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ChIP-seq of H3K27ac, H3K9me3, and HA tag for ATFs on MEF-derived cells

ABSTRACT: We tested a genome-scale artificial transcription factor (ATF) library in reprogramming mouse embryonic fibroblasts to induced pluripotent stem (iPS) cells. Three combinations of ATFs (C2, C3, and C4) could induce pluripotency when expressed with Sox2, Klf4, and c-Myc. The transcriptional profiles of ATF-induced iPS cells are similar to that of iPS cells induced with Oct4, Sox2, Klf4, and c-Myc and mouse embryonic stem cells, exhibiting up-regulation of pluripotency markers and down-regulation of fibroblast markers. We performed ChIP-seq on the active histone mark H3K27ac and the repressive histone mark H3K9me3 in C2+SKM iPS cells, C3+SKM iPS cells, in Oct4+SKM to validate the total transcriptome results. In addition, the ATFs from C2 were chromatin immunoprecipitated to determine where they are bound in the genome at an intermediate stage before being fully reprogrammed to iPS cells. This study provides a proof-of-principle that a gene-activating ATF library can be used to identify cell fate-defining transcriptional networks in an unbiased manner.

ORGANISM(S): Mus musculus

PROVIDER: GSE89220 | GEO | 2016/12/05

SECONDARY ACCESSION(S): PRJNA350709

REPOSITORIES: GEO

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Project description:Reprogramming of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility to generate patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcription factor quartet Oct4, Sox2, c-Myc, and Klf4. Considering that ectopic expression of c-Myc causes tumourigenicity in offspring and retroviruses themselves can cause insertional mutagenesis, the generation of iPS cells with a minimal number of factors may hasten the clinical application of this approach. Here, we show that adult mouse neural stem cells (NSCs) express higher endogenous levels of Sox2 and c-Myc than ES cells and that exogenous Oct4 together with either Klf4 or c-Myc are sufficient to generate iPS cells from NSCs. These two-factor (2F) iPS cells are similar to embryonic stem cells at the molecular level, contribute to development of the germline, and form chimeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors. Experiment Overall Design: 8 hybridizations in total. Experiment Overall Design: NSC derived iPS cells by 2 factors (Oct4 and Klf4) in triplicate: Experiment Overall Design: - iPS cell_2F_1 Experiment Overall Design: - iPS cell_2F_2 Experiment Overall Design: - iPS cell_2F_3 Experiment Overall Design: Embryonic Stem cells (ESC) in triplicate: Experiment Overall Design: - ESC_1 Experiment Overall Design: - ESC_2 Experiment Overall Design: - ESC_3 Experiment Overall Design: NSC cultures in duplicates: Experiment Overall Design: - NSC_2 Experiment Overall Design: - NSC_3 Experiment Overall Design: NSC derived iPS cells by 4 factors (Oct4, Sox2, c-Myc and Klf4) in triplicate: Experiment Overall Design: - iPS cell_4F_1 Experiment Overall Design: - iPS cell_4F_2 Experiment Overall Design: - iPS cell_4F_3

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ChIP-seq of H3K27ac, H3K9me3, and HA tag for ATFs on MEF-derived cells

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