Identification of Epigenetic Biomarkers of Lung Adenocarcinoma through Multi-Omics Data Analysis

Epigenetic mechanisms such as DNA methylation or histone modifications are essential for the regulation of gene expression and development of tissues. Alteration of epigenetic modifications can be used as an epigenetic biomarker for diagnosis and as promising targets for epigenetic therapy. A recent study explored cancer-cell specific epigenetic biomarkers by examining different types of epigenetic modifications simultaneously. However, it was based on microarrays and reported biomarkers that were also present in normal cells at a low frequency. Here, we first analyzed multi-omics data (including ChIP-Seq data of six types of histone modifications: H3K27ac, H3K4me1, H3K9me3, H3K36me3, H3K27me3, and H3K4me3) obtained from 26 lung adenocarcinoma cell lines and a normal cell line. We identified six genes with both H3K27ac and H3K4me3 histone modifications in their promoter regions, which were not present in the normal cell line, but present in ≥85% (22 out of 26) and ≤96% (25 out of 26) of the lung adenocarcinoma cell lines. Of these genes, NUP210 (encoding a main component of the nuclear pore complex) was the only gene in which the two modifications were not detected in another normal cell line. RNA-Seq analysis revealed that NUP210 was aberrantly overexpressed among the 26 lung adenocarcinoma cell lines, although the frequency of NUP210 overexpression was lower (19.3%) in 57 lung adenocarcinoma tissue samples studied and stored in another database. This study provides a basis to discover epigenetic biomarkers highly specific to a certain cancer, based on multi-omics data at the cell population level.     

Crosstalk Between DNA and Histones: Tet’s New Role in Embryonic Stem Cells

Embryonic stem (ES) cells are characterized by the expression of an extensive and interconnected network of pluripotency factors which are downregulated in specialized cells. Epigenetic mechanisms, including DNA methylation and histone modifications, are also important in maintaining this pluripotency program in ES cells and in guiding correct differentiation of the developing embryo. Methylation of the cytosine base of DNA blocks gene expression in all cell types and further modifications of methylated cytosine have recently been discovered. These new modifications, putative intermediates in a pathway to erase DNA methylation marks, are catalyzed by the ten-eleven translocation (Tet) proteins, specifically by Tet1 and Tet2 in ES cells. Surprisingly, Tet1 shows repressive along with active effects on gene expression depending on its distribution throughout the genome and co-localization with Polycomb Repressive Complex 2 (PRC2). PRC2 di- and tri-methylates lysine 27 of histone 3 (H3K27me2/3 activity), marking genes for repression. In ES cells, almost all gene loci containing the repressive H3K27me3 modification also bear the active H3K4me3 modification, creating “bivalent domains” which mark important developmental regulators for timely activation. Incorporation of Tet1 into the bivalent domain paradigm is a new and exciting development in the epigenetics field, and the ramifications of this novel crosstalk between DNA and histone modifications need to be further investigated. This knowledge would aid reprogramming of specialized cells back into pluripotent stem cells and advance understanding of epigenetic perturbations in cancer.     

Differences among brain tumor stem cell types and fetal neural stem cells in focal regions of histone modifications and DNA methylation,broad regions of modifications,and bivalent promoters

Background

Aberrational epigenetic marks are believed to play a major role in establishing the abnormal features of cancer cells. Rational use and development of drugs aimed at epigenetic processes requires an understanding of the range, extent, and roles of epigenetic reprogramming in cancer cells. Using ChIP-chip and MeDIP-chip approaches, we localized well-established and prevalent epigenetic marks (H3K27me3, H3K4me3, H3K9me3, DNA methylation) on a genome scale in several lines of putative glioma stem cells (brain tumor stem cells, BTSCs) and, for comparison, normal human fetal neural stem cells (fNSCs).

Results

We determined a substantial “core” set of promoters possessing each mark in every surveyed BTSC cell type, which largely overlapped the corresponding fNSC sets. However, there was substantial diversity among cell types in mark localization. We observed large differences among cell types in total number of H3K9me3+ positive promoters and peaks and in broad modifications (defined as >50 kb peak length) for H3K27me3 and, to a lesser extent, H3K9me3. We verified that a change in a broad modification affected gene expression of CACNG7. We detected large numbers of bivalent promoters, but most bivalent promoters did not display direct overlap of contrasting epigenetic marks, but rather occupied nearby regions of the proximal promoter. There were significant differences in the sets of promoters bearing bivalent marks in the different cell types and few consistent differences between fNSCs and BTSCs.

Conclusions

Overall, our “core set” data establishes sets of potential therapeutic targets, but the diversity in sets of sites and broad modifications among cell types underscores the need to carefully consider BTSC subtype variation in epigenetic therapy. Our results point toward substantial differences among cell types in the activity of the production/maintenance systems for H3K9me3 and for broad regions of modification (H3K27me3 or H3K9me3). Finally, the unexpected diversity in bivalent promoter sets among these multipotent cells indicates that bivalent promoters may play complex roles in the overall biology of these cells. These results provide key information for forming the basis for future rational drug therapy aimed at epigenetic processes in these cells.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-724) contains supplementary material, which is available to authorized users.     

Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells

Combinations of histone modifications have significant biological roles, such as maintenance of pluripotency and cancer development, but cannot be analyzed at the single cell level. Here, we visualized a combination of histone modifications by applying the in situ proximity ligation assay, which detects two proteins in close vicinity (∼30 nm). The specificity of the method [designated as imaging of a combination of histone modifications (iChmo)] was confirmed by positive signals from H3K4me3/acetylated H3K9, H3K4me3/RNA polymerase II and H3K9me3/H4K20me3, and negative signals from H3K4me3/H3K9me3. Bivalent modification was clearly visualized by iChmo in wild-type embryonic stem cells (ESCs) known to have it, whereas rarely in Suz12 knockout ESCs and mouse embryonic fibroblasts known to have little of it. iChmo was applied to analysis of epigenetic and phenotypic changes of heterogeneous cell population, namely, ESCs at an early stage of differentiation, and this revealed that the bivalent modification disappeared in a highly concerted manner, whereas phenotypic differentiation proceeded with large variations among cells. Also, using this method, we were able to visualize a combination of repressive histone marks in tissue samples. The application of iChmo to samples with heterogeneous cell population and tissue samples is expected to clarify unknown biological and pathological significance of various combinations of epigenetic modifications.     

Generation of disease-specific induced pluripotent stem cells from patients with rheumatoid arthritis and osteoarthritis

Introduction

Since the concept of reprogramming mature somatic cells to generate induced pluripotent stem cells (iPSCs) was demonstrated in 2006, iPSCs have become a potential substitute for embryonic stem cells (ESCs) given their pluripotency and “stemness” characteristics, which resemble those of ESCs. We investigated to reprogram fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) to generate iPSCs using a 4-in-1 lentiviral vector system.

Methods

A 4-in-1 lentiviral vector containing Oct4, Sox2, Klf4, and c-Myc was transduced into RA and OA FLSs isolated from the synovia of two RA patients and two OA patients. Immunohistochemical staining and real-time PCR studies were performed to demonstrate the pluripotency of iPSCs. Chromosomal abnormalities were determined based on the karyotype. SCID-beige mice were injected with iPSCs and sacrificed to test for teratoma formation.

Results

After 14 days of transduction using the 4-in-1 lentiviral vector, RA FLSs and OA FLSs were transformed into spherical shapes that resembled embryonic stem cell colonies. Colonies were picked and cultivated on matrigel plates to produce iPSC lines. Real-time PCR of RA and OA iPSCs detected positive markers of pluripotency. Immunohistochemical staining tests with Nanog, Oct4, Sox2, Tra-1-80, Tra-1-60, and SSEA-4 were also positive. Teratomas that comprised three compartments of ectoderm, mesoderm, and endoderm were formed at the injection sites of iPSCs. Established iPSCs were shown to be compatible by karyotyping. Finally, we confirmed that the patient-derived iPSCs were able to differentiate into osteoblast, which was shown by an osteoimage mineralization assay.

Conclusion

FLSs derived from RA and OA could be cell resources for iPSC reprogramming. Disease- and patient-specific iPSCs have the potential to be applied in clinical settings as source materials for molecular diagnosis and regenerative therapy.     

Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting

Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS) to isolate single cells expressing the cell surface marker signature CD13^NEGSSEA4^POSTra-1-60^POS on day 7–10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and “contaminating” partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential in vitro and in vivo. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral) or non- integrating (Sendai virus) reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.     

In situ histone landscape of nephrogenesis

In the developing kidney, self-renewing progenitors respond to inductive signaling from the adjacent branching ureteric bud by undergoing mesenchyme-to-epithelium transition. Nascent nephrons subsequently undergo elongation, segmentation, and differentiation into a mature renal epithelium with diverse functions. Epigenetic mechanisms have been implicated in impacting cell fate decisions during nephrogenesis; however, the chromatin landscape of nephron progenitors and daughter differentiating cells are largely unknown. Here, we examined the spatiotemporal expression patterns of histone H3 methylation and histone methyltransferases in E15.5 mouse kidneys. Kidney sections were probed with antibodies against histone modifications, enzymes, and markers of progenitors and differentiation. The results revealed that: (1) nephron progenitor cells exhibit a broad histone methylation signature that comprises both “active” and “repressive” marks (H3K4me3/K9me3/K27me3/R2me2/R17me2); (2) nascent nephrons retain high H3K4me3 but show downregulation of H3K9/K27me3 and; (3) maturing epithelial tubules acquire high levels of H3K79me2/3. Consistent with respective histone marks, the H3K4 methyltransferase, Ash2l, is expressed in progenitors and nascent nephrons, whereas the H3K9/K27 methyltransferases, G9a/Ezh2, are more enriched in progenitors than nascent nephrons. We conclude that combinatorial histone signatures correlate with cell fate decisions during nephrogenesis.     

Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation

Stem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G₂, mitotic, and G₁ phases of the cell cycle, we found striking variations in the levels of H3K4me3 through the G₂-M-G₁ transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. Interestingly, bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in the maintenance of pluripotency.     

INO80 promotes H2A.Z occupancy to regulate cell fate transition in pluripotent stem cells

The INO80 chromatin remodeler is involved in many chromatin-dependent cellular functions. However, its role in pluripotency and cell fate transition is not fully defined. We examined the impact of Ino80 deletion in the naïve and primed pluripotent stem cells. We found that Ino80 deletion had minimal effect on self-renewal and gene expression in the naïve state, but led to cellular differentiation and de-repression of developmental genes in the transition toward and maintenance of the primed state. In the naïve state, INO80 pre-marked gene promoters that would adopt bivalent histone modifications by H3K4me3 and H3K27me3 upon transition into the primed state. In the primed state, in contrast to its known role in H2A.Z exchange, INO80 promoted H2A.Z occupancy at these bivalent promoters and facilitated H3K27me3 installation and maintenance as well as downstream gene repression. Together, our results identified an unexpected function of INO80 in H2A.Z deposition and gene regulation. We showed that INO80-dependent H2A.Z occupancy is a critical licensing step for the bivalent domains, and thereby uncovered an epigenetic mechanism by which chromatin remodeling, histone variant deposition and histone modification coordinately control cell fate.     

Spatial Distribution of Epigenetic Modifications in Brachypodium distachyon Embryos during Seed Maturation and Germination

Seed development involves a plethora of spatially and temporally synchronised genetic and epigenetic processes. Although it has been shown that epigenetic mechanisms, such as DNA methylation and chromatin remodelling, act on a large number of genes during seed development and germination, to date the global levels of histone modifications have not been studied in a tissue-specific manner in plant embryos. In this study we analysed the distribution of three epigenetic markers, i.e. H4K5ac, H3K4me2 and H3K4me1 in ‘matured’, ‘dry’ and ‘germinating’ embryos of a model grass, Brachypodium distachyon (Brachypodium). Our results indicate that the abundance of these modifications differs considerably in various organs and tissues of the three types of Brachypodium embryos. Embryos from matured seeds were characterised by the highest level of H4K5ac in RAM and epithelial cells of the scutellum, whereas this modification was not observed in the coleorhiza. In this type of embryos H3K4me2 was most evident in epithelial cells of the scutellum. In ‘dry’ embryos H4K5ac was highest in the coleorhiza but was not present in the nuclei of the scutellum. H3K4me1 was the most elevated in the coleoptile but absent from the coleorhiza, whereas H3K4me2 was the most prominent in leaf primordia and RAM. In embryos from germinating seeds H4K5ac was the most evident in the scutellum but not present in the coleoptile, similarly H3K4me1 was the highest in the scutellum and very low in the coleoptile, while the highest level of H3K4me2 was observed in the coleoptile and the lowest in the coleorhiza. The distinct patterns of epigenetic modifications that were observed may be involved in the switch of the gene expression profiles in specific organs of the developing embryo and may be linked with the physiological changes that accompany seed desiccation, imbibition and germination.