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BackgroundDNA methylation and histone 3 lysine 9 (H3K9) methylation are considered as epigenetic marks that can be inherited through cell divisions. To explore the functional consequences and stability of these modifications, we employed targeted installment of DNA methylation and H3K9 methylation in the vascular endothelial growth factor A (VEGF-A) promoter using catalytic domains of DNA or H3K9 methyltransferases that are fused to a zinc finger protein which binds a site in the VEGF-A promoter.ResultsExpression of the targeted DNA and H3K9 methyltransferases caused dense deposition of DNA methylation or H3K9 di- and trimethylation in the promoter of VEGF-A and downregulation of VEGF-A gene expression. We did not observe positive feedback between DNA methylation and H3K9 methylation. Upon loss of the targeted methyltransferases from the cells, the epigenetic marks, chromatin environment, and gene expression levels returned to their original state, indicating that both methylation marks were not stably propagated after their installment.ConclusionsThe clear anti-correlation between DNA or H3K9 methylation and gene expression suggests a direct role of these marks in transcriptional control. The lack of maintenance of the transiently induced silenced chromatin state suggests that the stability of epigenetic signaling is based on an epigenetic network consisting of several molecular marks. Therefore, for stable reprogramming, either multivalent deposition of functionally related epigenetic marks or longer-lasting trigger stimuli might be necessary.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0002-z) contains supplementary material, which is available to authorized users.

The Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in many cancers including ovarian cancer and EpCAM overexpression correlates with decreased survival of patients. It was the aim of this study to achieve a targeted methylation of the EpCAM promoter and silence EpCAM gene expression using an engineered zinc finger protein that specifically binds the EpCAM promoter fused to the catalytic domain of the Dnmt3a DNA methyltransferase. We show that transient transfection of this construct increased the methylation of the EpCAM promoter in SKOV3 cells from 4–8% in untreated cells to 30%. Up to 48% methylation was observed in stable cell lines which express the chimeric methyltransferase. Control experiments confirmed that the methylation was dependent on the fusion of the Zinc finger and the methyltransferase domains and specific for the target region. The stable cell lines with methylated EpCAM promoter showed a 60–80% reduction of EpCAM expression as determined at mRNA and protein level and exhibited a significantly reduced cell proliferation. Our data indicate that targeted methylation of the EpCAM promoter could be an approach in the therapy of EpCAM overexpressing cancers.

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Late-stage colorectal cancer (CRC) is still a clinically challenging problem. The activity of the tumor suppressor p53 is regulated via posttranslational modifications (PTMs). While the relevance of p53 Cterminal acetylation for transcriptional regulation is well-defined, it is unknown whether this PTM controls mitochondrially mediated apoptosis directly. We used wild-type p53 or p53-negative human CRC cells, cells with acetylation-defective p53, transformation assays, CRC organoids, and xenograft mouse models to assess how p53 acetylation determines cellular stress responses. The topoisomerase-1 inhibitor irinotecan induces acetylation of several lysine residues within p53. Inhibition of histone deacetylaces (HDACs) with the class I HDAC inhibitor entinostat synergistically triggers mitochondrial damage and apoptosis in irinotecan-treated p53-positive CRC cells. This specifically relies on the C-terminal acetylation of p53 by CREB binding protein (CBP)/p300 and the presence of C-terminally acetylated p53 in complex with the pro-apoptotic BCL2 antagonist/killer (BAK) protein. This control of C-terminal acetylation by HDACs can mechanistically explain why combinations of irinotecan and entinostat represent clinically tractable agents for the therapy of p53-proficient CRC.

Organoids culture provides unique opportunities to study human diseases and to complement animal models. Several organs and tissues can be in vitro cultured in 3D structures resembling in vivo tissue organization. Organoids culture contains most of the cell types of the original tissue and are maintained by growth factors mimicking the in vivo state. However, the system is yet not fully understood, and specific in vivo features especially those driven by cell-extrinsic factors may be lost in culture. Here we show a comprehensive transcriptome-wide characterization of mouse gut organoids derived from different intestinal compartments and from mice of different gender and age. RNA-seq analysis showed that the in vitro culture strongly influences the global transcriptome of the intestinal epithelial cells (~ 60% of the total variance). Several compartment-, age- and gender-related transcriptome features are lost after culturing indicating that they are driven by niche or systemic factors. However, certain intrinsic transcriptional programs, for example, some compartment-related features and a minority of gender- and aging- related features are maintained in vitro which suggested possibilities for these features to be studied in this system. Moreover, our study provides knowledge about the cell-extrinsic or cell-intrinsic origin of intestinal epithelial transcriptional programs. We anticipated that our characterization of this in vitro system is an important reference for scientists and clinicians using intestinal organoids as a research model.

Acute rejection (AR) can lead to allograft dysfunction following renal transplantation, despite immunosuppressive treatments. Accumulating evidence points out a role for epigenetic modification in immune responses. However, the mechanism and contribution of DNA methylation in allograft survival remain unclear. In this study, we followed up patients who successively experienced end‐stage renal disease, renal transplantation with allograft function or dysfunction, and hemodialysis. Peripheral blood mononuclear cells were collected at different time points for analysis of the DNA methylation. Epigenetic modifier analysis was also performed to explore its effect of methylation in a mouse model of AR. Compared with the allograft‐stable cohort, patients who experienced AR‐induced allograft dysfunction demonstrated more changes in methylation patterns. Pathway analysis revealed that the hypermethylated areas in the allograft dysfunction group were associated with genes related to the mechanistic target of rapamycin (mTOR) signaling pathway. Moreover, in the mouse AR model, treatment with the DNA methyltransferase inhibitor—decitabine regulated the Th1/2/17/regulatory T cell (Treg cell) immune response via its demethylating role in the suppressing the activity of the mTOR pathway, which ultimately ameliorated renal allograft‐related inflammatory injuries. These results revealed that changes in methylation accompany AR‐induced allograft dysfunction after renal transplantation. Epigenetics may provide new insights into predicting and improving allograft survival.

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