Gene body methylation of the dimethylarginine dimethylamino-hydrolase 2 (Ddah2) gene is an epigenetic biomarker for neural stem cell differentiation

DNA methylation is an important epigenetic mark that is involved in the regulation of many cellular processes such as gene expression, genomic imprinting and silencing of repetitive elements. Because of their ability to cause and capture phenotypic plasticity, epigenetic marks such as DNA methylation represent potential biomarkers to distinguish between different types of tissues and stages of differentiation. Here, we have identified differential DNA methylation in the gene body of the nitric oxide inhibitor Ddah2 that discriminates embryonic stem cells from neural stem cells and is positively correlated with differential gene expression.     

Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells

With the emergence of new CRISPR/dCas9 tools that enable site specific modulation of DNA methylation and histone modifications, more detailed investigations of the contribution of epigenetic regulation to the precise phenotype of cells in culture, including recombinant production subclones, is now possible. These also allow a wide range of applications in metabolic engineering once the impact of such epigenetic modifications on the chromatin state is available.In this study, enhanced DNA methylation tools were targeted to a recombinant viral promoter (CMV), an endogenous promoter that is silenced in its native state in CHO cells, but had been reactivated previously (β-galactoside α-2,6-sialyltransferase 1) and an active endogenous promoter (α-1,6-fucosyltransferase), respectively. Comparative ChIP-analysis of histone modifications revealed a general loss of active promoter histone marks and the acquisition of distinct repressive heterochromatin marks after targeted methylation. On the other hand, targeted demethylation resulted in autologous acquisition of active promoter histone marks and loss of repressive heterochromatin marks. These data suggest that DNA methylation directs the removal or deposition of specific histone marks associated with either active, poised or silenced chromatin. Moreover, we show that de novo methylation of the CMV promoter results in reduced transgene expression in CHO cells. Although targeted DNA methylation is not efficient, the transgene is repressed, thus offering an explanation for seemingly conflicting reports about the source of CMV promoter instability in CHO cells.Importantly, modulation of epigenetic marks enables to nudge the cell into a specific gene expression pattern or phenotype, which is stabilized in the cell by autologous addition of further epigenetic marks. Such engineering strategies have the added advantage of being reversible and potentially tunable to not only turn on or off a targeted gene, but also to achieve the setting of a desirable expression level.     

Methylome sequencing for fibrolamellar hepatocellular carcinoma depicts distinctive features

With the goal of studying epigenetic alterations in fibrolamellar hepatocellular carcinoma (FLC) and establish an associated DNA methylation signature, we analyzed LINE-1 methylation in a cohort of FLC and performed next-generation sequencing of DNA methylation in a training set of pure-FLCs and non-cirrhotic hepatocellular carcinomas (nc-HCC). DNA methylation was correlated with gene expression. Furthermore, we established and validated an epigenetic signature differentiating pure-FLC from other HCCs. LINE-1 methylation correlated with shorter recurrence-free survival and overall survival in resected pure-FLC patients. Unsupervised clustering using CG sites located in islands distinguished pure-FLC from nc-HCC. Major DNA methylation changes occurred outside promoters, mainly in gene bodies and intergenic regions located in the vicinity of liver developmental genes (i.e., SMARCA4 and RXRA). Partially methylated domains were more prone to DNA methylation changes. Furthermore, we identified several putative tumor suppressor genes (e.g., DLEU7) and oncogenes (e.g., DUSP4). While ∼70% of identified gene promoters gaining methylation were marked by bivalent histone marks (H3K4me3/H3K27me3) in embryonic stem cells, ∼70% of those losing methylation were marked by H3K4me3. Finally, we established a pure FLC DNA methylation signature and validated it in an independent dataset. Our analysis reveals a distinct epigenetic signature of pure FLC as compared to nc-HCC, with DNA methylation changes occurring in the vicinity of liver developmental genes. These data suggest new options for targeting FLC based on cancer epigenome aberrations.     

Perspectives for epigenetic editing in crops

Site-specific nucleases (SSNs) have drawn much attention in plant biotechnology due to their ability to drive precision mutagenesis, gene targeting or allele replacement. However, when devoid of its nuclease activity, the underlying DNA-binding activity of SSNs can be used to bring other protein functional domains close to specific genomic sites, thus expanding further the range of applications of the technology. In particular, the addition of functional domains encoding epigenetic effectors and chromatin modifiers to the CRISPR/Cas ribonucleoprotein complex opens the possibility to introduce targeted epigenomic modifications in plants in an easily programmable manner. Here we examine some of the most important agronomic traits known to be controlled epigenetically and review the best studied epigenetic catalytic effectors in plants, such as DNA methylases/demethylases or histone acetylases/deacetylases and their associated marks. We also review the most efficient strategies developed to date to functionalize Cas proteins with both catalytic and non-catalytic epigenetic effectors, and the ability of these domains to influence the expression of endogenous genes in a regulatable manner. Based on these new technical developments, we discuss the possibilities offered by epigenetic editing tools in plant biotechnology and their implications in crop breeding.

     

To incise or not and where: SET-domain methyltransferases know

The concept of the histone code posits that histone modifications regulate gene functions once interpreted by epigenetic readers. A well-studied case is trimethylation of lysine 4 of histone H3 (H3K4me3), which is enriched at gene promoters. However, H3K4me3 marks are not needed for the expression of most genes, suggesting extra roles, such as influencing the 3D genome architecture. Here, we highlight an intriguing analogy between the H3K4me3-dependent induction of double-strand breaks in several recombination events and the impact of this same mark on DNA incisions for the repair of bulky lesions. We propose that Su(var)3–9, Enhancer-of-zeste and Trithorax (SET)-domain methyltransferases generate H3K4me3 to guide nucleases into chromatin spaces, the favorable accessibility of which ensures that DNA break intermediates are readily processed, thereby safeguarding genome stability.     

The re-expression of the epigenetically silenced e-cadherin gene by a polyamine analogue lysine-specific demethylase-1 (LSD1) inhibitor in human acute myeloid leukemia cell lines

Aberrant epigenetic silencing of tumor suppressor genes is a common feature observed during the transformation process of many cancers, including those of hematologic origin. Histone modifications, including acetylation, phosphorylation, and methylation, collaborate with DNA CpG island methylation to regulate gene expression. The dynamic process of histone methylation is the latest of these epigenetic modifications to be described, and the identification and characterization of LSD1 as a demethylase of lysine 4 of histone H3 (H3K4) has confirmed that both the enzyme and the modified histone play important roles as regulators of gene expression. LSD1 activity contributes to the suppression of gene expression by demethylating promoter-region mono- and dimethyl-H3K4 histone marks that are associated with active gene expression. As most post-translational modifications are reversible, the enzymes involved in the modification of histones have become targets for chemotherapeutic intervention. In this study, we examined the effects of the polyamine analogue LSD1 inhibitor 2d (1,15-bis{N ⁵-[3,3-(diphenyl)propyl]-N ¹-biguanido}-4,12-diazapentadecane) in human acute myeloid leukemia (AML) cell lines. In each line studied, 2d evoked cytotoxicity and inhibited LSD1 activity, as evidenced by increases in the global levels of mono- and di-methylated H3K4 proteins. Global increases in other chromatin modifications were also observed following exposure to 2d, suggesting a broad response to this compound with respect to chromatin regulation. On a gene-specific level, treatment with 2d resulted in the re-expression of e-cadherin, a tumor suppressor gene frequently silenced by epigenetic modification in AML. Quantitative chromatin immunoprecipitation analysis of the e-cadherin promoter further confirmed that this re-expression was concurrent with changes in both active and repressive histone marks that were consistent with LSD1 inhibition. As hematologic malignancies have demonstrated promising clinical responses to agents targeting epigenetic silencing, this polyamine analogue LSD1 inhibitor presents an exciting new avenue for the development of novel therapeutic agents for the treatment of AML.     

Chromatin dynamics controls epigenetic domain formation

In multicellular organisms, nucleosomes carry epigenetic information that defines distinct patterns of gene expression, which are inherited over multiple generations. The enhanced capacity for information storage arises by nucleosome modifications, which are triggered by enzymes. Modified nucleosomes can transfer the mark to others that are in proximity by a positive-feedback (modification begets modification) mechanism. We created a generic polymer model, referred to as 3DSpreader, in which each bead, representing a nucleosome, stochastically switches between unmodified (U) and modified (M) states depending on the states of the neighbors. Modification begins at a specific nucleation site (NS) that is permanently in the M state, and could spread to other loci that is dictated by chromatin dynamics. Transfer of marks among the non-nucleation loci occurs stochastically as chromatin evolves in time. If the spreading rate is slower than the chromatin relaxation rate, which is biologically pertinent, then finite-sized domains form, driven by contacts between nucleosomes through a three-dimensional looping mechanism. Surprisingly, simulations based on the 3DSpreader model result in finite bounded domains that arise without the need for any boundary elements. Maintenance of spatially and temporally stable domains requires the presence of the NS, whose removal eliminates finite-sized modified domains. The theoretical predictions are in excellent agreement with experimental data for H3K9me3 spreading in mouse embryonic stem cells.     

Epigenetic control of plant immunity

In eukaryotic genomes, gene expression and DNA recombination are affected by structural chromatin traits. Chromatin structure is shaped by the activity of enzymes that either introduce covalent modifications in DNA and histone proteins or use energy from ATP to disrupt histone–DNA interactions. The genomic ‘marks’ that are generated by covalent modifications of histones and DNA, or by the deposition of histone variants, are susceptible to being altered in response to stress. Recent evidence has suggested that proteins generating these epigenetic marks play crucial roles in the defence against pathogens. Histone deacetylases are involved in the activation of jasmonic acid‐ and ethylene‐sensitive defence mechanisms. ATP‐dependent chromatin remodellers mediate the constitutive repression of the salicylic acid‐dependent pathway, whereas histone methylation at the WRKY70 gene promoter affects the activation of this pathway. Interestingly, bacterial‐infected tissues show a net reduction in DNA methylation, which may affect the disease resistance genes responsible for the surveillance against pathogens. As some epigenetic marks can be erased or maintained and transmitted to offspring, epigenetic mechanisms may provide plasticity for the dynamic control of emerging pathogens without the generation of genomic lesions.     

Exploring and exploiting epigenetic variation in crops

This review addresses the mechanisms by which epigenetic variation modulates plant gene regulation and phenotype. In particular we explore the scope for harnessing such processes within the context of crop genetic improvement. We focus on the role of DNA methylation as an epigenetic mark that contributes to epiallelic diversity and modulation of gene regulation. We outline the prevalence and distribution of epigenetic marks in relation to eukaryote developmental processes, and in particular identify where this may be relevant to crop traits both in terms of specific developmental stages and in relation to physiological responses to environmental change. Recent whole genome surveys have identified specific characteristics of the distribution of DNA methylation within plant genomes. Together with greater understanding of the mode of action of different maintenance and de novo methyltransferases, this provides an opportunity to modulate DNA methylation status at specific loci as an intervention strategy in crop genetic improvement. We discuss alternative approaches that may be suitable for harnessing such induced epiallelic variation. Most of the discussion is associated with Brassica crops, which demonstrate considerable morphological plasticity, segmental chromosomal duplication, and polyploidy.     

Epigenetic Signatures Associated with Different Levels of Differentiation Potential in Human Stem Cells

Background

The therapeutic use of multipotent stem cells depends on their differentiation potential, which has been shown to be variable for different populations. These differences are likely to be the result of key changes in their epigenetic profiles.

Methodology/Principal Findings

to address this issue, we have investigated the levels of epigenetic regulation in well characterized populations of pluripotent embryonic stem cells (ESC) and multipotent adult stem cells (ASC) at the trancriptome, methylome, histone modification and microRNA levels. Differences in gene expression profiles allowed classification of stem cells into three separate populations including ESC, multipotent adult progenitor cells (MAPC) and mesenchymal stromal cells (MSC). The analysis of the PcG repressive marks, histone modifications and gene promoter methylation of differentiation and pluripotency genes demonstrated that stem cell populations with a wider differentiation potential (ESC and MAPC) showed stronger representation of epigenetic repressive marks in differentiation genes and that this epigenetic signature was progressively lost with restriction of stem cell potential. Our analysis of microRNA established specific microRNA signatures suggesting specific microRNAs involved in regulation of pluripotent and differentiation genes.

Conclusions/Significance

Our study leads us to propose a model where the level of epigenetic regulation, as a combination of DNA methylation and histone modification marks, at differentiation genes defines degrees of differentiation potential from progenitor and multipotent stem cells to pluripotent stem cells.