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An Unbiased Approach to Identifying Cellular Reprogramming-Inducible Enhancers [RNA-seq]

ABSTRACT: Cellular reprogramming of somatic cells towards induced Pluripotent Stem Cells (iPSCs) is achieved through the over-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM). Due to the high complexity of the process, the vast number of the involved molecules and its stochastic and inefficient nature, the molecular background of reprogramming is only partially revealed. The aim of this study is the identification of genomic regulatory elements which are induced during cellular reprogramming, the examination of their action and their association with the expression of important genes. First, we constructed a combined ChIP-seq OSKM dataset using experiments on MEFs (Mouse Embryonic Fibroblasts) from multiple published studies, in order to examine the OSKM binding upon the genome, irrespective of minor discrepancies originating from the different reprogramming systems and protocols used. We also performed detailed RNA-seq and ATAC-seq experiments in order to monitor the functional results of the OSKM genomic binding. Initially, we focused on the ESC-related sites (Embryonic Stem Cells, natural equivalent of iPSCs) and found out that almost 1/3 of them are bound by OSKM as early as at day 1, or since MEFs. Our analysis highlighted the significant role of Myc in reprogramming, which is often overlooked. We saw that, along with Klf4, they “mark” positions where Oct4 and/or Sox2 will bind after reprogramming initiation. Furthermore, we discovered that various genes are regulated by multiple genomic elements, where OSKM bind at different timepoints in reprogramming. For example, many genes involved in the MET pathway (Mesenchymal to Epithelial Transition) are located near both ESC-, MEF- and Transient-OSKM sites, resulting in stably high expression levels which temporarily increase during the intermediate timepoints. Finally, we observed that OSKM often bind on the chromatin in a dynamic fashion. This “on-and-off” association of OSKM with the genome is related to the need of chromatin reorganization to achieve pluripotency. It is logical to assume that part of the inherent stochasticity of cellular reprogramming is based on the low percentage of cells that will achieve to reproduce this dynamic OSKM binding to regulate efficiently their transcriptional programs and to finally transform to iPSCs.

ORGANISM(S): Mus musculus

PROVIDER: GSE274132 | GEO | 2024/12/05

REPOSITORIES: GEO

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Project description:Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4 and Myc (OSKM) into somatic cells. Though iPSCs are pluripotent, they frequently exhibit high variation in their quality as measured by chimera contribution and tetraploid (4n) complementation. Thus, improving the quality of iPSCs is an indispensable prerequisite for future iPSC-based therapy. Here we show that one major determinant for iPSCs quality is the combination of reprograming factor selected. Ectopic expression of Sall4, Nanog, Esrrb and Lin28 (SNEL) in MEFs efficiently generated high quality iPSCs as compared to other combinations of factors. SNEL-iPSCs produced approximately 5 times more efficiently “all-iPSC” mice compared to OSKM-iPSCs. While differentially methylated regions, transcript number of master regulators, establishment of ESC-specific super enhancers, and global aneuploidy were comparable between the lines, aberrant expression of 1,765 genes, trisomy of chromosome 8 and abnormal H2A.X deposition were frequently observed in poor quality OSK-iPSCs and OSKM-iPSCs. Whole-genome single-base resolution MethylC sequencing collected from a variety of Mouse pluripotent cells To reveal the DNA methylation signature associated with developmental competence, we selected the following groups of iPSC lines for whole-genome bisulfite sequencing: i) "Poor quality" iPSCs: This group included the three OSKM-iPSC lines Nanog-GFP OSKM#2, Oct4-GFP OSKM#2 and KH2 OSKM(Stadtfeld et al., 2010), that either did not produce fully developed pups or produced very low number of pups; ii) "Good quality" iPSCs: This group included BC_2 OSKM (Carey et al., 2011) and Nanog-GFP SNEL#3, both of which gave rise to live, normal pups that survived only few hours; iii) "High quality" iPSCs consisting of Nanog-GFP SNEL#2 and Oct4-GFP SNEL#1, both of which generated live pups that survived postnatally (representative pups from each iPSC group are shown in Figure S3A). iv) As controls we used Nanog-GFP, Oct4-GFP and KH2 (Beard et al., 2006) ESCs. Note that all cells were cultured in 2i medium but not in mES medium.

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An Unbiased Approach to Identifying Cellular Reprogramming-Inducible Enhancers [RNA-seq]

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