Epigenetic Stability Research Articles

Genetic and epigenetic stability of stem cells: Epigenetic modifiers modulate the fate of mesenchymal stem cells

Stem cell research has progressed widely and has been receiving a considerable attention for its advantages and drawbacks. Despite their extensive therapeutic potential in regenerative medicine, they are debatable for their genetic and epigenetic stability. In fact lineage specific differentiation is mediated via epigenetic changes in DNA methylation, acetylation, histone modifications etc. Thus epigenetics plays an important role in stem cell biology. For therapeutic interventions stem cells need to be genetically and epigenetically stable for their maximum paracrine secretions for bringing about expected tissue repair and regeneration. In this review we have focused on the current status of genetic and epigenetic stability in stem cells and their importance in regenerative medicine. We have also touched upon the possibility of considering tissue resident mesenchymal stem cells as epigenetic modifiers. This is likely to open a new era in stem cell therapeutic intervention by reversing disease inducing epigenetic changes.

Loss of Thymine DNA Glycosylase Causes Dysregulation of Bile Acid Homeostasis and Hepatocellular Carcinoma

Thymine DNA glycosylase (TDG) is a nuclear receptor coactivator that plays an essential role in the maintenance of epigenetic stability in cells. Here, we demonstrate that the conditional deletion of TDG in adult mice results in a male-predominant onset of hepatocellular carcinoma (HCC). TDG loss leads to a prediabetic state, as well as bile acid (BA) accumulation in the liver and serum of male mice. Consistent with these data, TDG deletion led to dysregulation of the farnesoid X receptor (FXR) and small heterodimer partner (SHP) regulatory cascade in the liver. FXR and SHP are tumor suppressors of HCC and play an essential role in BA and glucose homeostasis. These results indicate that TDG functions as a tumor suppressor of HCC by regulating a transcriptional program that protects against the development of glucose intolerance and BA accumulation in the liver.

Genetic and Epigenetic Stability in Rye Seeds under Different Storage Conditions: Ageing and Oxygen Effect.

Seed ageing is a complex process and can be described as the loss of viability or quality with time. It is important to elucidate whether genetic and epigenetic stability is altered in stored seeds and in seedlings produced from them. Non-stored and stored rye seeds at different stages of ageing were compared, as well as the seedlings obtained from them. Seeds were stored at 35 °C and 15% water content, under vacuum or air atmosphere. DNA of seeds and seedlings was isolated at three stages of the deterioration curve: P75 (13 days), P20 (29 days), and P0 (36 days). Genetic stability was assessed by RAPD technique, and epigenetic changes by MSAP markers. While seeds showed genetic stability after storage, the similarity of seedlings obtained from seeds stored for 29 days was lower (95%) when compared to seedlings from control seeds. Epigenetic changes were between 15% and 30% (both de novo methylation and demethylation) in the stored seeds compared to control seeds, with no differences between 13 and 29 days of storage with either air or vacuum atmospheres. In seedlings, epigenetic changes significantly increased with storage time. In conclusion, ageing increased epigenetic instability in both seeds and seedlings, when compared to controls.

Transcription-Associated Cyclin-Dependent Kinases as Targets and Biomarkers for Cancer Therapy.

Drugs targeting the cell cycle-regulatory cyclin-dependent kinase (CDK) 4 and 6 have been approved for the treatment of hormone receptor-positive breast cancer, and inhibitors targeting other cell-cycle CDKs are currently in clinical trials. Another class of CDKs, the transcription-associated CDKs, including CDK7, CDK8, CDK9, CDK12 and CDK13, are critical regulators of gene expression. Recent evidence suggests several novel functions of these CDKs, including regulation of epigenetic modifications, intronic polyadenylation, DNA-damage responses, and genomic stability. Here, we summarize our current understanding of the transcriptional CDKs, their utility as biomarkers, and their potential as therapeutic targets. SIGNIFICANCE: CDK inhibitors targeting CDK4 and CDK6 have been approved in hormone receptor-positive breast cancer, and inhibitors targeting other cell-cycle CDKs are currently in clinical trials. Several studies now point to potential therapeutic opportunities by inhibiting the transcription-associated CDKs as well as therapeutic vulnerabilities with PARP inhibitors and immunotherapy in tumors deficient in these CDKs.

P53 and The Immune Response: 40 Years of Exploration-A Plan for the Future.

The p53 field was born from a marriage of the techniques of cancer virus research and immunology. Over the past 40 years, it has followed the path of cancer research. Now cancer treatments are turning to immunotherapy, and there are many hints of the role of the p53 protein in both the regulation of the innate immune system and as an antigen in adaptive immune responses. The p53 gene and protein are part of the innate immune system, and play an important role in infectious diseases, senescence, aging, and the surveillance of repetitive DNA and RNAs. The mutant form of the p53 protein in cancers elicits both a B-cell antibody response (a tumor antigen) and a CD-8 killer T-cell response (a tumor-specific transplantation antigen). The future will take the p53-immune response field of research into cancer immunotherapy, autoimmunity, inflammatory responses, neuro-degeneration, aging, and life span, and the regulation of epigenetic stability and tissue regeneration. The next 40 years will bring the p53 gene and its proteins out of a cancer focus and into an organismic and environmental focus.

Rádioaktívna kontaminácia v Černobyle a (epi)genetická stabilita rastlín - Prehľad

Rapid industrial and agricultural development brought, besides the indisputable advances, also risks related to the environmental pollution. Widespread soil deterioration represents a global problem. Chernobyl area contaminated by radionuclides after nuclear accident in 1986 provides the opportunity to analyze in situ the impact of radiation on plant systems. Unlike animals and humans, plants are not able to move to another place with better living conditions. Therefore, plants are an ideal object to study adaptation to the conditions of environmental stress. Long- term exposure to the ionizing radiation causes widespread changes in plant genome and epigenome. Also, these alterations may result in changed phenotype. In particular, this review discusses the effect of ionizing radiation on genetic and epigenetic stability of plant genome.

Positioning Heterochromatin at the Nuclear Periphery Suppresses Histone Turnover to Promote Epigenetic Inheritance

In eukaryotes, heterochromatin is generally located at the nuclear periphery. This study investigates the biological significance of perinuclear positioning for heterochromatin maintenance and gene silencing. We identify the nuclear rim protein Amo1NUPL2 as a factor required for the propagation of heterochromatin at endogenous and ectopic sites in the fission yeast genome. Amo1 associates with the Rix1PELP1-containing RNA processing complex RIXC and with the histone chaperone complex FACT. RIXC, which binds to heterochromatin protein Swi6HP1 across silenced chromosomal domains and to surrounding boundary elements, connects heterochromatin with Amo1 at the nuclear periphery. In turn, the Amo1-enriched subdomain is critical for Swi6 association with FACT that precludes histone turnover to promote gene silencing and preserve epigenetic stability ofheterochromatin. In addition to uncovering conserved factors required for perinuclear positioning of heterochromatin, these analyses elucidate a mechanism by which a peripheral subdomain enforces stable gene repression and maintains heterochromatin in a heritable manner.

Mechanisms of DNA Replication and Repair: Insights from the Study of G-Quadruplexes.

G-quadruplexes are four-stranded guanine-rich structures that have been demonstrated to occur across the genome in humans and other organisms. They provide regulatory functions during transcription, translation and immunoglobulin gene rearrangement, but there is also a large amount of evidence that they can present a potent barrier to the DNA replication machinery. This mini-review will summarize recent advances in understanding the many strategies nature has evolved to overcome G-quadruplex-mediated replication blockage, including removal of the structure by helicases or nucleases, or circumventing the deleterious effects on the genome through homologous recombination, alternative end-joining or synthesis re-priming. Paradoxically, G-quadruplexes have also recently been demonstrated to provide a positive role in stimulating the initiation of DNA replication. These recent studies have not only illuminated the many roles and consequences of G-quadruplexes, but have also provided fundamental insights into the general mechanisms of DNA replication and its links with genetic and epigenetic stability.

Genetic and epigenetic stability of recovered mint apices after several steps of a cryopreservation protocol by encapsulation-dehydration. A new approach for epigenetic analysis

The genetic and epigenetic stability (analysis of DNA methylation using MSAP markers) of mint (Mentha x piperita L.) apices was studied after each step of a cryopreservation protocol, by encapsulation-dehydration. The effect of the addition of an antioxidant (ascorbic acid) during one of the protocol steps was also evaluated. Eight-week old in vitro recovered shoots from apices after each step of the protocol were genetically stable when compared to control in vitro shoots, using RAPD and AFLP markers. The addition of ascorbic acid in the medium with the highest sucrose concentration did not improve recovery and did not have any effect on stability. Apices sampled immediately after each step showed increased epigenetic differences as the protocol advanced, compared to in vitro control apices, in particular related to de novo methylation events. However, after one-day in vitro recovery, methylation status was similar to control apices. To improve the quality of methylation data interpretation, a simple and fast method for MSAP markers analysis, based on R programming, has been developed which allows the statistical comparison of treatments to control samples and its graphical representation.

Epigenetics and Neurological Disorders in ART

About 1–4% of children are currently generated by Assisted Reproductive Technologies (ART) in developed countries. These babies show only a slightly increased risk of neonatal malformations. However, follow-up studies have suggested a higher susceptibility to multifactorial, adult onset disorders like obesity, diabetes and cardiovascular diseases in ART offspring. It has been suggested that these conditions could be the consequence of epigenetic, alterations, due to artificial manipulations of gametes and embryos potentially able to alter epigenetic stability during zygote reprogramming. In the last years, epigenetic alterations have been invoked as a possible cause of increased risk of neurological disorders, but at present the link between epigenetic modifications and long-term effects in terms of neurological diseases in ART children remains unclear, due to the short follow up limiting retrospective studies. In this review, we summarize the current knowledge about neurological disorders promoted by epigenetics alterations in ART. Based on data currently available, it is possible to conclude that little, if any, evidence of an increased risk of neurological disorders in ART conceived children is provided. Most important, the large majority of reports appears to be limited to epidemiological studies, not providing any experimental evidence about epigenetic modifications responsible for an increased risk.

Human rDNA copy number is unstable in metastatic breast cancers

ABSTRACTChromatin-mediated silencing, including the formation of heterochromatin, silent chromosome territories, and repressed gene promoters, acts to stabilize patterns of gene regulation and the physical structure of the genome. Reduction of chromatin-mediated silencing can result in genome rearrangements, particularly at intrinsically unstable regions of the genome such as transposons, satellite repeats, and repetitive gene clusters including the rRNA gene clusters (rDNA). It is thus expected that mutational or environmental conditions that compromise heterochromatin function might cause genome instability, and diseases associated with decreased epigenetic stability might exhibit genome changes as part of their aetiology. We find the support of this hypothesis in invasive ductal breast carcinoma, in which reduced epigenetic silencing has been previously described, by using a facile method to quantify rDNA copy number in biopsied breast tumours and pair-matched healthy tissue. We found that rDNA and satellite DNA sequences had significant copy number variation – both losses and gains of copies – compared to healthy tissue, arguing that these genome rearrangements are common in developing breast cancer. Thus, any proposed aetiology onset or progression of breast cancer should consider alterations to the epigenome, but must also accommodate concomitant changes to genome sequence at heterochromatic loci.

Transcriptional activity of epigenetic remodeling genes declines in keratinocytes after in vitro expansion

PurposeIn vitro expansion is an invaluable method to obtain keratinocytes in amounts necessary for effective transplantation therapies. In vitro cell culturing provokes questions concerning potential epigenetic alterations occurring in expanded cells in the context of usefulness for transplantation and safety. The purpose of this study was to investigate as to whether keratinocyte expansion is associated with changes in the activity of genes responsible for the maintenance of epigenetic stability. Materials and methodsWe focussed on the transcriptional activity of genes involved in different epigenetic mechanisms including DNA methylation and histone modifications. We used quantitative real-time PCR to determine transcript levels of 16 epigenetic remodelling markers in 14 patients in the epidermal cells directly after collection and after in vitro expansion. ResultsWe observed a remarkable decline in the transcriptional activity of the epigenetic remodelling genes following in vitro expansion, while no further fall of expression with passaging. In whole skin, we found even higher expression levels of the epigenetic markers. ConclusionsTransmission to in vitro environment challenges cellular signalling and metabolism. The regulation of epigenetic remodelling maintains the balance between cellular plasticity and phenotype deviation. This preliminary research demonstrated reduced activity of genes responsible for epigenetic modifications of DNA and histones in in vitro expanded epidermal cells. This observation indicates that epigenome re-patterning in cultured epidermal cells is significantly less intensive than in the skin. Also, this observation may imply that after adaptation to in vitro conditions, the epigenome does not undergo extensive transformation during further cultivation.

Transient Stability of Epigenetic Population Differentiation in a Clonal Invader.

Epigenetic variation may play an important role in how plants cope with novel environments. While significant epigenetic differences among plants from contrasting habitats have often been observed in the field, the stability of these differences remains little understood. Here, we combined field monitoring with a multi-generation common garden approach to study the dynamics of DNA methylation variation in invasive Chinese populations of the clonal alligator weed (Alternanthera philoxeroides). Using AFLP and MSAP markers, we found little variation in DNA sequence but substantial epigenetic population differentiation. In the field, these differences remained stable across multiple years, whereas in a common environment they were maintained at first but then progressively eroded. However, some epigenetic differentiation remained even after 10 asexual generations. Our data indicate that epigenetic variation in alligator weed most likely results from a combination of environmental induction and spontaneous epimutation, and that much of it is neither rapidly reversible (phenotypic plasticity) nor long-term stable, but instead displays an intermediate level of stability. Such transient epigenetic stability could be a beneficial mechanism in novel and heterogeneous environments, particularly in a genetically impoverished invader.

Wnt/\u03b2-catenin signaling pathway safeguards epigenetic stability and homeostasis of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) are pluripotent and can differentiate into cells belonging to the three germ layers of the embryo. However, mESC pluripotency and genome stability can be compromised in prolonged in vitro culture conditions. Several factors control mESC pluripotency, including Wnt/β-catenin signaling pathway, which is essential for mESC differentiation and proliferation. Here we show that the activity of the Wnt/β-catenin signaling pathway safeguards normal DNA methylation of mESCs. The activity of the pathway is progressively silenced during passages in culture and this results into a loss of the DNA methylation at many imprinting control regions (ICRs), loss of recruitment of chromatin repressors, and activation of retrotransposons, resulting into impaired mESC differentiation. Accordingly, sustained Wnt/β-catenin signaling maintains normal ICR methylation and mESC homeostasis and is a key regulator of genome stability.

Intricate regulation on epigenetic stability of the subtelomeric heterochromatin and the centromeric chromatin in fission yeast.

In eukaryotes, the integrity of chromatin structure and organization is crucial to diverse key cellular processes from development to disease avoidance. To maintain the cell identity through mitotic cell generations, the genome (the genomic DNA sequence) as well as the epigenome (pertaining various forms of epigenetic information carriers, such as histone modifications, nucleosome positioning and the chromatin organization) is inherited with high fidelity. In comparison to the wealth of knowledge on genetic stability, we know much less on what may control the accuracy of epigenetic inheritance. In our recent work in the fission yeast Schizosaccharomyces pombe, by quantifying the epigenetic fidelity of CENP-A/Cnp1 or H3K9me2 nucleosome inheritance through cell divisions, we demonstrated that Ccp1, a homolog of histone chaperone Vps75 in budding yeast, participates in the modulation of centromeric nucleosomal epigenetic stability as well as proper heterochromatin organization. In this essay, we focus on discussing the uniquely high dynamicity of the subtelomeric heterochromatin regions and the complex mechanisms regulating epigenetic stability of centromeric chromatin.

Epigenetic stability in Saffron (Crocus sativus L.) accessions during four consecutive years of cultivation and vegetative propagation under open field conditions

Saffron (Crocus sativus L.) is a sterile species that is vegetatively propagated in the field, year by year, via the production of new corms. While Saffron’s genetic variability is extremely low, phenotypic variation is frequently observed in the field and epigenetics could be a possible origin of these alternative phenotypes. Present day knowledge on Saffron epigenetics is very low or absent. In the present paper, to deepen existing knowledge, we focused on the epigenetic differences and stability among 17 Saffron accessions, of different geographic origin, during four consecutive years of vegetative propagation under open field conditions. Before the analysis, the selected accessions have been cultivated in the same field for at least three consecutive years. Despite the low genetic variability and the prolonged co-cultivation in the same environment, Methylation-Sensitive Amplified Fragment Length Polymorphism (MS-AFLP) analysis revealed a very high epigenetic difference among accessions, making it possible to discriminate them based on the epigenetic profiles. During the four years of the study, a little variation has been observed within accessions following different patterns, slightly modifying the accession epigenotypes but not enough to even them to a more uniform profile. These results confirm that, under natural conditions, Saffron epigenotypes are highly stable, supporting a role for epigenetics in phenotypic variability.

Whole Genome Incorporation and Epigenetic Stability in a Newly Synthetic Allopolyploid of Gynogenetic Gibel Carp.

Allopolyploidization plays an important role in speciation, and some natural or synthetic allopolyploid fishes have been extensively applied to aquaculture. Although genetic and epigenetic inheritance and variation associated with plant allopolyploids have been well documented, the relative research in allopolyploid animals is scarce. In this study, the genome constitution and DNA methylation inheritance in a newly synthetic allopolyploid of gynogenetic gibel carp were analyzed. The incorporation of a whole genome of paternal common carp sperm in the allopolyploid was confirmed by genomic in situ hybridization, chromosome localization of 45S rDNAs, and sequence comparison. Pooled sample-based methylation sensitive amplified polymorphism (MSAP) revealed that an overwhelming majority (98.82%) of cytosine methylation patterns in the allopolyploid were inherited from its parents of hexaploid gibel carp clone D and common carp. Compared to its parents, 11 DNA fragments in the allopolyploid were proved to be caused by interindividual variation, recombination, deletion, and mutation through individual sample-based MSAP and sequencing. Contrast to the rapid and remarkable epigenetic changes in most of analyzed neopolyploids, no cytosine methylation variation was detected in the gynogenetic allopolyploid. Therefore, the newly synthetic allopolyploid of gynogenetic gibel carp combined genomes from its parents and maintained genetic and epigenetic stability after its formation and subsequently seven successive gynogenetic generations. Our current results provide a paradigm for recurrent polyploidy consequences in the gynogenetic allopolyploid animals.

Ccp1 modulates epigenetic stability at centromeres and affects heterochromatin distribution in Schizosaccharomyces pombe

Distinct chromatin organization features, such as centromeres and heterochromatin domains, are inherited epigenetically. However, the mechanisms that modulate the accuracy of epigenetic inheritance, especially at the individual nucleosome level, are not well-understood. Here, using ChIP and next-generation sequencing (ChIP-Seq), we characterized Ccp1, a homolog of the histone chaperone Vps75 in budding yeast that functions in centromere chromatin duplication and heterochromatin maintenance in fission yeast (Schizosaccharomyces pombe). We show that Ccp1 is enriched at the central core regions of the centromeres. Of note, among all histone chaperones characterized, deletion of the ccp1 gene uniquely reduced the rate of epigenetic switching, manifested as position effect variegation within the centromeric core region (CEN-PEV). In contrast, gene deletion of other histone chaperones either elevated the PEV switching rates or did not affect centromeric PEV. Ccp1 and the kinetochore components Mis6 and Sim4 were mutually dependent for centromere or kinetochore association at the proper levels. Moreover, Ccp1 influenced heterochromatin distribution at multiple loci in the genome, including the subtelomeric and the pericentromeric regions. We also found that Gar2, a protein predominantly enriched in the nucleolus, functions similarly to Ccp1 in modulating the epigenetic stability of centromeric regions, although its mechanism remained unclear. Together, our results identify Ccp1 as an important player in modulating epigenetic stability and maintaining proper organization of multiple chromatin domains throughout the fission yeast genome.

Automethylation-induced conformational switch in Clr4 (Suv39h) maintains epigenetic stability.

Histone H3 lysine 9 methylation (H3K9me) mediates heterochromatic gene silencing and is important for genome stability and regulation of gene expression1–4. The establishment and epigenetic maintenance of heterochromatin involve the recruitment of H3K9 methyltransferases to specific sites on DNA followed by the recognition of pre-existing H3K9me by the methyltransferase and methylation of proximal histone H35-11. This positive feedback loop must be tightly regulated to prevent deleterious epigenetic gene silencing. Extrinsic anti-silencing mechanisms involving histone demethylation or boundary elements help limit inappropriate H3K9me spreading12–15. However, how H3K9 methyltransferase activity is locally restricted or prevented from initiating random H3K9me leading to aberrant gene silencing and epigenetic instability is not fully understood. Here we reveal an autoinhibited conformation in the conserved fission yeast S. pombe H3K9 methyltransferase Clr4/Suv39h that plays a critical role in preventing aberrant heterochromatin formation. Biochemical and X-ray crystallographic data show that an internal loop in Clr4 inhibits its catalytic activity by blocking the histone H3K9 substrate-binding pocket, and that automethylation of specific lysines in this loop promotes a conformational switch that enhances Clr4 H3K9 methylation activity. Mutations predicted to disrupt this regulation lead to aberrant H3K9me, loss of heterochromatin domains, and growth inhibition, demonstrating the importance of Clr4 intrinsic inhibition and auto-activation in regulating H3K9me deposition and preventing epigenetic instability. Conservation of the Clr4 autoinhibitory loop in other H3K9 methyltransferases, and automethylation of a corresponding lysine in the human SUV39H2 homolog16, suggest that the mechanism described here is broadly conserved.

MSX2 Initiates and Accelerates Mesenchymal Stem/Stromal Cell Specification of hPSCs by Regulating TWIST1 and PRAME

SummaryThe gap in knowledge of the molecular mechanisms underlying differentiation of human pluripotent stem cells (hPSCs) into the mesenchymal cell lineages hinders the application of hPSCs for cell-based therapy. In this study, we identified a critical role of muscle segment homeobox 2 (MSX2) in initiating and accelerating the molecular program that leads to mesenchymal stem/stromal cell (MSC) differentiation from hPSCs. Genetic deletion of MSX2 impairs hPSC differentiation into MSCs. When aided with a cocktail of soluble molecules, MSX2 ectopic expression induces hPSCs to form nearly homogeneous and fully functional MSCs. Mechanistically, MSX2 induces hPSCs to form neural crest cells, an intermediate cell stage preceding MSCs, and further differentiation by regulating TWIST1 and PRAME. Furthermore, we found that MSX2 is also required for hPSC differentiation into MSCs through mesendoderm and trophoblast. Our findings provide novel mechanistic insights into lineage specification of hPSCs to MSCs and effective strategies for applications of stem cells for regenerative medicine.