Although Mcl-1 is a known substrate for Mule’s E3 ubiq- uitin ligase activity, only slightly more Mcl-1 protein was present in BMKO B cells than in control B cells at steady state (Fig. 9 D). To investigate whether stress-induced Mcl-1 degradation is mediated by Mule, we treated purified splenic control and BMKO B cells with etoposide and analyzed lysates of these cells by immunoblotting. Mcl-1 protein degra- dation was highly efficient in stressed control B cells but largely inhibited in stressed BMKO B cells (Fig. 9 D), indi- cating that the E3 ubiquitin ligase activity of Mule is critical for controlling Mcl-1 protein levels in response to genotoxic stress. Consistent with their impaired Mcl-1 degradation and decreased p53 accumulation and phosphorylation, BMKO B cells treated with etoposide for 4–6 h showed a two to three- fold reduction in caspase-3 and -9 cleavage (Fig. 9 E and not depicted) and were consequently resistant to etoposide- induced apoptosis (Fig. 9 F). However, BMKO B cells were just as sensitive as control B cells to apoptosis induced by dexamethasone (Fig. 9 F), which is a p53-independent stim- ulus. Thus, Mule is required for Mcl-1 degradation and proper ATM-mediated p53 responses to genotoxic stress. In summary, our results show that, in addition to target- ing Mcl-1 and p53 for polyubiquitination and degradation (Chen et al., 2005; Zhong et al., 2005), Mule is required for the regulation of ATM and its substrates. Importantly, this regulation is required to maintain cellular homeostasis under both steady-state and genotoxic stress conditions. DISCUSSION Protein ubiquitination plays key roles in the transcriptional regulation and signal transduction that control the balance between cell survival and cell death (Liu et al., 2005a). Mule has been identified as an E3 ubiquitin ligase that polyubiqui- tinates p53 and targets it for proteasomal degradation (Chen et al., 2005). In this study, we present data from examinations of Mule-deficient B cells and MEFs which indicate that Mule is essential for cellular homeostasis under both resting and stress conditions. We found that B cell development, proliferation, homeo- stasis, and immune responses were impaired in BMKO mice, and that Mule maintains B cell homeostasis at steady state by regulating p53 activity. Elevated p53 transactivation is largely responsible for the blockade of B cell development and disturbed B cell functions observed in unstressed BMKO mice, as indicated by the rescue of these phenotypes upon genetic ablation of p53. Given that Mule-deficient B cells expressed a basal low level of phospho–ATM-S1987 that was not observed in control cells, we speculate that this activated ATM may constitutively phosphorylate p53 and activate it sufficiently to at least partially account for the two to fourfold increase in p53 transactivation seen in the mutant cells. In addition to increasing apoptosis, this elevated p53 transactiva- tion could trigger cell cycle arrest. This hypothesis is sup- ported by our observation of decreased BrdU incorporation by BMKO splenic B cells compared with controls. We rec- ognize that further investigation is needed to clarify the extent 182 to which cell cycle arrest contributes to the B cell deficit caused by loss of Mule. However, the abrogation of the ab- normal increase in Puma and Noxa mRNAs in BMKO B cells upon genetic ablation of p53 argues in favor of the interpretation that p53–mediated apoptosis is responsible for the B cell defects seen in BMKO mice. Genetic ablation of Puma in BMKO mice through genetic breeding should re- solve this issue clearly. Finally, although BMKO B cells ex- hibited elevated p53 transactivation, total p53 protein levels remained below the limit of detection by immunoblot. Con- sequently, we cannot be certain that p53 protein levels were coordinately increased in these cells, although elevated p53 protein was detected in both Mule-deficient MEFs and ES cells under the same conditions. In any case, these data sug- gest that Mule may influence the transactivation and func- tions of p53 in a manner that is not exclusively dependent on p53 polyubiquitination and degradation. Modulation of p53–Mdm2 interaction is essential for normal p53 activity because genetic ablation of p53 prevents the lethal- ity of Mdm2 KO mice (Brooks and Gu, 2006). B cell–specific Mdm2 deletion prevents any B cell development in these animals (unpublished data), a more severe phenotype than that resulting from B cell–specific loss of Mule. Because both Mule and Mdm2 target p53 for polyubiquitination and degradation, it is reason- able to speculate that Mule might compensate for the loss of Mdm2 in Mdm2-deficient mice and vice versa. However, the embryonic lethality of both Mule-null and Mdm2-null mice, and the defective B cell development in B cell–specific Mule and B cell–specific Mdm2 KO mutants, suggest that Mule and Mdm2 have nonoverlapping functions in maintaining p53 levels. Thus, both Mdm2 and Mule are needed to maintain p53 protein at the appropriate low levels at steady state. Although p53 ablation rescued the defective B cell devel- opment, proliferation, and homeostasis in BMKO mice, it failed to restore normal B1a cells, CSR, or serum Ig levels. We speculate that the latter two deficits may be related to the impaired ATM activation in BMKO B cells. ATM-deficient mice show defects in B cell CSR and serum Ig levels that par- tially mirror those in BMKO mice (Xu et al., 1996; Lumsden et al., 2004). Because ATM stabilizes the DNA double-strand breaks required for Ig gene assembly (Bredemeyer et al., 2006), it is possible that some of the effects of Mule deficiency on B cell development are mediated through ATM. In addi- tion, given that siRNA knockdown of Mule leads to increased DNA repair in manner dependent on DNA polymerase  (Parsons et al., 2009), loss of Mule in B cells could quite con- ceivably lead to defects in RAG-mediated V(D)J recombina- tion. However, BMKO and control mice showed comparable percentages of intracellular Ig + cells among B220+CD43+ BM B cells (unpublished data). Consistent with this finding, the Ig -to- light chain ratio in newly formed splenic B cells is not altered in BMKO mice. These results suggested that neither RAG-mediated V(D)J recombination of the Ig H locus and its expression nor the  light chain usage are affected by loss of Mule. Thus, Mule is unlikely to be required for events associated with DNA recombination in B cells. E3 ligase Mule is essential for B cell homeostasis | Hao et al.