Paf1C regulates the Neurospora circadian clock by promoting the transcription elongation of frequency.

The circadian rhythm is crucial for organisms to adapt timely to external environmental changes, and the operation of the circadian oscillator relies on the precise transcriptional regulation of clock genes, a process that is highly conserved across species. The activation and repression of transcriptional initiation of clock genes have been extensively studied. However, the regulation of transcriptional elongation remains largely unexplored. Here, we showed that the RNA Polymerase II Associated Factor 1 complex (Paf1C) is required for maintaining the normal circadian rhythm in Neurospora. The loss of PAF-1, CTR-9 or RTF-1 subunit of Paf1C led to a shorter circadian period and advanced phase. Mechanistically, the PAF-1 and CTR-9 subunits promote the transcription of the clock gene frequency (frq) by enhancing the enrichment of not only histone H2B ubiquitination (H2BK131ub), but also the phosphorylation of Ser2 in RNA Polymerase II CTD and H3K36 trimethylation (H3K36me3) at the frq ORF region. Moreover, the other subunit RTF-1 promotes frq transcription by controlling global H2BK131ub through interaction with the RAD-6/BRE-1 ubiquitin conjugase-ligase complex. Surprisingly, a highly conserved region within RTF-1 nearly rescues global H2BK131ub and the circadian clock defects in rtf-1KO strains. Taken together, these results indicate that Paf1C regulates the Neurospora circadian clock by promoting histone H2B ubiquitination and facilitating transcription elongation of the frq gene.