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 Dbp. Our earlier genome-wide analysis of the mouse hippocampus-derived cell line HT22 showed that KLF9 associates in chromatin with Per1, Per3, Dbp, Tef, Bhlhe40, Bhlhe41, Nr1d1, and Nr1d2. 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. Klf9 mRNA showed circadian oscillation in synchronized HT22 cells, mouse hippocampus, and liver. At the clock-output gene Dbp, KLF9 exhibited circadian rhythmicity in its association in chromatin in HT22 cells and hippocampus. Forced expression of KLF9 in HT22 cells repressed basal Dbp transcription and strongly inhibited CLOCK+BMAL1-dependent transcriptional activation of a transfected Dbp reporter. Mutational analysis showed that this action of KLF9 depended on 2 intact KLF9-binding motifs within the Dbp locus that are in close proximity to E-boxes. Knockout of Klf9 or the paralogous gene Klf13 using CRISPR/Cas9 genome editing in HT22 cells had no effect on Dbp expression, but combined knockout of both genes strongly impaired circadian Dbp mRNA 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 on Dbp transcription. 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.

一个复杂的转录-翻译反馈回路 (TTFL) 控制着哺乳动物的细胞昼夜节律。在这里，我们报告锌指转录因子 Krüppel 样因子 9 (KLF9) 受此 TTFL 调节，它与核心生物钟和时钟输出基因的染色质结合，并调节时钟输出的转录基因Dbp。我们早期对小鼠海马衍生细胞系 HT22 的全基因组分析表明，KLF9 在染色质中与Per1、Per3、Dbp、Tef、Bhlhe40、Bhlhe41、Nr1d1和Nr1d2 相关联. 在 HT22 细胞中鉴定的 3514 个 KLF9 峰中，1028 个包含转录激活因子 CLOCK 和 BMAL1 可能结合的 E-box 序列，其频率明显高于偶然预期的频率。Klf9 mRNA 在同步的 HT22 细胞、小鼠海马和肝脏中显示出昼夜节律振荡。在时钟输出基因Dbp中，KLF9 在 HT22 细胞和海马的染色质中表现出昼夜节律性。KLF9 在 HT22 细胞中的强制表达抑制了基础Dbp转录并强烈抑制了转染的Dbp报告基因的 CLOCK+BMAL1 依赖性转录激活。突变分析表明，KLF9 的这种作用取决于Dbp中的 2 个完整的 KLF9 结合基序靠近电子盒的位置。在 HT22 细胞中使用 CRISPR/Cas9 基因组编辑敲除Klf9或旁系同源基因Klf13对Dbp表达没有影响，但联合敲除这两个基因会严重损害昼夜节律Dbp mRNA 振荡。与 KLF9 一样，KLF13 在染色质中也显示出与时钟和时钟输出基因的关联，并且 KLF13 的强制表达抑制了 CLOCK+BMAL1 对Dbp转录的作用。我们的研究结果表明，KLF9 和 KLF13 在通过涉及与核心 TTFL 直接相互作用的机制调节细胞生物钟输出方面具有新颖且部分重叠的作用。