Abstract: Sirtuins are conserved proteins implicated in myriad key processes including gene control, aging, cell survival, metabolism, and DNA repair. In Saccharomyces cerevisiae, the sirtuin Sir2 promotes silent chromatin formation, suppresses recombination between repeats, and inhibits senescence. We performed a genome-wide screen for factors that negatively regulate Sir activity at a reporter gene placed immediately outside a silenced region. After linkage analysis, assessment of Sir-dependency, and knockout tag verification, 40 loci were identified, including 20 of which have not been previously described to regulate Sir. In addition to chromatin-associated factors known to prevent ectopic silencing (Bdf1, SAS-I complex, Rpd3L complex, Ku), we identified the Rtt109 DNA repair-associated histone H3 lysine 56 acetyltransferase as an anti-silencing factor. Our findings indicate that Rtt109 functions independently of its proposed effectors, the Rtt101 cullin, Mms1 and Mms22, and demonstrate unexpected interplay between H3-K56 and H4-K16 acetylation. The screen also identified subunits of mediator (Soh1, Srb2, and Srb5) and mRNA metabolism factors (Kem1, Ssd1), thus raising the possibility that weak silencing affects some aspect of mRNA structure. Finally, several factors connected to metabolism were identified. These include the PAS-domain metabolic sensor kinase Psk2, the mitochondrial homocysteine detoxification enzyme Lap3, and the Fe-S cluster protein maturase Isa2. We speculate that PAS kinase may integrate metabolic signals to control sirtuin activity.