07-449 Sigma-AldrichAnti-trimethyl-Histone H3 (Lys27) Antibody

The adaptation of the lungs to air breathing at birth requires the fine orchestration of different processes to control lung morphogenesis and progenitor cell differentiation. However, there is little understanding of the role that epigenetic modifiers play in the control of lung development. We found that the histone methyl transferase Ezh2 plays a critical role in lung lineage specification and survival at birth. We performed a genome-wide transcriptome study combined with a genome-wide analysis of the distribution of H3K27 tri-methylation marks to interrogate the role of Ezh2 in lung epithelial cells. Lung cells isolated from Ezh2-deficient and control mice at embryonic day E16.5 were sorted into epithelial and mesenchymal populations based on EpCAM expression. This enabled us to dissect the transcriptional and epigenetic changes induced by the loss of Ezh2 specifically in the lung epithelium. Here we provide a detailed description of the analysis of the RNA-seq and ChIP-seq data, including quality control, read mapping, differential expression and differential binding analyses, as well as visualisation methods used to present the data. These data can be accessed from the Gene Expression Omnibus database (super-series accession number GSE57393).

The paradigm of developmental regulation by Polycomb group (PcG) proteins posits that they maintain silencing outside the spatial expression domains of their target genes, particularly of Hox genes, starting from mid embryogenesis. The Enhancer of zeste [E(z)] PcG protein is the catalytic subunit of the PRC2 complex, which silences its targets via deposition of the H3K27me3 mark. Here, we studied the ascidian Ciona intestinalis counterpart of E(z). Ci-E(z) is detected by immunohistochemistry as soon as the 2- and 4-cell stages as a cytoplasmic form and becomes exclusively nuclear thereafter, whereas the H3K27me3 mark is detected starting from the gastrula stage and later. Morpholino invalidation of Ci-E(z) leads to the total disappearance of both Ci-E(z) protein and its H3K27me3 mark. Ci-E(z) morphants display a severe phenotype. Strikingly, the earliest defects occur at the 4-cell stage with the dysregulation of cell positioning and mitotic impairment. At later stages, Ci-E(z)-deficient embryos are affected by terminal differentiation defects of neural, epidermal and muscle tissues, by the failure to form a notochord and by the absence of caudal nerve. These major phenotypic defects are specifically rescued by injection of a morpholino-resistant Ci-E(z) mRNA, which restores expression of Ci-E(z) protein and re-deposition of the H3K27me3 mark. As observed by qPCR analyses, Ci-E(z) invalidation leads to the early derepression of tissue-specific developmental genes, whereas late-acting developmental genes are generally down-regulated. Altogether, our results suggest that Ci-E(z) plays a major role during embryonic development in Ciona intestinalis by silencing early-acting developmental genes in a Hox-independent manner.

DNA methylation loss can produce inheritable active epialleles in plants. The mechanism involved in the stable transmission of hypomethylated epimuations is presently not clear. Here we show that maintenance of a stably hypomethylated active epiallele in rice required a CHD3 protein (CHR729) and that over-expression of an H3K4me3 demethylase (JMJ703) or H3K27me3 methyltransferase (SDG711) could stably resilence the epiallele. CHR729 and JMJ703 have antagonistic function in H3K4me3 in maintaining the active state of the epiallele, whereas SDG711-mediated H3K27me3 was sufficient to stably repress the locus. The data suggest that H3K4me3 and H3K27me3 controlled by these chromatin regulators may be involved in stable transmission/resetting of epigenetic variation in rice.

JMJD3 (KDM6B) is an H3K27me3 demethylases and emerges as an important player in developmental processes. Although some evidence indicated the involvement of JMJD3 in osteoblast differentiation in vitro, its role as a whole in osteoblast differentiation and bone formation in vivo remains unknown. Here we showed that homozygous deletion of Jmjd3 resulted in severe delay of osteoblast differentiation and bone ossification in mice. By biochemical and genetical methods, we demonstrated that JMJD3 mediated RUNX2 transcriptional activity and cooperated with RUNX2 to promote osteoblast differentiation and bone formation in vivo. These results strongly demonstrated that JMJD3 is required for osteoblast differentiation and bone formation in mice.

Cytomixis is a poorly studied process of nuclear migration between plant cells. It is so far unknown what drives cytomixis and what is the functional state of the chromatin migrating between cells. Using immunostaining, we have analyzed the distribution of posttranslational histone modifications (methylation, acetylation, and phosphorylation) that reflect the functional state of chromatin in the tobacco microsporocytes involved in cytomixis. We demonstrate that the chromatin in the cytomictic cells does not differ from the chromatin in intact microsporocytes according to all 14 analyzed histone modification types. We have also for the first time demonstrated that the migrating chromatin contains normal structures of the synaptonemal complex (SC) and lacks any signs of apoptosis. As has been shown, the chromatin migrating between cells in cytomixis is neither selectively heterochromatized nor degraded both before its migration to another cell and after it enters a recipient cell as micronuclei. We also showed that cytomictic chromatin contains marks typical for transcriptionally active chromatin as well as heterochromatin. Moreover, marks typical for chromosome condensation, SC formation and key proteins required for the formation of bivalents were also detected at migrated chromatin.

Transcriptional gene silencing (TGS) can serve as an innate immunity against invading DNA viruses throughout Eukaryotes. Geminivirus code for TrAP protein to suppress the TGS pathway. Here, we identified an Arabidopsis H3K9me2 histone methyltransferase, Su(var)3-9 homolog 4/Kryptonite (SUVH4/KYP), as a bona fide cellular target of TrAP. TrAP interacts with the catalytic domain of KYP and inhibits its activity in vitro. TrAP elicits developmental anomalies phenocopying several TGS mutants, reduces the repressive H3K9me2 mark and CHH DNA methylation, and reactivates numerous endogenous KYP-repressed loci in vivo. Moreover, KYP binds to the viral chromatin and controls its methylation to combat virus infection. Notably, kyp mutants support systemic infection of TrAP-deficient Geminivirus. We conclude that TrAP attenuates the TGS of the viral chromatin by inhibiting KYP activity to evade host surveillance. These findings provide new insight on the molecular arms race between host antiviral defense and virus counter defense at an epigenetic level.

Aberrant epigenetic silencing of tumor suppressor genes has been recognized as a driving force in cancer. Epigenetic drugs such as the DNA methylation inhibitor decitabine reactivate genes and are effective in myeloid leukemia, but resistance often develops and efficacy in solid tumors is limited. To improve their clinical efficacy, we searched among approved anti-cancer drugs for an epigenetic synergistic combination with decitabine.We used the YB5 cell line, a clonal derivative of the SW48 colon cancer cell line that contains a single copy of a hypermethylated cytomegalovirus (CMV) promoter driving green fluorescent protein (GFP) to screen for drug-induced gene reactivation and synergy with decitabine. None of the 16 anti-cancer drugs tested had effects on their own. However, in combination with decitabine, platinum compounds showed striking synergy in activating GFP. This was dose dependent, observed both in concurrent and sequential combinations, and also seen with other alkylating agents. Clinically achievable concentrations of carboplatin at (25 μM) and decitabine reactivated GFP in 28 % of the YB5 cells as compared to 15 % with decitabine alone. Epigenetic synergy was also seen at endogenously hypermethylated tumor suppressor genes such as MLH1 and PDLIM4. Genome-wide studies showed that reactivation of hypermethylated genes by the combination was significantly better than that induced by decitabine alone or carboplatin alone. Platinum compounds did not enhance decitabine-induced hypomethylation. Rather, we found significantly inhibited HP1α expression by carboplatin and the combination. This was accompanied by increased histone H3 lysine 4 (H3K4) trimethylation and histone H3 lysine 9 (H3K9) acetylation at reactivated genes (P less than  0.0001) and reduced occupancy by methyl-binding proteins including MeCP2 and methyl-CpG-binding domain protein 2 (MBD2) (P less than  0.0001).Our results suggest that the combination of decitabine with platinum analogs shows epigenetic synergy that might be exploited in the treatment of different cancers.

The genome-wide hyperacetylation of chromatin caused by histone deacetylase inhibitors (HDACi) is surprisingly well tolerated by most eukaryotic cells. The homeostatic mechanisms that underlie this tolerance are unknown. Here we identify the transcriptional and epigenomic changes that constitute the earliest response of human lymphoblastoid cells to two HDACi, valproic acid and suberoylanilide hydroxamic acid (Vorinostat), both in widespread clinical use.Dynamic changes in transcript levels over the first 2 h of exposure to HDACi were assayed on High Density microarrays. There was a consistent response to the two different inhibitors at several concentrations. Strikingly, components of all known lysine acetyltransferase (KAT) complexes were down-regulated, as were genes required for growth and maintenance of the lymphoid phenotype. Up-regulated gene clusters were enriched in regulators of transcription, development and phenotypic change. In untreated cells, HDACi-responsive genes, whether up- or down-regulated, were packaged in highly acetylated chromatin. This was essentially unaffected by HDACi. In contrast, HDACi induced a strong increase in H3K27me3 at transcription start sites, irrespective of their transcriptional response. Inhibition of the H3K27 methylating enzymes, EZH1/2, altered the transcriptional response to HDACi, confirming the functional significance of H3K27 methylation for specific genes.We propose that the observed transcriptional changes constitute an inbuilt adaptive response to HDACi that promotes cell survival by minimising protein hyperacetylation, slowing growth and re-balancing patterns of gene expression. The transcriptional response to HDACi is mediated by a precisely timed increase in H3K27me3 at transcription start sites. In contrast, histone acetylation, at least at the three lysine residues tested, seems to play no direct role. Instead, it may provide a stable chromatin environment that allows transcriptional change to be induced by other factors, possibly acetylated non-histone proteins.

Myeloid-derived suppressor cells (MDSCs) were recently found to accumulate in the lungs during Pneumocystis pneumonia (PcP). Adoptive transfer of these cells caused lung damage in recipient mice, suggesting that MDSC accumulation is a mechanism of pathogenesis in PcP. In this study, the phagocytic activity of alveolar macrophages (AMs) was found to decrease by 40% when they were incubated with MDSCs from Pneumocystis-infected mice compared to those incubated with Gr-1(+) cells from the bone marrow of uninfected mice. The expression of the PU.1 gene in AMs incubated with MDSCs also was decreased. This PU.1 downregulation was due mainly to decreased histone 3 acetylation and increased DNA methylation caused by MDSCs. MDSCs were found to express high levels of PD-L1, and alveolar macrophages (AMs) were found to express high levels of PD-1 during PcP. Furthermore, PD-1 expression in AMs from uninfected mice was increased by 18-fold when they were incubated with MDSCs compared to those incubated with Gr-1(+) cells from the bone marrow of uninfected mice. The adverse effects of MDSCs on AMs were diminished when the MDSCs were pretreated with anti-PD-L1 antibody, suggesting that MDSCs disable AMs through PD-1/PD-L1 ligation during PcP.

Alterations of epigenetic modifications are promising targets for cancer therapy, and several epigenetic drugs are now being clinically utilized. At the same time, individual epigenetic modifications have physiological functions in normal cells, and cancer cell specificity is considered difficult to achieve using a drug against a single epigenetic modification. To overcome this limitation, a combination of epigenetic modifications specifically or preferentially present in cancer cells is a candidate target. In this study, we aimed to demonstrate (i) the presence of a cancer cell-specific combination of epigenetic modifications by focusing on DNA methylation and trimethylation of histone H3 lysine 27 (H3K27me3) and (ii) the therapeutic efficacy of a combination of DNA demethylation and EZH2 inhibition. Analyses of DNA methylation and H3K27me3 in human colon, breast and prostate cancer cell lines revealed that 24.7±4.1% of DNA methylated genes had both DNA methylation and H3K27me3 (dual modification) in cancer cells, while it was 11.8±7.1% in normal cells. Combined treatment with a DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC) and an EZH2 inhibitor, GSK126, induced marked re-expression of genes with the dual modification, including known tumor-suppressor genes such as IGFBP7 and SFRP1, and showed an additive inhibitory effect on growth of cancer cells in vitro. Finally, an in vivo combined treatment with 5-aza-dC and GSK126 inhibited growth of xenograft tumors more efficiently than a single treatment with 5-aza-dC. These results showed that the dual modification exists specifically in cancer cells and is a promising target for cancer cell-specific epigenetic therapy.