Given that Mule has multiple substrates, it is not surpris- ing that Mule has different regulatory effects on different cell types. In neurons, Mule controls differentiation and prolifer- ation in vivo by destabilizing its substrate N-Myc (Zhao et al., 2008). In our study, we have shown that Mule regulates B cell development, proliferation, and homeostasis through the ATM–p53 axis. Although Mule was previously identified as an ATM substrate in screening studies, the biological impli- cations of Mule–ATM interaction were not explored (Matsuoka et al., 2007; Mu et al., 2007). Our study has unearthed the intriguing possibility that Mule plays opposite roles in ATM phosphorylation depending on whether the cell is at steady state or has experienced DNA damage. We theorize that, under steady-state conditions, Mule is required to suppress ATM phosphorylation, thereby preventing excessive activa- tion of p53 that could disrupt cellular homeostasis. However, during responses to genotoxic stress, Mule protein quickly accumulates and binds to phospho-ATM. The importance of this Mule–ATM interaction is highlighted by the im- paired ATM accumulation and phosphorylation observed in both Mule-deficient B cells and MEFs treated with DNA- damaging agents. Without Mule, neither of the ATM sub- strates phospho-p53 nor Brca1 can be efficiently induced. Thus, Mule is directly or indirectly involved in the ATM- mediated response to DNA damage. In support of this hy- pothesis, we have found that the numbers of ATM foci and 53BP1 foci induced by DNA damage are greatly reduced in Mule-deficient MEFs compared with controls (unpublished data). Whether Mule itself participates in the DNA damage response through its interaction with ATM, or whether it is Mule’s ubiquitination of its known substrates (such as his- tones [Liu et al., 2005b], TopBP1 [Herold et al., 2008], and/or DNA polymerase [Parsons et al., 2009]) that is im- portant, remains to be determined. In conclusion, we have demonstrated crucial functions for Mule in regulating the ATM–p53 axis under both steady- state and genotoxic stress conditions. Disruption of Mule function in various cell types leads to developmental and functional deficits that implicate this E3 ubiquitin ligase as a key player in a new molecular network required for the maintenance of cellular homeostasis. MATERIALS AND METHODS Mice. Mule floxed (Mulefl/fl (y)) mice were generated using Cre/LoxP recom- bination and the gene targeting and screening strategies outlined in Fig. 1. The experimental procedures used for the culture, transfection, and selection of E14K ES cells (129/Ola) have been previously described (Hao and Rajewsky, 2001). Chimeric mice were crossed to Flpe-deleter mice (Rodríguez et al., 2000) to generate Mulefl/fl (y) mice. To generate BMKO mice (Mule deleted specifically in B cells), Mulefl/fl (y) mice were bred with Mb1Cre mice (C57BL/6; Hobeika et al., 2006). The resulting Mulefl/fl (y)Mb1Cre mice were backcrossed for 6–10 generations to C57BL/6. p53 was deleted in Mulefl/fl (y)Mb1Cre mice by crossing to P53fl/fl mice (Jonkers et al., 2001). The resulting Mulefl/+ (y)P53fl/+ F1 mice, with or without Mb1Cre, were intercrossed to generate Mulefl/fl (y)P53fl/fl Mb1Cre mice (BDKO mice; mixed genetic background). Because Cre expression can be toxic to cells (Schmidt-Supprian and Rajewsky, 2007), we included Mulefl/+ Mb1Cre, P53fl/+ Mb1Cre and Mulefl/+ P53fl/+ Mb1Cre mice in all initial JEM Vol. 209, No. 1 A r t i c l e analyses of the corresponding mutants. These control mice were phenotypi- cally indistinguishable from the control Mulefl/fl (y) animals used in this study, as judged by cellularity and size of spleen. Mice used in experiments were ≥2 mo old unless otherwise specified. All animal experiments were approved by the University Health Network Animal Care Committee. Immunoblotting and immunoprecipitation. Cells were lysed in 0.5% NP-40 lysis buffer (20 mM Tris-HCl, pH 8, 137 mM NaCl, 10% glycerol, 0.5% NP-40, and 2.5 mM EDTA), or RIPA buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.5% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS), with protease inhibitor and phosphatase inhibitor (Roche) freshly added. Lysates were incubated on ice for 30 min and cleared by centrifugation. Pro- tein concentrations in lysates were determined using the BCA protein assay (Thermo Fisher Scientific). Cell lysates (20–100 µg) were loaded onto 4–12% bis-Tris gels to detect proteins below 200 kD, or onto 3–8% Tris- acetate gels to detect Mule. Fractionated proteins were transferred to a nitro- cellulose membrane by i-Blot according to the manufacturer’s instructions (Invitrogen). The membranes were immunoblotted using Abs recognizing the following: ATM (GeneTex); Ser1987-ATM (Rockland); Brca1 (Santa Cruz Biotechnology, Inc.); p53 (Santa Cruz Biotechnology, Inc.); Ser18- p53 (R&D Systems); and phospho-tyrosine (Millipore). Infrared dye-labeled secondary Abs (anti–rabbit Alexa Fluor 680 [Invitrogen] and anti–mouse IR800 [LI-COR Biosciences]) were visualized with an Odyssey scanner (LI-COR Biosciences). For immunoprecipitations, cell lysates prepared from 108 MEFs were incubated with anti-Mule Ab and protein G–Sepharose beads at 4°C over- night. Immunocomplexes were washed five times with lysis buffer, followed by standard immunoblotting analysis as described in the previous paragraph. Flow cytometry. Single cell suspensions were prepared from spleen or BM preparations that had been treated to lyse red blood cells. Cells (1–2 × 106) were preincubated with Fc block for 15 min at 4°C and immunostained with Abs recognizing the following: IgM (II/41), IgD (11-26c.2a), IgG1 (X56), IgG3 (R40-82), Ig  light chain (187.1), Ig  light chain (R26-46), CD5 (53–7.3), CD9 (KMC8), CD16/CD32 (2.4G2), CD21 (7G6), CD23 (B3B4), CD24 (M1/69), CD25 (PC61), CD38 (90), CD43 (S7), CD45R/ B220 (RA3-6B2), CD69 (H1.2F3), CD86 (GL1), CD93 (AA4.1), or CD117/ cKit (2B8; all obtained from BD or eBioscience unless otherwise specified). A mouse mAb against bacterially expressed Mule (aa 2375–2491) was gener- ated by ProMab and used to detect Mule protein in immunoblotting and in- tracellular flow cytometry experiments. This anti-Mule mAb recognizes a specific 480-kD band in mouse cell extracts, as confirmed in Mule-deficient MEFs. Fluorescein-labeled PNA (peanut agglutinin) was used to detect GCs (Vector Laboratories). Flow cytometry data were acquired using either a FACSCalibur (BD) or FACSCanto (BD) flow cytometer, and analyzed with either the CellQuest software (BD) or the FlowJo analysis program (Tree Star). Intracellular staining of Mule was performed as described previously (Hao et al., 2008). In brief, BM cells were fixed with 1.6% paraformaldehyde and incubated for 30 min at RT. After one wash in PBS, ice-cold methanol (100%) was added dropwise and cells were incubated on ice for 30 min. Washed cells were incubated for 30 min with Abs recognizing B220 (RA3- 6B2), CD43 (S7), IgM (II/4.1), or Mule. Immunostained cells were incubated with Alexa Fluor 488– or Alexa Fluor 647–conjugated rabbit anti–mouse IgG and analyzed by flow cytometry. For intracellular Ig  staining, BM cells were first surface stained with the following fluorescent Abs recognizing B220, IgM, CD43, or CD25. B220+IgMCD43+CD25 cells were sorted by flow cytometry, fixed, and permeabilized using the Cytofix/Cytoperm kit according to the manufac- turer’s instructions (BD). Fixed cells were stained with FITC–anti-IgM (II/4.1) and analyzed by flow cytometry. For BrdU staining, mice were supplied with BrdU-containing drink- ing water (1 mg/ml) for 3.5 d. Total splenic B cells were isolated and immunostained as described in the previous paragraph, and sorted into TS (B220+IgM+CD93+), MZ (B220+IgM+CD21+CD23low), and FO 183