Interactions between retroelements and host cells: From nuclear import to chromosomal integration – NiCiTy

LTR-retrotransposons are ubiquitous in eukaryotic genomes, and important understandings on retroviral biology have been gained by their study in yeast. Therefore, we will use the Ty1 LTR-retrotransposon, as working model.
1) Our first goal is to study the influence of transcription on Ty1 integration site selection.
We will:
• Set up advanced in vitro integration systems coupling Ty1 integration to an active transcription;
• Develop innovative strategies to identify in vivo, new Ty1 co-factors involved in nuclear import and integration, and characterize their impact on integration site selection in genome-wide studies.
2) Our second goal is to determine the impact of chromosome architecture on retroviral integration, as well as the influence of retroviral insertion on 3D nuclear organization.
We will use the most modern tools to localize loci, follow them in living cells, and statistically map their position in two dimensions in the nuclear space, in order to:
• study the localization of distinct Ty1 genomic loci in the nuclear space;
• compare the position of different loci before and after Ty1 integration.
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3) Our third goal is to decipher the contribution of the Spindle Pole Body (the yeast centrosome) and the microtubule network in Ty1 replication.

We will:
• analyze the localization of Ty1 and SPB proteins by immunofluorescence microscopy and at super resolution;
• determine whether a fully functional SPB is required for Ty1 retrotransposition, using genetic and genomic approaches.

The Ty1 retrotransposon of S. cerevisiae integrates upstream of RNA polymerase III (Pol III)-transcribed genes, yet the primary determinant of target specificity has remained elusive. We have identified an interaction between Ty1 IN and the AC40 subunit of Pol III, which is essential for Ty1 targeting to Pol III promoters. Lack of an integrase-AC40 interaction dramatically alters target site choice, leading to a redistribution of Ty1 insertions in the genome, mainly to chromosome ends.
Understanding the molecular mechanism of targeted integration has been a Holy Grail of mobile genetic element research for decades. Indeed, the integration sites of mobile genetic elements have profound influence on genome stability, expression and evolution. The problem is also important as it relates to both retroviruses and genomic retrotransposons as well. Although retroviruses were initially thought to integrate into euchromatic regions relatively randomly, it is fairly clear now that this is not the case. Therefore, our work on the characterization of Ty1 IN integration targeting mechanism has been a breakthrough in the field.
We have constructed several expressing vectors suitable for IN complexes purifications. However in our attempt to purify Ty1 IN and its cofactors, we discovered that the protein is largely insoluble. Therefore we decided to mainly focus on the proteomic approaches after in vivo crosslink that could be performed under denaturing conditions. Concerning the modified TAp-tag strategy, we have now developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link. We are currently adapting the technique to identify the proteins associated with Ty1 IN. On the other hand, all the tools required for the capture of a plasmid and its associated proteins have been constructed. We are also developing a novel strategy to label Ty loci. On the other hand, we have developed analytical tools allowing for dynamic behavior of labeled loci.

The outcome of this project will be to:
• identify new Ty1 cellular partners;
• decipher at the molecular level the connection between the transcriptional machinery and the preintegration complex, and its role in the integration process;
• determine the impact of the chromosomes spatial organization on the integration process;
• get insight into the contribution of the SPB in Ty1 lifecycle.

Furthermore, this project should open new directions to explore the interplay between cellular machineries and mammal retroviruses, which should have valuable implications in clinical therapy for the treatment of retroviral infections and for the development of novel retroviruses-based vectors for gene therapy.

A) PUBLICATIONS
1) Bridier-Nahmias A., Tchalikian-Cosson A., Baller J., Menouni R., Fayol H., Flores A., Saïb A., Werner M., Voytas DF and Lesage P. (2015) An RNA polymerase III subunit determines sites of retrotransposon integration. Science. 2015 May 1;348(6234):585-8.
2) Curcio MJ., Lutz S. & Lesage P. (2015). The Ty1 LTR- retrotransposon of budding yeast, Saccharomyces?Microbiol Spectr. 2015 Apr;3(2).1-35.
3) Nguyen NT, Saguez C, Conesa C, Lefebvre O, Acker J. Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification. Gene. 2015 Feb 1;556(1):51-60.
4) Spichal, M., S. Herbert, A. Cournac, C. Zimmer, R. Koszul, and E. Fabre. (2015). Evidence for actin dual roles in regulating chromosome organization and dynamics in yeast J. Cell Science (revision).
B) COMMUNICATIONS
2014
1) Keystone meeting Mobile DNA, Santa Fe, USA. An essential role of Pol III subunit AC40 in Ty1 integration targeting, P. Lesage (oral) ?
2) 5th International Conference on Retroviral Integration, Asilomar, USA. An essential role for an RNA polymerase III subunit in determining sites of Ty1 retrotransposon integration. P. Lesage (oral) ?
3) FEBS-EMBO 2014 joint conference for life science. An essential role of Pol III subunit AC40 in Ty1 integration targeting, A. Bridier-Nahmias and P. Lesage (Poster)
4) Levures Modèles outils LMO11, Bordeaux: An essential role of Pol III subunit AC40 in Ty1 integration targeting. A. Bridier-Nahmias (oral)?
5) GDR Les Eléments Génétiques Mobiles : du mécanisme aux populations, une approche intégrative. Paris. An essential role of Pol III subunit AC40 in Ty1 integration targeting. A. Bridier-Nahmias (oral).

2015
1) Colloque des 3R. Presqu’ile de Giens. An RNA polymerase III subunit determines sites of retrotransposon integration, A. Bridier-Nahmias (oral).

SCIENTIFIC BACKGROUND AND OBJECTIVES
Retroelements (retroviruses and long terminal repeats (LTR)-retrotransposons) replicate by reverse transcription of their RNA genome into a cDNA that is next integrated into host-cell chromosomal DNA, by the retroelement-encoded integrase. To accomplish a productive replication, the cDNA must traffic through the cytoplasm, cross the nuclear membrane barrier with the integrase, and access a site of integration that allows active transcription of the viral genes. Whereas important advances have been made on the traffic of viral particles within the cytoplasm, the events occurring at the interface between the cytoplasm and the nucleus, until their integration into the host DNA remain largely unknown. This question is fundamental since cDNA integration is an essential step to achieve productive infection and long-term viral persistence.
The goal of this project is to address the fundamental question of the interplay between the replication of retroelements and the nuclear architecture, from viral nuclear import to its genomic integration.

DESCRIPTION OF THE PROJECT/METHODOLOGY
LTR-retrotransposons are ubiquitous in eukaryotic genomes, and important understandings on retroviral biology have been gained by their study in yeast. Therefore, we will use yeast Ty1 LTR-retrotransposon, as working model.
Integration does not occur randomly throughout the host-cell genome in vivo, revealing a retroelement-specific pattern of preferred sites, which depends in part on transcription determinants.
Our first goal is to study the influence of transcription on Ty1 integration site selection.
We will:
- Set up advanced in vitro integration systems coupling Ty1 integration to an active transcription;
- Develop innovative strategies to identify in vivo, new Ty1 co-factors involved in nuclear import and integration, and characterize their impact on integration site selection in genome-wide studies.
In the nucleus, chromosomes and individual loci display dynamic but non-random spatial positional preferences, which appear to influence biological process linked to DNA metabolism.
Our second goal is to determine the impact of chromosome architecture on retroviral integration, as well as the influence of retroviral insertion on 3D nuclear organization.
We will use the most modern tools to localize loci, follow them in living cells, and statistically map their position in two dimensions in the nuclear space, in order to:
- study the localization of distinct Ty1 genomic loci in the nuclear space;
- compare the position of different loci before and after Ty1 integration.
On their nuclear import journey, cDNA and viral proteins of several retroviruses accumulate at the centrosome but the nature of events occurring at this organelle and their impact on nuclear import and integration is unknown.
Our third goal is to decipher the contribution of the Spindle Pole Body (the yeast centrosome) and the microtubule network in Ty1 replication.
We will:
- analyze the localization of Ty1 and SPB proteins by immunofluorescence microscopy and at super resolution;
- determine whether a fully functional SPB is required for Ty1 retrotransposition, using genetic and genomic approaches.

EXPECTED RESULTS
The outcome of this project will be to:
- identify new Ty1 cellular partners;
- decipher at the molecular level the connection between the transcriptional machinery and the preintegration complex, and its role in the integration process;
- determine the impact of the chromosomes spatial organization on the integration process;
- get insight into the contribution of the SPB in Ty1 lifecycle.

Furthermore, this project should open new directions to explore the interplay between cellular machineries and mammal retroviruses, which should have valuable implications in clinical therapy for the treatment of retroviral infections and for the development of novel retroviruses-based vectors for gene therapy.