Project description:Krueppel-like factor 9 (Klf9) is a DNA-binding transcription factor that has been implicated in neuronal development and regeneration. We used mouse hippocampus-derived HT22 cell line to identify genes differentially regulated by forced Klf9 expression. We stably transfected HT22 cells with pCDNA6:TR (encoding the Tet repressor) and pCDNA6:TO-Klf9 (encoding Klf9 downstream of the Tet operator), allowing doxycycline(dox)-inducible expression of Klf9. We chose a stably transfected clone with baseline Klf9 mRNA level similar to that of untransfected HT22 cells (parent line) and approximately 10-fold induction after dox treatment. We conducted RNA-seq on stably transfected HT22 cells treated with or without dox for 8 hours; we also conducted RNA-seq on parent cells subjected to the same dox treatment to separate nonspecific effects of dox from the effects of forced Klf9 expression. Analysis of RNA-seq data found 217 gene repressed and 21 upregulated by forced Klf9 expression (FDR-adjusted p<.005; because fold changes were small we used a stringent p value cutoff to ensure that we were studying genes likely to be bona fide targets), showing that Klf9 functions primarily as a transcriptional repressor. Gene ontology analysis showed that Klf9-repressed genes were enriched in cytoskeletal and Wnt signaling-related genes.
Project description:Krueppel-like factor 9 (Klf9) is a DNA-binding transcription factor that has been implicated in neuronal development and regeneration, but none of its genomic targets are known in neurons. We used the mouse hippocampus-derived cell line HT22 to identify Klf9’s genomic binding sites in neurons. We stably transfected HT22 cells with the biotin ligase BirA with or without co-transfection of a Klf9 fusion protein with an n-terminal FLAG tag and a biotin ligase recognition peptide. This allowed us to precipitate Klf9 in chromatin using streptavidin. This permits the use of more stringent wash conditions, improving signal-to-noise ratio. We conducted high-throughput sequencing on DNA precipitated from these lines and identified 3,378 regions where Klf9 associated in chromatin. We found that Klf9 associated near transcription start sites and that its genomic targets were enriched for genes related to cytoskeletal regulation and apoptosis.
