Identification of Transcriptional Targets of Krüppel-like Factor 9 in Mouse Hippocampal Neurons

Presentation Number: MON-0390
Date of Presentation: June 23rd, 2014

Joseph Riley Knoedler*1 and Robert J Denver2
1University of Michigan, Ann Arbor, MI, 2Univ of MI, Ann Arbor, MI

Abstract

Krüppel-like factor 9 (KLF9) is a DNA-binding transcription factor that is expressed in the mammalian brain, most prominently in the hippocampus and cerebellum. In rodents its expression is low at birth but rises postnatally in parallel with rising plasma T3 titers. KLF9 is a direct target of glucocorticoid and thyroid hormone receptors, and it may mediate actions of T3 on maturation, morphogenesis and survival of neurons and oligodendrocytes. However, its transcriptional targets and genomic binding sites in the brain are unknown. We used the immortalized mouse hippocampal cell line HT22 as a model system to discover KLF9-regulated genes in differentiated neurons. We generated stably transfected HT22 cells to allow for tetracycline (TET)-inducible KLF9 expression by co-transfecting cells with a plasmid expressing the TET repressor and a TET-responsive expression plasmid encoding KLF9.  We selected one stable clone for analysis that had low basal KLF9 expression that was increased ~5-fold after TET induction (similar to that achievable with hormone treatment). Forced expression of KLF9 reduced cell proliferation measured by MTT cell viability assay, and reduced neurite outgrowth. We conducted RNA-sequencing on the parent cell line and the stable TET-inducible line treated +/- 0.5 mg/ml TET for 8 hr (3 replicates/treatment). Data analysis using the Tuxedo suite (Bowtie, Cuffdiff and CummeRbund) identified ~550 downregulated and ~120 upregulated genes, suggesting that KLF9 functions predominantly as a repressor of gene transcription in these cells. We validated the relative expression levels of 18 genes by RT-qPCR in independent TET-treated samples and transiently transfected HT22 cells. Gene ontology (GO) analysis by the DAVID pipeline (http://david.abcc.ncifcrf.gov/) showed that among the most enriched functional categories of repressed genes were cytoskeletal and actin-binding proteins and cell adhesion molecules, suggesting a physiological basis for the reduction in neuritogenesis following forced KLF9 expression. Other strongly enriched categories included kinase signaling and regulation of transcription, suggesting that KLF9 may influence many downstream physiological and gene-regulatory processes. Promoter analysis using the PAINT motif discovery web tool (http://www.dbi.tju.edu/dbi/tools/paint/) found overrepresentation of Sp1-sites (GC-boxes that can be bound by KLFs) in the proximal promoters of KLF9-repressed genes, compared to their frequency in the promoters of mouse genes as a whole. This is consistent with direct repression of these genes by KLF9 binding to their promoters. Our results are the first to identify regulatory targets of KLF9 in neurons to explain its role in neuronal development and nuclear receptor signaling. 

 

Nothing to Disclose: JRK, RJD