Nucleoporin Nup358 drives the differentiation of myeloid-biased multipotent progenitors by modulating HDAC3 nuclear translocation

Abstract

Nucleoporins, the components of nuclear pore complexes (NPCs), can play cell type- and tissue-specific functions. Yet, the physiological roles and mechanisms of action for most NPC components have not yet been established. We report that Nup358, a nucleoporin linked to several myeloid disorders, is required for the developmental progression of early myeloid progenitors. We found that Nup358 ablation in mice results in the loss of myeloid-committed progenitors and mature myeloid cells and the accumulation of myeloid-primed multipotent progenitors (MPPs) in bone marrow. Accumulated MPPs in Nup358 knockout mice are greatly restricted to megakaryocyte/erythrocyte-biased MPP2, which fail to progress into committed myeloid progenitors. Mechanistically, we found that Nup358 is required for histone deacetylase 3 (HDAC3) nuclear import and function in MPP2 cells and established that this nucleoporin regulates HDAC3 nuclear translocation in a SUMOylation-independent manner. Our study identifies a critical function for Nup358 in myeloid-primed MPP2 differentiation and uncovers an unexpected role for NPCs in the early steps of myelopoiesis.

Fig. 1.. Nup358 is required for myeloid cell development.

(A) Total bone marrow cells in Nup358^fl/fl (control) and Nup358^fl/flCreER^T2 (Nup358 knockout) mice treated with tamoxifen. (B) Schematic illustration of hematopoiesis. HSCs, hematopoietic stem cells; MPPs, multipotent progenitors; LSK, Lin⁻Sca1⁺cKit⁺ cells; MyP, myeloid progenitors; CMP, common myeloid progenitors; MEP, megakaryocyte erythrocyte progenitors, GMP, granulocyte monocyte progenitors; LMPP, lymphoid-myeloid primed progenitors; CLP, common lymphoid progenitors; Meg, megakaryocytes; Er, erythrocytes; Mo, monocytes; Ne, neutrophils; DC, dendritic cells; B, B lymphocytes, T, T lymphocytes; NK, natural killer cells. (C to H) Flow cytometry analysis of bone marrow cells from control and Nup358 knockout mice. Mature immune cells (Lin⁺) and hematopoietic progenitors (Lin⁻) numbers (C), Lin⁺ and Lin⁻ percentage of viable cells (D). Number of MyP (E), LMPP and CLP (F). Number of monocytes (Mon), neutrophils (Neu), and macrophages (Mac) (G), NK cells, and B cells (H). (I) Number of CD3⁺, CD4⁺, and CD8⁺ T lymphocytes in spleen of control and Nup358 knockout mice. (J) Colony formation assay of bone marrow cells from control and Nup358 knockout mice in conditions promoting myeloid differentiation. (K) Representative images of macrophages differentiated in vitro from bone marrow cells of tamoxifen-treated Nup358^fl/fl and Nup358^fl/flCreER^T2 mice. Scale bars, 75 μm. (L) Representative images macrophages differentiated in vitro from bone marrow cells of Nup358^fl/fl and Nup358^fl/flCreER^T2 mice treated with tamoxifen after differentiation. Scale bars, 75 μm. (M) Viability of differentiated macrophages from (L). Data are means ± SD **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 by either unpaired Student’s t test [(A), (E), and (M)] or multiple unpaired Student’s t test with Holm-Sidak method to correct for multiple comparisons [(C) and (D) and (F) to (I)]. Data are pooled from eight (A), six [(C) and (D)], three [(E) and (F)], two [(G) to (I)], and four (I) independent experiments with at least n = 3 mice of each genotype.

Fig. 2.. Nup358 knockouts have increased the number of megakaryocyte erythroid-primed MPP (MPP2).

(A) Schematic illustration of the LSK subpopulations and MPP lineage priming. (B) Number of LSK cells in bone marrows of control and Nup358 knockout mice. (C) LSK cells as percentage of Lin⁻ cells. (D) Number of HSC^LT and HSC^ST in bone marrow of control and Nup358 knockout mice. (E) HSC^LT and HSC^ST as percentage of LSK cells. (F) Number of MPP2, MPP3, and MPP4 in bone marrows of control and Nup358 knockout mice. (G) Percentage of MPP2, MPP3, and MPP4 in the LSK population. Data are means ± SD. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 by either unpaired Student’s t test [(B) and (C)] or multiple unpaired Student’s t test with Holm-Sidak method to correct for multiple comparisons [(D) to (G)]. Data are pooled from six [(B) and (C)] and three [(D) to (G)] independent experiments with at least n = 3 mice of each genotype.

Fig. 3.. Nup358 role in myeloid development is intrinsic to hematopoietic progenitors.

(A) Schematic illustration of the bone marrow transplantation experiments. (B) Number of neutrophils (NE), monocytes (MO), and lymphocytes (LY) per milliliter in peripheral blood of CD45.1 wild-type mice transplanted with bone marrow cells from either Nup358^fl/fl or Nup358^fl/fl-CreER^T2 mice after 12 weeks of reconstitution and 5 days after tamoxifen administration. (C) Total bone marrow cells from CD45.1 wild-type mice transplanted with bone marrow cells from either Nup358^fl/fl or Nup358^fl/fl-CreER^T2 mice at 12 days after tamoxifen administration. (D to G) Flow cytometry analysis on bone marrow cells isolated from CD45.1 wild-type mice transplanted with bone marrow cells from either Nup358^fl/fl or Nup358^fl/fl-CreER^T2 mice after 12 weeks of reconstitution and 12 days after tamoxifen administration. Lin⁺ and Lin⁻ cells as number (D) or percentage of viable bone marrow cells (E), and HSC^LT, HSC^ST MPP2, MPP3, and MPP4 as number (F) or percentage of LSK cells (G) at 12 days after tamoxifen injection. (H and I) Flow cytometry analysis on splenocytes isolated from CD45.1 wild-type mice transplanted with bone marrow cells from either Nup358^fl/fl or Nup358^fl/fl-CreER^T2 mice after 12 weeks of reconstitution and 12 days after tamoxifen administration. Total CD3⁺ T lymphocytes (H), CD4⁺ T lymphocytes, and CD8⁺ T lymphocytes (I). Data are means ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, *****P ≤ 0.00001 by unpaired Student’s t test [(C) and (H)] or multiple unpaired Student’s t test with Holm-Sidak method to correct for multiple comparisons [(D) to (G) and (I)].

Fig. 4.. scRNA-seq analysis of Nup358^−/− LSK cells.

(A) Uniform Manifold Approximation and Projection plot of control and Nup358 knockout cells showing 15 clusters identified by dimensionality reduction and unsupervised clustering. (B) Distribution of control (blue) and Nup358 knockout (red) cells among clusters. (C) Violin plots representing the aggregated expression of genes associated with neutrophils, megakaryocytes/erythrocytes, and lymphocytes in the identified clusters. (D) Distribution of control and Nup358 knockout cells in unprimed and primed clusters. (E) Distribution of control and Nup358 knockout cells in neutrophil-primed (N-primed), megakaryocytes/erythrocyte-primed (ME-primed), and lymphocyte-primed (L-primed) clusters. (F) Heatmaps showing genes differentially expressed between control and Nup358 knockout LSK cells from clusters 4 and 9 (fold change > ±1.25; P < 0.05). (G) Volcano plot of single-cell transcriptome data showing the differentially expressed genes in Nup358 knockout cells from clusters 4 and 9. Genes are organized by log₂ fold change and –log₁₀ P value. Genes below the cutoff (dashed line, P < 0.05) are shown in gray. (H) Ten most significantly altered pathways in Nup358 knockout LSK cells from cluster 4 and cluster 9 ranked by P value. (I) Cell cycle analysis on MPP2 in control (Nup358^fl/fl) and Nup358 knockout (Nup358^fl/flCreER^T2) by flow cytometry.

Fig. 5.. Nup358 is required for HDAC3 function and nuclear translocation.

(A) Upstream transcriptional regulators (P < 0.05) predicted to be altered in Nup358 knockout LSK cells belonging to cluster 4 and cluster 9 ranked by P value. (B) The intracellular localization of HDAC3, E2F1, RB1, p53, RBL2, DMTF1, and GTF2I was analyzed by immunofluorescence in 293T cells transfected with control or Nup358-specific siRNAs. Scale bars, 25 μm. (C) HDAC3 intracellular localization was analyzed by immunofluorescence in 293T cells transfected with control or Nup358-specific siRNAs. Cells were costained with antibodies against nucleoporins Nup358 and Nup98. Scale bars, 25 μm. (D) Quantification of HDAC3 nuclear/cytoplasmic signal ratio in control or Nup358 knockdown cells. Data are expressed as mean ± SD. ****P ≤ 0.0001. (E) Protein levels of HDAC3 and Nup358 in cells transfected with scramble control or Nup358-specific siRNAs were analyzed by Western blot. HSP90 was used as loading control.

Fig. 6.. Nup358 regulates HDAC3 nuclear import in a SUMOylation-independent manner.

(A) 293T cells were transduced with lentivirus carrying scramble control or Nup358-targeting shRNAs and treated with doxycycline to induce down-regulation of Nup358. HDAC3 was immunoprecipitated, and Nup358, Nup210, and Nup96 were analyzed by Western blot. (B) 293T cells were transfected with scramble control or Ubc9-specific siRNAs, and the localization of HDAC3 was analyzed after 120 hours by immunofluorescence. Cells were coimmunostained with an antibody against nucleoporin Nup358. Scale bars, 25 μm. (C) Ubc9 levels in 293T cells treated with control or Ubc9-specific siRNAs were analyzed by immunofluorescence 120 hours after transfection. Cells were coimmunostained with mAb414 antibody against NPCs. Scale bars, 25 μm. (D) 293T cells were treated for 72 hours with the selective SUMOylation inhibitor 2-D08 (25 μM), and the localization of HDAC3 was analyzed by immunofluorescence. Cells were costained with an antibody against nucleoporin Nup358. Scale bars, 25 μm.

Fig. 7.. Nup358 and HDAC3 work together to regulate MPP2 homeostasis.

(A) Schematic illustration of the in vitro myeloid differentiation of primary hematopoietic progenitors. (B) The total number of viable cells in cultures of Nup358^fl/fl and Nup358^fl/flCreER^T2 hematopoietic progenitors treated with tamoxifen in vitro was measured over time. (C to F) Flow cytometry analysis of hematopoietic progenitor cultures 96 hours after treatment with tamoxifen. Fold change relative to vehicle-treated cells. (G to J) Cell growth and viability of Nup358^fl/fl and Nup358^fl/flCreER^T2 hematopoietic progenitor cultures treated with either tamoxifen or vehicle for 96 hours and HDAC3 inhibitor RGFP966 or vehicle for 48 hours. (K to M) Flow cytometry analysis of hematopoietic progenitor cultures treated with either tamoxifen or vehicle for 96 hours and HDAC3 inhibitor RGFP966 or vehicle for 48 hours. Data are means ± SD *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, *****P ≤ 0.00001 by unpaired Student’s t test (D) or multiple unpaired Student’s t test with Holm-Sidak method to correct for multiple comparisons [(C), (E), and (F)] or analysis of variance (ANOVA) with Turkey correction for multiple comparison [(K) to (M)]. Data in (B) to (F) are representative of two independent experiments with at least n = 3 biological samples of each genotype.