Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression changes upon Dot1L knockdown and Dot1L inhibitor treatment during reprogramming

ABSTRACT: Through a loss-of-function approach, we identified that inhibition of the histone methyltransferase, Dot1L, accelerated somatic cell reprogramming, significantly increased the yield of induced pluripotent stem (iPS) cell colonies, and substituted for Klf4 and c-Myc in the reprogramming cocktail. To understand the mechanism by which Dot1L inhibition results in these phenotypes, we carried out gene expression profiling using Affymetrix microarrays. GSM723207-GSM723224: Embryonic stem cell-derived fibroblasts (dH1fs) were retrovirally transduced in culture with vectors expressing either a control shRNA or an shRNA targeting Dot1L. These cells were then superinfected with either Oct4, Sox2, Klf4 and c-Myc (OSKM) retroviruses or Oct4, Sox2 and c-Myc (OSM) retroviruses. Total RNA was harvested 6 days later. There were three biologic replicates for each condition. GSM880675-GSM880682: dH1fs were treated with 10uM Dot1L inhibitor (EPZ004777) and then superinfected with Oct4, Sox2, Klf4 and c-Myc (OSKM) retroviruses. Total RNA was harvested 6 days later. There were two biologic replicates for each condition.

ORGANISM(S): Homo sapiens

SUBMITTER: Tamer Onder

PROVIDER: E-GEOD-29253 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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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Gene expression changes upon Dot1L knockdown and Dot1L inhibitor treatment during reprogramming

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