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DNA and chromatin modification networks distinguish stem cell pluripotent ground states.

ABSTRACT: Pluripotent stem cells are capable of differentiating into all cell types of the body and therefore hold tremendous promise for regenerative medicine. Despite their widespread use in laboratories across the world, a detailed understanding of the molecular mechanisms that regulate the pluripotent state is currently lacking. Mouse embryonic (mESC) and epiblast (mEpiSC) stem cells are two closely related classes of pluripotent stem cells, derived from distinct embryonic tissues. Although both mESC and mEpiSC are pluripotent, these cell types show important differences in their properties suggesting distinct pluripotent ground states. To understand the molecular basis of pluripotency, we analyzed the nuclear proteomes of mESCs and mEpiSCs to identify protein networks that regulate their respective pluripotent states. Our study used label-free LC-MS/MS to identify and quantify 1597 proteins in embryonic and epiblast stem cell nuclei. Immunoblotting of a selected protein subset was used to confirm that key components of chromatin regulatory networks are differentially expressed in mESCs and mEpiSCs. Specifically, we identify differential expression of DNA methylation, ATP-dependent chromatin remodeling and nucleosome remodeling networks in mESC and mEpiSC nuclei. This study is the first comparative study of protein networks in cells representing the two distinct, pluripotent states, and points to the importance of DNA and chromatin modification processes in regulating pluripotency. In addition, by integrating our data with existing pluripotency networks, we provide detailed maps of protein networks that regulate pluripotency that will further both the fundamental understanding of pluripotency as well as efforts to reliably control the differentiation of these cells into functional cell fates.

SUBMITTER: Song J

PROVIDER: S-EPMC3494154 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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DNA and chromatin modification networks distinguish stem cell pluripotent ground states.

Song Jing J Saha Sudipto S Gokulrangan Giridharan G Tesar Paul J PJ Ewing Rob M RM

Molecular & cellular proteomics : MCP 20120722 10

Pluripotent stem cells are capable of differentiating into all cell types of the body and therefore hold tremendous promise for regenerative medicine. Despite their widespread use in laboratories across the world, a detailed understanding of the molecular mechanisms that regulate the pluripotent state is currently lacking. Mouse embryonic (mESC) and epiblast (mEpiSC) stem cells are two closely related classes of pluripotent stem cells, derived from distinct embryonic tissues. Although both mESC ...[more]

PMID: 22822199

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Project description:RATIONALE:Regulatory DNA elements in the human genome play important roles in determining the transcriptional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is not well known how chromatin marks in regulatory DNA elements are modulated to establish cell type-specific gene expression in the human heart. OBJECTIVE:We aimed to decipher the cell type-specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression. METHODS AND RESULTS:We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing; n=84) in human endothelial cells (CD31+CD144+), cardiac progenitor cells (Sca-1+), fibroblasts (DDR2+), and their respective induced pluripotent stem cells. We uncovered 2 classes of regulatory DNA elements: class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas class II was enriched with H3K4me1 and H3K4me3 in a cell type-specific manner. Both class I and class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, class I promoters displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human induced pluripotent stem cells and somatic cells from the heart selected their preferential regulatory elements to maintain cell type-specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells and identified a few enhancers that could possibly regulate the cardiac-specific gene expression. CONCLUSIONS:Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering the epigenetic modulation of regulatory DNA elements that fine-tune spatiotemporal gene expression in human cardiac development and diseases.

Project description:Pluripotent stem cell lines derived from embryos of different stages have distinct pluripotent ground states, but similar levels of the transcription factor Oct4. Epiblast-derived pluripotent stem cells (EpiSCs), in contrast to embryonic stem (ES) cells, cannot form chimeras. We show that EpiSCs express lower levels of the transcription factors Sox2 and Klf4 than ES cells and have limited reprogramming potential, as shown by cell fusion. Sox2 overexpression dramatically increases the reprogramming potential, chimera formation, and germline contribution of EpiSCs. Therefore, although Oct4 is essential for reprogramming, the level of Sox2 defines both the reprogramming capability and the pluripotent ground states. RNA samples to be analyzed on microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 hrs of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was then hybridized for 18 hrs onto MouseRef-8 v2 expression BeadChips (Illumina) according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were hybridized as biological replicates. 12 sample types were analyzed, each of them in duplicate. ESCm: Mouse ESC male; ESCf: Mouse ESC OG2 female; F9 EC: F9 EC (mouse embryonic carcinoma cell); F9-Sox2: F9 EC (mouse embryonic carcinoma cell) overexpressing wild type Sox2; EpiSCf: Mouse EpiSC OG2 female; Epi-Sox2f: Mouse EpiSC Sox2 (OG2 female) overexpressing wild type Sox2; P19 EC: P19 EC (mouse embryonic carcinoma cell); P19-Sox2: P19 EC (mouse embryonic carcinoma cell) overexpressing wild type Sox2; EpiSCm: Mouse EpiSC (GOF18 male) (duplicates); EpiSox2mL2: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in condition EpiSC medium (CM); EpiSox2mE1: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in ESC medium (ESC like1); EpiSox2mE2: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in ESC medium (ESC like2).

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DNA and chromatin modification networks distinguish stem cell pluripotent ground states.

Publications

DNA and chromatin modification networks distinguish stem cell pluripotent ground states.

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