AbstractHistone acetyltransferase Gcn5 plays an important role in transcription activation, DNA replication-coupled nucleosome assembly and nucleotide excision repair (NER). However, its functions on the heterochromatin are unexplored. Here, we find that removal of Gcn5 leads to more condensed heterochromatin structure, as revealed by topology analysis ofHMLcircles. Importantly, the altered heterochromatin structure is restored by re-expression of Gcn5 in thegcn5Δcells. As a result of the more compact heterochromatin, gene silencing at theHMLlocus is increased and NER efficiency atHMLis impaired in the absence of Gcn5. Interestingly, while the association of SIR complex withHMLis enhanced in cells lacking Gcn5, the altered compaction ofHMLheterochromatin is also observed due to the deletion of Gcn5 from Sir−cells. These findings reveal a role of Gcn5 in the regulation of heterochromatin structure, gene silencing and NER efficiency at the heterochromaticHMLlocus in yeast.