Borrowing Nuclear DNA Helicases to Protect Mitochondrial DNA

In normal cells, mitochondria are the primary organelles that generate energy, which is critical for cellular metabolism. Mitochondrial dysfunction, caused by mitochondrial DNA (mtDNA) mutations or an abnormal mtDNA copy number, is linked to a range of human diseases, including Alzheimer’s disease, premature aging‎ and cancer. mtDNA resides in the mitochondrial lumen, and its duplication requires the mtDNA replicative helicase, Twinkle. In addition to Twinkle, many DNA helicases, which are encoded by the nuclear genome and are crucial for nuclear genome integrity, are transported into the mitochondrion to also function in mtDNA replication and repair. To date, these helicases include RecQ-like helicase 4 (RECQ4), petite integration frequency 1 (PIF1), DNA replication helicase/nuclease 2 (DNA2) and suppressor of var1 3-like protein 1 (SUV3). Although the nuclear functions of some of these DNA helicases have been extensively studied, the regulation of their mitochondrial transport and the mechanisms by which they contribute to mtDNA synthesis and maintenance remain largely unknown. In this review, we attempt to summarize recent research progress on the role of mammalian DNA helicases in mitochondrial genome maintenance and the effects on mitochondria-associated diseases.     

Epigenetic effects of environmental chemicals bisphenol a and phthalates

The epigenetic effects on DNA methylation, histone modification, and expression of non-coding RNAs (including microRNAs) of environmental chemicals such as bisphenol A (BPA) and phthalates have expanded our understanding of the etiology of human complex diseases such as cancers and diabetes. Multiple lines of evidence from in vitro and in vivo models have established that epigenetic modifications caused by in utero exposure to environmental toxicants can induce alterations in gene expression that may persist throughout life. Epigenetics is an important mechanism in the ability of environmental chemicals to influence health and disease, and BPA and phthalates are epigenetically toxic. The epigenetic effect of BPA was clearly demonstrated in viable yellow mice by decreasing CpG methylation upstream of the Agouti gene, and the hypomethylating effect of BPA was prevented by maternal dietary supplementation with a methyl donor like folic acid or the phytoestrogen genistein. Histone H3 was found to be trimethylated at lysine 27 by BPA effect on EZH2 in a human breast cancer cell line and mice. BPA exposure of human placental cell lines has been shown to alter microRNA expression levels, and specifically, miR-146a was strongly induced by BPA treatment. In human breast cancer MCF7 cells, treatment with the phthalate BBP led to demethylation of estrogen receptor (ESR1) promoter-associated CpG islands, indicating that altered ESR1 mRNA expression by BBP is due to aberrant DNA methylation. Maternal exposure to phthalate DEHP was also shown to increase DNA methylation and expression levels of DNA methyltransferases in mouse testis. Further, some epigenetic effects of BPA and phthalates in female rats were found to be transgenerational. Finally, the available new technologies for global analysis of epigenetic alterations will provide insight into the extent and patterns of alterations between human normal and diseased tissues. In vitro models such as human embryonic stem cells may be extremely useful in bettering the understanding of epigenetic effects on human development, health and disease, because the formation of embryoid bodies in vitro is very similar to the early stage of embryogenesis.     

Chemo-Enzymatic Fluorescence Labeling Of Genomic DNA For Simultaneous Detection Of Global 5-Methylcytosine And 5-Hydroxymethylcytosine**

5-Methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. We present a new, simple, and high-throughput platform for multi-color epigenetic analysis. The novelty of our approach is the ability to multiplex methylation and de-methylation signals in the same assay. We utilize an engineered methyltransferase enzyme that recognizes and labels all unmodified CpG sites with a fluorescent cofactor. In combination with the already established labeling of the de-methylation mark 5-hydroxymethylcytosine via enzymatic glycosylation, we obtained a robust platform for simultaneous epigenetic analysis of these marks. We assessed the global epigenetic levels in multiple samples of colorectal cancer and observed a 3.5-fold reduction in 5hmC levels but no change in DNA methylation levels between sick and healthy individuals. We also measured epigenetic modifications in chronic lymphocytic leukemia and observed a decrease in both modification levels (5-hydroxymethylcytosine: whole blood 30 %; peripheral blood mononuclear cells (PBMCs) 40 %. 5-methylcytosine: whole blood 53 %; PBMCs 48 %). Our findings propose using a simple blood test as a viable method for analysis, simplifying sample handling in diagnostics. Importantly, our results highlight the assay‘s potential for epigenetic evaluation of clinical samples, benefiting research and patient management.     

Epigenetic disregulation in oral cancer

Squamous cell carcinoma of the oral region (OSCC) is one of the most common and highly aggressive malignancies worldwide, despite the fact that significant results have been achieved during the last decades in its detection, prevention and treatment. Although many efforts have been made to define the molecular signatures that identify the clinical outcome of oral cancers, OSCC still lacks reliable prognostic molecular markers. Scientific evidence indicates that transition from normal epithelium to pre-malignancy, and finally to oral carcinoma, depends on the accumulation of genetic and epigenetic alterations in a multistep process. Unlike genetic alterations, epigenetic changes are heritable and potentially reversible. The most common examples of such changes are DNA methylation, histone modification, and small non-coding RNAs. Although several epigenetic changes have been currently linked to OSCC initiation and progression, they have been only partially characterized. Over the last decade, it has been demonstrated that especially aberrant DNA methylation plays a critical role in oral cancer. The major goal of the present paper is to review the recent literature about the epigenetic modifications contribution in early and later phases of OSCC malignant transformation; in particular we point out the current evidence of epigenetic marks as novel markers for early diagnosis and prognosis as well as potential therapeutic targets in oral cancer.     

Mitochondrial Aminoacyl-tRNA Synthetase and Disease: The Yeast Contribution for Functional Analysis of Novel Variants

In most eukaryotes, mitochondrial protein synthesis is essential for oxidative phosphorylation (OXPHOS) as some subunits of the respiratory chain complexes are encoded by the mitochondrial DNA (mtDNA). Mutations affecting the mitochondrial translation apparatus have been identified as a major cause of mitochondrial diseases. These mutations include either heteroplasmic mtDNA mutations in genes encoding for the mitochondrial rRNA (mtrRNA) and tRNAs (mttRNAs) or mutations in nuclear genes encoding ribosomal proteins, initiation, elongation and termination factors, tRNA-modifying enzymes, and aminoacyl-tRNA synthetases (mtARSs). Aminoacyl-tRNA synthetases (ARSs) catalyze the attachment of specific amino acids to their cognate tRNAs. Differently from most mttRNAs, which are encoded by mitochondrial genome, mtARSs are encoded by nuclear genes and then imported into the mitochondria after translation in the cytosol. Due to the extensive use of next-generation sequencing (NGS), an increasing number of mtARSs variants associated with large clinical heterogeneity have been identified in recent years. Being most of these variants private or sporadic, it is crucial to assess their causative role in the disease by functional analysis in model systems. This review will focus on the contributions of the yeast Saccharomyces cerevisiae in the functional validation of mutations found in mtARSs genes associated with human disorders.     

Epigenetic Distribution of Recombinant Plant Chromosome Fragments in a Human–Arabidopsis Hybrid Cell Line

Methylation systems have been conserved during the divergence of plants and animals, although they are regulated by different pathways and enzymes. However, studies on the interactions of the epigenomes among evolutionarily distant organisms are lacking. To address this, we studied the epigenetic modification and gene expression of plant chromosome fragments (~30 Mb) in a human–Arabidopsis hybrid cell line. The whole-genome bisulfite sequencing results demonstrated that recombinant Arabidopsis DNA could retain its plant CG methylation levels even without functional plant methyltransferases, indicating that plant DNA methylation states can be maintained even in a different genomic background. The differential methylation analysis showed that the Arabidopsis DNA was undermethylated in the centromeric region and repetitive elements. Several Arabidopsis genes were still expressed, whereas the expression patterns were not related to the gene function. We concluded that the plant DNA did not maintain the original plant epigenomic landscapes and was under the control of the human genome. This study showed how two diverging genomes can coexist and provided insights into epigenetic modifications and their impact on the regulation of gene expressions between plant and animal genomes.     

Could the Epigenetics of Eosinophils in Asthma and Allergy Solve Parts of the Puzzle?

Epigenetics is a field of study investigating changes in gene expression that do not alter the DNA sequence. These changes are often influenced by environmental or social factors and are reversible. Epigenetic mechanisms include DNA methylation, histone modification, and noncoding RNA. Understanding the role of these epigenetic mechanisms in human diseases provides useful information with regard to disease severity and development. Several studies have searched for the epigenetic mechanisms that regulate allergies and asthma; however, only few studies have used samples of eosinophil, a proinflammatory cell type known to be largely recruited during allergic or asthmatic inflammation. Such studies would enable us to better understand the factors that influence the massive recruitment of eosinophils during allergic and asthmatic symptoms. In this review, we sought to summarize different studies that aimed to discover differential patterns of histone modifications, DNA methylation, and noncoding RNAs in eosinophil samples of individuals with certain diseases, with a particular focus on those with asthma or allergic diseases.     

Methylated DNA and microRNA in Body Fluids as Biomarkers for Cancer Detection

Epigenetic alterations including DNA methylation and microRNAs (miRNAs) play important roles in the initiation and progression of human cancers. As the extensively studied epigenetic changes in tumors, DNA methylation and miRNAs are the most potential epigenetic biomarkers for cancer diagnosis. After the identification of circulating cell-free nuclear acids, increasing evidence demonstrated great potential of cell-free epigenetic biomarkers in the blood or other body fluids for cancer detection.     

Progress on the Physiological Function of Mitochondrial DNA and Its Specific Detection and Therapy

Mitochondrial DNA (mtDNA) is the genetic information of mitochondrion, and its structure is circular double-stranded. Despite the diminutive size of the mitochondrial genome, mtDNA mutations are an important cause of mitochondrial diseases which are characterized by defects in oxidative phosphorylation (OXPHOS). Mitochondrial diseases are involved in multiple systems, particularly in the organs that are highly dependent on aerobic metabolism. The diagnosis of mitochondrial disease is more complicated since mtDNA mutations can cause various clinical symptoms. To realize more accurate diagnosis and treatment of mitochondrial diseases, the detection of mtDNA and the design of drugs acting on it are extremely important. Over the past few years, many probes and therapeutic drugs targeting mtDNA have been developed, making significant contributions to fundamental research including elucidation of the mechanisms of mitochondrial diseases at the genetic level. In this review, we summarize the structure, function, and detection approaches for mtDNA. The most current topics in this field, such as mechanistic exploration and treatment of mtDNA mutation-related disorders, are also reviewed. Specific attention is given to discussing the design and development of these probes and drugs for mtDNA. We hope that this review will provide readers with a comprehensive understanding of the importance of mtDNA, and promote the development of effective molecules for theragnosis of mtDNA mutation-related diseases.     

Kaiso Regulates DNA Methylation Homeostasis

Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA methylation and demethylation. Previously, it was shown that methyl-DNA protein Kaiso may attract NCoR, SMRT repressive complexes affecting histone modifications. On the other hand, the deficiency of Kaiso resulted in reduced methylation of ICR in H19/Igf2 locus and Oct4 promoter in mouse embryonic fibroblasts. However, nothing is known about how Kaiso influences DNA methylation at the genome level. Here we show that deficiency of Kaiso led to whole-genome hypermethylation, using Kaiso deficient human renal cancer cell line obtained via CRISPR/CAS9 genome editing. However, Kaiso serves to protect genic regions, enhancers, and regions with a low level of histone modifications from demethylation. We detected hypomethylation of binding sites for Oct4 and Nanog in Kaiso deficient cells. Kaiso immunoprecipitated with de novo DNA methyltransferases DNMT3a/3b, but not with maintenance methyltransferase DNMT1. Thus, Kaiso may attract methyltransferases to surrounding regions and modulate genome methylation in renal cancer cells apart from being methyl DNA binding protein.     

Cancer Development,Progression, and Therapy: An Epigenetic Overview

Carcinogenesis involves uncontrolled cell growth, which follows the activation of oncogenes and/or the deactivation of tumor suppression genes. Metastasis requires down-regulation of cell adhesion receptors necessary for tissue-specific, cell–cell attachment, as well as up-regulation of receptors that enhance cell motility. Epigenetic changes, including histone modifications, DNA methylation, and DNA hydroxymethylation, can modify these characteristics. Targets for these epigenetic changes include signaling pathways that regulate apoptosis and autophagy, as well as microRNA. We propose that predisposed normal cells convert to cancer progenitor cells that, after growing, undergo an epithelial-mesenchymal transition. This process, which is partially under epigenetic control, can create a metastatic form of both progenitor and full-fledged cancer cells, after which metastasis to a distant location may occur. Identification of epigenetic regulatory mechanisms has provided potential therapeutic avenues. In particular, epigenetic drugs appear to potentiate the action of traditional therapeutics, often by demethylating and re-expressing tumor suppressor genes to inhibit tumorigenesis. Epigenetic drugs may inhibit both the formation and growth of cancer progenitor cells, thus reducing the recurrence of cancer. Adopting epigenetic alteration as a new hallmark of cancer is a logical and necessary step that will further encourage the development of novel epigenetic biomarkers and therapeutics.     

Advances and Opportunities in Epigenetic Chemical Biology

The study of epigenetics has greatly benefited from the development and application of various chemical biology approaches. In this review, we highlight the key targets for modulation and recent methods developed to enact such modulation. We discuss various chemical biology techniques to study DNA methylation and the post-translational modification of histones as well as their effect on gene expression. Additionally, we address the wealth of protein synthesis approaches to yield histones and nucleosomes bearing epigenetic modifications. Throughout, we highlight targets that present opportunities for the chemical biology community, as well as exciting new approaches that will provide additional insight into the roles of epigenetic marks.     

Extranuclear Inheritance of Mitochondrial Genome and Epigenetic Reprogrammability of Chromosomal Telomeres in Somatic Cell Cloning of Mammals

The effectiveness of somatic cell nuclear transfer (SCNT) in mammals seems to be still characterized by the disappointingly low rates of cloned embryos, fetuses, and progeny generated. These rates are measured in relation to the numbers of nuclear-transferred oocytes and can vary depending on the technique applied to the reconstruction of enucleated oocytes. The SCNT efficiency is also largely affected by the capability of donor nuclei to be epigenetically reprogrammed in a cytoplasm of reconstructed oocytes. The epigenetic reprogrammability of donor nuclei in SCNT-derived embryos appears to be biased, to a great extent, by the extranuclear (cytoplasmic) inheritance of mitochondrial DNA (mtDNA) fractions originating from donor cells. A high frequency of mtDNA heteroplasmy occurrence can lead to disturbances in the intergenomic crosstalk between mitochondrial and nuclear compartments during the early embryogenesis of SCNT-derived embryos. These disturbances can give rise to incorrect and incomplete epigenetic reprogramming of donor nuclei in mammalian cloned embryos. The dwindling reprogrammability of donor nuclei in the blastomeres of SCNT-derived embryos can also be impacted by impaired epigenetic rearrangements within terminal ends of donor cell-descended chromosomes (i.e., telomeres). Therefore, dysfunctions in epigenetic reprogramming of donor nuclei can contribute to the enhanced attrition of telomeres. This accelerates the processes of epigenomic aging and replicative senescence in the cells forming various tissues and organs of cloned fetuses and progeny. For all the above-mentioned reasons, the current paper aims to overview the state of the art in not only molecular mechanisms underlying intergenomic communication between nuclear and mtDNA molecules in cloned embryos but also intrinsic determinants affecting unfaithful epigenetic reprogrammability of telomeres. The latter is related to their abrasion within somatic cell-inherited chromosomes.     

Chromatin Profiling Techniques: Exploring the Chromatin Environment and Its Contributions to Complex Traits

The genetic architecture of complex traits is multifactorial. Genome-wide association studies (GWASs) have identified risk loci for complex traits and diseases that are disproportionately located at the non-coding regions of the genome. On the other hand, we have just begun to understand the regulatory roles of the non-coding genome, making it challenging to precisely interpret the functions of non-coding variants associated with complex diseases. Additionally, the epigenome plays an active role in mediating cellular responses to fluctuations of sensory or environmental stimuli. However, it remains unclear how exactly non-coding elements associate with epigenetic modifications to regulate gene expression changes and mediate phenotypic outcomes. Therefore, finer interrogations of the human epigenomic landscape in associating with non-coding variants are warranted. Recently, chromatin-profiling techniques have vastly improved our understanding of the numerous functions mediated by the epigenome and DNA structure. Here, we review various chromatin-profiling techniques, such as assays of chromatin accessibility, nucleosome distribution, histone modifications, and chromatin topology, and discuss their applications in unraveling the brain epigenome and etiology of complex traits at tissue homogenate and single-cell resolution. These techniques have elucidated compositional and structural organizing principles of the chromatin environment. Taken together, we believe that high-resolution epigenomic and DNA structure profiling will be one of the best ways to elucidate how non-coding genetic variations impact complex diseases, ultimately allowing us to pinpoint cell-type targets with therapeutic potential.     

Histone Methylation Regulation in Neurodegenerative Disorders

Advances achieved with molecular biology and genomics technologies have permitted investigators to discover epigenetic mechanisms, such as DNA methylation and histone posttranslational modifications, which are critical for gene expression in almost all tissues and in brain health and disease. These advances have influenced much interest in understanding the dysregulation of epigenetic mechanisms in neurodegenerative disorders. Although these disorders diverge in their fundamental causes and pathophysiology, several involve the dysregulation of histone methylation-mediated gene expression. Interestingly, epigenetic remodeling via histone methylation in specific brain regions has been suggested to play a critical function in the neurobiology of psychiatric disorders, including that related to neurodegenerative diseases. Prominently, epigenetic dysregulation currently brings considerable interest as an essential player in neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), Amyotrophic lateral sclerosis (ALS) and drugs of abuse, including alcohol abuse disorder, where it may facilitate connections between genetic and environmental risk factors or directly influence disease-specific pathological factors. We have discussed the current state of histone methylation, therapeutic strategies, and future perspectives for these disorders. While not somatically heritable, the enzymes responsible for histone methylation regulation, such as histone methyltransferases and demethylases in neurons, are dynamic and reversible. They have become promising potential therapeutic targets to treat or prevent several neurodegenerative disorders. These findings, along with clinical data, may provide links between molecular-level changes and behavioral differences and provide novel avenues through which the epigenome may be targeted early on in people at risk for neurodegenerative disorders.     

Identification of Mitochondrial DNA (NUMTs) in the Nuclear Genome of Daphnia magna

This is the first study in which the Daphnia magna (D. magna) nuclear genome (nDNA) obtained from the GenBank database was analyzed for pseudogene sequences of mitochondrial origin. To date, there is no information about pseudogenes localized in D. magna genome. This study aimed to identify NUMTs, their length, homology, and location for potential use in evolutionary studies and to check whether their occurrence causes co-amplification during mitochondrial genome (mtDNA) analyses. Bioinformatic analysis showed 1909 fragments of the mtDNA of D. magna, of which 1630 were located in ten linkage groups (LG) of the nDNA. The best-matched NUMTs covering >90% of the gene sequence have been identified for two mt-tRNA genes, and they may be functional nuclear RNA molecules. Isolating the total DNA in mtDNA studies, co-amplification of nDNA fragments is unlikely in the case of amplification of the whole tRNA genes as well as fragments of other genes. It was observed that TRNA-MET fragments had the highest level of sequence homology, thus they could be evolutionarily the youngest. The lowest homology was found in the D-loop-derived pseudogene. It may probably be the oldest NUMT incorporated into the nDNA; however, further analysis is necessary.     

Structure and function of mammalian DNA methyltransferases

DNA methylation plays an important role in epigenetic signalling, having an impact on gene regulation, chromatin structure, development and disease. Here, we review the structures and functions of the mammalian DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b, including their domain structures, catalytic mechanisms, localisation, regulation, post-translational modifications and interaction with chromatin and other proteins, summarising data obtained in genetic, cell biology and enzymatic studies. We focus on the question of how the molecular and enzymatic properties of these enzymes are connected to the dynamics of DNA methylation patterns and to the roles the enzymes play in the processes of de novo and maintenance DNA methylation. Recent enzymatic and genome-wide methylome data have led to a new model of genomic DNA methylation patterns based on the preservation of average levels of DNA methylation in certain regions, rather than the methylation states of individual CG sites.     

Exploring Epigenetic Marks by Analysis of Noncovalent Interactions

Epigenetic marks are modest chemical modifications on DNA and histone proteins that regulate the activation or silencing of genes through modulation of the intermolecular interactions between the DNA strands and the protein machinery. The process is complex and not always well understood. One of the systems studied in greater detail is the epigenetic mark on H3K9: lysine 9 of histone 3. The degree of methylation or acetylation of this histone is linked to silencing or activation of the corresponding gene, but it is not clear which effect each mark has in gene expression. We shed light on this particular methylation process by using density functional theory (DFT) calculations. First, we built a model consisting of a DNA double strand containing three base pairs and a sequence of three amino acids of the histone's tail. Then, we computed the modulation introduced into the intermolecular interactions by each epigenetic modification: from mono- to trimethylation and acetylation. The calculations show that whereas acetylation and trimethylation result in a reduction of the DNA-peptide interaction; non-, mono-, and dimethylation increase the intermolecular interactions. Such observations compare well with the findings reported in the literature, and highlight the correlation between the balance of intermolecular forces and biological properties, simultaneously advancing quantum-mechanical studies of large biochemical systems at molecular level through the use of DFT methods.     

Epigenetic Mechanisms in Penile Carcinoma

Penile carcinoma (PeCa) represents an important public health problem in poor and developing countries. Despite its unpredictable behavior and aggressive treatment, there have only been a few reports regarding its molecular data, especially epigenetic mechanisms. The functional diversity in different cell types is acquired by chromatin modifications, which are established by epigenetic regulatory mechanisms involving DNA methylation, histone acetylation, and miRNAs. Recent evidence indicates that the dysregulation in these processes can result in the development of several diseases, including cancer. Epigenetic alterations, such as the methylation of CpGs islands, may reveal candidates for the development of specific markers for cancer detection, diagnosis and prognosis. There are a few reports on the epigenetic alterations in PeCa, and most of these studies have only focused on alterations in specific genes in a limited number of cases. This review aims to provide an overview of the current knowledge of the epigenetic alterations in PeCa and the promising results in this field. The identification of epigenetically altered genes in PeCa is an important step in understanding the mechanisms involved in this unexplored disease.