E2A proteins maintain the hematopoietic stem cell pool and promote the maturation of myelolymphoid and myeloerythroid progenitors

Abstract

Hematopoiesis is a tightly controlled process maintained by a small pool of hematopoietic stem cells (HSCs). Here, we demonstrate that the LT-HSC, MPP, premegakaryocytic/erythroid, Pre CFU-E, Pre GM, MkP, and granulocyte-macrophage compartments were all significantly reduced in E2A-deficient bone marrow. Despite a severe depletion of erythroid progenitors, the erythrocyte and megakaryocyte compartments were equivalent in E2A-deficient bone marrow as compared with wild-type mice. E2A-deficient HSCs also failed to efficiently maintain the HSC pool on serial transplantation, and we demonstrate that the E2A proteins regulate cell cycle progression of HSCs by regulating the expression of p21(Cip1), p27(Kip1), and the thrombopoietin receptor, known regulators of HSC self-renewal activity. Based on these observations, we propose that the E2A proteins promote the developmental progression of the entire spectrum of early hematopoietic progenitors and to suppress an erythroid specific program of gene expression in alternative cell lineages. Last, the data mechanistically link E2A, cell cycle regulators, and the maintenance of the HSC pool in a common pathway.

Fig. 1.

Effects of E2A deletion on the numbers of HSCs in adult mouse bone marrow (BM). Total BM cells from wild-type, E2A +/−, and E2A −/− mice were harvested and prepared for analysis by flow cytometry. (A) Representative staining profiles for LSK cells and the LT-HSC, ST-HSC, and MPP subpopulations. The small gate in the MPP quadrant is representative of the LMPP population. (B) Reduced HSC numbers in the BM of E2A −/− mice. Shown are the absolute numbers of the LSK, LT-HSC, ST-HSC, and MPP populations in the BM of wild-type, E2A +/−, and E2A −/− mice (n = 6). Horizontal bars show the mean values. Statistical significance determined by unpaired t test, 2-tailed, between E2A −/− and wild type.

Fig. 2.

Effects of E2A deletion on the numbers of myeloerythroid progenitors in adult mouse BM. Total BM cells from wild-type, E2A +/−, and E2A −/− mice were harvested and prepared for analysis by flow cytometry. (A) Representative staining profiles for myeloerythroid progenitors. (B) Reduced numbers of myeloerythroid progenitor subsets in the BM of E2A −/− mice. Shown are the absolute numbers of the myeloerythroid progenitor subsets in the BM of wild-type, E2A +/−, and E2A −/− mice (n = 5). Horizontal bars show the mean values. Statistical significance determined by unpaired t test, 2-tailed, between E2A −/− and wild type.

Fig. 3.

Increased cycling by E2A-deficient HSCs. To investigate cycling of E2A −/− HSCs, BrdU was administered to 6 week-old wild-type and E2A −/− mice and BrdU incorporation by the LT-HSC, ST-HSC, and MPP cell fractions was analyzed by flow cytometry. (A) Representative staining profiles for the LT-HSC (LSK/CD150⁺/Flk2⁻) fraction. (B) Increased incorporation of BrdU by E2A −/− HSCs. Shown are the percentages of BrdU incorporation by the LT-HSC (LSK/CD150⁺/Flk2⁻), ST-HSC (LSK/CD150⁻/Flk2⁻), and MPP (LSK/CD150⁻/Flk2⁺) fractions in the bone marrow of wild-type and E2A −/− mice (n = 4). Statistical significance determined by unpaired t test, 2-tailed, between E2A −/− and wild type. (C) Cell cycle distribution in E2A −/− HSCs. Shown are the percentages of LSKFlk2⁺ and LSKFlk2⁻ fractions in G₀, G₁, and SG₂M in the BM of wild-type and E2A −/− mice (n = 3). Statistical significance determined by 2-sided Student's t test, E2A −/−, compared with wild-type. (D) Schematic representation depicting the roles of E2A in early hematopoiesis. The blue arrow indicates the importance of E2A proteins in HSC self-renewal. Decreases in the indicated hematopoietic populations detected in E2A −/− mice are shown by solid red down arrows. In the absence of E2A, significant decreases in LMPPs, CLPs, and GMPs are detected. Also, the Pre MegE, Pre CFU-E, and MkP compartments are significantly decreased. However, E2A −/− mice have near wild-type levels of Pre GMs and CFU-Es. We propose that the E2A proteins act to promote the development of the LMPPs and the Pre MegE progenitors, and to suppress the development of the Pre GM stage into CFU-Es. Data represent the mean ± SD. Statistical significance determined by unpaired t test, 2-tailed, between E2A −/− and wild type.