David Shore, Ph.D.
Professor
Department of Molecular Biology, Room 3067
University of Geneva, Sciences III
30, quai Ernest-Ansermet
CH-1211, Geneva 4, Switzerland
Tel: 41 22 379 61 83
Fax: 41 22 379 68 68
E-mail: David.Shore@unige.ch
Our general area of interest is the relationship between chromosome structure and the processes of gene regulation, DNA replication, repair and recombination, and chromosome segregation. We use the budding yeast Saccharomyces cerevisiae as our experimental system and employ genetic, molecular, biochemical and cell biological approaches to address specific mechanistic questions. At present, we are carrying out projects in three different areas: telomere biology, growth regulation of transcription involved in ribosomal biogenesis, and gene silencing. These diverse areas are unified by the common involvement of a remarkably multi-functional DNA-binding protein called Rap1.
Telomere replication and 'capping'
Telomeres, the extremities of eukaryotic chromosomes, carry out two essential functions: chromosome end protection ('capping') and end replication. The telomere capping function plays an important role in promoting genome stability, yet the mechanism(s) by which cells hide telomeres from DNA damage surveillance ('checkpoint') and recombination machineries is still unclear.
Telomere DNA replication requires an unusual mechanism, due to the requirement for an RNA primer by all known cellular DNA polymerases to copy the very ends of linear chromosomes. This "end replication problem" is solved in almost all eukaryotes from yeast to human by a specialized reverse transcriptase, called telomerase, that carries its own RNA template. Telomerase synthesizes the TG-rich simple repeat sequence that provides the DNA platform for its own recruitment and for assembly of the telomere cap. In budding yeast, the Rap1 protein the duplex part, of the telomeric TG repeats, and the Cdc13 protein binds to a 3’ TG-rich single-stranded extension that is a common feature of all telomeres. These two essential proteins organize the telomere cap and regulate the action of telomerase at telomeres so that the TG-repeat tracts are kept within a narrow range of lengths. We proposed the existence of a negative feedback system (often referred to as the "counting model") in which the number of telomere-bound Rap1 molecules, together with the Rap1-interacting Rif proteins, generates a tract-length dependent signal controlling telomerase action (see Fig. 1).
We are currently employing two different experimental systems to address the molecular mechanisms underlying both capping and telomere length regulation. In one system (see Fig. 2), we introduce a DNA double-strand break (DSB) in a controlled fashion at a single pre-determined site in the genome, using a galactose-regulated version of the endogenous HO endonuclease. This experimental set-up, combined with very sensitive chromatin immunoprecipiation (ChIP) assays to measure binding of telomeric factors to the DSB, has revealed a novel capping function of the Rap1 protein (see Negrini et al., 2007). The mechanism underlying this phenomenon is currently under investigation.
A second experimental system developed in our laboratory utilizes the Cre-LoxP site-specific recombination system to rapidly shorten the TG tract of a unique, engineered telomere in yeast (see Fig. 3). This system, together with the ChIP assay, allows us to test specific models to explain how the cell senses telomere length and uses this information to regulate telomerase action. A novel and unanticipated finding that emerged from these studies is that TG tract length can modulate that activity and timing of sub-telomeric origins of DNA replication (see Bianchi & Shore, 2007). Further studies have more recently provided evidence in support of a model in which TG tract length acts to modulate the association of the telomerase holoenzyme with chromosome ends (Bianchi & Shore, 2007) Genes Dev, in press). Ongoing studies are aimed at uncovering details of the mechanisms underlying these initial observations. Future studies will be aimed at extending these results to more complex eukaryotes, including humans.
Growth regulation of ribosome biogenesis
Ribosome biogenesis is the most energetically costly process involved in cell growth, and is thus tightly regulated in all organisms. This process is also unique in its requirement for the coordinated action of all three different RNA polymerase enzymes: RNA Pol II for the transcription of ribosomal protein genes, and RNA Pol I and Pol III for the transcription of ribosomal RNAs (rRNAs).
At present we are trying to understand how growth signals are integrated at the level of the ribosomal protein (RP) gene promoters. Our work has identified two key regulatory proteins, Fhl1 and Ifh1, whose combined action we propose to be crucial for the regulated activation of these genes (see Schawalder et al, 2004; Fig. 4).
Data from our laboratory and several other groups indicates that the Fhl1-Ifh1 regulators receive signals from several different growth-promoting pathways, including the highly conserved TOR and Ras-PKA pathways. Nevertheless, the mechanisms involved remain obscure and are the subject of present studies. The role of an additional DNA-binding regulator, the Sfp1 protein, is also being actively pursued. Finally, we are investigating the mechanisms underlying a recently revealed connection between the regulation of Pol I transcription of the 35S rRNA gene and control of RP gene transcription.
Gene silencing, the Sir2 deacetylase, genome stability and aging
Our laboratory has been investigating mechanisms of gene silencing in yeast since its inception in 1987. These studies are now focused on the highly conserved Sir2 deacetylase enzyme, involved in the three known forms of silencing in budding yeast that affect mating-type genes, telomeres, and the rDNA repeats. Recent work from our laboratory has uncovered a remarkable relationship between Sir2 and the control of rDNA repeat stability (see Michel et al., 2005). In brief, we found that the tandem rDNA repeats (present in ~200 copies in wild-type strains) are subject to spontaneous deletion events that can remove over half of the copies. These deletion events lead to a highly specific transcriptional down-regulation of SIR2, in an apparently auto-regulatory process that is currently under investigation. These studies may have important implications for basic mechanisms underlying aging, since the evolutionarily conserved Sir2 protein has been implicated in aging not only in yeast, but in multicellular eukaryotes as well.