Non-coding (nc)RNAs are key players in numerous biological processes such as 
gene regulation, chromatin domain formation and genome stability. Large ncRNAs 
interact with histone modifiers and are involved in cancer development, 
X-chromosome inactivation and autosomal gene imprinting. However, despite recent 
evidence showing that pervasive transcription is more widespread than previously 
thought, only a few examples mediating gene regulation in eukaryotes have been 
described. In Saccharomyces cerevisiae, the bona-fide regulatory ncRNAs are 
destabilized by the Xrn1 5'-3' RNA exonuclease (also known as Kem1), but the 
genome-wide characterization of the entire regulatory ncRNA family remains 
elusive. Here, using strand-specific RNA sequencing (RNA-seq), we identify a 
novel class of 1,658 Xrn1-sensitive unstable transcripts (XUTs) in which 66% are 
antisense to open reading frames. These transcripts are polyadenylated and RNA 
polymerase II (RNAPII)-dependent. The majority of XUTs strongly accumulate in 
lithium-containing media, indicating that they might have a role in adaptive 
responses to changes in growth conditions. Notably, RNAPII chromatin 
immunoprecipitation followed by DNA sequencing (ChIP-seq) analysis of 
Xrn1-deficient strains revealed a significant decrease of RNAPII occupancy over 
273 genes with antisense XUTs. These genes show an unusual bias for H3K4me3 
marks and require the Set1 histone H3 lysine 4 methyl-transferase for silencing. 
Furthermore, abolishing H3K4me3 triggers the silencing of other genes with 
antisense XUTs, supporting a model in which H3K4me3 antagonizes antisense ncRNA 
repressive activity. Our results demonstrate that antisense ncRNA-mediated 
regulation is a general regulatory pathway for gene expression in S. cerevisiae.