Base-Resolution Analysis of 5‑Hydroxymethylcytosine by One-Pot Bisulfite-Free Chemical Conversion with Peroxotungstate

5-Hydroxymethylcytosine (^hmC) is an essential intermediate in the active DNA demethylation pathway. Here we report a new base-resolution method for measuring ^hmC by combining peroxotungstate-mediated oxidation and sequencing analysis. We reveal that an oxidized product of ^hmC, trihydroxylated thymine (^thT), tolerated the incorporation of dATP as a substrate in the process of DNA polymerase elongation. By comparing the results of Sanger sequencing before and after the oxidation, we observed that ^hmC sites on single-stranded DNAs could be discriminated from unmethylated cytosines. We found that a thermal cycle condition during peroxotungstate treatment enhanced the oxidation reaction of ^hmC in double-stranded DNA. Furthermore, Illumina sequencing analysis of ^hmC-containing synthetic genome fragments enabled us to identify simultaneously the positions of ^hmC in base resolution. This bisulfite-free simple ^hmC detection technique could facilitate the acquisition of epigenomic information

Genome-Wide Analysis of the Chromatin Composition of Histone H2A and H3 Variants in Mouse Embryonic Stem Cells

<div><p>Genome-wide distribution of the majority of H2A and H3 variants (H2A, H2AX, H2AZ, macroH2A, H3.1, H3.2 and H3.3) was simultaneously investigated in mouse embryonic stem cells by chromatin immunoprecipitation sequencing. Around the transcription start site, histone variant distribution differed between genes possessing promoters of high and low CpG density, regardless of their expression levels. In the intergenic regions, regulatory elements were enriched in H2A.Z and H3.3, whereas repeat elements were abundant in H2A and macroH2A, and H3.1, respectively. Analysis of H2A and H3 variant combinations composing nucleosomes revealed that the H2A.Z and H3.3 combinations were present at a higher frequency throughout the genome than the other combinations, suggesting that H2A.Z and H3.3 associate preferentially with each other to comprise the nucleosomes independently of genome region. Finally, we found that chromatin was unstable only in regions where it was enriched in both H2A.Z and H3.3, but strongly quantified stable in regions in which only H3.3 was abundant. Therefore, histone variant composition is an important determinant of chromatin structure, which is associated with specific genomic functions.</p></div

The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

<div><p>NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.</p> </div

Identification of Novel HLA-A*24:02-Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies

<div><p>The homeobox protein, PEPP2 (RHOXF2), has been suggested as a cancer/testis (CT) antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs) is unknown. In this study, we revealed that <i>PEPP2</i> gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2^271-279). The CTLs induced by PEPP2^271-279 recognized PEPP2-positive target cells in an HLA-A*24:02-restricted manner. We also found that a demethylating agent, 5-aza-2’-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2’-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients.</p></div

Enrichment of histone variants in repeat elements.

<p>Bars represent the ratios of the number of ChIP reads to that of input reads in repeat elements.</p

Down-regulation of NF-κB activity and undifferentiated markers upon monolayer differentiation.

<p>Representative photographs of monolayer differentiated cells (Days 2 and 7: original magnification, x200) (A). Representative electrophoretic mobility shift assays (EMSA) shows NF-κB binding activity in the undifferentiated iPS cells (FGF2+ and feeder+) and monolayer differentiated cells (FGF2- and feeder-). Proximal tubular epithelial cells (PTECs) were used as control cells (differentiated cells) for the EMSA. Results of 2 independent experiments are shown. In the last lane, the competition assays were performed using the undifferentiated iPS cells (B). Western blot analysis of actin, Oct3/4, NANOG, WT-1, and Pax-2 in the undifferentiated iPS cells (FGF2+ and feeder+) and monolayer differentiated cells (FGF2- and feeder-). Feeder cells (SNL) were used as negative control cells. Results of 2 independent experiments are shown (C).</p

Enrichment of histone variants in repeat elements.

<p>Bars represent the ratios of the number of ChIP reads to that of input reads in repeat elements.</p

H3K4me3 level in H2A.Z- and H3.3-enriched regions.

<p>The genome was divided into 150 nucleotide bins, and the bins in which a particular variant was significantly enriched was determined as described in Materials and Methods (<i>see </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092689#pone.0092689.s004" target="_blank">Figure S4</a>). (A) Boxplot of H3K4me3 levels in H2A-and-H3-variant-enriched bins. (B) Boxplot of H3K4me3 levels in H2A.Z- or H3.3-, and both H2A.Z and H3.3 (H2A.Z/H3.3)-enriched bins. The bottom and top of the box are the 25th and 75th percentile, respectively, and the upper and lower whiskers represent 2.5th and 97.5th percentile, respectively. <i>P</i>-values were calculated using Mann–Whitney U-tests; (*) <i>P</i><0.001.</p

Downregulation of Oct3/4 and Nanog by knockdown of NF-κB.

<p>Western blot analysis of actin, NF-κB p65, IκBα, Oct3/4, Nanog, WT-1, and Pax-2 in human iPS cells treated with siRNA (A). Immunohistochemical staining for NF-κB p65 (B–D) and Oct3/4 (E–G) in human iPS cells treated with siRNA. No treatment (B and E), treatment with control siRNA (C and F), and treatment with p65 siRNA (D and G). Negative control staining without a primary antibody is shown (H) (Original magnification, x200).</p

Concurrent Activation of Acetylation and Tri-Methylation of H3K27 in a Subset of Hepatocellular Carcinoma with Aggressive Behavior

<div><p>Analysis of acetylation and tri-methylation of the same residue of histone molecules might identify a subset of hepatocellular carcinoma (HCC) with aggressive behavior. In the present study, we examined acetylation and tri-methylation of lysine 27 on histone H3 (H3K27ac and H3K27me3, respectively) because these two modifications are known to exhibit opposite effects (enhancing and silencing) on gene expression. Neoplastic and non-neoplastic tissues from 198 HCC cases were immunostained with specific monoclonal antibodies against H3K27ac and H3K27me3. The stained tissues were evaluated by an image analyzing program to generate histological scores (H-scores, range 0–300), which were determined by multiplying the percentage of positive-stained cells with the classified immunohistochemical marker intensity (0–3). HCC tissues showed significantly higher H3K27ac (156.7±86.8) and H3K27me3 H-scores (151.8±78.1) compared with the background liver (40.3±33.0 and 64.7±45.6, respectively) (both <i>P</i><0.001). The cases with H-scores of high-H3K27ac/high-H3K27me3 (n = 54) showed significant correlation with poor differentiation of morphology (<i>P</i><0.01) and p53-positive staining (<i>P</i><0.05), and poor prognosis (<i>P</i><0.01). Confocal microscopy revealed segregated intranuclear localization of both modifications in the individual cancer cells: H3K27ac localization in central euchromatin regions and H3K27me3 in peripheral heterochromatin regions. Concurrent acetylation and methylation at H3K27 occurs in HCC cells in association with p53 abnormalities. These findings demonstrate that image analyzer-assisted H-scores of H3K27ac and H3K27me3 identified an aggressive subgroup of HCC, and could serve as a prognostic marker for HCC.</p></div