P53 Family Members Modulate the Expression of PRODH, but Not PRODH2, via Intronic p53 Response Elements

Klaus Roemer,Ivan Raimondi,Yari Ciribilli,Alessandra Bisio,Paola Monti,Alberto Inga,Gilberto Fronza,Paola Campomenosi,Loredano Pollegioni

doi:10.1371/journal.pone.0069152

Abstract

The tumor suppressor p53 was previously shown to markedly up-regulate the expression of the PRODH gene, encoding the proline dehydrogenase (PRODH) enzyme, which catalyzes the first step in proline degradation. Also PRODH2, which degrades 4-hydroxy-L-proline, a product of protein (e.g. collagen) catabolism, was recently described as a p53 target. Here, we confirmed p53-dependent induction of endogenous PRODH in response to genotoxic damage in cell lines of different histological origin. We established that over-expression of TAp73β or TAp63β is sufficient to induce PRODH expression in p53-null cells and that PRODH expression parallels the modulation of endogenous p73 by genotoxic drugs in several cell lines. The p53, p63, and p73-dependent transcriptional activation was linked to specific intronic response elements (REs), among those predicted by bioinformatics tools and experimentally validated by a yeast-based transactivation assay. p53 occupancy measurements were validated in HCT116 and MCF7 human cell lines. Conversely, PRODH2 was not responsive to p63 nor p73 and, at best, could be considered a weak p53 target. In fact, minimal levels of PRODH2 transcript induction by genotoxic stress was observed exclusively in one of four p53 wild-type cell lines tested. Consistently, all predicted p53 REs in PRODH2 were poor matches to the p53 RE consensus and showed very weak responsiveness, only to p53, in the functional assay. Taken together, our results highlight that PRODH, but not PRODH2, expression is under the control of p53 family members, specifically p53 and p73. This supports a deeper link between proteins of the p53-family and metabolic pathways, as PRODH modulates the balance of proline and glutamate levels and those of their derivative alpha-keto-glutarate (α-KG) under normal and pathological (tumor) conditions.

Highlights

The p53 protein exerts its tumour suppressive function acting primarily as a transcription factor, that controls the expression of a large and ever increasing number of target genes in response to a variety of stresses [1,2,3]
The identification of putative p53 response elements (REs) in the proline dehydrogenase (PRODH) and PRODH2 genes and the experimental analysis of their functionality are described. These genes have been previously described as p53 targets
Both proteins are capable of inducing apoptosis via ROS production [16,19]

Summary

Introduction

The p53 protein exerts its tumour suppressive function acting primarily as a transcription factor, that controls the expression of a large and ever increasing number of target genes in response to a variety of stresses [1,2,3]. P53 represents the founder member of the p53 family, to which p63 and p73 belong. These proteins share the highest level of homology in the DNA binding domain and often recognize the same REs in the promoter of target genes. There is a complex transactivation network within the p53 family and between the p53 family and other transcription factors in the regulation of target genes [10,11]. Noncanonical binding sites have been identified comprising halfsites and three quarter sites, expanding the universe of potential downstream target genes which may be regulated by p53 [13]

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