Abstract: The conserved Paf1 complex negatively regulates the expression of numerous genes, yet the mechanisms by which it represses gene expression are not well understood. In this study, we use the ARG1 gene as a model to investigate the repressive functions of the Paf1 complex in Saccharomyces cerevisiae. Our results indicate that Paf1 mediates repression of the ARG1 gene independently of the gene-specific repressor, ArgR/Mcm1. Rather, by promoting histone H2B lysine 123 ubiquitylation, Paf1 represses the ARG1 gene by negatively affecting Gcn4 occupancy at the promoter. Consistent with this observation, Gcn5 and its acetylation sites on histone H3 are required for full ARG1 derepression in paf1? cells, and the repressive effect of Paf1 is largely maintained when the ARG1 promoter directs transcription of a heterologous coding region. Derepression of the ARG1 gene in paf1? cells is accompanied by small changes in nucleosome occupancy, although these changes are subtle in comparison to those that accompany gene activation through amino acid starvation. Additionally, conditions that stimulate ARG1 transcription, including PAF1 deletion, lead to increased antisense transcription across the ARG1 promoter. This promoter-associated antisense transcription positively correlates with ARG1 sense transcription. Finally, our results indicate that Paf1 represses other genes through mechanisms similar to those used at the ARG1 gene.