MtDNA-Depleted Mitochondria Form Sites of Contact with the Nucleus and Alter the Cellular Epigenome

BackgroundModifications of DNA and histones in various combinations are correlated with many cellular processes. In this study, we investigated the possible relationship between histone H4 tetraacetylation, DNA methylation and histone H3 dimethylation at lysine 9 during mitosis in maize root meristems.ResultsTreatment with trichostatin A, which inhibits histone deacetylases, resulted in increased histone H4 acetylation accompanied by the decondensation of interphase chromatin and a decrease in both global H3K9 dimethylation and DNA methylation during mitosis in maize root tip cells. These observations suggest that histone acetylation may affect DNA and histone methylation during mitosis. Treatment with 5-azacytidine, a cytosine analog that reduces DNA methylation, caused chromatin decondensation and mediated an increase in H4 acetylation, in addition to reduced DNA methylation and H3K9 dimethylation during interphase and mitosis. These results suggest that decreased DNA methylation causes a reduction in H3K9 dimethylation and an increase in H4 acetylation.ConclusionsThe interchangeable effects of 5-azacytidine and trichostatin A on H4 acetylation, DNA methylation and H3K9 dimethylation indicate a mutually reinforcing action between histone acetylation, DNA methylation and histone methylation with respect to chromatin modification. Treatment with trichostatin A and 5-azacytidine treatment caused a decrease in the mitotic index, suggesting that H4 deacetylation and DNA and H3K9 methylation may contain the necessary information for triggering mitosis in maize root tips.

Hybrid organisms may fail to develop, be sterile or they may be more vigorous than either of the parents. Examples of hybrid vigour or hybrid necrosis in the F1 are often not inherited stably in subsequent generations if they are associated with overdominance. There can also be transgressive phenotypes that are inherited stably in these later generations, but the underlying mechanisms are not well understood. Here we have investigated the possibility that stable transgressive phenotypes in the progeny of crosses between cultivated tomato (Solanum lycopersicum cv. M82) and a wild relative (Solanum pennellii, accession LA716) are associated with micro or small interfering(si) RNAs. We identified loci from which these small(s)RNAs were more abundant in hybrids than in either parent and we show that accumulation of such transgressive sRNAs correlated with suppression of the corresponding target genes. In one instance this effect was associated with hypermethylation of the corresponding genomic DNA. Our results illustrate a potential role of transgressive sRNAs in plant breeding and in natural evolution with wild plants.

BackgroundPatient-derived tumour xenografts are an attractive model for preclinical testing of anti-cancer drugs. Insights into tumour biology and biomarkers predictive of responses to chemotherapeutic drugs can also be gained from investigating xenograft models. As a first step towards examining the equivalence of epigenetic profiles between xenografts and primary tumours in paediatric leukaemia, we performed genome-scale DNA methylation and gene expression profiling on a panel of 10 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) tumours that were stratified by prednisolone response.ResultsWe found high correlations in DNA methylation and gene expression profiles between matching primary and xenograft tumour samples with Pearson’s correlation coefficients ranging between 0.85 and 0.98. In order to demonstrate the potential utility of epigenetic analyses in BCP-ALL xenografts, we identified DNA methylation biomarkers that correlated with prednisolone responsiveness of the original tumour samples. Differential methylation of CAPS2, ARHGAP21, ARX and HOXB6 were confirmed by locus specific analysis. We identified 20 genes showing an inverse relationship between DNA methylation and gene expression in association with prednisolone response. Pathway analysis of these genes implicated apoptosis, cell signalling and cell structure networks in prednisolone responsiveness.ConclusionsThe findings of this study confirm the stability of epigenetic and gene expression profiles of paediatric BCP-ALL propagated in mouse xenograft models. Further, our preliminary investigation of prednisolone sensitivity highlights the utility of mouse xenograft models for preclinical development of novel drug regimens with parallel investigation of underlying gene expression and epigenetic responses associated with novel drug responses.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-416) contains supplementary material, which is available to authorized users.

Mesenchymal cells' biology has been an important investigative tool to maximize bone regeneration through tissue engineering. Here we used mesenchymal cells from periodontal ligament (PDLCs) with high (h-) and low (l-) osteogenic potential, isolated from different donors, to investigate the impact of the individual epigenetic and transcriptional profiles on the osteogenic potential. Genome-wide and gene-specific DNA (hydroxy) methylation, mRNA expression and immunofluorescence analysis were carried out in h- and l-PDLCs at DMEM (non-induced to osteogenesis) and OM (induced-3rd and 10th days of osteogenic differentiation) groups in vitro. Genome-wide results showed distinct epigenetic profile among PDLCs with most of the differences on 10th day of OM; DMEMs showed higher concentrations (xOM) of differentially methylated probes in gene body, intronic and open sea (3rd day), increasing this concentration in TSS200 and island regions, at 10 days. At basal levels, h- and l-PDLCs showed different transcriptional profiles; l-PDLCs demonstrated higher levels of NANOG/OCT4/SOX2, BAPX1, DNMT3A, TET1/3, and lower levels of RUNX2 transcripts, confirmed by NANOG/OCT4 and RUNX2 immunofluorescence. After osteogenic induction, the distinct transcriptional profile of multipotentiality genes was maintained among PDLCs. In l-PDLCs, the anti-correlation between DNA methylation and gene expression in RUNX2 and NANOG indicates methylation could play a role in modulating both transcripts. Epigenetic and transcriptional distinct profiles detected at basal levels among PDLCs were maintained after osteogenic induction. We cannot discard the existence of a complex that represses osteogenesis, suggesting the individual donors' characteristics have significant impact on the osteogenic phenotype acquisition.

Triple negative breast cancer (TNBC) is an aggressive disease with a high degree of genomic instability. TNBC patients have poor prognosis and the risk of metastasis and death is increased for women who relapse within four years of treatment compared to other breast cancer subtypes. Currently, there are no reliable prognostic markers to identify which population is at risk for early relapse and the molecular mechanism for disease recurrence is not well understood. Epigenetic changes, especially DNA methylation, are common in breast cancer and have been found to be associated with increased metastatic capability. Therefore, the methylation states of genes in TNBC tumors compared to non-tumor breast tissue was examined in order to identify potential biomarkers and therapeutic targets in TNBC. Matched tumor and adjacent non-tumor tissue from patients with TNBC were obtained following surgical resection and genomic DNA was extracted. Epigenetic profiling of TNBC tumors and non-tumor tissue was performed using a highly sensitive and quantitative analytics platform, which utilizes methylation sensitive restriction endonucleases to detect changes in methylation of CpG sites. Millions of CpG sites exist within the human genome and many of these are altered with tumor formation and progression, therefore, changes in methylation profiles of CpG sites may be useful as a diagnostic and/or prognostic biomarker in TNBC patients. Non-metric multidimensional scaling ordination analysis of the CpG sites revealed highly distinct methylation patterns between tumor and non-tumor tissue. Approximately 326 sites had a significant methylation score difference (p&lt0.0025) between TNBC tumor and non-tumor tissue, with most of the CpG sites having greater than 2-fold change in methylation status. Analysis of functional gene classes using KEGG classifiers revealed a significant change in methylation patterns of genes involved in response to infections and other immune related functions. Additionally, the top ten genes with hyper- or hypomethylated sites within TNBC tumors when compared to non-tumor tissue were identified. Interestingly, Gli-1 was one of the top hypomethylated genes. Gli-1 has been shown in our lab and others to have significance in chemoresistance and recurrence in TNBC patients. Immunohistochemical analysis of TNBC tumors revealed Gli-1 overexpression in TNBC tumors compared to non-tumor tissue. Expression levels of Gli-1 in TNBC tumors correlated with stage (p&lt0.001) and recurrence free (p&lt0.0213) and overall survival (p&lt0.017). Thus, analysis of CpG methylated sites in TNBC tumors and non-tumor tissue revealed differences in epigenetic profiles allowing for distinction between tumor and non-tumor tissue. Genes in TNBC tumors with significant changes in methylation status may be potential candidate genes that serve as a diagnostic or prognostic biomarker for TNBC. Citation Format: Kimberly M. Arnold, Adam G. Marsh, Jennifer Sims-Mourtada. Epigenetic DNA methylation profiling of triple negative breast cancer: a quantitative NGS approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3369. doi:10.1158/1538-7445.AM2017-3369

Epigenetic variations have been shown to reveal vulnerability to diabetes and its complications. Although it has become clear that metabolic derangements, especially hyperglycemia, can impose a long-term metabolic memory that predisposes to diabetic complications, the underlying mechanisms remain to be understood. It has been suggested that epigenetics (e.g., histone modification, DNA methylation, and non-coding RNAs) help link metabolic disruption to aberrancies related to diabetic kidney disease (DKD). In this review, we discuss the key findings and advances made in the epigenetic risk profile of DKD and provide perspectives on the emerging topics that implicate epigenetics in DKD. Epigenetic profiles can be profoundly altered in patients with diabetes, in circulating blood cells as well as in renal tissues. These changes provide useful insight into the mechanisms of diabetic kidney injury and progressive kidney dysfunction. Increasing evidence supports the role of epigenetic regulation in DKD. More studies are needed to elucidate the mechanism and importance of epigenetic changes in the initiation and progression of DKD and to further explore their diagnostic and therapeutic potential in the clinical management of patients with diabetes who have a high risk for DKD.

A7.5 Combined Analysis of Epigenetic and Transcriptional Profiles in Different Immune Cells Identifies Hot Spots of Gene Regulation by DNA Methylation

Alpine habitats are shaped by harsh abiotic conditions and cold climates. Plant life in such habitats is challenging, as environmental influence can alter the conditions for development and reproduction. More specifically, phenotypic plasticity of morphological traits can be influenced by temperature stress. Temperature stress can also affect epigenetic and gene expression profiles, which may have an impact on acclimation and adaptation of the species. Polyploidy seems to affect the DNA methylation profiles, while distribution patterns suggest that it could be advantageous under cold conditions. Nevertheless, little is known about non -model plants, whether temperature stress can induce methylation changes depending on the cytotypes of the individuals, to what extent a treatment shift can induce epigenetic responses and how they are depicted in phenotypic plasticity and reproduction of the species. Furthermore, it remains vague how cold stress is translated in gene expression changes under different cytotypes and how such a putative response is framed through gene set pathways and epigenetic control. The perennial alpine plant Ranunculus kuepferi was utilized to investigate the correlations of cold stress with polyploidy, mode of reproduction, phenotypic plasticity, epigenetics, gene expression and geographical parthenogenesis . The species is mainly found in the wild with diploid and autotetraploid cytotypes, which are mostly sexual and facultative apomicts, respectively. Diploid and autotetraploid individuals were placed in two climate chambers and exposed to cold (+7°C day/+2°C night, -1°C cold shocks for three nights per week) and warm (control) (+15°C day/+10°C night) temperature treatments in climate growth chambers for four consecutive flowering periods and shifted from one condition to the other after the first flowering period. Methylation-sensitive amplified fragment- length polymorphism markers were applied for the first two years, to screen possible genome-wide methylation alterations triggered by temprerature treatments and treatment shifts. For the second year of t emperature treatments, morphological traits (height, leaves and flowers) and the proportion of well- developed seeds were measured as fitness indicators, while flow cytometric seed screening (FCSS) was utilized to determine the reproduction mode. Subsequently, comparisons with patterns of methylation-sensitive amplified fragment- length polymorphisms (MS-AFLPs/MSAPs) regarding the same year of treatment were conducted. Finally, for the last year of treatment, both cytotypes were investigated for their gene expression profiles via transcriptome sequencing and qRT-PCR. The datasets were analyzed for four predefined groups with respect to treatment (Cold/Warm) and ploidy level (Diploid/Tetraploid). DNA methylation profiles showed temperature sensitivity and propose a ploidy effect for both years of analysis. Likewise, the treatment shift had an impact on both cytotypes, resulting in significantly less epiloci, regardless of the shift’s direction. Such correlations of ploidy level and epigenetic profiles may reflec t DNA methylation dynamics during cold acclimation. The AMOVA results are in line with the hypothesis of cold stress influencing the epigenetic patterns, while they also depict the DNA methylation dynamics of tetraploids, as a response to temperature treatment shift. Concerning the phenotypic plasticity of the species under temperature treatments, the potential of acclimation under environmental conditions is underlined, as diploids grow better under warm conditions and tetraploids perform better in cold treatments, while the expressed morphological traits are linked with epigenetic patterns. Moreover, cold stress reduced the reproduction fitness but did not induce apomixis in diploid individuals. These results confirm the different niche preferences of cytotypes in natural populations and empower the geographical parthenogenesis scenario, which is previously proposed for the species. Cold acclimation of the cytotypes is further indicated by gene expression profiles. Overall, diploid individuals altered more gene set pathways than tetraploid ones, and suppressed pathways involved in ion/cation homeostasis. Gene Set Pathways mostly activated under tetraploids are related to cell wall and plasma membrane. Thus, tetraploids seem to be better acclimated to cold conditions, enabling them to colonize colder climatic areas in the Alps. Finally, an epigenetic background for gene regulation in response to temperature conditions is indicated.

Cellular mechanotransduction plays a central role in fibroblast activation during fibrotic disease progression, leading to increased tissue stiffness and reduced organ function. While the role of epigenetics in disease mechanotransduction has begun to be appreciated, little is known about how substrate mechanics, particularly the timing of mechanical inputs, regulate epigenetic changes such as DNA methylation and chromatin reorganization during fibroblast activation. In this work, we engineered a hyaluronic acid hydrogel platform with independently tunable stiffness and viscoelasticity to model normal (storage modulus, G' ∼ 0.5 kPa, loss modulus, G'' ∼ 0.05 kPa) to increasingly fibrotic (G' ∼ 2.5 and 8 kPa, G'' ∼ 0.05 kPa) lung mechanics. Human lung fibroblasts exhibited increased spreading and nuclear localization of myocardin-related transcription factor-A (MRTF-A) with increasing substrate stiffness within 1 day, with these trends holding steady for longer cultures. However, fibroblasts displayed time-dependent changes in global DNA methylation and chromatin organization. Fibroblasts initially displayed increased DNA methylation and chromatin decondensation on stiffer hydrogels, but both of these measures decreased with longer culture times. To investigate how culture time affected the responsiveness of fibroblast nuclear remodeling to mechanical signals, we engineered hydrogels amenable to in situ secondary crosslinking, enabling a transition from a compliant substrate mimicking normal tissue to a stiffer substrate resembling fibrotic tissue. When stiffening was initiated after only 1 day of culture, fibroblasts rapidly responded and displayed increased DNA methylation and chromatin decondensation, similar to fibroblasts on static stiffer hydrogels. Conversely, when fibroblasts experienced later stiffening at day 7, they showed no changes in DNA methylation and chromatin condensation, suggesting the induction of a persistent fibroblast phenotype. These results highlight the time-dependent nuclear changes associated with fibroblast activation in response to dynamic mechanical perturbations and may provide mechanisms to target for controlling fibroblast activation.

The mutagenic activity of N-nitrosodimethylamine (NDMA) in the laboratory mice was studied usingthe chromosome aberration test. It was established that NDMA with intraperitoneal single administration(acute experience) in doses of 2.0; 4,0 and 8,0 mg/kg induced chromosomal aberrations in the mouse bonemarrow cells with a frequency statistically significantly exceeding the control level. With an increase in thedose of xenobiotics, the frequency of aberrant cells increased by 2.23 (p <0.05); 3.00 (p <0.05) and 3.89(p <0.001) times, respectively. The dose dependence of the level of induced mutagenesis was revealed(r = 0.97, p = 0.03). A statistically significant increase in the level of aneuploid and polyploid cells wasestablished, however, no dose dependence was found (r = 0.85, p = 0.29). Prolonged intoxication ofNDMA (subacute experience, intoxication within 10 days) of experimental animals resulted in a statistically significant increase in the frequency of aberrant bone marrow cells and the number of chromosomalaberrations per 100 metaphase compared to intact animals and animals of acute experience. The dose ofNDMA 8 mg/kg, equal to 1/5 LD50, with repeated administration was lethal for all individuals. With repeated administration of NDMA at doses of 2.0 and 4.0 mg/kg, the frequency of aberrant cells increasedstatistically significantly in comparison with a single injection of 1.70 (p <0.001) and 1.60 (p <0.01), respectively, and the number of chromosomal aberrations per 100 metaphase is 1.73 (p <0.001) and 1.51 (p<0.01) times. With prolonged exposure to xenobiotic, the frequency of cells with genomic mutations alsoincreased statistically. The increase in the overall frequency of chromosomal aberrations occurred mainlydue to chromatin-type disorders. The mutagenic effect of N-nitrosodimethylamine on mice, established inour studies, may be due to an increase in the level of active forms of oxygen and the accumulation of lipidperoxidation products in the tissues of the body. Possible mechanisms of mutagenic and genotoxic actionof NDMA can be the enhancement of free radical processes and DNA methylation.Key words: N-nitrosodimethylamine, mutagenic effect, acute and subacute effects, chromosomalaberrations, genomic mutations.

DNA Methylation Plasticity of Human Adipose-Derived Stem Cells in Lineage Commitment

Epigenetic Profile of Treg-like Cells Induced By Mesenchymal Stem Cells in Vitro Resembles That of Natural Treg

In 2014, Facebook and Apple announced that they would pay for female employees to have their oocytes frozen to allow them to delay having children and instead focus on their careers. Whatever motivated the companies to make their offers, the fact that they did so highlights a prevalent problem faced by many young women: Their most fertile years are also a crucial period for building a career, when time off work may disadvantage them. > … cryopreservation is known to affect cell survival after thawing, which can have an impact on the subsequent clinical applications of frozen cells. To fulfill their offers, Facebook and Apple will need to offer their employees access to cryopreservation technologies that profoundly change the dynamics of family planning. Such technologies are not new, but work over the past decades has been aimed at increasing safety and efficacy and has reduced costs to the point that companies can now offer cryopreservation as a way to attract and retain female workers. Of course, the potential of cryopreservation goes far beyond freezing the eggs or sperm of ambitious young technology workers—it is a ubiquitous technology used in research and medicine for a wide variety of applications (Fig 1). For example, cryopreservation is used to store and transport biological material, including adult stem cells or stem cells from umbilical cord blood or bone marrow—both of which can later be used to treat disease or extend lifespan in the same patient—blood donations, especially of rare blood types, tissues, and organs. It is also offered as a crucial service for cancer patients to preserve their gametes before they undergo therapy that may render them infertile and, generally used in assisted reproduction to store oocytes, fertilized eggs, or embryos. Cryopreservation can contribute to environmental preservation efforts, where it is used to conserve the …

Normal lymphocytes were found to adhere strongly to monolayer cultures of fibroblasts deficient in the lysosomal enzyme, beta-glucuronidase. During this co-culture, the fibroblasts acquired from the lymphocytes substantial amounts of this enzyme, which often accumulated at sites of contact between the two types of cell. Enzyme transfer was prevented by addition to the co-cultures either of purified lymphocyte plasma membranes or of antibody raised against such plasma membranes, but it was not inhibited by the addition of antibody raised against lymphocyte-derived beta-glucuronidase. An active role for the lymphocyte in this contact-dependent process was suggested by interference contrast, immunofluorescence and scanning electron-microscopic studies. These revealed extensive arrays of projections of the lymphocyte that ramified over the fibroblast cell surface. By transmission electron microscopy, conspicuous clusters of micropinocytotic vesicles were evident in the cytoplasm of the 'recipient' fibroblasts, subjacent to the surface in regions closely apposed to adherent lymphocytes. Such high frequencies of these vesicles were restricted to sites of lymphocyte-fibroblast contact, suggesting that they may play an important part in the transfer of enzyme between these two types of cell.

Folate is an essential mediator in one-carbon metabolism, which provides methyl groups for DNA synthesis and methylation. The availability of active methyl groups can be influenced by the uptake of folic acid. We conducted a randomized intervention trial to test the influence of folic acid supplementation on DNA methylation in an unfortified population in Germany. A total of 16 healthy male volunteers (age range 23–61 y) were randomized to receive either 400 μg (n = 9) or 800 μg (n = 7) folic acid supplements daily for 8 weeks. Infinium Human Methylation 450K BeadChip Microarrays were used to assay site-specific DNA methylation across the genome. Microarray analyses were conducted on PBL DNA. We estimated several epigenetic clocks and mean DNA methylation across all autosomal probes on the array. AgeAccel was estimated as the residual variation in each metric. In virtually all participants, both serum and red blood cell (RBC) folate increased successively throughout the trial period. Participants with a larger increase in RBC folate had a larger increase in DNAmAge AgeAccel (Spearman Rho: 0.56, p-value = 0.03). No notable changes in the methylome resulting from the folic acid supplementation emerged. In this population with adequate folate levels derived from diet, an increase in RBC folate had a modest impact on the epigenetic clock predicting chronologic age.