conserved such that the human and mouse forms share >90% amino acid identity. From a functional viewpoint, the fact that Mule ubiquitinates p53 and other apoptotic molecules suggests that Mule may contribute to the maintenance of cellular homeostasis by preventing inappropriate cell death. In this study, we examined the consequences of Mule deficiency in mouse B cells and mouse embryonic fibroblasts (MEFs). Our analyses reveal a novel mechanism by which Mule acts through the ATM–p53 axis to regulate cellular homeostasis under both steady-state and genotoxic stress conditions. RESULTS Generation of conditional Mule KO mice We generated a conditional Mule allele (Mulefl ) in which exons 76 and 77 (encoding the HECT domain) were flanked by loxP sites (Fig. 1). Mulefl/y male mice were created using Flpe-deleter mice (Rodríguez et al., 2000) and bred with DeleterCre females (Schwenk et al., 1995) to produce Muledel/+ females. According to Mendelian principles, 50% of the male progeny from a cross between a WT male and a Muledel/+ female should be Muledel/y (Mule KO) mice. However, only Mule+/y males were born alive, implying that Muledel/y mice die before birth (unpublished data). Analysis of Muledel/y embryos at embryonic day 9.5 (E9.5) and 10.5 revealed various developmental anomalies, including enlarged heart, reduced somites, and greatly decreased body size JEM Vol. 209, No. 1 A r t i c l e Figure 2. Blockade of B cell development and impaired homeostasis in BMKO mice. (A) Southern blot of lysates of thymocytes (Thy) and splenic B cells from Mulefl/fl(y)Mb1Cre (BMKO) mice. The floxed (8 kb) and deleted (6.9 kb) Mule alleles are indicated. (B, left) Immunoblot of purified splenic B cells from control (Mulefl/fl(y)) and BMKO mice detecting Mule pro- tein. -Tub, loading control. (B, right) Mule expression in con- trol and BMKO pro– and pre–B cells as determined by flow cytometry. (C, left) Flow cytometric analysis of BM from litter- mate control and BMKO mice showing the pro–B cell (B220lowIgMcKit+; top) and pre–B cell (B220lowIgMCD25+; bottom) compartments. Numbers are the percentage of the gated IgM population. (C, right) Absolute numbers of pro–B cells and pre–B cells in BM isolated from both hind leg femurs (mean ± SD; *, P = 0.006). (D, top) Percentage of apoptotic pre–B cells as determined by flow cytometric analysis of Annexin V expression. (D, bottom) Apoptotic pre–B cells (mean ± SD; *, P = 0.01). Data are from a single experiment involving four to five mice per genotype. (E and F) Flow cytometric analysis of B cell populations in littermate control and BMKO mice. (E, top) Percentage of B220+IgM+ B cells among gated lymphocytes in spleen. (E, bottom) Percentage of CD5+B220+ B1a cells among gated lymphocytes in the peritoneal cavity. Numbers are the percentage among total lymphocytes. (F, left) Splenic B cell cellularity (mean ± SD; ***, P = 1.125). (F, right) Percent- age of B1a cells in peritoneum (mean ± SD; **, P = 0.0019). Results are representative of two to three (A–C) and three to four (E and F) independent experiments involving one to four mice per genotype. compared with controls (unpublished data). The mutant embryos were dead by E12.5, indicating that Mule is essential for mouse embryogenesis. Loss of Mule impairs B cell development and homeostasis We chose to characterize the effects of Mule deletion in B lymphocytes in adult mice because these cells are known to accumulate p53 and succumb to apoptosis under conditions of genotoxic stress (Murray-Zmijewski et al., 2008). Mulefl/y (male) and Mulefl/fl (female) mice showed normal B lympho- cyte development and subset distribution as determined by flow cytometry (unpublished data). To generate mutants in which Mule was selectively deleted in B cells (B cell–specific Mule KO [BMKO] mice), Mulefl/fl (y) mice (male or female) were crossed to Mb1Cre mice in which Cre recombinase is controlled by the B cell–specific Mb1 promoter (Hobeika et al., 2006). Southern blotting of DNA from purified splenic B cells of BMKO mice confirmed efficient Cre-mediated dis- ruption of Mule in B cells but not in thymocytes (Fig. 2 A). Immunoblotting demonstrated the absence of Mule protein in purified splenic BMKO B cells (Fig. 2 B, left). Flow cytometric analysis revealed that Mule was also deleted in a large fraction of BMKO pro–B cells and pre–B cells (Fig. 2 B, right). The main stages of B cell development in the BM are: pro–B (B220lowIgMcKit+), pre–B (B220lowIgMCD25+), immature B (B220lowIgM+), and recirculating mature B (B220highIgM+). Al- though the pro–B cell compartment of BMKO BM was normal, 175