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The underlying mechanisms of circadian clocks are highly conserved across species, driven by endogenous transcriptional\u2013translational feedback loops. CLOCK, a central transcription factor in circadian regulation, requires coregulatory factors and histone modification dynamics to regulate downstream clock-controlled gene expression. In this study, we identify the Drosophila polymerase-associated factor 1 complex (Paf1C) as a previously unrecognized regulator of the circadian transcriptional machinery. We found that knocking down different subunits of Paf1C in pacemaker neurons lengthens the circadian locomotion period. Specifically, downregulation of rtf1, a core subunit of Paf1C, significantly dampens the amplitude of circadian rhythms and extends the locomotor period. This effect is primarily attributed to a reduction in the pacemaker protein PERIOD (PER), mediated through decreased per transcription. Indeed, the overexpression of per in pacemaker neurons rescues the circadian defects caused by rtf1 downregulation. Mechanistically, we identified that rtf1 enhances CLK-mediated per transcription activation. RTF1 physically interacts with CLK, thus promoting its occupancy on the promoters of per and other clock genes. Furthermore, the H3K4me3 methyltransferase SET1 forms complex with CLK and RTF1, facilitates their interaction, and thereby increases H3K4me3 levels at the per\/tim promoter to promote their expression. Notably, we discovered that human RTF1 physically interacts with BMAL1\/CLOCK and affects the circadian rhythms in U2OS cells, indicating a potentially conserved mechanism in mammals. Together, our results demonstrate that RTF1 regulates circadian rhythms by modulating CLK occupancy and H3K4me3 levels at pacemaker gene promoters.<\/jats:p>","DOI":"10.1083\/jcb.202501221","type":"journal-article","created":{"date-parts":[[2025,8,20]],"date-time":"2025-08-20T06:21:01Z","timestamp":1755670861000},"update-policy":"https:\/\/doi.org\/10.1083\/jcb.crossmarkpolicy","source":"Crossref","is-referenced-by-count":2,"title":["RTF1 enhances CLK occupancy and histone methylation at key circadian clock pacemaker gene loci"],"prefix":"10.1083","volume":"224","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-5208-4229","authenticated-orcid":true,"given":"Xu","family":"Liu","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05t8y2r12","id-type":"ROR","asserted-by":"publisher"}],"name":"Jiangsu Key Laboratory of Drug Discovery and Translational Research for Brain Diseases, Cambridge-Suda Genomic Resource Center, The Fourth Affiliated Hospital, Suzhou Medical College, 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