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First published online July 19, 2007
Stem Cells Vol. 25 No. 10 October 2007, pp. 2567 -2574
doi:10.1634/stemcells.2007-0131; www.StemCells.com
© 2007 AlphaMed Press

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EMBRYONIC STEM CELLS

Global Epiproteomic Signatures Distinguish Embryonic Stem Cells from Differentiated Cells

Bo Daia,b, Theodore P. Rasmussena,b,c

aCenter for Regenerative Biology, University of Connecticut, Storrs, Connecticut, USA;
bDepartment of Animal Science, University of Connecticut, Storrs, Connecticut, USA;
cDepartment of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA

Key Words. Embryonic stem cells • Chromatin • Epigenetics • Pluripotency • Proteomics • Epiproteomics • Nucleosome Post-translational modification

Correspondence: Theodore Rasmussen, Ph.D., Center for Regenerative Biology, University of Connecticut, Storrs, Connecticut 06269-4243, USA. Telephone: 860-486-8339; Fax: 860-486-8809; e-mail: theodore.rasmussen{at}uconn.edu

Received on February 19, 2007; accepted for publication on July 6, 2007.

First published online in STEM CELLS EXPRESS  July 19, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Complex organisms contain a variety of distinct cell types but only a single genome. Therefore, cellular identity must be specified by the developmentally regulated expression of a subset of genes from an otherwise static genome. In mammals, genomic DNA is modified by cytosine methylation, resulting in a pattern that is distinctive for each cell type (the epigenome). Because nucleosomal histones are subject to a wide variety of post-translational modifications (PTMs), we reasoned that an analogous "epiproteome" might exist that could also be correlated with cellular identity. Here, we show that the quantitative evaluation of nucleosome PTMs yields epiproteomic signatures that are useful for the investigation of stem cell differentiation, chromatin function, cellular identity, and epigenetic responses to pharmacologic agents. We have developed a novel enzyme-linked immunosorbent assay-based method for the quantitative evaluation of the steady-state levels of PTMs and histone variants in preparations of native intact nucleosomes. We show that epiproteomic responses to the histone deacetylase inhibitor trichostatin A trigger changes in histone methylation as well as acetylation, and that the epiproteomic responses differ between mouse embryonic stem cells and mouse embryonic fibroblasts (MEFs). ESCs subjected to retinoic acid-induced differentiation contain reconfigured nucleosomes that include increased content of the histone variant macroH2A and other changes. Furthermore, ESCs can be distinguished from embryonal carcinoma cells and MEFs based purely on their epiproteomic signatures. These results indicate that epiproteomic nucleosomal signatures are useful for the investigation of stem cell identity and differentiation, nuclear reprogramming, epigenetic regulation, chromatin dynamics, and assays for compounds with epigenetic activities.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Many biological processes of keen interest are profoundly controlled or influenced by epigenetics. In broad terms, epigenetics is concerned with influences on gene expression that are independent of DNA sequence per se. Complex multicellular organisms contain a plethora of specialized cell types that are assembled into three-dimensional configurations, thus forming tissues and organs. Yet this remarkable complexity and organization must arise from the developmentally regulated proliferation of progenitor cells that arise from a single totipotent zygote. Although humans contain over 200 distinct cell types, all somatic cells contain essentially the same content of genes. Therefore, the specification of cell type must be controlled by the regulated expression (or silencing) of sets of genes present within an otherwise fixed genome, suggesting a major role for epigenetic mechanisms in developmental processes. For these reasons, epigenetics has been an area of intense interest in the field of stem cell research, since directed stem cell differentiation procedures attempt to recapitulate development in vitro. Recent successes demonstrate that terminally differentiated mammalian cells can be reprogrammed to a pluripotent state using a variety of approaches [1]. Mammalian reprogramming was first demonstrated by transfer of differentiated nuclei into oocytes from which the nuclear DNA was removed [2, 3] but has now been shown to occur by many other mechanisms such as fusion of somatic cells with ESCs [1, 4], introduction of transgenes with pluripotency function into somatic cells [5], and the use of reprogramming extracts and cocktails [6]. All of these reprogramming processes are thought to involve epigenetic reconfigurations. Epigenetics is also of major importance in disease etiologies. For instance, aberrant silencing of tumor-suppressor genes is thought to be a common mechanism leading to neoplastic transformations, and alterations of centric heterochromatin can lead to chromosomal instability. Finally, the loss of epigenetic checks on gene expression is thought to be an important mechanism of aging.

Though the importance of epigenetics is now widely appreciated, few methods exist that can expediently assess the overall epigenetic status of cells. Nucleosomes are the smallest macromolecular complex that can be isolated from cells in which histones are preserved in their native modification states in contact with cognate genomic DNA. Indeed, much evidence now exists to support the proposition that the transcriptional status of DNA that is associated with a given nucleosome is influenced by the modification status of the histones within that same nucleosome [7–9]. On a molecular level, epigenetic regulation is thought to be mediated by the placement of a wide variety of small molecule adducts (acetylations, methylations, phosphorylations, ubiquitinations, etc.) onto chromatin [10]. Many eukaryotic genomes are modified by methylation of genomic DNA. In mammals, each cell type contains a distinctive distribution of 5-methylcytosine present within CpG dinucleotides. Because the pattern of methylated CpG dinucleotides differs from cell type to cell type, each cell is thought to contain an idiosyncratic epigenome that is defined by a distribution of CpG methylation events. We reasoned that an analogous "epiproteome" might exist that is composed of unique patterns of post-translational modifications (PTMs) present upon histones embedded within intact nucleosomes. Since the nucleosome can reasonably be considered the fundamental unit of chromatin, we reasoned that nucleosomes could serve as informative antigens in enzyme-linked immunosorbent assays (ELISAs). Furthermore, since the modification status of nucleosomes is part of a molecular-epigenetic mechanism that can influence the transcriptional status of associated genomic DNA, we surmised that methods designed to interrogate nucleosomal PTMs might form the basis of novel assays that could serve to indicate the global epigenetic status of cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cell Culture and Nucleosome Preparations
J1 (129Sv/Jae) and V6.5 (129Sv/Jae x C57BL/6) murine ESCs were grown in Dulbecco's modified Eagle's medium (DMEM)-based medium supplemented with 15% fetal bovine serum (FBS) and 500 U/ml leukemia inhibitory factor (LIF). Mouse embryonic fibroblasts (MEFs) (BALB/c x 129Sv/Jae) were grown in DMEM-based medium supplemented with 10% FBS. MEFs were grown in triplicate cultures with or without 10 ng/ml trichostatin A (TSA) for 48 hours. Feeder-free ESCs were cultured with or without 10 ng/ml TSA for 24 hours, after which time they had reached near confluence, and were then harvested for nucleosome extraction. P19 embryonal carcinoma (EC) cells (#CRL-1825; American Type Culture Collection, Manassas, VA, http://www.atcc.org) were grown in the same medium as MEFs described above.

Nucleosomes were prepared essentially as described [11]. Briefly, 108 cells were disrupted with 20 strokes of a type-B pestle in a Dounce homogenizer. (This large preparation of nucleosomes allowed us to prepare a series of identically loaded plates for subsequent analysis. In practice, many fewer cells could be used for analyses of more limited scope.) Nuclei were separated from cytoplasm and cellular debris by centrifugation through a 30% (wt/vol) sucrose cushion. Chromatin was digested in situ by adding 2 U of micrococcal nuclease (MNase; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) for 12 minutes at 37°C then stopped by adding Na-EDTA to a 10-mM concentration. Nucleosomes were extracted from nuclei with two 1-hour hypotonic incubations on ice, and the resulting nucleosome-rich supernatants were pooled.

Nucleosome ELISA
Serial twofold dilutions of mononucleosomes were prepared in coating buffer (32 mM Na2CO3, 68 mM NaHCO3) and added to wells of Nunc (Rochester, NY, http://www.nuncbrand.com) MaxiSorp plates overnight at 4°C. The plates were then washed four times with 200 microliters per well phosphate-buffered saline (PBS)/0.5% Tween 20 at room temperature (RT) for a total of 10 minutes then blocked in 100 microliters per well PBS with 0.05% Tween 20/5% bovine serum albumin (BSA) for 1 hour at RT. Blocking buffer was then aspirated, and 50 microliters per well of the 1° antibody (Ab) in PBS with 0.05% Tween 20/5% BSA were added and incubated at RT for 1 hour. (All PTMs are described by Brno nomenclature [12], and anti-PTM Abs are described in supplemental online Table 1.) After four 2.5-minute washes, 100 microliters per well horseradish peroxidase (HRP)-conjugated 2° Abs (diluted 1:5,000 in PBS/0.05% Tween 20/5% BSA) were added and incubated for 1 hour at RT. Wells were again washed four times for 2.5 minutes in PBS/Tween 20, then aspirated and developed by adding 50 microliters per well 1-Step Ultra 3,3',5,5'-tetramethyl benzidine (TMB)-ELISA reagent (catalog number 34028; Pierce, Rockford, IL, http://www.piercenet.com) for 10 minutes at RT. Development was stopped by adding 50 microliters per well of 2 N H2SO4, and the plates were read on a Bio-Rad (Hercules, CA, http://www.bio-rad.com) model 680 Microplate Reader at 450 nm.

Statistical Methods
All assays were performed on nucleosomes obtained from at least three independent cultures of each cell line, and each nucleosome sample was loaded in triplicate dilutions (technical replicates) on ELISA plates. Raw absorbance values in the linear response range of assays were collected from each technical replicate, and these values were corrected by subtracting background (obtained from control wells with no nucleosomes). The background-corrected values were then averaged. PTM values were then normalized for nucleosome loading by dividing them by loading-controlled values obtained from identically prepared ELISA plates probed with an anti-histone H2A Ab, which detects nucleosomes independently of their modification status. Finally, values corresponding to three normalized biological replicates (each corresponding to nucleosomes from three independent cultures) were processed to determine the overall mean and standard deviation of nucleosome ELISA (NU-ELISA) PTM values for each culture. All error bars are standard deviations.

Retinoic Acid-Induced Differentiation
J1 ESCs were grown on mitotically arrested feeder cells. After ESCs reached 60%–70% confluency, cultures were trypsinized and passed through an 18.5-gauge needle, and feeders were panned by adherence to ungelatinized 10-cm dishes for 30 minutes. ESCs were plated on six 145-mm plates coated with 0.2% gelatin in differentiation medium (regular embryonic stem [ES] medium lacking LIF supplemented with 10–7 M all-trans retinoic acid [RA]) for 8 days [13].

Western Blot Analysis
Nucleosomes were prepared from J1 ESCs as described above and subjected to Western blot analysis. Nucleosomal proteins were separated by SDS-polyacrylamide gel electrophoresis on 12% polyacrylamide gels and then transferred to Hybond ECL membrane at 10 V overnight. The membranes were incubated with PTM-specific 1° Abs recognizing H3K4me3, H3K9me3, H4K12ac, and H4K20me3 at the same dilutions used for NU-ELISA (supplemental online Table 1). Membranes were then incubated with HRP-conjugated 2° Abs (Pierce) and detected by the ECL Detection Kit (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com) according to the manufacturer's instructions.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
ELISA-Based Analysis of Post-Translational Modifications of Total Cellular Nucleosomes from ESCs and MEFs
We isolated chromatin from mammalian cells using established methods that yield chromatin preparations rich in native mononucleosomes [11]. To do this, we disrupted cells by Dounce homogenization then separated nuclei from cytoplasm and cellular debris by centrifugation through a sucrose cushion. Chromatin was then digested in situ by treatment of isolated nuclei with MNase. MNase activity was then quenched by adding Na-EDTA, and chromatin consisting mostly of mononucleosomes was driven from nuclei by two sequential extractions of soluble nuclear material under hypotonic conditions. To monitor the quality of chromatin preparations, we retained samples at each stage of the preparation and assessed them for their content of DNA (Fig. 1A) and protein (Fig. 1B). We found that the vast majority of total cellular chromatin could be isolated in the supernatants (S1 and S2) generated by centrifugation after each extraction. Results showed that approximately 75% of the chromatin was extracted in the form of mononucleosomes, with a smaller portion of dinucleosomes, and minor amounts of polynucleosomes of higher order. Free nucleosomes are not subject to steric hindrance normally imposed by higher-order chromatin structure. Therefore, the use of free nucleosomes allowed us to assess total chromatin obtained from different cell types that vary in their content of compacted chromatin. Our preparations of soluble chromatin contained abundant content of low molecular weight proteins that resolved as bands diagnostic of the four histones, H3, H2A, H2B, and H4 (Fig. 1B). Nucleosome-rich supernatants S1 and S2 were pooled for further analysis.


Figure 1
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  		Figure 1. Chromatin preparation. (A): DNA content over the course of a representative chromatin extraction is shown. Lanes are as follows: mouse embryonic fibroblasts, residual cytoplasm after Dounce homogenization, nuclei, first supernatant after micrococcal nuclease digestion in situ, residual pellet, pooled supernatants after first and second chromatin extractions, and final residual pellet. (B): Protein content from the same samples shown in (A) resolved by SDS-polyacrylamide gel electrophoresis. Abbreviations: bp, base pairs; C, cytoplasm; MEF, mouse embryonic fibroblast; N, nuclei; P, pellet; S, supernatant.

 
We wished to see whether our preparations containing native, intact mononucleosomes could be subjected to ELISA assays to quantitatively determine endogenous levels of histone PTMs. In order to develop and optimize this method (hereafter called NU-ELISA), we initially utilized chromatin samples with differences in histone PTM content that were known a priori. To produce such samples, we treated J1 ESCs and MEFs with TSA, a potent inhibitor of histone deacetylase activity [14]. We also mock-treated both cell types to yield control chromatin samples with normal acetylation levels for purposes of comparison. We subjected chromatin from TSA-treated and mock-treated cells to ELISA and optimized assays that proved to be both sensitive and quantitative. We first quantitated chromatin preparations by spectrophotometry and diluted samples to equal concentrations based on absorbance readings. We then prepared twofold dilution series of chromatin from TSA-treated and mock-treated ESCs and MEFs, which were then loaded into 96-well microtiter plates. We found it useful to prepare a series of identical microtiter plates that were identically loaded with the four types of chromatin. This approach allowed us to interrogate the status of PTMs by applying a series of antibodies to identical plates. We desired to develop an approach to internally control the content of nucleosomes deposited within each well. We reasoned that the absolute amount of nucleosomes within each well could be quantitated by probing the wells with an Ab that generally binds to histones regardless of their modification status. We probed one plate from each series with an anti-H2A Ab (which binds to H2A independently of its modification status) to provide a precise indication of the nucleosome content present within each well. Bound 1° Ab content was then detected with a HRP-conjugated 2° Ab and colorimetric detection using the HRP substrate TMB (Fig. 2A). Signal intensity within each well was then quantitated by measuring the absorbance at 450 nm within each well using a Bio-Rad model 680 Microtiter Plate Reader. This approach (Fig. 2B) allowed us to precisely determine the total amount of chromatin from TSA-treated and mock-treated MEFs and ESCs that was loaded into individual wells based on H2A content (Fig. 2C, 2D). We then probed a second, identically prepared plate with a 1° Ab that is specific for histone H3 acetylated at lysine 18 (H3K18ac) and detected the signal (Fig. 2E). We corrected the raw H3K18ac readings for variations in nucleosome loading as judged by H2A reactivity so that samples from different sources could be directly compared (Fig. 2E, 2F). We readily detected the predicted increase in H3K18ac in TSA-treated samples.


Figure 2
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  		Figure 2. Nucleosome enzyme-linked immunosorbent assay (NU-ELISA). (A): Schematic representation of NU-ELISA. Quantitation of histone PTM content is based on the specificity of the 1° antibody (Ab). (B): Flow chart summarizing NU-ELISA data analysis. (C): Representative assay performed on serially diluted nucleosomes from MEFs and ESCs that were treated with the histone deacetylase inhibitor trichostatin A (+TSA) or mock-treated (–TSA) probed with 1° Ab with general specificity for histone H2A content. Chromatin was prepared in triplicate from cultures subjected to each treatment, and serial dilutions of each sample were coated in vertical columns. A graphical representation of A450 readings of the plate is provided. (D): Relative amounts of chromatin from TSA-treated and untreated MEFs and ESCs as judged by H2A content. (E): NU-ELISA for H3K18ac performed on replicate ELISA plate with identical content of that in (C). A graph of relative H3K18ac PTM content is shown. (F): Relative content of H3K18ac PTM in TSA-treated and untreated MEFs and ESCs after adjusting for chromatin loading based on H2A reactivity. Abbreviations: E, ESC; ES, embryonic stem; HRP, horseradish peroxidase; M, mouse embryonic fibroblast; MEF, mouse embryonic fibroblast; PTM, post-translational modification; Rel, relative; TMB, 3,3',5,5'-tetramethyl benzidine; TSA, trichostatin A.

 
We performed side-by-side comparisons of NU-ELISA and Western blotting to directly compare their thresholds of detection. Nucleosomes from J1 ES cells were serially diluted then subjected to Western analysis (Fig. 3A), and NU-ELISA (Fig. 3B) Abs specific for H3K4me3, H3K9me3, H4K12ac, and H4K20me were used at identical dilutions in both assays. The threshold of detection by Western blotting ranged from 1.25 µg to 10 µg for these Abs (Fig. 3A). We conservatively designated the threshold of detection by NU-ELISA as three standard deviations above background. The thresholds of detection by NU-ELISA ranged from 16–125 ng using the same Ab dilutions (Fig. 3B). Thus, we estimate that NU-ELISA is from 200–3,200 times more sensitive than Western blotting.


Figure 3
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  		Figure 3. Side-by-side comparison of Western analysis and NU-ELISA. A single preparation of nucleosomes from ESCs was subjected to Western analysis and NU-ELISA. (A): Western blot assays performed on serially diluted nucleosomes probed with antibodies for the indicated histone post-translational modifications. (B): NU-ELISA assays of the same material used in (A). (Micrograms of chromatin loaded per lane or well are indicated. Note that higher dilutions are used for NU-ELISA, indicating greater sensitivity.) Abbreviation: NU-ELISA, nucleosome enzyme-linked immunosorbent assay.

 
Together, the results indicated that the assay was highly sensitive, quantitative, and yielded very small variances in estimates of PTM content for each sample. Our results indicate that the center of the linear range of detection occurs when approximately 25 ng of total cellular chromatin is loaded into an individual well. This corresponds to the amount of chromatin that can be extracted from approximately 1,250 cells. We found that the limit of detection for nucleosomal modifications of average abundance occurred at 3–6 ng of total cellular nucleosomes per well, corresponding to chromatin from approximately 150–300 cells.

Differential Responses of ESCs and MEFs to TSA-Induced Hyperacetylation of Histones
The pluripotent nature of ESCs and their ability to differentiate are controlled in part by epigenetic mechanisms that involve the management of histone PTMs [15, 16]. We therefore were interested in whether histone PTM metabolism differed between pluripotent ESCs and differentiated cells (MEFs). To explore this question, we prepared additional series of identically loaded plates containing TSA-treated and untreated chromatin extracted from ESCs and MEFs. We then probed these plates with a battery of Abs specific for a variety of histone PTMs (supplemental online Table 1). As before, we corrected all PTM values for relative differences in total chromatin content as judged by general H2A reactivity. We found a robust increase in H3 acetylation at K9 and K18 in TSA-treated chromatin as compared with mock-treated chromatin from both cell types (Fig. 4A; supplemental online Table 2). In addition, an Ab that simultaneously recognizes H3 that is dually acetylated at K9 and K14 also indicated increased acetylation levels in the chromatin from TSA-treated cells. We found that all acetylation increases were of comparable magnitude in comparisons restricted by cell type (approximately sixfold in ESCs and two- to threefold in MEFs).


Figure 4
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  		Figure 4. Response of specific histone PTMs to TSA-induced hyperacetylation. Nucleosome enzyme-linked immunosorbent assay (NU-ELISA) results from chromatin extracted from TSA-treated and untreated J1 ESCs and MEFs using 1° antibodies specific for the indicated PTMs are shown. (A): Quantitative responses of specific acetylated lysines to TSA-induced hyperacetylation are shown. Ratios were derived by dividing NU-ELISA results of TSA-treated chromatin by results from mock-treated cells. (B): Responses of specific histone methylation sites to TSA-treatment in J1 ESCs and MEFs. In (A, B), the dotted line indicates the expected ratio for no effect of TSA treatment (a ratio of 1:1, treated/untreated). Abbreviations: ES, embryonic stem; MEF, mouse embryonic fibroblast; PTM, post-translational modification; TSA, trichostatin A.

 
We also wanted to see if NU-ELISA could detect differences in histone methylation in response to TSA treatment. We probed additional plates (from the same plate series used in Fig. 4A) with a panel of anti-methyl-lysine histone Abs (Fig. 4B). NU-ELISA analysis indicated that mono-, di-, and trimethylation of H3K4 (H3K4me1, H3K4me2, H3K4me3) were increased in both ESCs and MEFs after TSA treatment (Fig. 4B), although the increase of H3K4me3 appeared to be of significantly lesser magnitude in ESCs as compared with MEFs. In contrast, methylation levels for H3K9 were not greatly affected by TSA treatment in either cell type except for a modest reduction of H3K9me2 in ESCs. Interestingly, we found that H3K27me2 was increased approximately twofold in TSA-treated MEFs, but a similar effect was not observed in ESCs. However, both cell types contained increased levels of H4K20me3 in response to TSA treatment. The results indicate that histone methylation responses to TSA treatment vary considerably since certain lysine residues respond to TSA with increased methylation and some with decreased methylation, whereas others are relatively insensitive. Furthermore, the results indicate that TSA-induced histone methylation responses are also dependent upon the identity of the treated cells. These findings are consistent with other reports that describe "crosstalk" between histone acetylation and methylation in which histone methylation can be perturbed by alteration of histone acetylation [17, 18].

Global Changes in Histone Modifications in ESCs Subjected to RA-Induced Differentiation
RA is a teratogen and morphogen that can affect the expression of Hox [19] and Cdx [20] genes in vivo, findings that implicate RA in the establishment of anterior/posterior patterning of developing embryos. RA is also a potent inducer of differentiation of ESCs and EC cells in vitro [21, 22]. Exposure of ESCs to RA leads to the semisynchronous production of differentiated cells [22]. We hypothesized that if cellular identity is correlated with distinct global configurations of histone PTMs, then NU-ELISA signatures of undifferentiated and RA-differentiated ESCs might differ.

We subjected J1 ESCs to RA-induced differentiation. After 2 days, we observed morphological changes in the cells, and by 8 days, the cells had adopted completely new morphologies (Fig. 5). Previous studies of ESCs found that 8 days of RA exposure led to the production of cultures consisting entirely of differentiated cells that had undergone synchronous, step-wise chromatin remodelling as judged by the establishment of stably inactivated X chromosomes [13, 22, 23]. We extracted nucleosomes from undifferentiated and 8-day RA-differentiated J1 ESCs and analyzed these by NU-ELISA using 14 Abs. We found that H3K4me2 was decreased approximately twofold in RA-differentiated ESCs as compared with undifferentiated ESCs, whereas H3K9me1 and H3K79me2 were modestly increased (Fig. 5A). We also found that levels of the heterochromatin-specific histone variant macroH2A were dramatically increased in RA-differentiated cells using an affinity purified Ab [24]. Collectively, these results show that RA-differentiated cells adopt a nucleosomal signature defined by altered levels of selected histone PTMs and macroH2A.


Figure 5
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  		Figure 5. Response of histone PTMs and macroH2A in nucleosomes from RA-differentiated ESCs. (A): Nucleosome enzyme-linked immunosorbent assay (NU-ELISA) results from chromatin extracted from RA-treated J1 ESCs at day 8 and untreated J1 ESCs using 1° antibodies specific for the indicated PTMs are shown. Ratios were calculated by dividing NU-ELISA results from differentiated ESCs by NU-ELISA results from undifferentiated ESCs. (The dotted line indicates no effect, corresponding to a ratio of 1:1, differentiated/undifferentiated.) *, p ≤ .05, significantly different from the no-effect value. (B): Colony morphologies of undifferentiated J1 ESCs and J1 ESCs after 8 days of RA-induced differentiation. Abbreviations: ES, embryonic stem; PTM, post-translational modification; RA, retinoic acid.

 
Epiproteomic Signatures of Cellular Identity
The above results showed that global patterns of nucleosome modification and histone variant content change when cells are treated with TSA or RA. We therefore wondered whether cellular identity could be associated with unique epiproteomic signatures defined by the overall content of histone PTMs and histone variants in isolates of total cellular nucleosomes. To investigate this possibility, we performed comparative NU-ELISA analyses of two mouse ESC lines (J1 and V6.5), an EC line (P19), and MEFs (Fig. 6).


Figure 6
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  		Figure 6. Nucleosome enzyme-linked immunosorbent assay (NU-ELISA) profiles distinguish cellular identity. (A): Steady-state levels of 11 nucleosome PTMs on histone H3 were determined by NU-ELISA for two ESC lines (J1 and V6.5), P19 EC cells, and MEFs. All bars indicate steady-state levels of specific PTMs standardized to total H2A content. (B): Steady-state levels of two PTMs on histone H4 were determined by NU-ELISA for the same cell lines. (C): Relative levels of the histone variant macroH2A in nucleosomes from J1 ESCs, differentiated J1 ESCs (after 8 days of retinoic acid-induced differentiation), and MEFs. Abbreviations: EC, embryonal carcinoma; MEF, mouse embryonic fibroblast; PTM, post-translational modification.

 
As before, we prepared plates containing chromatin extracted from three independent cultures of each cell line and subjected each chromatin sample to triplicate assays for each Ab (resulting in three biological replicates, each consisting of three technical replicates) and normalized the content to H2A levels. We probed these plates with a variety of anti-PTM Abs and found that the two ESC lines, J1 and V6.5, were quite similar in their global PTM pattern (Fig. 6A, 6B; Table 1). EC cells exhibited some differences as compared with ESCs. P19 EC cells contained H3K4me1 at levels approximately 10-fold less than the ESCs, and there were modest increases in H3K9me1, H3K9me3, and H3K79me2 (Fig. 6A; Table 1). MEFs differed substantially in their steady-state content of certain nucleosomal PTMs (Fig. 6A, 6C; Table 1), whereas levels of H3K9ac were similar among all cell lines H3K9acK14ac, and H3K18ac were reduced in MEFs as compared with ESCs and EC cells (Fig. 6A; Table 1). We found higher steady-state levels of H3K9me2 and H3K9me3 and lower levels of H3K4me1 in MEFs as compared with ESCs (Fig. 6A; Table 1). Levels of the histone variant macroH2A were high in MEFs as compared with J1 ESCs, and the differentiation of ESCs led to the accumulation of increased amounts of macroH2A to levels similar to those found in MEFs (Fig. 6C). We conclude that epiproteomic signatures defined by the steady-state content of selected nucleosomal PTMs and macroH2A are sufficient to distinguish among ESCs, EC cells, and MEFs.


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  		Table 1. Epiproteomic profiles of cell lines

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
NU-ELISA provides an expedient means to assemble a composite epiproteomic signature by assay of PTM levels present within the total cellular pool of nucleosomes. We found that NU-ELISA analysis with 14 Abs specific for eight lysines within H3 and H4 N-terminal tail were sufficient to distinguish ESCs from EC cells and MEFs. The use of additional Abs would further refine these epiproteomic signatures. Nucleosomes are highly diverse in terms of the spectrum of possible small molecule modifications as compared with DNA. In addition, distinct PTMs may occur in a large number of combinations upon any single nucleosome, an observation that points to the existence of a remarkably complex epiproteome within eukaryotic cells, a system that likely impacts the regulated expression of almost every gene in all cell types. Thus, NU-ELISA may provide a useful way to assess this complexity and hasten investigations of the histone code hypothesis [8, 9].

Mass spectrometry approaches have also been of great utility for the investigation of histone PTMs [25, 26]. Our approach differs from mass spectrometry approaches in that NU-ELISA can be performed on relatively crude chromatin preparations containing intact nucleosomes without the need to perform proteolytic digests of purified histones and is also free from the need to prepare chemically derivatized histones. Unlike mass spectrometry approaches, each PTM must be assessed with an individual NU-ELISA assay. However, we feel that the relative simplicity of NU-ELISA with regard to chromatin preparatory steps may allow its use in a wide variety of laboratory settings in which comprehensive profiles of modifications are desired without extensive preparation and costly instrumentation. The association of nucleosome PTMs with cognate DNA sequence is necessary to complete an exhaustive picture of the epigenetically managed genome. The recent and widespread use of chromatin immunoprecipitation (ChIP) reflects the recognition of the importance of molecular-epigenetics to investigate gene regulation as it correlates to cellular identity. NU-ELISA results can serve as an excellent way to identify sensitive PTMs so as to inform the choice of Abs for use in ChIP experiments.

Our studies showed that MEFs and ESCs differ in their responses to inhibition of histone deacetylase activity and in their relative levels of nucleosome PTMs. These findings indicate that chromatin is configured differently in these two divergent cell types. We interrogated the behavior of the cellular chromatin system in these divergent cell types by exposing them to TSA to generally inhibit histone deacetylase activity. Remarkably, the responses to this epigenetic interrogation were quite distinct in these two cell types, suggesting that specialized regulatory pathways exist that intersect both histone acetylation and methylation pathways in these two cell types. Based on this simple observation, it seems likely that rules for nucleosome modification differ between these cell types. Indeed, a recent report showed that in ESCs, the chromatin of genes encoding key developmentally regulated transcription factors is configured to contain regions of H3K27me3 with smaller embedded regions of H3K4me3 in ESCs [27]. This modification state may predispose these genes for derepression upon differentiation. We find that H3K4me1, H3K4me2, and H3K4me3 increased in response to TSA treatment in MEFs, whereas H3K4me1 and H3K4me2, but not H3K4me3, increased due to treatment of ESCs with TSA. In addition, we found that H3K27me3 levels failed to respond to TSA treatment in ESCs but responded robustly in MEFs. Additionally, a recent report showed that TSA treatment can generally inhibit the ability of ESCs to undergo differentiation [28]. Based on these recent reports and our results, we suspect that subsets of nucleosome PTMs may be poised for rapid reconfiguration in ESCs as they undergo differentiation. This proposition is supported by the recent demonstration that the structure of ESC chromatin is especially relaxed and maintained in a state in which architectural proteins are loosely bound [16, 29]. In our analyses of RA-differentiated ESCs, we found that differentiation occurs in conjunction with dramatic changes in nucleosome configuration. Notably, we found that ESC differentiation led to nucleosomes with increased content of macroH2A (Fig. 5A). We also found high levels of macroH2A in nucleosomes from MEFs (Fig. 6C). A number of studies have implicated the histone variant macroH2A in transcriptional silencing and heterochromatin homeostasis [30–33]. These findings suggest a scenario in which differentiation of ESCs leads to the broad incorporation of macroH2A into chromatin.

Assessing the status of the epiproteome with methods such as NU-ELISA may advance several areas of intensive research. A great deal of effort is currently focused on the investigation of mechanisms of reprogramming, either through somatic cell nuclear transfer or through a variety of alternative methods. It is now clear that the nuclear genome of somatic cells can be reconfigured to a state of pluripotency in a matter of hours during the reprogramming process. Since successful reprogramming is equivalent to a rapid and massive epigenetic resetting to an early developmental state, assays for global epiproteomic reconfigurations will be needed to understand reprogramming on a mechanistic level. In addition, NU-ELISA may provide an excellent assay to screen pharmaceutical agents for epigenetic reactivity. In this study, we showed that NU-ELISA quantitatively detected changes in nucleosome PTM content induced by TSA and RA. Therefore, NU-ELISA should be generally useful to identify molecules with epigenetic modulatory activities. NU-ELISA may prove to be a useful tool to understand mechanisms of neoplastic transformation and tumor progression and to assist in the development of treatment modalities for cancer patients. Indeed, a recent report showed that patient-specific variation in global histone modification patterns as judged by immunohistochemistry of malignant prostate glandular cells can serve as a useful prognostic indicator [34]. The utilization of a highly quantitative method such as NU-ELISA to assess global nucleosome modifications may therefore lead to significant advances in cancer therapy. Furthermore, since epigenetic insult can lead to neoplastic transformations [35] and teratogenic effects, cell-based assays coupled with NU-ELISA may be useful as expedient screens for epigenetic toxicity.


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosure of Potential...
 Acknowledgments
 References
 
NU-ELISA provides an expedient method to quantitatively assess the status of PTMs and histone variants present within the total cellular nucleosome pool. The results indicate that assessment of the steady-state levels of PTMs and macroH2A yields an epiproteomic signature that can distinguish between ESCs, EC cells, and MEFs. Furthermore, epiproteomic nucleosome signatures change in response to exposure of cells to small molecules such as RA and TSA and over the course of ESC differentiation. Therefore, assessment of global nucleosomal epiproteomic profiles provides a useful tool for the investigation of differentiation and cellular identity and will yield insights into studies of pluripotency, differentiation, reprogramming, and screens for molecules with epigenetic activity.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Dr. Lawrence Silbart and Cassandra Godman for useful discussions concerning ELISA and immunodetection methodologies and Therese Doherty for proofreading and editing of this manuscript. This research was supported in part by NIH Grant RO1AG023687 and the Cooperative State Research Service, USDA, under project CONS00812.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 

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P. Rodriguez-Collazo, S. H. Leuba, and J. Zlatanova
Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications
Nucleic Acids Res., June 1, 2009; 37(11): e81 - e81.
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