An intricate transcription-translation feedback loop (TTFL) governs cellular circadian rhythms in mammals. Here, we report that the zinc finger transcription factor Krüppel-like factor 9 (KLF9) is regulated by this TTFL, it associates in chromatin at the core circadian clock and clock-output genes, and it acts to modulate transcription of the clock-output gene. Our earlier genome-wide analysis of the mouse hippocampus-derived cell line HT22 showed that KLF9 associates in chromatin with,,,,,,, and. Of the 3514 KLF9 peaks identified in HT22 cells, 1028 contain E-box sequences to which the transcriptional activators CLOCK and BMAL1 may bind, a frequency significantly greater than expected by chance.mRNA showed circadian oscillation in synchronized HT22 cells, mouse hippocampus, and liver. At the clock-output gene, KLF9 exhibited circadian rhythmicity in its association in chromatin in HT22 cells and hippocampus. Forced expression of KLF9 in HT22 cells repressed basaltranscription and strongly inhibited CLOCK+BMAL1-dependent transcriptional activation of a transfectedreporter. Mutational analysis showed that this action of KLF9 depended on 2 intact KLF9-binding motifs within thelocus that are in close proximity to E-boxes. Knockout ofor the paralogous geneusing CRISPR/Cas9 genome editing in HT22 cells had no effect onexpression, but combined knockout of both genes strongly impaired circadianmRNA oscillation. Like KLF9, KLF13 also showed association in chromatin with clock- and clock-output genes, and forced expression of KLF13 inhibited the actions of CLOCK+BMAL1 ontranscription. Our results suggest novel and partly overlapping roles for KLF9 and KLF13 in modulating cellular circadian clock output by a mechanism involving direct interaction with the core TTFL. Dbp Per1Per3Dbp Tef Bhlhe40Bhlhe41Nr1d1Nr1d2Klf9Dbp Dbp Dbp Dbp Klf9Klf13Dbp Dbp Dbp
