Prep1 Directly Regulates the Intrinsic Apoptotic Pathway by Controlling Bcl-XL Levels

Prep1 transcription factor DNA independently heterodimerizes with Pbx family members (4, 8, 22), which allows its nuclear localization and activity (1, 2). Prep1 is essential for embryonic development: in particular, Prep1 null embryos die before gastrulation (L. C. Fernandez-Diaz and F. Blasi, unpublished data), while Prep1 hypomorphic mutant mouse (Prep1i/i) embryos, which express 2 to 3% of Prep1 mRNA and up to 10% of the protein, show a leaky embryonic-lethal phenotype and defects in angiogenesis, hematopoiesis, and eye development. The molecular basis of the Prep1i/i phenotype depends, at least in part, on reduction of the Pbx protein level (13, 15, 29). Programmed cell death may be triggered by external signals mediated by specific cell surface receptors (17) or by damage or stress-generated intrinsic signals mediated by mitochondria (12). Pathway-specific caspase cascade activation, in turn, induces characteristic biochemical and morphological changes and, ultimately, cell death (27). Proteins of the Bcl-2 family are central regulators of apoptosis, with antiapoptotic (Bcl-2-like survival factors: Bcl-2, Bcl-XL, Bcl-w, and Mcl1), as well as proapoptotic, members. These, in turn, can be distinguished as follows: Bax-like death factors (Bax, Bak, and Bcl-XS) and BH3-only death factors (BimL, Bad, Bid, Noxa, Puma, and Bik) (18). Proapoptotic members of the Bcl-2 family increase the permeability of the outer mitochondrial membrane, whereas antiapoptotic members inhibit their action and maintain mitochondrial homeostasis (12). Thus, the balance of pro- and antiapoptotic proteins at the mitochondrial outer membrane determines a cell's fate (5). The p53 tumor suppressor regulates the balance between these proteins and thus controls the apoptotic destiny of a cell. Upon genotoxic stress, p53 accumulates in the nucleus (26, 32) and transcriptionally activates genes that promote apoptosis, in particular, the proapoptotic members of the Bcl-2 family, such as Bax (16, 19). The increased ratio of proapoptotic to antiapoptotic Bcl-2 proteins at the outer mitochondrial membrane favors the release of apoptogenic proteins and the activation of caspases, ultimately tipping the balance toward cell death. Here, we report that hypomorphic Prep1i/i embryos display substantial generalized apoptosis and that Prep1i/i mouse embryo fibroblasts (MEFs) have increased basal apoptosis compared to those of wild-type (WT) littermates and respond faster than the WT to intrinsic, but not extrinsic, apoptotic stimuli. Endogenous p53 mRNA and protein levels are only marginally affected, as is the genotoxic-stress-induced p53 response. However, Prep1i/i MEFs have decreased levels of endogenous Bcl-XL protein, a regulator of mitochondrial-membrane permeability (35). Transient-transfection and chromatin immunoprecipitation (ChIP) analyses showed that Bcl-x is a direct target of Prep1 and that restoring Bcl-XL levels rescues the apoptotic phenotype of Prep1i/i MEFs. Therefore, Prep1 affects apoptosis by directly modulating mitochondrial homeostasis through the control of Bcl-x gene expression.