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Comparative Study
. 2012 Oct;11(10):1036-47.
doi: 10.1074/mcp.M111.011114. Epub 2012 Jul 22.

DNA and chromatin modification networks distinguish stem cell pluripotent ground states

Jing Song  1 , Sudipto Saha, Giridharan Gokulrangan, Paul J Tesar, Rob M Ewing
Affiliations
Comparative Study

DNA and chromatin modification networks distinguish stem cell pluripotent ground states

Jing Song et al. Mol Cell Proteomics. 2012 Oct.

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.

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Figures

Fig. 1.
Fig. 1.
Experimental workflow. Sample preparation and comparative microarray and proteomics analysis of mouse embryonic stem cells (mESC) and mouse epiblast stem cells (mEpiSC) by LC-MS/MS.
Fig. 2.
Fig. 2.
Biological trends in mESC and mEpiSC nuclear proteomes. A, Top functional categories represented in combined mESC and mEpiSC data sets (using all identified proteins) showing significant representation of proteins involved in embryogenesis and development. B, Proteins with significantly increased expression (p < 0.1; Student's t test) in either mESC and mEpiSC show significant differences in representation of several functional categories. C, Analysis of mutant phenotypes of genes corresponding to mESC and mEpiSC proteins (proteins differentially expressed in mESCs or mEpiSCs with p < 0.1; Student's t test). mESC proteins represent genes with embryonic lethal phenotypes at earlier developmental stages than mEpiSC proteins. Phenotypes are arranged according to developmental stage: (1) Prenatal lethality, (2) embryonic lethality before implantation (E0-E4.5), (3) embryonic lethality before somite formation (E4.5-E8), (4) embryonic lethality before turning of embryo (E8-E9), (5) embryonic lethality during organogenesis (E19-E14), (6) postnatal lethality, (7) absent somites, (8) open neural tube.
Fig. 3.
Fig. 3.
Intersecting proteomics studies with existing pluripotency networks. A, Overall overlap of all identified proteins in union set of proteins identified from gel-free and gel-based proteomics studies (“union set all” - 2 peptides; Scaffold probability ≥ 99%) with PluriNet and PluriNetWork. B, The intersection between proteomics data and PluriNet (analyzed using Ingenuity Pathways Analysis) - 125 proteins. C, The intersection between proteomics data and PluriNetwork - 94 proteins. Each node in (B) and (C) represents a protein identified in mESC or mEpiSC samples (red shaded nodes indicate mESC>mEpiSC protein expression with p < 0.1 (Student's t test), green shaded indicate mEpiSC>mESC protein expression with p < 0.1 (Student's t test) and gray shaded nodes indicate identified protein in either mESC or mEpiSC but without statistically significant quantitative difference.
Fig. 4.
Fig. 4.
Selected mESC- and mEpiSC-associated protein networks. Ingenuity Pathway Analysis (IPA) reveals differentially expressed subnetworks including (A) Polycomb-group (PcG), DNA methyltransferase, Nucleosome remodeling/histone deacetylase (NuRD), OCT4-SOX2-NANOG and SWI/SNF BAF complexes, B, Protein networks functioning in DNA replication and Genome surveillance/DNA repair, (C) Transcription factor complexes, and (D) Homeobox protein complex. All proteins shown were identified as being either more abundant in mESC (red nodes) or more abundant in mEpiSC (green nodes) at p < 0.1 (Student's t test).
Fig. 5.
Fig. 5.
Immunoblot analyses of proteins with significantly different mESC/mEpiSC expression. A, Immunoblot analyses of selected DNA methyltransferase and Swi/Snf-related Baf complex components. Nuclear protein extracts (20 μg) from mESC and mEpiSC were loaded on 1D SDS-PAGE gel and transferred to membrane followed by immunoblotting using native antibodies. Mr refers to standard protein marker and α-tubulin was used as a loading control. B, Immunoblot quantification of two replicates of each protein verified. Bands for same protein in two replicated immunoblots were measured for intensity and the mean log ratio of mESC/mEpiSC fold change (the height of column) and the standard deviation (the error bar) between two blots were plotted.
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