Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Defining a molecular roadmap of cellular reprogramming into iPS cells [mRNA profiling]

ABSTRACT: Factor induced reprogramming is a slow and inefficient process with only rare cells progressing towards induced pluripotent stem cells (iPSCs). Owing to these restraints, mechanistic studies have been limited to analyses of heterogeneous bulk populations undergoing reprogramming and partially reprogrammed cell lines. Here, by combining surface markers (Thy1, SSEA1) and an Oct4-GFP fluorescent reporter allele, we analyzed defined intermediate cell populations poised to becoming iPSCs at the transcriptional and epigenetic levels using genome-wide and single cell technologies. We found that factor-induced reprogramming elicits two discernible transcriptional waves that are characterized by the initial extinction of the somatic gene expression program and the concomitant acquisition of an ESC-like proliferative and metabolic state, followed by the activation of an embryonic pluripotent state primed for differentiation. The first wave is mostly driven by gene activation through c-Myc and gene repression by Klf4, whereas the second wave is a result of gradually activated Oct4/Sox2 targets in cooperation with Klf4 targets and other downstream regulators. While microRNA expression and enrichment for individual histone modifications (H3K4me3 or H3K27me3 enriched promoters) mirrored the observed biphasic transcriptional pattern, the establishment of bivalent domains (H3K4me3/H3K27me3 enriched promoters) occurred more gradually. In contrast, changes in DNA methylation took place predominantly at the end of reprogramming when cells assumed a stable pluripotent state. Cells that became refractory to reprogramming activated the first but failed to initiate the second transcriptional wave. However, introduction of additional copies of the reprogramming transgenes into these cells rescued their ability to form iPSCs, indicating that suboptimal transcription factor levels are a limiting factor for efficient iPSC formation. This integrative analysis allowed us to identify novel genes and microRNAs that enhance reprogramming and surface markers that further subdivide intermediate cell populations. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming and provide a valuable resource of molecules that may act as roadblocks during iPSC formation. Time series design with samples sorted into subpopulations according to surface markers.

ORGANISM(S): Mus musculus

SUBMITTER: Ben Wittner

PROVIDER: E-GEOD-42379 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

A molecular roadmap of reprogramming somatic cells into iPS cells.

Polo Jose M JM Anderssen Endre E Walsh Ryan M RM Schwarz Benjamin A BA Nefzger Christian M CM Lim Sue Mei SM Borkent Marti M Apostolou Effie E Alaei Sara S Cloutier Jennifer J Bar-Nur Ori O Cheloufi Sihem S Stadtfeld Matthias M Figueroa Maria Eugenia ME Robinton Daisy D Natesan Sridaran S Melnick Ari A Zhu Jinfang J Ramaswamy Sridhar S Hochedlinger Konrad K

Cell 20121201 7

Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcript ...[more]

PMID: 23260147

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Project description:Factor induced reprogramming is a slow and inefficient process with only rare cells progressing towards induced pluripotent stem cells (iPSCs). Owing to these restraints, mechanistic studies have been limited to analyses of heterogeneous bulk populations undergoing reprogramming and partially reprogrammed cell lines. Here, by combining surface markers (Thy1, SSEA1) and an Oct4-GFP fluorescent reporter allele, we analyzed defined intermediate cell populations poised to becoming iPSCs at the transcriptional and epigenetic levels using genome-wide and single cell technologies. We found that factor-induced reprogramming elicits two discernible transcriptional waves that are characterized by the initial extinction of the somatic gene expression program and the concomitant acquisition of an ESC-like proliferative and metabolic state, followed by the activation of an embryonic pluripotent state primed for differentiation. The first wave is mostly driven by gene activation through c-Myc and gene repression by Klf4, whereas the second wave is a result of gradually activated Oct4/Sox2 targets in cooperation with Klf4 targets and other downstream regulators. While microRNA expression and enrichment for individual histone modifications (H3K4me3 or H3K27me3 enriched promoters) mirrored the observed biphasic transcriptional pattern, the establishment of bivalent domains (H3K4me3/H3K27me3 enriched promoters) occurred more gradually. In contrast, changes in DNA methylation took place predominantly at the end of reprogramming when cells assumed a stable pluripotent state. Cells that became refractory to reprogramming activated the first but failed to initiate the second transcriptional wave. However, introduction of additional copies of the reprogramming transgenes into these cells rescued their ability to form iPSCs, indicating that suboptimal transcription factor levels are a limiting factor for efficient iPSC formation. This integrative analysis allowed us to identify novel genes and microRNAs that enhance reprogramming and surface markers that further subdivide intermediate cell populations. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming and provide a valuable resource of molecules that may act as roadblocks during iPSC formation. Time series design with samples sorted into subpopulations according to surface markers. 18 Samples total

Project description:4C-Seq has proven to be a powerful technique to identify genome-wide interactions with a single locus of interest (or "bait") that can be important for gene regulation. However, analysis of 4C-Seq data is complicated by the many biases inherent to the technique. An important consideration when dealing with 4C-Seq data is the differences in resolution of signal across the genome that result from differences in 3D distance separation from the bait. This leads to the highest signal in the region immediately surrounding the bait and increasingly lower signals in far-cis and trans. Another important aspect of 4C-Seq experiments is the resolution, which is greatly influenced by the choice of restriction enzyme and the frequency at which it can cut the genome. Thus, it is important that a 4C-Seq analysis method is flexible enough to analyze data generated using different enzymes and to identify interactions across the entire genome. Current methods for 4C-Seq analysis only identify interactions in regions near the bait or in regions located in far-cis and trans, but no method comprehensively analyzes 4C signals of different length scales. In addition, some methods also fail in experiments where chromatin fragments are generated using frequent cutter restriction enzymes. Here, we describe 4C-ker, a Hidden-Markov Model based pipeline that identifies regions throughout the genome that interact with the 4C bait locus. In addition, we incorporate methods for the identification of differential interactions in multiple 4C-seq datasets collected from different genotypes or experimental conditions. Adaptive window sizes are used to correct for differences in signal coverage in near-bait regions, far-cis and trans chromosomes. Using several datasets, we demonstrate that 4C-ker outperforms all existing 4C-Seq pipelines in its ability to reproducibly identify interaction domains at all genomic ranges with different resolution enzymes. 4C-Seq experiments from Igh and Cd83 bait in activated B cells and Tcrb (Eb) bait in double negative T cells and immature B cells. RNA-Seq and ATAC-Seq experiments in DN and Immature B cells.

Dataset Information

Defining a molecular roadmap of cellular reprogramming into iPS cells [mRNA profiling]

Publications

A molecular roadmap of reprogramming somatic cells into iPS cells.

Similar Datasets

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