An Unbiased Approach to Identifying Cellular Reprogramming-Inducible Enhancers [ChIP-seq]
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1145012 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR30151614.fastq.gz | Fastqsanger.gz | |||
| SRR30151615.fastq.gz | Fastqsanger.gz |
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Project description:Cellular reprogramming of somatic cells towards induced pluripotency is a multistep stochastic process mediated by the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM), which orchestrate global epigenetic and transcriptional changes. We performed a large-scale analysis of integrated ChIP-seq, ATAC-seq and RNA-seq data and revealed the spatiotemporal highly dynamic pattern of OSKM DNA binding during reprogramming. We found that OSKM show distinct temporal patterns of binding to different classes of pluripotency-related enhancers. Genes involved in reprogramming are regulated by the coordinated activity of multiple enhancers, which are sequentially bound by OSKM for strict transcriptional control. Based on these findings, we developed an unbiased approach to identify Reprogramming-Inducible Enhancers (RIEs), constructed enhancer-traps and isolated cells undergoing reprogramming in real time. We used a representative RIE taken from the Upp1 gene fused to GFP and isolated cells at different time-points during reprogramming and found that they have unique developmental capacities as they are reprogrammed with high efficiency due to their distinct molecular signatures. In conclusion, our experiments have led to the development of an unbiased method to identify and isolate reprogrammable cells in real time by exploiting the functional dynamics of OSKM, which can be used as efficient reprogramming biomarkers.
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Project description:Cellular reprogramming of somatic cells towards induced pluripotency is a multistep stochastic process mediated by the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM), which orchestrate global epigenetic and transcriptional changes. We performed a large-scale analysis of integrated ChIP-seq, ATAC-seq and RNA-seq data and revealed the spatiotemporal highly dynamic pattern of OSKM DNA binding during reprogramming. We found that OSKM show distinct temporal patterns of binding to different classes of pluripotency-related enhancers. Genes involved in reprogramming are regulated by the coordinated activity of multiple enhancers, which are sequentially bound by OSKM for strict transcriptional control. Based on these findings, we developed an unbiased approach to identify Reprogramming-Inducible Enhancers (RIEs), constructed enhancer-traps and isolated cells undergoing reprogramming in real time. We used a representative RIE taken from the Upp1 gene fused to GFP and isolated cells at different time-points during reprogramming and found that they have unique developmental capacities as they are reprogrammed with high efficiency due to their distinct molecular signatures. In conclusion, our experiments have led to the development of an unbiased method to identify and isolate reprogrammable cells in real time by exploiting the functional dynamics of OSKM, which can be used as efficient reprogramming biomarkers.
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Project description:Cellular reprogramming of somatic cells towards induced pluripotency is a multistep stochastic process mediated by the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM), which orchestrate global epigenetic and transcriptional changes. We performed a large-scale analysis of integrated ChIP-seq, ATAC-seq and RNA-seq data and revealed the spatiotemporal highly dynamic pattern of OSKM DNA binding during reprogramming. We found that OSKM show distinct temporal patterns of binding to different classes of pluripotency-related enhancers. Genes involved in reprogramming are regulated by the coordinated activity of multiple enhancers, which are sequentially bound by OSKM for strict transcriptional control. Based on these findings, we developed an unbiased approach to identify Reprogramming-Inducible Enhancers (RIEs), constructed enhancer-traps and isolated cells undergoing reprogramming in real time. We used a representative RIE taken from the Upp1 gene fused to GFP and isolated cells at different time-points during reprogramming and found that they have unique developmental capacities as they are reprogrammed with high efficiency due to their distinct molecular signatures. In conclusion, our experiments have led to the development of an unbiased method to identify and isolate reprogrammable cells in real time by exploiting the functional dynamics of OSKM, which can be used as efficient reprogramming biomarkers.
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Project description: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.
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