RENT complex

Abstract: The MET3 promoter (MET3pr) inserted into the silenced chromosome in budding yeast can overcome Sir2-dependent silencing upon induction and activate transcription in every single cell among a population. Despite the fact that MET3pr is turned on in all the cells, its activity still shows very high cell-to-cell variability. To understand the nature of such "gene expression noise," we followed the dynamics of the MET3pr-GFP expression inserted into ribosomal DNA (rDNA) using time-lapse microscopy. We found that the noisy "on" state is comprised of multiple substable states with discrete expression levels. These intermediate states stochastically transition between each other, with "up" transitions among different activated states occurring exclusively near the mitotic exit and "down" transitions occurring throughout the rest of the cell cycle. Such cell cycle dependence likely reflects the dynamic activity of the rDNA-specific RENT complex, as MET3pr-GFP expression in a telomeric locus does not have the same cell cycle dependence. The MET3pr-GFP expression in rDNA is highly correlated in mother and daughter cells after cell division, indicating that the silenced state in the mother cell is inherited in daughter cells. These states are disrupted by a brief repression and reset upon a second activation. Potential mechanisms behind these observations are further discussed.

Abstract: Protein sumoylation is a reversible posttranslational modification that controls multiple processes during cell cycle progression. Frequently, SUMO synergistically targets various subunits in a protein complex to modulate its function, leading to what has been defined as protein group sumoylation. Different subunits in the RENT (regulator of nucleolar silencing and telophase) complex, including Net1, Sir2, and Cdc14, can be coupled to SUMO, making it difficult to ascertain the role of this modification. Here we describe a method to downregulate sumoylation in RENT, consisting in the fusion of a catalytic domain of the Ulp1 SUMO protease (Ulp Domain; UD) to the C-terminus of members in the complex using epitope tags as linkers. Targeting of the UD to specific loci can be simplified by transformation of PCR-amplified cassettes. The presence of the UD in the complex allows the concurrent downregulation of sumoylated species in the RENT complex, what can be easily monitored by pull-down of SUMO conjugates. This methodology can be applied to other protein complexes exhibiting group sumoylation.

Abstract: Gene silencing in the budding yeast Saccharomyces cerevisiae requires the enzymatic activity of the Sir2 protein, a highly conserved NAD-dependent deacetylase. In order to study the activity of native Sir2, we purified and characterized two budding yeast Sir2 complexes: the Sir2/Sir4 complex, which mediates silencing at mating-type loci and at telomeres, and the RENT complex, which mediates silencing at the ribosomal DNA repeats. Analyses of the protein compositions of these complexes confirmed previously described interactions. We show that the assembly of Sir2 into native silencing complexes does not alter its selectivity for acetylated substrates, nor does it allow the deacetylation of nucleosomal histones. The inability of Sir2 complexes to deacetylate nucleosomes suggests that additional factors influence Sir2 activity in vivo. In contrast, Sir2 complexes show significant enhancement in their affinities for acetylated substrates and their sensitivities to the physiological inhibitor nicotinamide relative to recombinant Sir2. Reconstitution experiments showed that, for the Sir2/Sir4 complex, these differences stem from the physical interaction of Sir2 with Sir4. Finally, we provide evidence that the different nicotinamide sensitivities of Sir2/Sir4 and RENT in vitro could contribute to locus-specific differences in how Sir2 activity is regulated in vivo.