Differential Roles for the E2A Activation Domains in B Lymphocytes and Macrophages 1 Savita Bhalla,* Christina Spaulding,* Rachel L. Brumbaugh,* Derek E. Zagort,* Mark E. Massari, ‡ Cornelis Murre, ‡ and Barbara L. Kee 2 * † The E2A gene encodes two E protein/class I basic helix-loop-helix transcription factors, E12 and E47, that are essential for B lymphopoiesis. In addition to the DNA-binding and protein dimerization domain, the E proteins share two highly conserved transcription activation domains. In this study, we show that both activation domains are required for optimal E2A-dependent transcription. Surprisingly, however, neither activation domain is required for E2A to rescue B lymphopoiesis from E2A / hemopoietic progenitors, although the N terminus of E2A, which harbors some transcription capacity, is required. Therefore, the E protein activation domains function redundantly in promoting B cell development. In contrast, the N-terminal activation domain, AD1, is required for a newly described ability of E2A to suppress macrophage development in vitro. Our findings demonstrate distinct functionalities for the E protein activation domains in B lymphocytes and macrophages. The Journal of Immunology, 2008, 180: 1694 –1703. B lymphocytes develop from hemopoietic stem cells in the embryonic liver and adult bone marrow (1). This devel- opmental program is marked by the progressive loss of myeloid differentiation potential followed by activation of lym- phoid-associated genes and commitment to the B lymphocyte lin- eage (2). The mechanisms underlying the loss of myeloid potential and induction of the B cell gene program are not fully understood but appear to involve the activation of transcription regulatory pro- teins that promote or repress gene expression. Activation of the B cell gene program is dependent on numerous transcription factors including Bcl11a, E2A, early B cell factor (EBF1), 3 Pax5, Pu.1, and Sox4 (2– 4). E2A, EBF1, and Pax5 are activated in a tran- scriptional hierarchy with E2A promoting expression of Ebf1 and EBF1 regulating expression of Pax5, although recent studies re- vealed multiple levels of feedback control within this simple net- work (2, 5). Nonetheless, the essential function of E2A in this differentiation program is induction of Ebf1 as demonstrated by the ability of ectopic EBF1 to rescue B cell lineage specification and commitment from E2A -/- progenitors (6). E2A cooperates with multiple factors including Pu.1, Ets1, Stat5, and Pax5 to promote Ebf1 transcription (5, 7–9). Although E2A DNA-binding se- quences have been identified in the Ebf1 promoter, precisely how E2A activates Ebf1 transcription remains to be determined (5, 10). The E2A proteins, E12 and E47, are basic helix-loop-helix (bHLH) transcription factors that differ only in the use of an al- ternatively spliced exon encoding the bHLH domain. Their bHLH domains share 80% amino acid identity and bind to the same DNA sequence (an E box) and dimerize with the same proteins (11). E2A proteins are related to two additional E proteins in mam- mals, E2-2 and HEB, and all four proteins have overlapping patterns of expression within the hemopoietic system (12). The E proteins function redundantly and expression of HEB from the E2A gene is sufficient to rescue B lymphopoiesis in E2A -/- mice (13). Although HEB and E2-2 are not essential for B lymphocyte development, they are expressed in these cells and compound E protein-deficient mice (i.e., E2A +/- HEB +/- and E2A +/- E2-2 +/- ) reveal some function for all E proteins in B cell development (14, 15). Indeed, the ability of EBF1 to rescue B cell development from E2A -/- fetal liver multi- potent progenitors (FL MPPs) is dependent on the activity of these other E proteins because complete inhibition of E protein activity in pro-B cells results in growth arrest and apoptosis (6, 16). Therefore, E2A specifically is required for induction of Ebf1 but activation of B cell genes in collaboration with EBF1 may occur with the lower levels of E protein activity conferred by HEB and E2-2 in the absence of E2A. E2A, HEB, and E2-2 share homology (40%) within two well- defined transcription activation domains (ADs), referred to as AD1 and AD2, or the loop-helix (LH) AD (17–20). When fused to a Gal4 DNA-binding domain AD1 and AD2 are able to activate transcription from a reporter construct containing 5 Gal4 DNA- binding sites upstream of a minimal promoter (17, 19). However, the requirement for these ADs has not been investigated in the context of a full-length E protein. In this regard, it is interesting that the Drosophila homolog of E2A, daughterless, has a sequence similar to AD2 but does not contain an AD1-related domain (17). The HEB gene also produces an alternatively spliced form (HEBAlt) that lacks the AD1 domain and instead encodes a novel sequence that confers unique properties on HEB in T lymphocytes (21). In addition, isoforms of E2A that lack AD1 have been de- scribed (22). Interestingly, AD1- and AD2-Gal4 fusion proteins were shown to have cell type- specific activity in zebrafish sug- gesting that they may interact with distinct coactivator complexes *Department of Pathology and † Committees on Immunology, Cancer Biology and Developmental Biology, University of Chicago, Chicago, IL 60637; and ‡ Department of Biology, University of California at San Diego, La Jolla, CA 92190 Received for publication July 18, 2007. Accepted for publication November 28, 2007. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the National Institutes of Health (R01 CA099978, to B.L.K.). 2 Address correspondence and reprint requests to Dr. Barbara L. Kee, Department of Pathology, University of Chicago, 5841 South Maryland Avenue, MC1089, Chicago, IL 60637. E-mail address: bkee@bsd.uchicago.edu 3 Abbreviations used in this paper: EBF, early B cell factor; bHLH, basic helix-loop- helix; FL MPP, fetal liver multipotent progenitor; AD, activation domain; LH, loop helix; IRES, internal ribosomal entry site; QPCR, quantitative real-time PCR; WT, wild type; FSC, forward scatter; SSC, side scatter; MFI, mean fluorescence intensity. Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 The Journal of Immunology www.jimmunol.org