HDAC3 is essential for DNA replication in hematopoietic progenitor cells

Abstract

Histone deacetylase 3 (HDAC3) contributes to the regulation of gene expression, chromatin structure, and genomic stability. Because HDAC3 associates with oncoproteins that drive leukemia and lymphoma, we engineered a conditional deletion allele in mice to explore the physiological roles of Hdac3 in hematopoiesis. We used the Vav-Cre transgenic allele to trigger recombination, which yielded a dramatic loss of lymphoid cells, hypocellular bone marrow, and mild anemia. Phenotypic and functional analysis suggested that Hdac3 was required for the formation of the earliest lymphoid progenitor cells in the marrow, but that the marrow contained 3-5 times more multipotent progenitor cells. Hdac3(-/-) stem cells were severely compromised in competitive bone marrow transplantation. In vitro, Hdac3(-/-) stem and progenitor cells failed to proliferate, and most cells remained undifferentiated. Moreover, one-third of the Hdac3(-/-) stem and progenitor cells were in S phase 2 hours after BrdU labeling in vivo, suggesting that these cells were impaired in transit through the S phase. DNA fiber-labeling experiments indicated that Hdac3 was required for efficient DNA replication in hematopoietic stem and progenitor cells. Thus, Hdac3 is required for the passage of hematopoietic stem/progenitor cells through the S phase, for stem cell functions, and for lymphopoiesis.

Figure 1. Hdac3 is required for lymphopoiesis.

(A) Western blot of HDAC3 expression in total bone marrow. (B) Gross morphology of thymus from 6-week-old mice. (C) FACS analysis of B220 and CD3 expression in spleens. (D) Analysis of myelopoiesis in total bone marrow samples using anti–MAC-1 and anti–GR-1. Numbers in each box represent the relative percentage of the cells in the indicated gated population. All FACS plots shown are representative of at least 5 mice. Graph at the right is a summation of biological replicates using 4 WT and 6 null mice first gated on GFP⁺ cells (MAC-1⁺/GR-1^–, P= 0.002; MAC-1⁺/GR-1⁺, P= 0.007; MAC-1^–/GR-1^–, P= 0.005; MAC-1^–/GR-1⁺, P= 0.008).

Figure 2. Inactivation of Hdac3 increases early stem and progenitor cells and blocks progression to the FLT3⁺ stage.

FACS analysis of LIN^– bone marrow to examine the (A) LSK population using SCA1 and c-KIT antibodies. (B) FACS analysis of LIN^– and c-KIT⁺ cells in A using FcγR and CD34 antibodies to distinguish GMP, CMP, and MEP populations. (C) Further analysis of LSK population (small plots on the left) using FLT3 antibody and (D) CD34 antibody. Representative FACS plots from at least 5 mice are shown. In D, the shaded curves designate the control mice and the open curves are the null mice. (E) Immunophenotypic analysis of stem and MPP cells. Top panels show the scheme for the flow cytometric analysis and the markers used. Graph at right shows the actual number of cells per leg (STHSC, P= 0.0375; MPP1, P= 0.0069; MPP2, P= 0.04; LMPP, P= 0.0295). Numbers in each box indicate the relative percentage of the cells in the indicated gated population. *P < 0.05; **P < 0.007. LTHSC, long-term HSC; STHSC, short-term HSC.

Figure 3. Hdac3 is required for lymphoid priming of bone marrow stem cells.

(A) Heat plot of lineage-specific genes from gene expression analysis of FACS-purified LSK/FLT3^– cells pooled from 2 groups of 5 null mice were compared with LSK/FLT3^– or LSK/FLT3⁺ cells pooled from 30 WT mice. (B) Heat plot diagram of granulocyte/monocyte- and megakaryocyte-primed genes associated with the LMPP population.

Figure 4. Loss of Hdac3 disrupts hematopoietic progenitor and stem cell functions.

(A) Myeloid progenitor cell colony formation assays. Bone marrow (2.5 × 10⁴) cells were plated in methylcellulose containing IL-3, IL-6, Epo, and SCF, and colonies were quantified after 8 to 10 days in culture. Representative images of the plates are shown. (B) BFU-E was determined using methylcellulose containing Epo. (C) B cell progenitor colony formation assays. CFU pre–B cells that expanded in IL-7 were quantified after 7 days in culture. (D) Survival curves of a bone marrow transplant experiment using 1Å~ 106 WT (gray line) or Hdac3-null bone marrow cells (hatched line) and irradiation controls (black line). CFU-S were assessed on day 8 (E) or day 12 (F). Quantification of each CFU-S was calculated for the number of colonies per spleen and is shown as a bar graph (WT: n = 5; Hdac3-null: n = 5; ***P = 0.0005). Con, control.

Figure 5. Hdac3 is required for stem cell functions.

(A) Competitive repopulation assay in which 90% control (empty bars) or Hdac3^–/– (filled bars) CD45.2⁺ bone marrow cells were coinjected with 10% WT CD45.1⁺ cells, and peripheral blood was analyzed by FACS. Data shown are the mean ± SEM at times after transplantation (***P ≤ 0.001; n = 4). (B) FACS analysis of bone marrow to determine the percentage of cells that were CD45.2⁺ after a competitive repopulation assay. (C) FACS analysis of LSK/FLT3^– cells to determine the percentage of CD45.2⁺ cells that had repopulated the marrow 3 weeks after cBMT. (D) FACS analysis of bone marrow of control and Mx1-Cre:Hdac3^–/– mice for GFP⁺ cells (for D: WT = 60% GFP⁺, null = 78% GFP⁺) that were LIN^– and SCA1⁺ and c-KIT⁺. (E) Graphs of the numbers of colonies formed in methylcellulose at the indicated times from LTC-IC cultures of WT (open bars) and Hdac3^–/– (filled bars) bone marrow cells, or WT cells treated with the indicated HDAC inhibitors (***P = 0.001, n = 3; *P = 0.013, week 3, n = 3; **P = 0.006, week 2; P = 0.002, week 3; P = 0.019, week 4, n = 3). Representative results from 2 separate experiments are shown. The numbers in each box are the relative percentage of the cells in the indicated gated population. Untr, untreated.

Figure 6. Inactivation of Hdac3 impairs progenitor cell proliferation differentiation.

(A) 2,500 WT or Hdac3-null LSK/FLT3^– bone marrow cells were cultured on an OP9-GFP stromal layer in media containing IL-6, SCF, and LIF, and proliferation was monitored by counting the total number of cells 3–7 days after harvesting the bone marrow cells (day 3, P = 0.026; day 7, P ≤ 0.001; n = 3). (B) Flow cytometric analysis of day-7 cultures (from A) using a combination of anti-CD3, anti-B220, anti–GR-1, anti–MAC-1, and anti-Ter119 to distinguish mature cells from immature progenitor cells (top panel) or a combination of anti–GR-1 and anti–MAC-1 to quantify myeloid cell differentiation (bottom panel). (C) Flow cytometric analysis of WT LSK cells treated with DMSO, SAHA, or depsipeptide (Depsi) and cultured for 7 days as in (A). Representative plots show a combination of anti-CD3, anti-B220, anti–GR-1, anti–MAC-1, and anti-Ter119 to distinguish mature cells from immature progenitor cells (top panel) or a combination of anti–GR-1 and anti–MAC-1 to quantify myeloid cell differentiation (bottom panel). The numbers in each box indicate the relative percentage of the cells in the indicated gated population.

Figure 7. Hdac3-null stem cells are cycling and show defects in DNA replication.

(A) Cell cycle status of LSK/FLT3^– cells was analyzed using BrdU. A representative FACS plot is shown from an experiment performed with 4 mice that is consistent with other biological replicates. The numbers in each box are the relative percentage of the cells in the indicated gated population. (B) DNA fiber labeling with IdU (green) and CldU (red) was used to assess DNA replication fork progression in LSK/FLT3^– cells from control and Hdac3^–/– bone marrow. Graphical representation of the length of replication tracks taken from 100 DNA fibers is shown in the graph. (C) DNA fiber labeling of LSK/FLT3^– cells as in B, but using untreated controls or cells treated with depsipeptide for 5 minutes prior to the addition of IdU with continued treatment with depsipeptide throughout the labeling period. A nonparametric Mann-Whitney U test indicated that the differences were significant at the P = 0.001 level for B and C.