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Identification of Klf9-regulated genes in HT22 tissue culture cells [RNA-Seq]

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE87093 | GEO | 2019/09/19

REPOSITORIES: GEO

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Project description:The generation of induced pluripotent stem cells (iPSCs) by the forced expression of defined transcription factors in somatic cells holds great promise for the future of regenerative medicine. However, due to a poor understanding of the initial reprogramming mechanism, it is difficult to control the efficiency and quality of the iPSCs. Here we show that Zscan4, expressed transiently in 2-cell embryos and embryonic stem cells (ESCs), efficiently produces iPSCs from mouse embryo fibroblasts when coexpressed with Klf4, Oct4, and Sox2. Unlike other factors, Zscan4 is required only for the first few days of iPSC formation. Microarray analysis revealed transient and early induction of preimplantation-specific genes in a Zscan4-dependent manner. Our work indicates that Zscan4 is a previously unidentified potent natural factor that facilitates the reprogramming process through the recapitulation of the early embryonic program. Secondary MEFs were isolated from E13.5 embryos, which were harvested by tetraploid complementation. Secondary MEFs were plated on gelatin-coated 6-well plates at a density of 1x105 cells/well in the complete ES medium. After 24 h incubation, secondary MEFs were fed with complete ES medium with or without Dox (1.5 ?g/ml) and with or without 200 nM Tmx. Culture medium was changed every day. Withdrawal of drugs (Dox or Tmx) was always followed by 1x washing by PBS before changing culture medium. We cotransfected MEFs with PB-Tet-MKOS and either a PB-Tet-Zscan4 or PB-Tet-DsRed (control). These cells were subsequently cultured in the standard iPSC generation condition (Dox+). Unexpectedly, we found that MEFs transfected with a PB-Tet-Zscan4 show consistently higher efficiency of iPSC generation than MEFs transfected with a PB-Tet-DsRed (control). All selected iPSCs expressed pluripotent marker genes according to RT-PCR analysis and were positive for ALP, Nanog, and SSEA-1. These iPSCs also formed embryoid bodies in hanging drops without LIF and could be differentiated in vitro to cells of all three germ layers. We are currently studying whether the presence of Zscan4 has the beneficial effects on iPSCs in terms of genome stability.

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Identification of Klf9-regulated genes in HT22 tissue culture cells [RNA-Seq]

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