Underlying DNA Sequence Research Articles

Epigenetic studies in Developmental Origins of Health and Disease: pitfalls and key considerations for study design and interpretation.

The field of Developmental Origins of Health and Disease (DOHaD) seeks to understand the relationships between early-life environmental exposures and long-term health and disease. Until recently, the molecular mechanisms underlying these phenomena were poorly understood; however, epigenetics has been proposed to bridge the gap between the environment and phenotype. Epigenetics involves the study of heritable changes in gene expression, which occur without changes to the underlying DNA sequence. Different types of epigenetic modifications include DNA methylation, post-translational histone modifications and non-coding RNAs. Increasingly, changes to the epigenome have been associated with early-life exposures in both humans and animal models, offering both an explanation for how the environment may programme long-term health, as well as molecular changes that could be developed as biomarkers of exposure and/or future disease. As such, epigenetic studies in DOHaD hold much promise; however, there are a number of factors which should be considered when designing and interpreting such studies. These include the impact of the genome on the epigenome, the tissue-specificity of epigenetic marks, the stability (or lack thereof) of epigenetic changes over time and the importance of associating epigenetic changes with changes in transcription or translation to demonstrate functional consequences. In this review, we discuss each of these key concepts and provide practical strategies to mitigate some common pitfalls with the aim of providing a useful guide for future epigenetic studies in DOHaD.

Targeted Epigenetic Modulation of Gene Expression in the Brain.

Epigenetics defines a broad class of changes that occur in the genome without altering the underlying DNA sequence. These changes include covalent chemical modifications to histone terminal tails, direct chemical modification of DNA, regulation of RNA content by noncoding RNAs, and both local and

In Vivo Characterization of Linc-p21 Reveals Functional cis-Regulatory DNA Elements

The Linc-p21 locus, encoding a long non-coding RNA, plays an important role in p53 signaling, cell-cycle regulation, and tumor suppression. However, despite extensive study, confusion exists regarding its mechanism of action: is activity driven by the transcript acting in trans, in cis, or by an underlying functional enhancer? Here, using a knockout mouse model and a massively parallel enhancer assay, we delineate the functional elements at this locus. We observe that, even in tissues with no detectable Linc-p21 transcript, deletion of the locus significantly affects local gene expression, including of the cell-cycle regulator Cdkn1a. To characterize this RNA-independent regulatory effect, we systematically interrogated the underlying DNA sequence for enhancer activity at nucleotide resolution and confirmed the existence of multiple enhancer elements. Together, these data suggest that, invivo, the cis-regulatory effects mediated by Linc-p21, in the presence or absence of transcription, are due to DNA enhancer elements.

Teaching artificial intelligence to read electropherograms

Electropherograms are produced in great numbers in forensic DNA laboratories as part of everyday criminal casework. Before the results of these electropherograms can be used they must be scrutinised by analysts to determine what the identified data tells us about the underlying DNA sequences and what is purely an artefact of the DNA profiling process. A technique that lends itself well to such a task of classification in the face of vast amounts of data is the use of artificial neural networks. These networks, inspired by the workings of the human brain, have been increasingly successful in analysing large datasets, performing medical diagnoses, identifying handwriting, playing games, or recognising images. In this work we demonstrate the use of an artificial neural network which we train to ‘read’ electropherograms and show that it can generalise to unseen profiles.

An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C. elegans Germline

SummaryCells benefit from silencing foreign genetic elements but must simultaneously avoid inactivating endogenous genes. Although chromatin modifications and RNAs contribute to maintenance of silenced states, the establishment of silenced regions will inevitably reflect underlying DNA sequence and/or structure. Here we demonstrate that a pervasive non-coding DNA feature in Caenorhabditis elegans, characterized by 10-basepair periodic An/Tn-clusters (PATCs), can license transgenes for germline expression within repressive chromatin domains. Transgenes containing natural or synthetic PATCs are resistant to position effect variegation and stochastic silencing in the germline. Among endogenous genes, intron length and PATC-character undergo dramatic changes as orthologs move from active to repressive chromatin over evolutionary time, indicating a dynamic character to the An/Tn periodicity. We propose that PATCs form the basis of a cellular immune system, identifying certain endogenous genes in heterochromatic contexts as privileged while foreign DNA can be suppressed with no requirement for a cellular memory of prior exposure.

Multiplexing Genetic and Nucleosome Positioning Codes: A Computational Approach.

Eukaryotic DNA is strongly bent inside fundamental packaging units: the nucleosomes. It is known that their positions are strongly influenced by the mechanical properties of the underlying DNA sequence. Here we discuss the possibility that these mechanical properties and the concomitant nucleosome positions are not just a side product of the given DNA sequence, e.g. that of the genes, but that a mechanical evolution of DNA molecules might have taken place. We first demonstrate the possibility of multiplexing classical and mechanical genetic information using a computational nucleosome model. In a second step we give evidence for genome-wide multiplexing in Saccharomyces cerevisiae and Schizosacharomyces pombe. This suggests that the exact positions of nucleosomes play crucial roles in chromatin function.

Centromeres

Centromeres, chromosomal regions that become physically linked to the spindle during cell division, ensure equal division of genetic material between daughter cells. They are ubiquitous and essential in eukaryotic life. In this primer, we ask the questions 'What defines a functional centromere?' and 'What do all centromeres have in common?' To address these questions we highlight what is known about centromere size, centromere architecture, underlying DNA sequence and centromeric proteins. Studies from a variety of organisms reveal a vast diversity in centromere form and function that remains perplexing and largely unexplained.

Epigenetics in Paediatric Gastroenterology, Hepatology, and Nutrition: Present Trends and Future Perspectives.

Epigenetics can be defined as stable, potentially heritable changes in the cellular phenotype caused by mechanisms other than alterations to the underlying DNA sequence. As such, any observed phenotypic changes including organ development, aging, and the occurrence of disease could be driven by epigenetic mechanisms in the presence of stable cellular DNA sequences. Indeed, with the exception of rare mutations, the human genome-sequence has remained remarkably stable over the past centuries. In contrast, substantial changes to our environment as part of our modern life style have not only led to a significant reduction of certain infectious diseases but also seen the exponential increase in complex traits including obesity and multifactorial diseases such as autoimmune disorders. It is becoming increasingly clear that epigenetic mechanisms operate at the interface between the genetic code and our environment, and a large body of existing evidence supports the importance of environmental factors such as diet and nutrition, infections, and exposure to toxins on human health. This seems to be particularly the case during vulnerable periods of human development such as pregnancy and early life. Importantly, as the first point of contact for many of such environmental factors including nutrition, the digestive system is being increasingly linked to a number of "modern" pathologies. In this review article, we aim to give a brief introduction to the basic molecular principals of epigenetics and provide a concise summary of the existing evidence for the role of epigenetic mechanisms in gastrointestinal health and disease, hepatology, and nutrition.

Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis.

The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species—Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast.

Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response.

Nucleosome occupancy is critically important in regulating access to the eukaryotic genome. Few studies in human cells have measured genome-wide nucleosome distributions at high temporal resolution during a response to a common stimulus. We measured nucleosome distributions at high temporal resolution following Kaposi's-sarcoma-associated herpesvirus (KSHV) reactivation using our newly developed mTSS-seq technology, which maps nucleosome distribution at the transcription start sites (TSS) of all human genes. Nucleosomes underwent widespread changes in organization 24 hours after KSHV reactivation and returned to their basal nucleosomal architecture 48 hours after KSHV reactivation. The widespread changes consisted of an indiscriminate remodeling event resulting in the loss of nucleosome rotational phasing signals. Additionally, one in six TSSs in the human genome possessed nucleosomes that are translationally remodeled. 72% of the loci with translationally remodeled nucleosomes have nucleosomes that moved to positions encoded by the underlying DNA sequence. Finally we demonstrated that these widespread alterations in nucleosomal architecture potentiated regulatory factor binding. These descriptions of nucleosomal architecture changes provide a new framework for understanding the role of chromatin in the genomic response, and have allowed us to propose a hierarchical model for chromatin-based regulation of genome response.

From One Life to Another- The Stress That Defies Time

FROM ONE LIFE TO ANOTHER - THE STRESS THAT DEFIES TIME BY LEANDRA PADAYACHEE W ith accumulating academic, finan- cial, and personal responsibilities, no college student is a stranger to the idea of stress - that feeling of overwhelming anxiety constant- ly nagging the mind. However, could there be a chance that this “stress” was passed down from our parents, or even grandparents? The idea of gene modification from environmental influences has been studied numerous times, yet the proposal of this alteration being passed down to succeeding generations is a novel concept entirely. Recent re- search with mice and human subjects reveals that when negatively influenced by a certain external trauma, both species could genetically transfer these sensitivities to their offspring. Yet how can a parent’s “fear” be biologically passed down? According to conventionally accepted scientific information, the genetic sequences contained in DNA are the only way to transmit biological information across generations. Random DNA mutations, when beneficial, enable organisms to adapt to changing conditions, but this process typically occurs slowly over many generations.1 However, the theory of epigenetics, or the study of heritable changes in gene expression, does not involve changes to the underlying DNA sequence. Epigenetic change occurs from the addition of methyl (CH3) groups to certain locations on the DNA molecule, which in turn silences parts of the gene in a particular pattern and leads to a specific modification of the gene’s phenotype. The DNA code itself is not changing; it is the parts of the expressed DNA code expressed that undergo alteration. This process is called DNA methylation, and can ultimately be influenced by several factors including age, disease, and the environment.4 Although the epigenetic concept of DNA meth- ylation has been scientifically proven, the idea of transmitting these alterations from generation to generation is still debatable, especially in such complex subjects as humans. One controlled experiment with mice, however, found that when a mouse learns to become afraid of a certain odor, its pups will be more sensitive to that odor, even though the pups will have never encountered it themselves.3 Researchers Kerry Ressler and Brian Dias studied epigenetic inheritance by training laboratory mice to fear the scent of acetophenone through the pairing of odor exposure with electric shocks. This Pavlovian fear conditioning ulti- mately increased sensitivity in the mice’s olfactory bulb; however, the researchers also discovered that the naive adult offspring of the sensitized mice inherited the same behavioral sensitivity to the smell.2 According to Dias, “[t]he inheritance takes place even if the mice are conceived by in vitro fertilization, and the sensitivity even appears in the second generation (grandchildren). This indicates that somehow, information about the experience connected with the odor is being transmitted via the sperm or eggs.” 3 The researchers proposed that DNA methylation explains the inherited sensitiv- FALL 2016 | Berkeley Scientific Journal

Bridging the Genetical Environmental Gap in Parkinson's Disease through Epigenetics

Parkinson's Disease (PD) is a progressive neurodegenerative disorder affecting 2% of the population over 60 years old, yet the exact molecular mechanism underlying its pathogenesis remains elusive. PD is a multifactorial disease with genetic and environmental factors intricately associated. Recently, epigenetic mechanisms have been recognized as potential mediators of environmental factors participating in the pathogenesis of PD. Epigenetics refer to the heritable changes in gene expression that do not involve changes to the underlying DNA sequence. Altered epigenetic mechanisms have been attributed to PD, Alzheimer’s and Huntington’s disease. Several studies have shown that DNA methylation, histone modifications and non-coding RNAs mechanisms contribute to the pathogenesis of PD. Accumulation of toxic metals such as manganese and iron, due to abnormal environmental exposure or increased dietary intake, can impact varied components of the epigenetic machinery through free radical formation. Current pharmacological agents only provide symptomatic relief, of which levodopa still remains the gold standard. However, drugs that halt or delay progression of PD are still lacking. In recent years, there has been considerable progress in the development of epigenetic drugs as a novel therapeutic modality in the management of PD. Cell replacement therapy is a promising avenue for the treatment of PD with scientific research making great progress in the development of Induced Pluripotent Stem Cells (iPSCs) to produce midbrain dopamine phenotypes. With direct access to the neurons that are affected in PD, the pace of discovery should speed up and the cure for PD should become an attainable goal.

Comprehensive nucleosome mapping of the human genome in cancer progression.

Altered chromatin structure is a hallmark of cancer, and inappropriate regulation of chromatin structure may represent the origin of transformation. Important studies have mapped human nucleosome distributions genome wide, but the role of chromatin structure in cancer progression has not been addressed. We developed a MNase-Transcription Start Site Sequence Capture method (mTSS-seq) to map the nucleosome distribution at human transcription start sites genome-wide in primary human lung and colon adenocarcinoma tissue. Here, we confirm that nucleosome redistribution is an early, widespread event in lung (LAC) and colon (CRC) adenocarcinoma. These altered nucleosome architectures are consistent between LAC and CRC patient samples indicating that they may serve as important early adenocarcinoma markers. We demonstrate that the nucleosome alterations are driven by the underlying DNA sequence and potentiate transcription factor binding. We conclude that DNA-directed nucleosome redistributions are widespread early in cancer progression. We have proposed an entirely new hierarchical model for chromatin-mediated genome regulation.

The intrinsic combinatorial organization and information theoretic content of a sequence are correlated to the DNA encoded nucleosome organization of eukaryotic genomes.

Thanks to research spanning nearly 30 years, two major models have emerged that account for nucleosome organization in chromatin: statistical and sequence specific. The first is based on elegant, easy to compute, closed-form mathematical formulas that make no assumptions of the physical and chemical properties of the underlying DNA sequence. Moreover, they need no training on the data for their computation. The latter is based on some sequence regularities but, as opposed to the statistical model, it lacks the same type of closed-form formulas that, in this case, should be based on the DNA sequence only. We contribute to close this important methodological gap between the two models by providing three very simple formulas for the sequence specific one. They are all based on well-known formulas in Computer Science and Bioinformatics, and they give different quantifications of how complex a sequence is. In view of how remarkably well they perform, it is very surprising that measures of sequence complexity have not even been considered as candidates to close the mentioned gap. We provide experimental evidence that the intrinsic level of combinatorial organization and information-theoretic content of subsequences within a genome are strongly correlated to the level of DNA encoded nucleosome organization discovered by Kaplan et al Our results establish an important connection between the intrinsic complexity of subsequences in a genome and the intrinsic, i.e. DNA encoded, nucleosome organization of eukaryotic genomes. It is a first step towards a mathematical characterization of this latter 'encoding'. Supplementary data are available at Bioinformatics online. futro@us.ibm.com.

T140: Epigenetically active xenobiotics in cancer prevention and therapy optimization

Nowadays the term “epigenetics” is described as regulation of gene expression persisting from one cell division to the next, despite a lack of changes in the underlying DNA sequence. The “epigenome” refers to different epigenetic states of a cell recognized as heritable environment influence on genome. The main epigenetic phenomena in mammals are DNA methylation and histone modifications, which are tightly interdependent. Many authors classify microRNA regulation as a third epigenetic phenomenon. Moreover, recently the discussion has been open that many different factors modifying DNA conformation represent a new class of epigenetic agents. In response to various environmental stimuli, cells produce different epigenetic changes that determine either an active or a repressed chromatin state. Epigenetic perturbations have been shown to associate with exposure to a range of drugs and toxicants, including non-genotoxic carcinogens. Consequently, on one hand, potential impact of epigenomics on drug development is under consideration as even well-known pharmacological drugs were shown to cause epigenetic changes that may be beneficial or hazardous. In particular, epigenetic effects were described for synthetic estrogens and contraceptives, beta-blockers and fluoroquinolone antibiotics, neuroleptics and anesthetics, chemotherapeutics and statins. Drug influence on gene silencing might have some therapeutic advantage in addition to the unfavourable effects. In particular, valproate, hydralazine and procainamide might be utilized to induce gene expression in cancers, where activation of a methylated gene might be of benefit. Screening of xenobiotics for epigenetic activity might identify new potential drugs for some specific diseases. On the other hand, epigenetic aspects of drug safety are investigated intensively. Many recent reviews in this field of research were devoted to the test elaboration for revealing different short-term and longer-lasting epigenetic changes modifying gene expression. Most of the short-term epigenetic screening tests are based on revealing of xenobiotics to influence on specific enzymes participating in epigenetic modifications, especially DNA methylation and histone modifications. Recently a new model system for the screening of compounds reactivating epigenetically repressed genes was described. It represents HeLa cell population that harbors silent GFP-reporter gene which could be activated via different epigenetic mechanisms. Epigenetic studies of xenobiotic-induced mechanisms will reveal valuable information clarifying both mechanisms of desirable therapy and side effects and open potential drug design directions. The major challenge that remains is to determine how epigenetic control is regulated and how it might be possible to intervene in such processes.

Strategies for precision modulation of gene expression by epigenome editing: an overview.

Genome editing technology has evolved rather quickly and become accessible to most researchers. It has resulted in far reaching implications and a number of novel designer systems including epigenome editing. Epigenome editing utilizes a combination of nuclease-null genome editing systems and effector domains to modulate gene expression. In particular, Zinc Finger, Transcription-Activator-Like Effector, and CRISPR/Cas9 have emerged as modular systems that can be modified to allow for precision manipulation of epigenetic marks without altering underlying DNA sequence. This review contains a comprehensive catalog of effector domains that can be used with components of genome editing systems to achieve epigenome editing. Ultimately, the evidence-based design of epigenome editing offers a novel improvement to the limited attenuation strategies. There is much potential for editing and/or correcting gene expression in somatic cells toward a new era of functional genomics and personalized medicine.

Nano-mechanical measurements of protein-DNA interactions with a silicon nitride pulley.

Proteins adhere to DNA at locations and with strengths that depend on the protein conformation, the underlying DNA sequence and the ionic content of the solution. A facile technique to probe the positions and strengths of protein-DNA binding would aid in understanding these important interactions. Here, we describe a ‘DNA pulley’ for position-resolved nano-mechanical measurements of protein-DNA interactions. A molecule of λ DNA is tethered by one end to a glass surface, and by the other end to a magnetic bead. The DNA is stretched horizontally by a magnet, and a nanoscale knife made of silicon nitride is manipulated to contact, bend and scan along the DNA. The mechanical profile of the DNA at the contact with the knife is probed via nanometer-precision optical tracking of the magnetic bead. This system enables detection of protein bumps on the DNA and localization of their binding sites. We study theoretically the technical requirements to detect mechanical heterogeneities in the DNA itself.

Epigenetics in Cystic Fibrosis: Epigenetic Targeting of a Genetic Disease

Cystic fibrosis (CF) is a deadly genetic disease that affects the lungs and digestive system. A mutation in the CF transmembrane conductance regulator (CFTR) gene is the cause of the disease. How epigenetics contributes to CFTR expression is still poorly understood. Epigenetics is a mechanism that alters gene expression without changing the underlying DNA sequence. Epigenetic mechanisms include DNA methylation and histone modification. Both mechanisms have been implicated in CFTR gene regulation. Here we review epigenetic regulation of CFTR transcription while discussing potential epigenetic targeting strategies including DNA methyltransferase, histone deacetylase, and histone methyltransferase and demethylase inhibition. Because of the reversibility of epigenetics, targeting epigenetic mechanisms has been an attractive therapeutic approach. However, epigenetic targeting of CF disease is still at its infant stage.

Nucleosome Organization in Human Embryonic Stem Cells.

The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a “ground state” of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this “ground state” by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states) are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational analysis of nucleosome organization alone is sufficient to elucidate much of the circuitry of pluripotency. Our results, suggest that nucleosome organization is associated with numerous genomic and epigenomic processes and can be used to elucidate cellular identity.

Towards an understanding of the role of DNA methylation in rheumatoid arthritis: therapeutic and diagnostic implications.

The term 'epigenetics' loosely describes DNA-templated processes leading to heritable changes in gene activity and expression, which are independent of the underlying DNA sequence. Epigenetic mechanisms comprise of post-translational modifications of chromatin, methylation of DNA, nucleosome positioning as well as expression of noncoding RNAs. Major advances in understanding the role of DNA methylation in regulating chromatin functions have been made over the past decade, and point to a role of this epigenetic mechanism in human disease. Rheumatoid arthritis (RA) is an autoimmune disorder where altered DNA methylation patterns have been identified in a number of different disease-relevant cell types. However, the contribution of DNA methylation changes to RA disease pathogenesis is at present poorly understood and in need of further investigation. Here we review the current knowledge regarding the role of DNA methylation in rheumatoid arthritis and indicate its potential therapeutic implications.