Identification of Genomic Targets of Krüppel-like Factor 9 in Mouse Hippocampal Neurons: Evidence for a Role in Modulating Peripheral Circadian Clocks.

[摘要] Krueppel-like factor 9 (Klf9) is a transcription factor that has diverse roles in development and physiology. Earlier work showed that Klf9 functions in neuronal differentiation, but nothing was known of its target genes in neurons. I used the mouse hippocampus-derived cell line HT22 to identify Klf9 genomic targets. I engineered HT22 cells to express Klf9 under the control of the tet repressor, and used RNA sequencing to identify genes modulated by Klf9. I identified 567 genes repressed, and 201 induced by Klf9. I also engineered HT22 cells to co-express biotin ligase and a Klf9 fusion protein containing an N-terminal polypeptide that can be biotinylated. Using chromatin-streptavidin precipitation (ChSP) sequencing I identified 3,516 genomic regions where Klf9 associated. Seventy five percent were <1 kilobase from transcription start sites, and Klf9 associated in chromatin with 60% of repressed genes. Computer analysis revealed GC-rich consensus motifs at 98% of regions where Klf9 associated. Transient transfection assays showed that Klf9 repressed promoter activity, which was abrogated after mutation of GC-rich motifs. Expression analysis of a subset of Klf9 target genes in hippocampus of wild type and Klf9-null mice showed that all were dysregulated in the mutants. Gene ontology analysis revealed that Klf9 regulates genes involved in neuronal morphogenesis, neurotrophin signaling, apoptosis and cell division.Analysis of my ChSP-seq data showed that Klf9 associated in chromatin with genes involved in the cellular circadian clock. Klf9 mRNA showed circadian oscillation in synchronized HT22 cells, and in mouse liver and hippocampus in vivo. Klf9 showed rhythmic association with several clock-output genes in mouse liver, supporting a role in regulating circadian gene expression. Forced expression of Klf9 repressed transcription of the clock-output gene Dbp and inhibited activation of the Dbp promoter by CLOCK/Bmal1, supporting that Klf9 antagonizes the CLOCK/Bmal1 complex that forms the positive limb of the circadian oscillator. My dissertation work represents the first analysis of Klf9 genomic targets in neurons, and supports a predominant repressor role for Klf9 in gene transcription. My findings also support that Klf9 acts as a novel component of the negative limb of the circadian clock.

[发布机构] University of Michigan

[效力级别] Transcription factors

[关键词] Neuroscience;Transcription factors;Gene expression;Circadian rhythms;Molecular;Cellular and Developmental Biology;Science;Neuroscience