The histone acetyl transferase activity of monocytic leukemia zinc finger is critical for the proliferation of hematopoietic precursors

Abstract

The monocytic leukemia zinc finger (MOZ) gene encodes a large multidomain protein that contains, besides other domains, 2 coactivation domains for the transcription factor Runx1/acute myeloid leukemia 1 and a histone acetyl transferase (HAT) catalytic domain. Recent studies have demonstrated the critical requirement for the complete MOZ protein in hematopoietic stem cell development and maintenance. However, the specific function of the HAT activity of MOZ remains unknown, as it has been shown that MOZ HAT activity is not required either for its role as Runx1 coactivator or for the leukemic transformation induced by MOZ transcriptional intermediary factor 2 (TIF2). To assess the specific requirement for this HAT activity during hematopoietic development, we have generated embryonic stem cells and mouse lines carrying a point mutation that renders the protein catalytically inactive. We report in this study that mice exclusively lacking the HAT activity of MOZ exhibit significant defects in the number of hematopoietic stem cells and hematopoietic committed precursors as well as a defect in B-cell development. Furthermore, we demonstrate that the failure to maintain a normal number of hematopoietic precursors is caused by the inability of HAT(-/-) cells to expand. These results indicate a specific role of MOZ-driven acetylation in controlling a desirable balance between proliferation and differentiation during hematopoiesis.

Figure 1

Analyses of hematopoietic cell populations in HAT^+/+, HAT^+/−, and HAT^−/− mice. Flow cytometry analysis of T-cell, B-cell, erythroid, and macrophage populations in the bone marrow, thymus, and spleen of 3-month-old mice. FACS profiles shown in the figure correspond to 1 representative mouse of each phenotype.

Figure 2

Analyses of HSC and precursor populations in HAT^+/+, HAT^+/−, and HAT^−/− fetal liver. (A) Staining and (B) quantification of E14.5 fetal liver cells with the indicated antibodies to identify the HSC, CLP, GMP, CMP, MEP, and KLSF populations by flow cytometry. To analyze the HSC and CLP populations, 8 HAT^+/+, 8 HAT^+/−, and 8 HAT^−/− mice were used. To analyze the GMP, KLSF, CMP, and MEP populations, 8 HAT^+/+ 24 HAT^+/−, and 8 HAT^−/− mice were used. The values represent the percentage of cells in each cell population. Each sample is represented by a ▴, and the average of the samples is presented as a horizontal bar. *P < .5; **P < .01; ***P < .005.

Figure 3

Hematopoietic colonies and long-term repopulation potential of HAT^+/+, HAT^+/−, and HAT^−/− fetal liver cells. (A) Numbers of colonies generated by HAT^+/+, HAT^+/−, and HAT^−/− fetal liver cells from littermate animals. A total of 30 × 10³ cells/mL was replated in methylcellulose medium. Fetal liver cells were then genotyped, and myeloid, erythroid, and mixed lineage colonies were scored after 6 to 8 days. The average of the number of colonies generated is indicated by a horizontal bar. (B) Comparative size of representative HAT^+/+ and HAT^−/− colonies generated from day 14.5 fetal liver (magnification ×100). (C) Competitive long-term repopulation of irradiated mice. Data are expressed in log ratio between tested C57BL/6 fetal liver–derived (CD45.2⁺) and competitor PEP3 bone marrow–derived cells (CD45.1⁺). A total of 2 × 10⁵ fetal liver cells from HAT^+/+, HAT^+/−, and HAT^−/− embryos was transplanted into irradiated CD45.1⁺/CD45.2⁺ recipients with 4 × 10⁵ competitor bone marrow cells from PEP3 mice (CD45.1⁺). A total of 2 × 10⁶ cells from primary recipients was transplanted into secondary recipients 3 months after reconstitution. Bars represent standard error of the mean values. *P < .5; **P < .01; ***P < .005.

Figure 4

Blast colony potential and hematopoietic and smooth muscle/endothelial potential of blast colonies generated from HAT^+/+, HAT^+/−, and HAT^−/− ES cell lines. (A) Number of blast colonies generated by HAT^+/+, HAT^+/−, and HAT^−/− ES cell lines. Bars represent standard error of the mean number of colonies from at least 3 cultures. P values indicating that the differences between the number of colonies generated by the HAT^+/+ and the HAT^−/− are statistically significant are indicated. (B) Comparative size of HAT^+/+ and HAT^−/− individual blast colonies generated from day 3.5 EBs (magnification ×200). (C) Individual HAT^+/+, HAT^+/−, and HAT^−/− blast colonies were analyzed for their ability to generate hematopoietic and endothelial cells. Thirty-two blast colonies from each cell line were grown in liquid culture. The ability of each individual blast colony to expand and generate or not hematopoietic (H⁺ or H⁻) and/or endothelial (E⁺ or E⁻) cells was scored 4 days later. (D) Generation by individual blast colonies of nonadherent hematopoietic (white arrowhead) and adherent endothelial cells (black arrowhead). Note the limited proliferation of hematopoietic cells from HAT^−/− blast colonies.

Figure 5

Analysis of primitive and definitive hematopoietic potential of HAT^+/+, HAT^+/−, and HAT^−/− ES cells. (A) EBs were harvested from day 4 to day 11 of differentiation, and single-cell suspensions were replated in conditions supporting the generation of both primitive and definitive hematopoietic colonies. Primitive erythroid (EryP), definitive erythroid (EryD), macrophage (Mac), and mast cell (Mast) colonies were scored. Days of differentiation are indicated. Bars represent standard error of the mean number of colonies from at least 3 cultures. (B) Comparative size of primitive erythroid and macrophage colonies generated from HAT^+/+ and HAT^−/− day 6 EBs (magnification ×200).

Figure 6

Comparative analysis of the proliferation and differentiation potential of HAT^+/+ and HAT^−/− CD34⁺/c-kit⁺ hematopoietic progenitors. (A) Cell morphology. Sorted CD34⁺/c-Kit⁺ hematopoietic populations from HAT^+/+ and HAT^−/− ES cell lines were resuspended in serum-free proliferation media at a concentration of 2 × 10⁵ cells/mL, harvested at indicated time points, and stained by May-Grünwald-Giemsa for morphologic analysis. (B) Relative frequency of undifferentiated/blast cells (□) and macrophages (■) at different stages of maturation in the HAT^+/+ and HAT^−/− cultures. A total of 200 cells was scored for each sample. (C) Analysis of CD34⁺/CD45⁻ cell population in HAT^+/+ and HAT^−/− cell cultures. Days of differentiation are indicated. Numbers represent the percentage of total population. (D) Percentage of apoptotic cells in HAT^+/+ and HAT^−/− asynchronous cultures. Percentage of annexin V–positive cells is indicated for each time point. (E) Expansion of CD34⁺/c-Kit⁺ cells and fetal liver cells. Total number of cells (×10⁴) generated during the culture. CD34⁺/c-Kit⁺ cells isolated from day 6 EBs or total fetal liver cells from embryos were seeded at a density of 2 × 10⁶ or 10⁶ cells/mL, respectively. (F) Cell cycle status of HAT^+/+ and HAT^−/− CD34⁺/c-Kit⁺ hematopoietic progenitors in serum-free proliferation media. Asynchronous cultures of CD34⁺/c-Kit⁺ hematopoietic progenitors were stained with propidium iodide and analyzed by flow cytometry. Percentages of cells in G₁ and S/G₂ in HAT^+/+ and HAT^−/− at day 3 of culture are indicated.