Sin3a-associated Hdac1 and Hdac2 are essential for hematopoietic stem cell homeostasis and contribute differentially to hematopoiesis

Abstract

Class I histone deacetylases are critical regulators of gene transcription by erasing lysine acetylation. Targeting histone deacetylases using relative non-specific small molecule inhibitors is of major interest in the treatment of cancer, neurological disorders and acquired immune deficiency syndrome. Harnessing the therapeutic potential of histone deacetylase inhibitors requires full knowledge of individual histone deacetylases in vivo. As hematologic malignancies show increased sensitivity towards histone deacetylase inhibitors we targeted deletion of class I Hdac1 and Hdac2 to hematopoietic cell lineages. Here, we show that Hdac1 and Hdac2 together control hematopoietic stem cell homeostasis, in a cell-autonomous fashion. Simultaneous loss of Hdac1 and Hdac2 resulted in loss of hematopoietic stem cells and consequently bone marrow failure. Bone-marrow-specific deletion of Sin3a, a major Hdac1/2 co-repressor, phenocopied loss of Hdac1 and Hdac2 indicating that Sin3a-associated HDAC1/2-activity is essential for hematopoietic stem cell homeostasis. Although Hdac1 and Hdac2 show compensatory and overlapping functions in hematopoiesis, mice expressing mono-allelic Hdac1 or Hdac2 revealed that Hdac1 and Hdac2 contribute differently to the development of specific hematopoietic lineages.

Figure 1.

Hdac1 and Hdac2 are collectively essential for hematopoiesis. (A) Splenocyte and thymocyte counts of the indicated groups (n=3 per group). (B) Quantification of bone marrow cells after erythrocyte lysis. (C) The bone marrow composition of WT, MxCre⁺;Hdac1^Δ/Δ, MxCre⁺;Hdac2^Δ/Δ and DKO mice 8 days after the last pl:pC injection (n=3 per group). Bone marrow cells were isolated, quantified and stained with labeled antibodies against CD11b and Gr-1 (myeloid cells), B220 and CD19 (B cells), TER119 (erythroid cells), CD41 (megakaryocytes) and CD3 (T cells) and analyzed by flow cytometry. (D) Apoptosis in the bone marrow of control and DKO mice was analyzed by flow cytometry using a labeled antibody against caspase-3. Histograms are representative of three experiments per group; the average and standard deviation are presented on top. (E) Flow cytometric quantification of c-Kit⁺ cells (progenitors) and LSK (Lin⁻;c-Kit⁺;ScaI⁺) cells in the bone marrow of WT and DKO mice (n=3 per group). (F) The number of myeloid and pre-B-cell colonies from bone marrow of WT and DKO mice cultured in methylcellulose enriched with either 10 ng/mL interleukin–3, 10 ng/mL interleukin-6 and 50 ng/mL stem cell factor to induce myeloid differentiation or with 10 ng/mL interleukin-7 to induce differentiation into pre-B cells.

Figure 2.

Hdac1 and Hdac2 have cell autonomous functions in HSC maintenance. (A) Bone marrow cells from WT (Ly5.1⁺) and MxCre⁺;Hdac1^L/L (Ly5.1⁺/Ly5.2⁺) or MxCre⁻;Hdac1^L/L (control) mice were transplanted in a 1:1 ratio in lethally irradiated recipient mice. Bone marrow reconstitution of Ly5.1⁺ and Ly5.1⁺/Ly5.2⁺ populations was measured 8 weeks post-transplantation (left panel). Subsequently, mice were injected five times with pI;pC (arrows right panel). Ly5.1⁺/Ly5.2⁺ cells were monitored in the blood for 91 days (right panel). The mean value of Ly5.1⁺Ly5.2⁺ cells was set at 100% before pI;pC injection, (n=5 mice per group). (B) Bone marrow cells from WT (Ly5.1+) and MxCre⁺;Hdac1^L/LHdac2^L/L (Ly5.2⁺) or MxCre⁻; Hdac1^L/LHdac2^L/L (control) mice were transplanted in a 1:1 ratio in lethally irradiated recipient mice. Bone marrow reconstitution of Ly5.1⁺ and Ly5.2⁺ populations was measured 8 weeks post-transplantation (left panel). Subsequently, mice were injected five times with pI;pC (arrows right panel). Ly5.2⁺ cells were monitored in the blood for 105 days (right panel). The mean value of Ly5.2⁺ cells was set at 100% before pI;pC injection (n=5 mice per group). (C) At day 105 after the first pI:pC injection, mice were sacrificed and Ly5.2⁺ LSK in the bone marrow of the chimeras described in (B), were quantified by flow cytometry (n=3 per group). (D) After the competitive bone marrow transfer described in (C), genomic DNA was isolated from sorted Ly5.2⁺ cells and analyzed by polymerase chain reaction for the presence of floxed and Δ alleles (right panel). 1–3 represents bone marrow samples from three independent mice.

Figure 3.

Hdac1 and Hdac2 do not compensate for each other equally in hematopoiesis. (A) Western blot analysis for Hdac1 and Hdac2 protein levels in the bone marrow of the mice with the indicated genotypes. Pictures are representative images of the results from three or more independent mice. Tubulin was used a loading control. (B) Kaplan-Meier survival curves demonstrating the survival of WT, MxCre⁺;Hdac1^+/ΔHdac2^Δ/Δ and MxCre⁺;Hdac1^Δ/ΔHdac2^+/Δ mice after five injections of pI;pC (at least 3 mice per group). (C) Quantification of erythrolysed bone marrow of indicated genotypes (n=3 mice per group). (D) Number of bone marrow cells after erythrocyte lysis of WT, MxCre⁺;Hdac1^+/ΔHdac2^Δ/Δ and MxCre⁺;Hdac1^Δ/ΔHdac2^+/Δ mice (n=3 mice per group). Bone marrow cells were isolated, quantified and stained with labeled antibodies against CD11b and Gr-1 (myeloid cells), B220 and CD19 (B cells), TER119 (erythroid cells), CD41 (megakaryocytes) and CD3 (T cells) and analyzed by flow cytometry. (E) The number of pre-B-cell and myeloid colonies from bone marrow of WT, MxCre⁺;Hdac1^Δ/ΔHdac2^+/Δ and MxCre⁺;Hdac1^+/ΔHdac2^Δ/Δ mice cultured in methylcellulose enriched with either 10 ng/mL interleukin-3, 10 ng/mL interleukin-6 and 50 ng/mL stem cell factor to induce myeloid differentiation or with 10 ng/mL interleukin-7 to induce differentiation into pre-B cells.

Figure 4.

Erythropoiesis is severely affected only in MxCre⁺;Hdac1^Δ/ΔHdac2^+/Δ mice. (A) Three WT and three enlarged MxCre⁺;Hdac1^Δ/ΔHdac2^+/Δ spleens. (B) Splenocyte numbers in WT and MxCre⁺;Hdac1^Δ/ΔHdac2^+/Δ mice (n=3 mice per group). (C) After erythrocyte lysis, which only affects enucleated cells, the number of nucleated erythrocytes in spleens from WT and MxCre⁺;Hdac1^Δ/ΔHdac2^+/Δ mice was determined (n=3 mice per group). (D) The number of myeloid colonies from spleen of WT and MxCre⁺;Hdac1^Δ/ΔHdac2^+/Δ mice cultured in methylcellulose enriched with 10 ng/mL interleukin-3, 10 ng/mL interleukin-6 and 50 ng/mL stem cell factor to induce myeloid differentiation. (E) Hematoxylin-eosinstained histological liver sections of pI;pC-treated mice with indicated genotypes. Only MxCre⁺;Hdac1^Δ/ΔHdac2^+/Δ livers showed the presence of nucleated erythroid cells (F) Erythroid development in the bone marrow of WT, MxCre⁺;Hdac1^Δ/ΔHdac2^+/Δ mice was analyzed by flow cytometry using the labeled erythroid markers CD71 and Ter119. I, II, III and IV represent pro-erythroblasts, basophilic, polychromatic and orthochromatic erythroblasts, respectively (left panel). Absolute numbers are plotted (right panel).

Figure 5.

Sin3A is essential for hematopoiesis. (A) Northern blot analysis of Sin3A expression in thymus, brain and bone marrow of WT, MxCre⁺;Sin3A^+/Δ and MxCre⁺;Sin3A^Δ/Δ mice 48 h after three injections of pI;pC (B) Kaplan-Meier survival curves demonstrating survival of WT, MxCre⁺;Sin3A^+/Δ and MxCre⁺;Sin3A^Δ/Δ mice after five injections of pI;pC (at least 3 mice per group). (C) Erythrocyte and thrombocyte numbers in peripheral blood of mice with the indicated genotypes 1 week and 2 weeks after the first of five injections of pI;pC. (D) Splenocyte and thymocyte counts of mice with the indicated genotypes (n=3 per group). (E) Immunohistochemistry on paraffin-embedded tissue sections of thymi (cortex) from animals with the indicated genotypes, using antibodies against activated caspase-3. Sections were counterstained with hematoxylin.

Figure 6.

Sin3A has a cell-autonomous function in HSC maintenance. (A) Quantification of bone marrow cells from WT, MxCre⁺;Sin3A^+/Δ and MxCre⁺;Sin3A^Δ/Δ mice (n=3 mice per group) (left panel). Representative hematoxylin–eosin-stained paraffin-embedded tissue sections from bone marrow of mice with the indicated genotypes showing loss of cellularity in Sin3A-deficient bone marrow. (B) The bone marrow composition of the indicated genotypes (n=3 mice per group). Bone marrow cells were isolated, quantified and stained with labeled antibodies against CD11b and Gr-1 (myeloid cells), B220 and CD19 (B cells), TER119 (erythroid cells), CD41 (megakaryocytes) and CD3 (T cells) and analyzed by flow cytometry. (C) Flow cytometric quantification of c-kit⁺ cells (progenitors) in the bone marrow of animals with the indicated genotypes (n=3 per group). (D) Flow cytometric quantification of LSK cells (lin⁻c-Kit⁺ScaI⁺) in the bone marrow of animals with the indicated genotypes (n=3 per group). (E) The number of myeloid colonies from bone marrow of animals with the indicated genotypes cultured in methylcellulose enriched with 10 ng/mL interleukin-3, 10 ng/mL interleukin-6 and 50 ng/ml stem cell factor to induce myeloid differentiation. (F) Bone marrow cells from WT (Ly5.2⁺) and MxCre⁺;Sin3A^L/L (Ly5.1⁺) or MxCre⁻;Sin3A^L/L (control) mice were transplanted in a 1:1 ratio in lethally irradiated recipient mice. Bone marrow reconstitution of Ly5.1⁺ and Ly5.2⁺ populations was measured 8 weeks post-transplantation (left panel). Subsequently, mice were injected five times with pI;pC (arrows right panel). Ly5.2⁺ cells were monitored in the blood for 70 days (right panel). The mean value of Ly5.2⁺ cells was set at 100% before pI;pC injection, (n=5 mice per group). (G) Flow cytometric quantification of Ly5.1⁺ LSK in the bone marrow of the chimeras described in (E) (n=3 per group).

Figure 7.

Sin3A is essential for lymphocyte development. (A) Quantification of cells in the thymus from WT, LCre⁺;Sin3A^+/Δ and LCre⁺;Sin3A^Δ/Δ mice (n=3 mice per group). (B) Representative dot plots of flow cytometric analyses of thymocyte development in 6-week old mice with the indicated genotypes. (C) Quantification of thymic subsets of 6-week old mice with the indicated genotypes (n=3 mice per group). CD25⁻CD44⁺ = DN1; CD25⁺CD44⁺ = DN2; CD25⁺CD44⁻ = DN3; CD25⁻CD44⁻ = DN4. (D) Quantification of thymic subsets of 6-week old mice with the indicated genotypes (n=3 mice per group). DN = CD4⁻CD8⁻, DP = CD4⁺CD8⁺, CD4 SP = CD4⁺CD8⁻, CD8 SP = CD4⁻CD8⁺. (E) Representative flow cytometry plots of B lymphocytes in the spleen and bone marrow of WT, Mb1Cre;Sin3A^+/Δ_Δ and Mb1Cre;Sin3A^Δ/Δ mice, using the labeled B-cell markers CD19 and B220 (left panel). Quantification of B lymphocytes in spleen and bone marrow (n=3 mice per group) (right panel). (F) Quantification of pro-B, pre-B and IgM⁺ B lymphocytes in the bone marrow of mice with the indicated genotypes (n=3 mice per group).