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Identification of Klf9 genomic binding sites in HT22 tissue culture cells [ChIP-Seq]

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE87203 | GEO | 2019/09/19

REPOSITORIES: GEO

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

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.

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Identification of Klf9-regulated genes in HT22 tissue culture cells

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Molecular mechanisms for Krüppel-like factor 13 actions in hippocampal neurons [ChSP-seq]

Project description:Krüppel-like factors (KLFs) play key roles in nervous system development and function. Several KLFs are known to promote, and then maintain neural cell differentiation. Our previous work focused on the actions of KLF9 in mouse hippocampal neurons. Here we investigated genomic targets and functions of KLF9’s paralog KLF13, with the goal of understanding how these two closely related transcription factors influence hippocampal cell function, proliferation, survival and regeneration. We engineered the adult mouse hippocampus-derived cell line HT22 to control Klf13 expression with doxycycline. We also generated HT22 Klf13 knock out cells, and we analyzed primary hippocampal cells from wild type and Klf13-/- mice. RNA sequencing showed that KLF13, like KLF9, acts predominantly as a transcriptional repressor in hippocampal neurons and can regulate other Klf genes. Pathway analysis revealed that genes regulated by KLF13 are involved in cell cycle, cell survival, cytoarchitecture regulation, among others. Chromatin-streptavidin sequencing conducted on chromatin isolated from HT22 cells expressing biotinylated KLF13 identified 9506 genomic targets; 79% were located 1 kilobase upstream of transcription start sites. Transfection-reporter assays confirmed that KLF13 can directly regulate transcriptional activity of its target genes. Comparison of the target genes of KLF9 and KLF13 found that they share some functions that were likely present in their common ancestor, but have also acquired distinct functions during evolution. Flow cytometry showed that KLF13 promotes cell cycle progression, and it protects cells from glutamate-induced excitotoxic damage. Taken together, our findings establish novel roles and molecular mechanisms for KLF13 actions in mammalian hippocampal neurons.

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Expression data from J147-treated HT22 cells compared to untreated HT22 cells

Project description:The Neurotrophic Compound J147 Reverses Cognitive Impairment In Aged Alzheimer's Disease We used GeneChip® Mouse Genome 430 2.0 Arrays to probe global gene expression changes when adding J147 compound to HT22 cells and compared to untreated cells. The hippocampal cell line HT22 were treated with the J147 compound for 1 hour and compared to HT22 treated only with vehicle (DMSO) for 1 hour.

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Project description:ChIP-seq analyses were performed in MEL cells expressing BirA alone or BirA and FLAG-Biotin tagged BCL11A (XL isoform).

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Transcription profiling of jejunums from wild type and Klf9-/- mice to investigate the involvement of Klf9 in small intestine crypt-villus cell renewal and lineage determination.

Project description:Kruppel-like factor 9 (Klf9), a zinc-finger transcription factor, is implicated in the control of cell proliferation, cell differentiation and cell fate in brain and uterus. Using Klf9 null mutant mice, we have investigated the involvement of Klf9 in small intestine crypt-villus cell renewal and lineage determination. We report the predominant expression of Klf9 gene in small intestine smooth muscle (muscularis externa). Jejunums null for Klf9 have shorter villi, reduced crypt stem/transit cell proliferation, and altered lineage determination as indicated by decreased and increased numbers of Goblet and Paneth cells, respectively. A stimulatory role for Klf9 in villus cell migration was demonstrated by BrdU labeling. Results suggest that Klf9 controls the elaboration, from small intestine smooth muscle, of molecular mediator(s) of crypt cell proliferation and lineage determination, and of villus cell migration. Experiment Overall Design: Total RNA was extracted in parallel from the jejunums of five WT and five Klf9-/- male mice (PND 30) using TRIzol reagent (Invitrogen, Carlsbad, CA). Conversion of each RNA preparation to corresponding fragmented cRNA. Fifteen ug of each cRNA was hybridized for 16 hours to an Affymetrix mouse 430A GeneChip. Ten GeneChips (each corresponding to a single animal) were hybridized, washed and scanned in parallel. Following the wash, signal amplification, and signal detection steps, GeneChips were scanned (Agilent GeneArray laser scanner) and the resultant images quantified using Affymetrix MAS 5.0 software.

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