Decreased E12 and/or E47 Transcription Factor Activity in the Bone Marrow As Well As in the Spleen of Aged Mice 1 Daniela Frasca, Diep Nguyen, Richard L. Riley, and Bonnie B. Blomberg 2 The E2A-encoded transcription factors E12 and E47 are key regulators of B cell functions. They bind to the E-box site, found in regulatory regions of B cell-specific genes; promote cell survival of early pre-B cells; help to initiate Ig rearrangements; and are also involved in class switch in mature B cells in the periphery. We have investigated the expression and function of E47 and E12 in IL-7-expanded pro-B/pre-B cell precursors and in unstimulated or LPS-activated splenic B cells from young and old BALB/c mice. Results show that B cell precursors from the bone marrow of old mice exhibit a reduced expression of E2A proteins and a reduced ability to bind DNA, as compared with young mice. In the spleen, E2A protein expression and DNA binding are present in unstimulated B cells from young mice and, to a significantly lesser extent, from old mice. These are both strongly induced by activation in splenic B cells from young mice but only moderately induced in old mice, indicating that aging affects the expression and activity of E2A-encoded genes and also that DNA binding correlates with the amount of protein expression. The levels of E2A DNA binding in the spleen correlate with those in the bone marrow for individual mice. In splenic mature B cells, only E47/E47 complexes bind DNA; whereas in bone marrow B cell precursors, E47/E12 complexes participate in DNA binding. Only nuclear extracts of splenic mature B cells, but both nuclear and cytoplasmic extracts of bone marrow B cell precursors, exhibit DNA binding. The Journal of Immunology, 2003, 170: 719 –726. T he E2A gene encodes two class I basic helix-loop-helix (bHLH) 3 proteins, E12 and E47, which are RNA splice variants. E12 and E47 are multifunctional transcription fac- tors involved in a large variety of developmental processes, charac- terized by their broad tissue distribution and by their ability to form homodimers or heterodimers with tissue-restricted class II bHLH. The two proteins share identical N-terminal trans activation domains but have distinct dimerization and DNA-binding motifs (1– 4). E47 was first identified as a protein that binds Ig and insulin gene-regulatory elements (2, 5, 6). In B lymphocytes, the active DNA-binding complex consists of E47 homodimers (7–9); whereas in non-B cells, E47 is unable to bind DNA or it binds DNA as a heterodimer with cell-restricted bHLH proteins, such as MyoD or NeuroD (10, 11). The formation and function of the homodimer or heterodimer depend on the balance between the E2A-encoded proteins, other class I bHLH proteins (class I bHLH transcription factor and E2-2), and the E protein-inhibitory pro- teins, Id 1–3, which lack the DNA-binding domain and function as dominant negative inhibitors of E proteins (12). E2A-null mutant mice fail to generate precursors and mature B cells. The arrest of B cell development occurs at an early stage, given that no Ig DJ rearrangements or expression of the B lineage-specific marker B220 can be detected in homozygous mutant mice (13, 14). Re- constitution of normal numbers of peripheral mature B cells re- quires the presence of both E12 and E47 (15). E2A transcription factors have been shown to regulate the expression of several B lineage genes such as 5, early B cell factor, TdT, and recombi- nation-activating gene-1 (15–19), to regulate Ig rearrangements (20, 21), and to promote Ig class switch recombination (2). Aging is associated with decreased production of precursor B lineage cells in the bone marrow (22–27). However, despite the decrease in newly emerging cells from the bone marrow, the pop- ulation of mature splenic B cells is maintained because of in- creased life span (23, 28). Reduced diversification of B cells has also been reported to occur in the germinal center (29), where affinity maturation and isotype switching take place (30–32). We have previously demonstrated that pre-B cell numbers, as well as the surrogate L chain, composed of the variable-like region of the pre-B cell receptor (VpreB) and 5 peptides, which is critical for Ig variable heavy chain selection, cellular proliferation, and sur- vival at the pre-B cell stage, are significantly reduced in aged mice (22, 26, 33). We further showed that low surrogate L chain mRNA and protein levels, as measured in IL-7-expanded cultures of B cell precursors from aged mice, are associated with decreased protein levels of the E2A-encoded transcription factors E47 and E12 (26). Here, we have investigated the expression and function of the E2A-encoded transcription factors E12 and E47 in IL-7-expanded pro-B/pre-B cell precursors and in splenic B cells, unactivated or LPS activated, from aging mice. We examined whether the decrease seen in E2A protein levels in aged bone marrow could also be re- flected in functional DNA binding and if these measurements of E2A (E12/E47) would also be affected in splenic B cells from aged mice. Moreover, we investigated the nature of the complex, i.e., whether E12/E47 or E47/E47 dimers were involved in DNA binding in B cell precursors in the bone marrow and in mature B cells in the spleen. Materials and Methods Mice Male and female young (2– 4 mo of age) and old (24 –27 mo of age) BALB/c mice were obtained from the National Institute on Aging (Bethesda, MD). Department of Microbiology and Immunology, University of Miami School of Med- icine, Miami, FL 33136. Received for publication August 13, 2002. Accepted for publication November 8, 2002 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 National Institutes of Health Grants AG-17618 (to B.B.B.) and AG-15474 (to R.L.R.). 2 Address correspondence and reprint requests to Dr. Bonnie B. Blomberg, Depart- ment of Microbiology and Immunology, RMSB, No. II 3146A, University of Miami School of Medicine, 1600 Northwest 10th Avenue, Miami, FL 33136. E-mail address: bblomber@med.miami.edu. 3 Abbreviations used in this paper: bHLH, basic helix-loop-helix; BCR, B cell receptor; VpreB, variable-like region of the pre-B cell receptor. The Journal of Immunology Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00