Retrotransposons are a class of highly repetitive sequences, which are very abundant in the human genome. They disperse by an RNA-based copy-and-paste mechanism, called retrotransposition. This process can drive profound genome rearrangements. Although generally silent, they are expressed in germ cells, in the early embryo, and in embryonic stem cells, which occasionally results in genetic diseases. Retrotransposons are also massively re-expressed in the large majority of cancers, but the importance and consequences of retrotransposition in human tumors have been poorly studied. Somatic retrotransposition is difficult to track in human tissue due to the highly repetitive and dispersed nature of these elements. Thus the questions we wish to address in this research proposal are the following: (i) What cellular pathways control retrotransposon copy number? This will be achieved by combining functional genomics and proteomics approaches to identify positive and negative regulators of retrotransposition in humans. (ii) What are the molecular mechanisms of retrotransposons replication? To answer this question, we will develop a cell-free assay that will contain the complete retrotransposition machinery. (iii) How retrotransposons participate in the normal and pathological remodeling of the human genome? To this purpose we are currently developing innovative approaches to track retrotransposition events in clinical samples, especially in tumor samples. Since LINE-1 elements (L1) are the most active and autonomous retroelements in our genome, we focus, at the moment, our investigations on this family. Understanding how the activity of retrotransposons is controlled will impact our knowledge of the mechanisms that lead to new genetic diseases or to cancer progression. Since mobile genetic elements are becoming important tools in insertional mutagenesis or gene-transfer technologies in mammals, our work should also help to improve their use in mammalian functional genomics.