research

Perp, a p53/p63 target gene | p53 targets in apoptosis | p53 knock-in mice

Specific projects in the Attardi Lab include:

Analysis of Perp, an apoptosis and adhesion-associated p53/p63 target gene

In a screen to identify genes activated during p53-mediated apoptosis but not G1 arrest, we previously isolated a novel gene known as Perp (P53 apoptosis Effector Related to PMP-22). Perp induction correlates with p53-dependent apoptosis and not p53-independent cell death, and Perp overexpression can induce cell death in p53-deficient MEFs. We subsequently demonstrated that the requirement for Perp in p53-dependent apoptosis is dependent on the cellular context - while the absence of Perp partially compromises apoptosis in immature thymocytes and neurons, Perp is dispensable for apoptosis in E1A-MEFs. In addition to its role in apoptosis, analysis of the Perp knockout mouse revealed a second unexpected role for this protein in cell-cell adhesion. In the absence of DNA damage, Perp is expressed in the developing and mature stratified epithelia, and endogenous Perp protein localizes to the desmosome, a multiprotein complex required for normal cell-cell adhesion in these tissues. Perp null mice die within the first week of life, as a result of severe adhesion defects and blistering in the skin and oral mucosa, and exhibit highly abnormal desmosomes by electron microscopy. This essential role in adhesion appears to be p53-independent; rather, developmental expression of Perp is regulated by the related transcription factor p63, a master regulator of epithelial development.

The dual roles of Perp in adhesion and apoptosis argue strongly that this protein may be affected in diverse human diseases. Mutations in p63 underlie multiple human developmental disorders affecting the stratified epithelia. In addition, every identified desmosomal protein has been shown to be affected in one or more human blistering diseases affecting the skin, hair, or oral mucosa. Perp may therefore be mutated in blistering diseases of unknown genetic origin. Finally, emerging work from other labs on the roles of p53 family members p63 and p73 in tumor suppression suggests that loss of p63 may affect tumor spectrum and metastasis. Perp, as a gene that can be transactivated by p53 and p63 to affect both adhesion and apoptosis, may be an important part of the pathways preventing tumor progression and invasion.

We are currently characterizing Perp and its encoded protein using genetic, cell biological, and biochemical techniques. We are constructing a Perp conditional knockout mouse to further define the function of Perp in development and tumorigenesis. We are also dissecting the mechanisms by which Perp induces cell death and cell adhesion. Perp is a multipass transmembrane protein that could act either as a signalling molecule or as an ion channel. The generation and analysis of Perp mutants will be helpful for determining how Perp exerts its effects on known steps of the apoptotic process, thus defining a link between p53 and the execution of apoptosis.

Characterization of additional targets of p53 in apoptosis

Another research direction will to be to identify and characterize other components of the p53-activated cell death pathway. A comprehensive search for additional p53 apoptosis-specific target genes will be carried out by the hybridization of G1-arrested and apoptotic MEF RNA populations to DNA microarrays. Genes upregulated in apoptotic cells will be characterized similarly to Perp, first in vitro, by cell biological and biochemical approaches to identify those that are most interesting, and then ultimately in vivo by gene targeting approaches.

Generation of p53 knock-in mice and cells

A complete understanding of p53 relies on studying its function in vivo, where its activity in tumor suppression can be analyzed. To test the role of specific biochemically-defined activities of p53 in tumor suppression, we are constructing "knock-in" mouse strains in which the wild-type p53 gene is replaced with particular p53 mutant alleles. Besides examining the ability of these alleles to act as tumor suppressor genes in vivo, cells from these mice can be studied for their properties in classical apoptosis and transformation assays. Testing p53 function in this way, by expressing it at physiological levels, should provide definitive information about its mechanism of action, both as a tumor suppressor and as an inducer of apoptosis.

One area of particular interest is in understanding the role of p53-dependent transcriptional activation in its biological function in vivo. To address this question, we have generated mouse strains carrying mutations in the p53 transactivation domain.One of these mouse strains was engineered with two mutant residues in the transactivation domain, at codons 25 and 26, alterations that had been shown previously to render p53 hypomorphic as a transactivator.The initial characterization of the mouse strain expressing this mutant,termed p53^25,26 (also known as p53^QS), has provided us with a greater understanding of p53 function at the cellular level.While p53^25,26 is compromised in its ability to activate the transcription of many p53 target genes, it does retain wild-type transactivation capacity on a subset of p53 targets, including the pro-apoptotic gene bax, suggesting that p53 regulates gene expression via distinct, target–specific mechanisms.In response to DNA damaging agents, this mutant is only partially active in eliciting a G1 cell cycle arrest, corroborating previous studies by our lab and others which had shown, in an in vitro context, that transactivation is critical for the arrest function of p53.Strikingly, this p53 mutant shows stress-specific apoptotic activity.In oncogene-expressing MEFs treated with DNA damage, p53^25,26 is completely defective at inducing apoptosis. However, in response to serum deprivation, this mutant retains partial apoptotic activity, and intriguingly, upon exposure to a hypoxic environment, p53^25,26 induces significant levels of apoptosis. The finding that this mutant protein has stress-specific apoptotic capacity suggests that p53 acts through distinct pathways to induce apoptosis in response to different stimuli.Currently, we are beginning to evaluate the activity of this mutant in several in vivosettings previously shown to be sensitive to p53-dependent apoptosis to determine whether it displays cell type-specific apoptotic activity. We are also in the process of evaluating the ability of p53^25,26 to suppress tumorigenesis in several models of cancer, a study that will significantly enhance our understanding of the role of transactivation by p53 in the prevention of tumor growth.