Ebf1-mediated down-regulation of Id2 and Id3 is essential for specification of the B cell lineage Melissa A. Thala, Thiago L. Carvalhoa,TiHea, Hyung-Gyoon Kima, Hua Gaob, James Hagmanb, and Christopher A. Kluga,1 aDepartment of Microbiology, The University of Alabama-Birmingham, Birmingham, AL 35294; and bIntegrated Department of Immunology, National Jewish Medical and Research Center, Denver, CO 80206 Edited by Cornelis Murre, University of California, San Diego, La Jolla, CA, and accepted by the Editorial Board November 7, 2008 (received for review March 13, 2008) Gene knockout experiments in mice have suggested a hierarchical of E47 (13) or E12 (14) in non-B-lineage cell lines, suggest that model of early B cell commitment wherein E2A proteins (E47 and E2A activity is essential upstream of Ebf1. Similarly, the Pax5 E12) activate early B cell factor (Ebf1), which in turn activates promoter is bound by Ebf1 based on EMSA and Ebf1 can expression of the B cell commitment factor, Pax5. In IL-7 receptor transactivate the Pax5 promoter in transient co-transfection alpha (IL-7R␣) knockout mice, B cell development is blocked before assays (15, 16). Ebf1 is also present in Pax5-deficient pro-B cells B-lineage commitment at the prepro-B cell stage in adult animals. derived from Pax5Ϫ/Ϫ adult mice (17), suggesting that Ebf1 may In IL-7R␣؊/؊ prepro-B cells, E47 is expressed and yet is insufficient participate in the activation of Pax5 expression. Complicating the to transcriptionally activate the putative downstream target gene, simple hierarchical model where E2A induces expression of Ebf1. In this study, we show that further increases of E47 expres- Ebf1, which then activates Pax5, are observations showing that sion in IL-7R␣؊/؊ prepro-B cells fails to activate Ebf1, but rather up-regulation of E2A protein levels in B cell progenitors are leads to a dramatic induction of the E2A inhibitory factors, Id2 and dependent on Ebf1 (18). In addition, expression of Pax5 from the Id3. In contrast, enforced expression of Ebf1 in IL-7R␣؊/؊ bone endogenous Ikaros locus resulted in activation of Ebf1 and B cell marrow potently down-regulates Id2 and Id3 mRNA expression development in the thymus (19) and in the bone marrow of and restores B cell differentiation in vivo. Down-regulation of both E2A-deficient mice (20). Pax5 was also shown to directly bind Id2 and Id3 during B cell specification is essential in that overex- one of two distinct EBF promoters in vivo and could transacti- pression of either Id2 or Id3 in wild-type bone marrow blocks B cell vate this promoter in transient transfection assays (12). Collec- specification at the prepro-B cell stage. Collectively, these studies tively, these results demonstrate a complex regulatory circuitry suggest a model where Ebf1 induction specifies the B cell fate by controlling B cell specification and commitment from CLP. dramatically increasing activity of E47 at the posttranslational The loss of either the IL-7 receptor alpha (IL-7R␣) chain or level. the IL-7 ligand also results in a complete block in adult B lymphopoiesis at the prepro-B cell stage, with either modest or B cell specification ͉ E2A ͉ Ebf1 ͉ Id proteins ͉ IL-7 receptor no reduction in the numbers of CLP being present in IL-7 knockout animals (21–24). In IL-7R␣Ϫ or IL-7-deficient mice, he earliest steps in the development of antibody-secreting B E2A proteins are expressed at wild-type levels and yet Ebf1 and Tlymphocytes from common lymphoid progenitor cells (CLP) Pax5 are absent, which suggests that other factors participate in in adult mouse bone marrow are orchestrated by the concerted Ebf1 induction in addition to E2A, that E2A requires posttrans- activities of a number of B-lineage transcription factors and lational modification to activate Ebf1 expression, or that inhib- signals from growth factor and cytokine receptors. Gene knock- itory proteins suppress the ability of E2A to activate Ebf1 out experiments in mice have shown the absolute requirement expression in this context (23–25). The activation of Ebf1 seems for the transcription factors PU.1 and Ikaros in the generation to be the pivotal event in B cell fate specification in that Ebf1 can of CLP and the requirement for E2A and Ebf1 in the generation surprisingly rescue the B cell developmental program when overexpressed in the context of multiple gene knockout back- of the first specified B cell progenitors referred to as prepro-B grounds that completely lack B-lineage cells including PU.1 (8), cells (1–3). Prepro-B cells can be prospectively identified in ϩ Ϫ Ϫ Ϫ E2A (26), IL-7R␣ (23), and IL-7 (24). murine bone marrow as B220 CD19 IgM NK1.1 cells that To clarify the complex regulatory circuitry between E2A, either lack or have just initiated rearrangement of the D -J H H Ebf1, and Pax5 in the specification and commitment of the B cell gene segments of the Ig heavy chain (IgH) locus. These cells ϩ ϩ Ϫ Ϫ lineage, we have performed in vivo genetic complementation developmentally progress to B220 CD19 IgM NK1.1 pro- assays to address why Ebf1, but not E47, is sufficient to rescue genitor B (pro-B/pre-B) cells upon induction of the B cell the B cell developmental block in IL-7R␣-knockout animals. commitment factor, Pax5, which functions to maintain B cell Ϫ Ϫ Analysis of animals transplanted with IL-7R␣ / cells expressing identity by both positively regulating early B-lineage-associated either a control retroviral vector or vectors expressing IL-7R␣, gene expression and negatively regulating a large number of E47, Ebf1, or Pax5 showed that B cell development was rescued genes that promote development along alternative hematopoi- by Ebf1 in all cases and in 7/11 animals by Pax5. Expression of etic cell fates (4–7). B cell development is completely blocked at E47 did not activate Ebf1 mRNA but rather led to increased the pro-B cell stage in the absence of Pax5. expression of the E2A-inhibitory proteins Id2 and Id3. Expres- A number of studies have suggested that a complex hierar- chical relationship exists between E2A, Ebf1, and Pax5 in early B cell development (8). The E2A gene encodes two alternative Author contributions: M.A.T., T.L.C., and C.A.K. designed research; M.A.T., T.L.C., T.H., and mRNA splicing variants, E47 and E12, which can homo- or H.G.K. performed research; H.G. and J.H. contributed reagents and advice; M.A.T, T.L.C., heterodimerize via their helix–loop–helix domains (9). Both and C.A.K. analyzed data; and M.A.T. and C.A.K. wrote the paper. variants are able to rescue B cell development when expressed The authors declare no conflict of interest. from a pim-1 promoter/IgH enhancer transgene vector that was This article is a PNAS Direct Submission. C.M. is a guest editor invited by the Editorial Board. crossed onto an E2A knockout background (10). Observations 1To whom correspondence should be addressed. E-mail: [email protected]. showing that E2A is present (albeit at reduced levels) in Ebf1 This article contains supporting information online at www.pnas.org/cgi/content/full/ knockout animals (3) and that the Ebf1 promoter is bound by 0802550106/DCSupplemental. E47 in vivo (11, 12) and can be transactivated by overexpression © 2009 by The National Academy of Sciences of the USA 552–557 ͉ PNAS ͉ January 13, 2009 ͉ vol. 106 ͉ no. 2 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802550106 Downloaded by guest on October 3, 2021 sion of Ebf1 resulted in near complete inactivation of both Id2 A IL-7Rα and Id3 in prepro-B cells. This suggests that one major function E47 of Ebf1 in B-lineage specification is to down-regulate Id levels to Ebf1 allow for subsequent activation of E2A protein activity. Collec- Pax5 tively, these results provide important new insights into the significance of Ebf1 induction as the defining molecular event LTR IRES GFP LTR that determines B-lineage cell fate. Results B 0.07 Ebf1 and Pax5 Can Rescue B Cell Development in IL-7R␣-Knockout Mice. To characterize the mechanism by which Ebf1 can specify GFP 31.2 the B cell fate, we first transduced IL-7R␣Ϫ/Ϫ bone marrow cells 1.3 with retroviruses that co-expressed either E47, Ebf1, Pax5, or the IL-7R␣ chain along with a blue-excited green fluorescent pro- 0.2 tein variant (Bex, hereafter referred to as GFP) (Fig. 1A). E47 34.2 Transduced cells were then transplanted into lethally irradiated 1.4 C57BL/6 recipient mice and analyzed for reconstitution begin- 7.6 ning at 3 weeks post-transplantation (PT). As previously noted 14 (23), B cell development in IL-7R␣Ϫ/Ϫ animals is blocked at the ϩ Ϫ Ϫ Ϫ IL-7Rα prepro-B cell stage (B220 CD19 NK1.1 IgM ), which was 29.6 confirmed by analysis of GFPϩ control cells in bone marrow of 4.5 ϭ animals reconstituted at 6 weeks PT (n 15) (Fig. 1B). 2.3 Expression of E47 failed to promote B cell development to the 2.6 pro-B/pre-B cell stage (B220ϩCD19ϩNK1.1ϪIgMϪ) even though Ebf1 ϩ 16.1 E47 mRNA levels were increased in GFP prepro-B cells by 2.4 expression of the retroviral vector (Figs. 1 B and C). Expression ␣Ϫ/Ϫ 2.0 of Ebf1 or Pax5 rescued B cell differentiation in IL-7R bone 0.8 marrow cells but failed to restore absolute numbers of pro-B or Pax5 6.4 pre-B lymphocytes due to the absence of IL-7 signaling in IgM CD19 20.1 rescued cells (Fig.