Construction of a dynamic gene regulatory network required for cellular reprogramming

The generation of induced Pluripotent Stem Cells (iPSCs) from somatic cells provides an excellent example to study mechanisms of transcription factor-induced global alterations of the genome and the epigenome. Here, we have investigated the early transcriptional events of cellular reprogramming triggered by the co-expression of OSKM (Oct4, Sox2, Klf4 and c-Myc) in Mouse Embryonic Fibroblasts (MEFs) and mouse Hepatocytes (mHeps) and identified a novel gene regulatory network composed of 9 Transcriptional Regulators (TRs), which are directly targeted by OSKM. Functional studies using single and double shRNA knock-downs of any of these TRs caused disruption of the network and dramatic reductions in reprogramming efficiency, demonstrating that this novel gene regulatory network is essential for the induction and establishment of pluripotency. We demonstrate that that the stochastic co-expression of the 9TRs network components occurs in a remarkably small number of cells approximating the percentage of reprogrammed cells as the result of dynamic molecular events. Thus, the early binding patterns of OSKM and the subsequent stochastic co-expression of pivotal TRs in subpopulations of cells steer the reconstruction of a gene regulatory network crucial for the generation of iPSCs. In this study, we show that OSKM trigger the gradual establishment of the stemness phenotype by inducing the stochastic reconstruction of a novel 9TRs gene regulatory network that guides the acquisition to pluripotency. Affymetrix Mouse Genome 430 A 2.0 version was used for gene expression profiling of the reprogramming course. RNA was isolated from mESCs, miPSCs, control (naïve) MEFs and reprogrammable MEFs on day 0.5, 1, 1.5, 2, 2.5, 3, 6, 12, 18 of reprogramming. Additional RNA was isolated from mHeps at days 0, 3 and 6 of reprogramming. UPDATE: [08-11-2024] The titles and antibodies of the following Sample pairs were swapped: GSM4667681 and GSM4667683 GSM4667682 and GSM4667684 GSM4667685 and GSM4667687 GSM4667686 and GSM4667688 GSM4667693 and GSM4667694 The raw data and processed data associated with these Sample accession numbers did not change.