The Molecular Mechanisms Underlying The Regulation Of Neurospora Circadian Clock Gene Frequency By Histone Modifications

Circadian clocks are endogenous cellular timekeepers in the organisms living on the earth and circadian clock oscillators are conserved among different organisms. Neurospora crassa is one of the best model organisms for the study of circadian clock and its oscillator relies on the rhythmic transcription of frequency (frq). However, the mechanism of transcriptional regulation of frq is not fully understood.In order to test the role of epigenetic modifications in circadian clock regulation, the related factors were screened in Neurospora. We find that the histone H3K36 methyltransferase SET-2 is involved in the regulation of circadian clock. First, loss of histone H3K36 methylation, due to either deletion of SET-2 or H3K36R mutation, results in arrhythmic frq transcription and loss of overt rhythmicity. Second, SET-2 is rhythmically recruited to the frq gene body which leads to rhythmic H3K36me3 at frq locus. Furthermore, the histone deacetylase RPD-3 participates in frq transcription with SET-2. RPD-3 is the catalytic subunit of Rpd3S complex. SET-2-mediated H3K36me3 is recognized by EAF-3, the histone deacetylase complex Rpd3S subunit. Deletion of SET-2 or RPD-3 results in increased histone acetylation levels over frq ORF. Meanwhile,frq can be transcribed in a WC-independent manner in set-2KO, rpd-3KO or eaf-3KO mutants, the WC-independent frq disrupts the rhythmical WC-dependent frq transcription through interference the negative feedback loop. To test which histone acetylation sites are essential for circadian clock, the candidate RPD-3 target sites were identified. A mutant strain with three amino acid substitutions (histone H3 lysine 9,14, and 18 to glutamine) was generated to mimic the strain with hyperacetylation state of histone H3. H3K9QK14QK18Q mutant exhibits the same defective clock phenotype with rpd-3KO mutant. What’s more, WC-independent frq expression is detected in H3K9QK14QK18Q strains. These results suggest that H3K9, K14 and Kl 8 are the main target sites for RPD-3 in the regulation of frq transcription.Besides RPD-3, SET-2 requires the chromatin remodelers to regulate frq transcription. Our results show that SET-2 and the chromatin remodeler CHD-1 suppress WC-independent frq transcription by preventing histone exchange. Soluble histones are predominantly acetylated, SET-2 and CHD-1 suppress the incorporation of newly acetylated histones during transcription elongation.By genetic screen, we find that the histone deacetylase HDA-2 is also involved in the regulation of frq transcription. Different from the histone deacetylase RPD-3, HDA-2 regulates frq trsncription through two pathways. Firstly, HDA-2 regulates the amplitude of clock through its histone deacetylase activity. In hda-2KO, the frq transcription is increased but the period is not influenced. Secondly, HDA-2 activates the WC-independent frq transcription in the absence of its repressors. As WC-independent frq can be detected in chd-1 wc-1 double mutants, the WC-independent frq can not be transcripted in the chd-1KO wc-1RIPhda-2KO mutant.This study uncovers the relationship between histone modifications an frq gene transcription. Our study provides valuable information to understand Neurospora circadian clock regulation and may be instructive for related studies in other organisms.