Positive intergenic feedback circuitry, involving EBF1 and FOXO1, orchestrates B-cell fate Robert Manssona,b,1,2, Eva Welindera,c,1, Josefine Åhsbergd, Yin C. Lina, Christopher Bennere, Christopher K. Glasse, Joseph S. Lucasa, Mikael Sigvardssond, and Cornelis Murrea,2 Departments of aMolecular Biology and eCellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093; bDepartment of Laboratory Medicine, Karolinska Institute, 14186 Stockholm, Sweden; cDepartment of Experimental Medical Science, Lund University, 22242 Lund, Sweden; and dDepartment of Clinical and Experimental Sciences, Linköping University, 58183 Linköping, Sweden Edited* by Michael Levine, University of California, Berkeley, CA, and approved October 24, 2012 (received for review July 6, 2012) Recent studies have identified a number of transcriptional regu- transcript abundance. On examination, we found that EBF1 lators, including E2A, early B-cell factor 1 (EBF1), FOXO1, and and FOXO1 bind to enhancer regions that interact with paired box gene 5 (PAX5), that promote early B-cell development. promoter regions corresponding to the FOXO1 and EBF1 loci, However, how this ensemble of regulators mechanistically pro- demonstrating that EBF1 and FOXO1 drive the expression of motes B-cell fate remains poorly understood. Here we demon- one another. We have generated a global regulatory network from strate that B-cell development in FOXO1-deficient mice is arrested EBF1 and FOXO1 genome-wide transcription factor occupancy and transcription signatures derived from EBF1- and FOXO1- in the common lymphoid progenitor (CLP) LY6D+ cell stage. We deficient CLPs. This analysis showed that EBF1 and FOXO1 demonstrate that this phenotype closely resembles the arrest in fi act in a positive feedback circuitry to promote and stabilize B-cell development observed in EBF1-de cient mice. Consistent B-cell fate. with these observations, we find that the transcription signatures of FOXO1- and EBF1-deficient LY6D+ progenitors are strikingly sim- Results ilar, indicating a common set of target genes. Furthermore, we + B-Lymphoid Development in FOXO1-Depleted Mice Is Arrested at the found that depletion of EBF1 expression in LY6D CLPs severely Common Lymphoid Progenitor Cell Stage. Previous studies have affects FOXO1 mRNA abundance, whereas depletion of FOXO1 established that the E2A proteins directly activate Foxo1 ex- + − activity in LY6D CLPs ablates EBF1 transcript levels. We generated pression in LY6D CLPs (8). To evaluate how activation of a global regulatory network from EBF1 and FOXO1 genome-wide Foxo1 expression relates to B-cell specification, we compared − transcription factor occupancy and transcription signatures de- Foxo1 mRNA abundance in sorted LY6D and LY6D+ CLPs. rived from EBF1- and FOXO1-deficient CLPs. This analysis reveals As expected, we observed a substantial and significant increase in that EBF1 and FOXO1 act in a positive feedback circuitry to pro- Foxo1 expression in the LY6D+ compartment, consistent with mote and stabilize specification to the B-cell lineage. the developmental stage at which B-cell specification is initiated (Fig. 1A). To explore the role for FOXO1 in B-cell progenitors in greater detail, we generated mice conditionally deleted for -cell development is orchestrated by a complex network of Foxo1 across the entire hematopoietic compartment. Mice transcription factors whose activities are modulated by a di- f B fi carrying a conditional Foxo1 (Foxo1 ) allele were bred to mice verse set of signaling modules. The rst cells in the bone marrow expressing Cre placed under Vav1-regulatory elements (15). − − (BM) primed for a lymphoid cell fate are referred to as lymphoid- FOXO1f/f Vav1-iCre mice (referred to as FOXO1 / here) mice primed multipotent progenitors (LMPPs) (1). LMPPs have the did not show gross abnormalities and exhibited normal BM capacity to differentiate into common lymphoid progenitors cellularity, whereas the number of cells in the spleen was reduced (CLPs), which in turn have the ability to give rise to all lymphoid by a factor of twofold (Fig. 1B; Fig. S1A). Notably, the number of lineages (2, 3). The CLP compartment is heterogeneous, con- + + fi CD19 B220 cells in the BM was reduced more than 50-fold, sisting of cells with different de ned lineage potentials, as well as and only a few CD19+ cells could be detected in the spleen of − − cells already committed to the B-cell fate. The CLP compart- FOXO1 / mice (Fig. 1C; Fig. S1B). The few CD19+ cells − − ment can be segregated into two distinct populations based on identified in the BM of FOXO1 / lacked expression of IgM, the expression of the cell surface marker LY6D (4, 5). During IgD, and CD25, indicating a developmental arrest at the pro- the last two decades, a subset of transcriptional regulators have + fi B-cell stage (Fig. S1C). Interestingly, the LY6D CLP com- been identi ed that specify and promote B-cell fate. Prominent partment was substantially increased in the absence of FOXO1 among those are E2A, early B-cell factor 1 (EBF1), and paired (Fig. 1D). box gene 5 (PAX5), as well as effectors acting downstream of the fi Because it is well established that FOXO1 plays a critical role IL7-signaling cascade (6). E2A-de cient mice exhibit a complete in regulating cell cycle progression and cell survival, we examined − − block before the expression of LY6D in the CLP compartment for viability and the fraction of cycling cells in FOXO1 / (16). (4, 7, 8). In the CLP compartment, the E2A proteins act in As predicted, FOXO1-ablated CLPs showed a higher percentage concert with HEB to induce the expression of forkhead box O1 of actively cycling cells and lower levels of Annexin V staining (Foxo1) (8). Although it is well established that Foxo1 is critical (Fig. S1 D and E). Although the use of Vav1-iCre should ensure for normal B-cell development, the dynamic regulation of Foxo1 expression in the CLP compartment suggested roles for Foxo1 at the earliest progenitor cell stage (5, 9–12). To determine the mechanistic impact of FOXO1 in B-lineage Author contributions: R.M., E.W., and M.S. designed research; R.M., E.W., J.Å., and J.S.L. fi fi performed research; R.M., E.W., Y.C.L., C.B., C.K.G., M.S., and C.M. analyzed data; and speci cation, we examined FOXO1-de cient mice for defects in R.M., E.W., and C.M. wrote the paper. early hematopoiesis. FOXO1-ablated BM exhibited an almost fl complete lack of CD19+ B cells and an accumulation of cells at The authors declare no con ict of interest. the LY6D+ CLP stage, resembling the phenotype observed in *This Direct Submission article had a prearranged editor. EBF1-deficient mice (12–14). Furthermore, we found that Data deposition: The data reported in this paper have been deposited in the Gene Ex- FOXO1-deficient LY6D+ CLPs lacked the expression of genes pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE41931). associated with the B-cell lineage and identified striking simi- 1R.M. and E.W. contributed equally to this work. larities in the transcription signatures derived from FOXO1- and 2To whom correspondence may be addressed. E-mail: [email protected] or robert. EBF1-ablated CLPs. Interestingly, we found that FOXO1-de- [email protected]. ficient progenitors display severely reduced Ebf1 transcript levels, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. whereas EBF1-deficient progenitors showed reduced Foxo1 1073/pnas.1211427109/-/DCSupplemental. 21028–21033 | PNAS | December 18, 2012 | vol. 109 | no. 51 www.pnas.org/cgi/doi/10.1073/pnas.1211427109 Downloaded by guest on September 26, 2021 A B C E WT FOXO1-/- FSC/SSC, Singlets, PIneg + + Foxo1 Total BM CD19 B220 105 43.8 105 65.4 105 104 104 WT 104 3 10 CLP 103 103 0 + 28.6 0 0 3 4 5 cells 01010 10 cells 0103 104 105 0103 104 105 6 6 IL7ra 105 , CD45.2 -/- LY6D neg x 10 FOXO1104 x 10 105 105 3 65.3 0.42 10 , LIN 0 neg 104 104 Relative expression (Hprt1) 0.67 0 3 104 105 3 3 -/- B220 10 -/- 10 10 WT FOXO1 WT FOXO1 BM B CLP CD19 0 0 0103 104 105 0103 104 105 D FSC/SSC, Singlets, Pineg, LINneg CLP 105 105 105 8.97 105 105 - + 57 0.31 LY6D LY6D FSC/SSC, Singlets, PI 50.2 104 104 4 CLP 35.2 WT 10 104 85.2 104 3 3 103 10 10 103 103 0 0 Spleen B 0 0 0 0103 104 105 0103 104 105 0103 104 105 0 103 104 105 0102 103 104 105 B220 cells 6 CD19 5 105 11.3 105 10 -/- -/- -/- -/- -/- 14.8 -/- 75.6 -/- FOXO1 4 4 4 x 10 F 10 10 87.5 10 -/- IL7 E2A PU.1 PAX5 3 EBF1 10 3 3 10 FOXO1 10 IKAROS 0 0 0 2 3 4 5 3 4 5 KIT 3 4 5 KIT KIT 10 FLT3 FLT3 01010 10 010 10 10 010 10 10 -/- -/- WT FOXO1 WT FOXO1 - + IL7ra SCA1 LY6D LMPP LY6D CLP LY6D CLP Pro-B − Fig. 1. FOXO1 plays an essential role in B-cell development. (A)LY6D and LY6D+ CLPs were analyzed by real-time PCR for the abundance of Foxo1 mRNA. Values were normalized to HPRT expression and shown as mean ± SEM, using cells from two independent sorts. (B) Total number of BM cells in sex- and age- − − matched WT and FOXO1 / mice. Data shown are pooled from two independent experiments. (C)(Left) Representative FACS plots of CD19+B220+ cells derived − − − − from WT and FOXO1 / BM. (Right) Total numbers of CD19+B220+ B cells isolated from WT and FOXO1 / BM. Data shown are pooled from two independent experiments.