Coevolution of expression of prion-protein and doppel genes

Abstract

Gene duplication is a major mechanism for the origin of new genes. Subsequent function divergence and acquisition of new regulatory elements is the necessary step for survival of a newly duplicated gene. However, despite of existence of several models, it remains unclear how paralogs evolve after the duplication and develop new functions. An example of evolution by gene duplication is PRNP, coding for prion protein (PrP), and its downstream duplicate, PRND, coding for doppel (Dpl). Recent studies have shown that PrP possesses neuroprotective functions while Dpl is neurotoxic and induces apoptosis. Expression of PrP and Dpl is altered in various neurodegenerative disorders, such as Alzheimer's, as well as after brain injury and during development; this suggests their possible antagonistic co-regulation by a so far unknown mechanism. The aims of the work described in this thesis are to examine the evolution of the PRNP and PRND genes using three models: frog (Xenopus laevis), marsupial opossum (Monodelphis domestica) and mouse, and to define the molecular mechanisms underlying the antagonism in the functions of their proteins. Previously unreported amphibian and marsupial PRND transcripts were cloned and characterized. Tissue-expression analysis demonstrated that in frog PRND relies primarily on regulatory DNA elements of the PRNP gene and is expressed in brain as a chimeric transcript, sharing the first two 5' UTR exons of the PRNP gene. PRND acquires its own promoter in mammals and develops new tissue specificity. Conservation of the chimeric transcript in mouse and its translation (shown by polysome binding assay) in mice embryos suggests possible functional significance in development. Transcription factors that might regulate the antagonistic relationship of PrP and Dpl were predicted by bioinformatics and comparative genomics analysis of the two genes within vertebrates. The predicted FAC1/BPTF binding site, situated next to the second non-coding exon of PRNP, was selected for experimental analysis based on the literature-deduced relationship between FAC1/BPTF, PrP, and Dpl. Reporter gene assays demonstrated upregulation of luciferase expression in constructscontaining both a Prnp conserved non-coding DNA region with the predicted FAC1/BPTF binding site and a Prnd promoter region without FAC1/BPTF binding site; the latter might be explained by an indirect effect. Binding sites for the transcription factors E2F1, CREB, AP2, USF1, and NFY cluster were predicted in the PRND proximal promoter by computational analysis. Consequently, a model of PrP/Dpl co-regulation is proposed that includes recruitment of BPTF by USF1, phosphorylation of retinoblastoma protein Rb induced by FAC1/BPTF followed by E2F1 de-repression leading to activation of the PRND promoter. Discovery and characterisation of previously undetected mouse Bptf transcript variants suggests tissue-specific modulations of this mechanism, as well as having wide implications for the functions of "BPTF" and "FAC1" not previously suspected Overall, the results presented are important for understanding of evolution by gene duplication in general. For the first time, gene development is compared across three evolutionarily distant vertebrate species: amphibians, marsupials, and mammals. The proposed mechanism for PrP and Dpl co-regulation by FAC1/BPTF provides novel insights into neuronal death.