E2F4 Modulates Differentiation and Gene Expression in Hematopoietic Progenitor Cells during Commitment to the Lymphoid Lineage This information is current as of September 27, 2021. Megan E. Enos, Simona A. Bancos, Timothy Bushnell and Ian N. Crispe J Immunol 2008; 180:3699-3707; ; doi: 10.4049/jimmunol.180.6.3699 http://www.jimmunol.org/content/180/6/3699 Downloaded from References This article cites 32 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/180/6/3699.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology E2F4 Modulates Differentiation and Gene Expression in Hemopoietic Progenitor Cells during Commitment to the Lymphoid Lineage1 Megan E. Enos,* Simona A. Bancos,† Timothy Bushnell,‡ and Ian N. Crispe2* The E2F4 protein is involved in gene repression and cell cycle exit, and also has poorly understood effects in differentiation. We analyzed the impact of E2F4 deficiency on early steps in mouse hemopoietic development, and found defects in early hemopoietic progenitor cells that were propagated through common lymphoid precursors to the B and T lineages. In contrast, the defects in erythromyeloid precursor cells were self-correcting over time. This suggests that E2F4 is important in early stages of commitment to the lymphoid lineage. The E2F4-deficient progenitor cells showed reduced expression of several key lymphoid-lineage genes, and overexpression of two erythromyeloid lineage genes. However, we did not detect Downloaded from effects on cell proliferation. These findings emphasize the significance of E2F4 in controlling gene expression and cell fate. The Journal of Immunology, 2008, 180: 3699–3707. emopoietic stem cells (HSC)3 must choose between self- expression of cyclin E, DNA polymerase ␣, thymidine kinase, renewal and differentiation; if they differentiate they can and other genes that advance the cell cycle (10). In contrast, the become common myeloid progenitors (CMP) or com- repressing E2Fs, namely E2F3b, E2F4 and E2F5, have the abil- H http://www.jimmunol.org/ mon lymphoid progenitors (CLP). It is still unclear how environ- ity to bind similar promoter regions to those bound by the ac- mental signals (1) and lineage-specific transcription factors work tivating E2Fs (11), but are simultaneously bound by pocket pro- together to control the frequency with which dividing HSC either teins (pRb, p107, or p130) that physically prevent interaction undergo self-renewal or commit to one or the other lineage. Tran- with the transcriptional machinery (12). However, phosphory- scription factors expressed in HSC can drive commitment to either lation of the pocket proteins bound to E2F3b-5 can de-repress the lymphoid or the myeloid lineage (2). For example, factors of the transcription of the promoter regions to which they bind. the Ikaros family specifically favor differentiation down the lym- The final subset of E2Fs, E2F6, E2F7, and E2F8 lack a trans- phoid pathway (3), whereas other factors, such as GATA-1 and activation domain and do not interact with pocket proteins, ␣ C/EBP , favor differentiation down the myeloid pathway (4, 5). and therefore have the potential to silence promoter regions by guest on September 27, 2021 We are particularly interested in mechanisms that influence (13, 14). the choice between self-renewal and differentiation. Thus we The repressing E2Fs could favor differentiation simply by pro- study the E2F family of transcription factors, which promotes moting cell cycle exit (7). Among these, E2F4 is found at high cell cycle progression and exit; the latter is associated with concentrations in hemopoietic cells (15). Overexpression of E2F4 terminal differentiation in many cell types (6, 7). The E2F fam- promotes terminal differentiation of cells in vitro (16), whereas ily includes eight genes, and several of their products are sus- E2F4-deficient animals show reduced neonatal viability, with very ceptible to differential splicing, generating distinct isoforms (8). few animals surviving to adulthood (15, 17). These animals died The activating E2Fs, namely E2F1, E2F2, and E2F3a, promote due to opportunistic infections and anemia, but they were also the G1-to-S phase transition during cell cycle progression (9), smaller than either heterozygous or wild-type (WT) littermates interacting with the basal transcriptional machinery to enhance (15, 17). Recently, E2F4 has been shown to play a role during fetal erythropoiesis, explaining the anemia that is seen in the fetuses and neonates (18). However, the impact of E2F4 deficiency on thymo- *Department of Microbiology and Immunology, and the David H Smith Center for Vaccine Biology and Immunology, †Department of Environmental Medicine, Lung cyte development has been controversial (15, 17). Both the ery- Biology and Disease Program, and ‡Department of Pediatrics, Center for Pediatric throid and the lymphoid defects in E2F4-deficient mice could be Biomedical Research, The James P. Wilmot Cancer Center, University of Rochester, Rochester, NY 14620 explained if the defect occurred at the level of the HSC, which gives rise to both lineages. Received for publication January 8, 2008. Accepted for publication January 8, 2008. In these studies we focused extensively on two populations of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance cells. The first is a subset that is enriched for HSC activity, which with 18 U.S.C. Section 1734 solely to indicate this fact. we identified using the phenotype first proposed by Spangrude Ϫ 1 This work was supported by Grant R01GM056689 from the National Institutes of et al. (19); they are cells lacking multiple lineage-specific (Lin ) Health (to I.N.C.). markers, but expressing stem cell Ag-1 (sca-1) and the stem cell 2 Address correspondence and reprint requests to Dr. Ian N. Crispe, David H Smith factor receptor c-kit. This cell population overlaps functionally Center for Vaccine Biology and Immunology, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14620. E-mail address: [email protected] with HSC, but because in these experiments we are identifying Ϫ 3 Ϫ ϩ them by phenotype, we use the term LSK cells to denote the Lin , Abbreviations used in this paper: HSC, hemopoietic stem cell; LSK, Lin sca-1 c- ϩ ϩ kitϩ; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, sca-1 , c-kit phenotype. This term is previously used to denote granulocyte-macrophage progenitor; MEP, megakaryocyte-erythroid progenitor; DN, this population. The second population of cells is one that has double negative; WT, wild type. been proposed to correspond to bone marrow B cell precursors Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 but that also has shown pro-T potential, rendering them CLP www.jimmunol.org 3700 E2F4 AND LYMPHOID DEVELOPMENT A Live Cell Gate Lineage Negative Lineage Negative 105 105 HSC-like 105 0.63 104 104 104 IL7Rα+ 3 3 3 6.14 10 c-kit 10 10 Lineage CD127 102 102 102 0 0 0 α 10.3 42.2 IL7R - 0102 103 104 105 0102 103 104 105 0102 103 104 105 DAPI sca-1 Lineage IL7Rα+ IL7Rα- 105 105 37 105 12.5 CLP-like 6.55 104 104 104 GMP-like 3 c-kit 3 3 AA4.1 10 10 10 CMP-like Downloaded from CD16/32 4.88 FIGURE 1. Identification of pro- 102 102 102 genitors in fetal liver. A, Identification 0 0 0 57.9 MEP-like of HSC-like, CLP-like, and CMP-like 0102 103 104 105 0102 103 104 105 0102 103 104 105 cells in WT fetal liver. Multicolor sca-1 sca-1 CD34 flow cytometry and a sequential gat- Ϫ ing strategy identify first Lin viable http://www.jimmunol.org/ cells, then cells coexpressing c-kit and B Live Cell Gate Lineage Negative Lineage Negative 5 5 5 sca-1. B, The same subsets of cells ap- 10 10 10 HSC-like pear in E2F4-deficient fetal liver. 2.21 4 4 10 10 104 6.8 IL7Rα+ 3 3 10 10 3 c-kit 10 Lineage CD127 102 2 10 102 0 0 IL7Rα- 0 24.6 10.6 by guest on September 27, 2021 2 3 4 5 2 3 4 5 010 10 10 10 2 3 4 5 010 10 10 10 010 10 10 10 DAPI sca-1 Lineage IL7Rα+ IL7Rα- 5 10 5 5 10 10 9.3 39.2 104 4 CLP-like 4 10 10 GMP-like 8.42 c-kit 3 AA4.1 10 3 10 103 CMP-like CD16/32 7.32 102 2 10 102 0 0 0 35.8 MEP-like 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 2 3 4 5 sca-1 010 sca-110 10 10 CD34 (20). These cells are also Lin Ϫ but express the IL-7R ␣-chain CMP-like cells overexpressed gata1 and gata2. This argues that and the marker AA4.1, together with an intermediate level of E2F4 acts during hemopoiesis by controlling the expression of sca-1.