorted LSKs of one fetal liver. Each circle in (C, E, G, I, and K) represents one lane. Data in all graphs are displayed as mean ± SEM, analyzed by one-way ANOVA with Tukey post hoc correction (B and C) or Student’s t test (D–K). MFI: median fluorescence intensity.

Figure 6

Loss and gain of KAT6B affect the expression of HSC genes (A) Representative FACS plot of the LSK cell population used in RNA-seq (N = 4 fetuses per genotype). Annotated protein-coding genes were examined. (B) Mean-difference plots comparing Tg(Kat6b) with Kat6b^{+/+FVBxBALB/c} and Kat6b^−/−, Kat6a^+/−, and Kat6a^+/−Kat6b^+/− double heterozygotes to Kat6b^+/+C57BL/6 LSK cells. Significantly downregulated genes are depicted in blue and significantly upregulated genes depicted in red. (C) Genes differentially expressed in Kat6b^−/− LSK cells relative to controls. (D) Multidimensional scaling plot showing distances between transcriptional profiles. Distances on the plot represent “leading log2 fold-change” between each pair of samples. (E) Barcode enrichment plot showing correlation between HSC-associated genes based on UniProtKB annotation: "hematopoietic stem cells" and gene expression changes in Tg(Kat6b) LSK cells vs. Kat6b^{+/+FVBxBALB/c} controls. Vertical lines represent "hematopoietic stem cell" genes. Red and blue shaded areas are differentially expressed genesin the Tg(Kat6b) vs. Kat6b^+/+ LSK cells. (F) Expression changes of specific hematopoietic regulators in Tg(Kat6b) LSK cells vs. wild-type. (G) Barcode enrichment plot showing correlation between HSC-associated genes and gene expression changes in Kat6a^+/− LSK cells vs. wild-type cells. (H) Expression changes of specific hematopoietic regulators in Kat6a^+/− LSK cells vs. wild-type cells. (I) Barcode enrichment plot showing an inverse correlation between genes differentially expressed in leukemia cells compared with HSCs from dataset GSE20377 (Wang et al., 2010) and genes differentially expressed in Kat6a^+/− LSK cells vs. wild-type cells. (J) Barcode enrichment plot showing correlation between HSC-associated genes and gene expression changes in Kat6b^+/−Kat6a^+/− LSK cells vs. wild-type controls. (K) Expression changes of specific hematopoietic regulators in Kat6b^+/−Kat6a^+/− LSK cells vs. wild-type cells. (L) Barcode enrichment plot showing an inverse correlation between genes differentially expressed in leukemia compared with HSCs from dataset GSE20377 (Wang et al., 2010) and genes differentially expressed in Kat6b^+/−Kat6a^+/− LSK cells vs. wild-type cells. (M–P) Chromatin immunoprecipitation (ChIP) qPCR IP/Input assessment of H3K9ac levels at the promoter region of genes differentially expressed in Kat6b^−/− (M), Tg(Kat6b) (N), Kat6a^+/− (O) and Kat6b^+/−Kat6a^+/− (P) LSKs vs. genetic background-matched controls. Data were analyzed as described under RNA-sequencing analysis in the methods section (A–L). Data in (M)–(P) presented as mean ± SEM and analyzed using multiple t tests with Benjamini-Hochberg corrections for multiple testing. Each circle in (M)–(P) represents IP/Input of LSKs derived from an individual fetal liver per genotype.

Figure 7

Combined heterozygosity at Kat6b and Kat6a results in the absence of long-term repopulating HSCs in the fetal liver (A and B) Percentage HSCs within the LSK population of E14.5 fetal livers (A) and adult bone marrow (B) from wild-type, Kat6a^+/− heterozygous, Kat6b^+/− heterozygous, and Kat6a^+/−Kat6b^+/− compound heterozygous animals. (C) Experimental design. (D and E) Percentage contribution of CD45.2⁺ donor cells at 15 weeks post-transplantation in the peripheral blood (D) and bone marrow (E and F). Bone marrow populations were separated by CD48 vs. CD150 (E) and CD34/CD16/CD32 or IL7R $\propto$ staining (F). N = 5–7 (A), 4–5 (B), and 3–5 fetal liver donor mice (D–F) per genotype. Data are presented as mean ± SEM and were analyzed using a one-way ANOVA with Tukey post hoc correction (A and B) or Welch t tests (D–G). Each circle represents a single fetal liver (A), bone marrow from a single adult animal (B), or the average of transplant recipients that received cells from a single fetal liver donor (D–F). Abbreviation as in Figure 3.