Underlying DNA Sequence Research Articles

Recurrent establishment of de novo centromeres in the pericentromeric region of maize chromosome 3.

Centromeres can arise de novo from non-centromeric regions, which are often called "neocentromeres." Neocentromere formation provides the best evidence for the concept that centromere function is not determined by the underlying DNA sequences, but controlled by poorly understood epigenetic mechanisms. Numerous neocentromeres have been reported in several plant and animal species. However, it has been elusive how and why a specific chromosomal region is chosen to be a new centromere during the neocentromere activation events. We report recurrent establishment of neocentromeres in a pericentromeric region of chromosome 3 in maize (Zea mays). This latent region is located in the short arm and is only 2Mb away from the centromere (Cen3) of chromosome 3. At least three independent neocentromere activation events, which were likely induced by different mechanisms, occurred within this latent region. We mapped the binding domains of CENH3, the centromere-specific H3 histone variant, of the three neocentromeres and analyzed the genomic and epigenomic features associated with Cen3, the de novo centromeres and an inactivated centromere derived from an ancestral chromosome. Our results indicate that lack of genes and transcription and a relatively high level of DNA methylation in this pericentromeric region may provide a favorable chromatin environment for neocentromere activation.

Centromere inheritance through the germline.

The centromere directs chromosome segregation and genetic inheritance but is not itself heritable in a canonical, DNA-based manner. In most species, centromeres are epigenetically defined by the presence of a histone H3 variant centromere protein A (CENP-A), independent of underlying DNA sequence. Therefore, centromere inheritance depends on maintaining the CENP-A nucleosome mark across generations. Experiments in cycling somatic cells have led to a model in which centromere identity is maintained by a cell cycle-coupled CENP-A chromatin assembly pathway. However, the processes of animal gametogenesis pose unique challenges to centromere inheritance because of the extended cell cycle arrest and the massive genome reorganization in the female and male germline, respectively. Here, we review our current understanding of germline centromere inheritance and highlight outstanding questions.

Optical determination of the electronic coupling and intercalation geometry of thiazole orange homodimer in DNA.

Sequence-selective bis-intercalating dyes exhibit large increases in fluorescence in the presence of specific DNA sequences. This property makes this class of fluorophore of particular importance to biosensing and super-resolution imaging. Here we report ultrafast transient anisotropy measurements of resonance energy transfer (RET) between thiazole orange (TO) molecules in a complex formed between the homodimer TOTO and double-stranded (ds) DNA. Biexponential homo-RET dynamics suggest two subpopulations within the ensemble: 80% intercalated and 20% non-intercalated. Based on the application of the transition density cube method to describe the electronic coupling and Monte Carlo simulations of the TOTO/dsDNA geometry, the dihedral angle between intercalated TO molecules is estimated to be 81° ± 5°, corresponding to a coupling strength of 45 ± 22 cm-1. Dye intercalation with this geometry is found to occur independently of the underlying DNA sequence, despite the known preference of TOTO for the nucleobase sequence CTAG. The non-intercalated subpopulation is inferred to have a mean inter-dye separation distance of 19 Å, corresponding to coupling strengths between 0 and 25 cm-1. This information is important to enable the rational design of energy transfer systems that utilize TOTO as a relay dye. The approach used here is generally applicable to determining the electronic coupling strength and intercalation configuration of other dimeric bis-intercalators.

Epigenetic changes in neurology: DNA methylation in multiple sclerosis

IntroductionEpigenetics is defined as the study of the mechanisms that regulate gene expression without altering the underlying DNA sequence. The best known is DNA methylation. Multiple sclerosis (MS) is a disease with no entirely known aetiology, in which it is stated that the involvement of environmental factors on people with a genetic predisposition, may be key to the development of the disease. It is at this intersection between genetic predisposition and environmental factors where DNA methylation may play a pathogenic role. DevelopmentA literature review of the effects of environmental risk factors for the development of MS can have on the different epigenetic mechanisms as well as the implication that such changes have on the development of the disease. ConclusionKnowledge of epigenetic modifications involved in the pathogenesis of MS, opens a new avenue of research for identification of potential biomarkers, as well as finding new therapeutic targets.

An artificial neural network system to identify alleles in reference 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 them about the underlying DNA sequences and what is purely an artefact of the DNA profiling process. This process of interpreting the electropherograms can be time consuming and is prone to subjective differences between analysts. Recently it was demonstrated that artificial neural networks could be used to classify information within an electropherogram as allelic (i.e. representative of a DNA fragment present in the DNA extract) or as one of several different categories of artefactual fluorescence that arise as a result of generating an electropherogram. We extend that work here to demonstrate a series of algorithms and artificial neural networks that can be used to identify peaks on an electropherogram and classify them. We demonstrate the functioning of the system on several profiles and compare the results to a leading commercial DNA profile reading system.

Epigenetics of Childhood Obesity

We sought to describe how epigenetics, the study of potentially heritable changes in gene expression that do not involve changes in the underlying DNA sequence, has promise in the development of effective preventive interventions for childhood obesity. Three main mechanisms cause epigenetic changes: DNA methylation, histone and chromatin modification, and post-translational regulation, including that by microRNAs (miRNAs). These mechanisms may be altered by a variety of exposures, and evidence suggests they act at multiple time points prior to conception, during pregnancy, and during infancy and childhood. Epigenetic changes, particularly DNA methylation, have been associated with birth weight, early childhood, and teenage obesity. Each epigenetic mechanism raises the possibility of therapeutics. DNA methylation may be affected by maternal and paternal diet, exercise, and supplements. Histone and chromatin modification can be altered by histone deacetylase inhibitors and other small molecules. miRNAs are already being studied in cancer and type 2 diabetes. Although epigenetics is in its infancy, it could provide an important link between maternal exposures and childhood metabolic outcomes.

DNA Methylation: Basic Biology and Application to Traumatic Brain Injury.

This article reviews the literature pertinent to epigenetic changes, and in particular, DNA methylation following traumatic brain injury (TBI). TBI is a heterogeneous disease that is a major cause of death and long-term disability. The links between TBI and epigenetics, the process by which environmental factors alter gene expression without changing the underlying DNA sequence, is an expanding area of research that may have profound consequences for understanding the disease, and for clinical care. There are various epigenetic changes that may occur as a direct result of TBI, including DNA methylation, histone modification, and changes in the levels of non-coding RNA. This review focuses on DNA methylation, its potential to alter the degree of injury, and the extent of recovery, including development of post-traumatic neurodegeneration, response to therapies, and the hereditable consequences of injury. The functional consequences of non-coding RNA and histone modifications are well described in the literature; however, the mechanism by which these three mechanisms interact are often overlooked. Here, we briefly describe the interaction of DNA methylation with the two other key epigenetic changes, and highlight key work being performed to understand the functional relevance of those mechanisms. The field of epigenetics is rapidly advancing as a result of the advent of less invasive and more versatile methods for measuring epigenetic proteins and their functional impact on cells; however, the evidence specific to TBI is limited. This review identifies several important outstanding questions that remain from the work already conducted, and highlights directions for the future.

Noise reduction in single time frame optical DNA maps.

In optical DNA mapping technologies sequence-specific intensity variations (DNA barcodes) along stretched and stained DNA molecules are produced. These “fingerprints” of the underlying DNA sequence have a resolution of the order one kilobasepairs and the stretching of the DNA molecules are performed by surface adsorption or nano-channel setups. A post-processing challenge for nano-channel based methods, due to local and global random movement of the DNA molecule during imaging, is how to align different time frames in order to produce reproducible time-averaged DNA barcodes. The current solutions to this challenge are computationally rather slow. With high-throughput applications in mind, we here introduce a parameter-free method for filtering a single time frame noisy barcode (snap-shot optical map), measured in a fraction of a second. By using only a single time frame barcode we circumvent the need for post-processing alignment. We demonstrate that our method is successful at providing filtered barcodes which are less noisy and more similar to time averaged barcodes. The method is based on the application of a low-pass filter on a single noisy barcode using the width of the Point Spread Function of the system as a unique, and known, filtering parameter. We find that after applying our method, the Pearson correlation coefficient (a real number in the range from -1 to 1) between the single time-frame barcode and the time average of the aligned kymograph increases significantly, roughly by 0.2 on average. By comparing to a database of more than 3000 theoretical plasmid barcodes we show that the capabilities to identify plasmids is improved by filtering single time-frame barcodes compared to the unfiltered analogues. Since snap-shot experiments and computational time using our method both are less than a second, this study opens up for high throughput optical DNA mapping with improved reproducibility.

Nutriepigenomics and malnutrition.

Epigenetics is defined as the modulation of gene expression without changes to the underlying DNA sequence. Epigenetic alterations, as a consequence of in utero malnutrition, may play a role in susceptibility to develop adulthood diseases and inheritance. However, the mechanistic link between epigenetic modifications and abnormalities in nutrition remains elusive. This review provides an update on the association of suboptimal nutritional environment and the high propensity to produce adult-onset chronic illnesses with a particular focus on modifications in genome functions that occur without alterations to the DNA sequence. We will mention the drivers of the phenotype and pattern of epigenetic markers set down during the reprogramming along with novel preventative and therapeutic strategies. New knowledge of epigenetic alterations is opening a gate toward personalized medicine.

Making antibodies minus the cells

Monoclonal antibodies (mAbs)—proteins engineered to bind to particular antigens—can serve as medications for cancer, autoimmune diseases, and other conditions. During mAb development, researchers often tweak the antibody’s underlying DNA sequence and screen for variants that give the highest yields when expressed in cells. Chinese hamster ovary (CHO) cells are typically used to produce these variants for screening, but that process takes a minimum of seven days to express the proteins. Researchers have now taken cells out of the equation to develop a faster, cell-free synthesis platform for making mAbs by modifying the formulation of a commercially available CHO cell extract (ACS Synth. Biol. 2017, DOI: 10.1021/acssynbio.7b00001). Michael C. Jewett of Northwestern University, Varnika Roy of biologics company MedImmune, and colleagues added a glutathione buffer solution and isomerase enzymes to the CHO extract to ensure the correct formation of the disulfide bonds that hold together the antibodies’ four subunits. They then

Epigenetics in the Primary Biliary Cholangitis and Primary Sclerosing Cholangitis.

AbstractEpigenomics, the study of modifications to genetic material that do not alter the underlying DNA sequence, is generating increasing interest as a means to help clarify disease pathogenesis and outcomes. Although genome-wide association studies have identified several potential candidate genes that may be implicated in primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), it is estimated that these genes explain less than 20% of the heritability of these diseases. Thus, to date, the origins of “missing heritability” for PBC and PSC remain elusive. The epigenome may provide a potentially elegant solution to this phenomenon, as it can be modified by both internal and external exposures (coined the “exposome”). This may explain differences in disease presentation, treatment response, and rates of progression between individuals. Epigenetic changes may also provide a framework for discovering potential biomarkers for diagnosis and screening of PBC and PSC. Importantly, because the epigenome is modifiable, it may also highlight novel pathways for therapeutic discovery and interventions of these diseases.

Bidirectional terminators in Saccharomyces cerevisiae prevent cryptic transcription from invading neighboring genes

Transcription can be quite disruptive for chromatin so cells have evolved mechanisms to preserve chromatin integrity during transcription, thereby preventing the emergence of cryptic transcripts from spurious promoter sequences. How these transcripts are regulated and processed remains poorly characterized. Notably, very little is known about the termination of cryptic transcripts. Here, we used RNA-Seq to identify and characterize cryptic transcripts in Spt6 mutant cells (spt6-1004) in Saccharomyces cerevisiae. We found polyadenylated cryptic transcripts running both sense and antisense relative to genes in this mutant. Cryptic promoters were enriched for TATA boxes, suggesting that the underlying DNA sequence defines the location of cryptic promoters. While intragenic sense cryptic transcripts terminate at the terminator of the genes that host them, we found that antisense cryptic transcripts preferentially terminate near the 3΄-end of the upstream gene. This finding led us to demonstrate that most terminators in yeast are bidirectional, leading to termination and polyadenylation of transcripts coming from both directions. We propose that S. cerevisiae has evolved this mechanism in order to prevent/attenuate spurious transcription from invading neighbouring genes, a feature that is particularly critical for organisms with small compact genomes.

Epigenetic approach for angiostatic therapy: promising combinations for cancer treatment.

Cancer cells are often dependent on epigenetic pathways for their survival. Consequently, drugs that target the epigenome, rather than the underlying DNA sequence, are currently attracting considerable attention. In recent years, the first epigenetic drugs have been approved for cancer chemotherapy, mainly for hematological applications. Limitations in single-drug efficacies have thus far limited their application in the treatment of solid tumors. Nevertheless, promising activity for these compounds has been suggested when combined with other, distinctly targeted agents. In this review, we discuss the anti-angiogenic activity of histone deacetylase and DNA methyltransferase inhibitors and their combinations with other targeted (anti-angiogenic) therapeutics in treatment of solid tumors. The role that these inhibitors play in the inhibition of tumor angiogenesis, particularly in combination with other targeted agents, and the advantages they present over broad acting anticancer agents, are critically discussed.

Centromere location in Arabidopsis is unaltered by extreme divergence in CENH3 protein sequence.

During cell division, spindle fibers attach to chromosomes at centromeres. The DNA sequence at regional centromeres is fast evolving with no conserved genetic signature for centromere identity. Instead CENH3, a centromere-specific histone H3 variant, is the epigenetic signature that specifies centromere location across both plant and animal kingdoms. Paradoxically, CENH3 is also adaptively evolving. An ongoing question is whether CENH3 evolution is driven by a functional relationship with the underlying DNA sequence. Here, we demonstrate that despite extensive protein sequence divergence, CENH3 histones from distant species assemble centromeres on the same underlying DNA sequence. We first characterized the organization and diversity of centromere repeats in wild-type Arabidopsis thaliana. We show that A. thaliana CENH3-containing nucleosomes exhibit a strong preference for a unique subset of centromeric repeats. These sequences are largely missing from the genome assemblies and represent the youngest and most homogeneous class of repeats. Next, we tested the evolutionary specificity of this interaction in a background in which the native A. thaliana CENH3 is replaced with CENH3s from distant species. Strikingly, we find that CENH3 from Lepidium oleraceum and Zea mays, although specifying epigenetically weaker centromeres that result in genome elimination upon outcrossing, show a binding pattern on A. thaliana centromere repeats that is indistinguishable from the native CENH3. Our results demonstrate positional stability of a highly diverged CENH3 on independently evolved repeats, suggesting that the sequence specificity of centromeres is determined by a mechanism independent of CENH3.

Adolescent depression linked to socioeconomic status? Molecular approaches for revealing premorbid risk factors.

The means by which social environmental exposures influence risk of mental disorders is a persistent and still open question. A key candidate mechanism for the biologic mediation of environmental effects involves epigenetic factors, which regulate gene function without altering underlying DNA sequence. Recent work has shown that environmental exposures such as childhood abuse, family history of mental disorder, and low socioeconomic status (SES) associate with differential DNA methylation (5mC) - a relatively stable, but modifiable, epigenetic factor. However, the longitudinal relation among SES, 5mC, brain function, and risk of depression remains to be elucidated. Here, we briefly review literature relevant to these associations and discuss recent findings that, for the first time, prospectively demonstrate sequential links between low SES, changes in 5mC, changes in brain function, and risk of depression in a cohort of adolescents.

Epigenetics in Gastrointestinal Health and Disease: Spotlight on DNA Methylation in the Intestinal Epithelium.

Epigenetics can be defined as stable, potentially heritable changes in cellular phenotype caused by mechanisms other than alterations in the underlying DNA sequence. DNA methylation is amongst the most intensely studied epigenetic mechanisms and has been shown to play a major role in regulating fundamental aspects of cell biology including cellular differentiation, organ development, and cell type-specific gene expression. Importantly, it is becoming increasingly clear that epigenetic mechanisms operate at the interface between the genetic code and our environment and are able to mediate environmental changes into stable phenotypic alterations. Given existing evidence supporting the important effects of environmental factors (e.g., diet, nutrition, and infections) on human health, epigenetic mechanisms provide a plausible mechanistic framework for the development of many multifactorial diseases including inflammatory bowel disease (IBD). Impaired function of the intestinal epithelium has been implicated in IBD pathogenesis, yet underlying mechanisms remain ill defined. The work of our group focuses on investigating the role of DNA methylation in regulating cellular function of the human intestinal epithelium during gastrointestinal health and IBD. In addition to performing an analysis of primary human intestinal epithelium, we utilize human intestinal organoid culture systems allowing us to perform functional analysis in a patient-derived ex vivo model.

Effects of DNA methyltransferase inhibition on pattern separation performance in mice

Introduction: Schizophrenia and anxiety disorders place a great burden on the patients suffering from it and on society. The cognitive symptoms of both diseases can be caused partly by impaired pattern separation. Pattern separation is the ability to make distinct representations out of highly overlapping information. Improving pattern separation could slow down the progression of the above mentioned disorders and thus improve the quality of life of the patients. Alterations in gene expression can lead to differences in pattern separation performance. Methylation of DNA is one of the epigenetic changes controlled by DNA methyltransferases (DNMT) that can alter such gene expression levels without changing the underlying DNA sequence. In this study, the DNMT inhibitor RG108 was given to mice in order to investigate its effect on pattern separation performance. Methods: 36 male C57BL/6 mice were used. Pattern separation performance was measured using the novel Object Pattern Separation task. It consists of two trials with two identical objects. In the second trial one of the objects is replaced along a vertical axis, while the second object stays in the same position. For the assessment of pattern separation the object is replaced at increasingly distinct locations. The most suitable inter-trial interval and position were assessed and pattern separation performance was measured after an intraperitoneal injection of either saline, 0.1mg/kg RG108 or 0.3mg/kg RG108. After 48 hours mice received a saline injection and pattern separation performance was measured again to check for carry-over effects. Lastly, a 24 hour inter-trial interval was used to unravel RG108 effects on long-term memory. After the behavioral task, the mice were decapitated and the dorsal hippocampus was dissected to quantify the expression of six target genes with qPCR. Results: RG108 leads to an acute, dose-dependent increase in pattern separation. This effect vanishes after 48 hours. Furthermore, RG108 administration is not sufficient to 89 enhance pattern separation in such a way that animals can still remember the object’s location after 24 hours. Whereas BDNF4, BDNF9, GRIA1, HDAC2 and HEY1 expressions do not change after acute administration of RG108, BDNF1 expression increases and can be the underlying reason for the improvement in pattern separation. Future studies are needed to reveal the possible methylation changes and to study the effect of chronic treatment with this drug.

A dynamic mode of mitotic bookmarking by transcription factors.

During mitosis, transcription is shut off, chromatin condenses, and most transcription factors (TFs) are reported to be excluded from chromosomes. How do daughter cells re-establish the original transcription program? Recent discoveries that a select set of TFs remain bound on mitotic chromosomes suggest a potential mechanism for maintaining transcriptional programs through the cell cycle termed mitotic bookmarking. Here we report instead that many TFs remain associated with chromosomes in mouse embryonic stem cells, and that the exclusion previously described is largely a fixation artifact. In particular, most TFs we tested are significantly enriched on mitotic chromosomes. Studies with Sox2 reveal that this mitotic interaction is more dynamic than in interphase and is facilitated by both DNA binding and nuclear import. Furthermore, this dynamic mode results from lack of transcriptional activation rather than decreased accessibility of underlying DNA sequences in mitosis. The nature of the cross-linking artifact prompts careful re-examination of the role of TFs in mitotic bookmarking.

Nucleosome fragility is associated with future transcriptional response to developmental cues and stress in C. elegans.

Nucleosomes have structural and regulatory functions in all eukaryotic DNA-templated processes. The position of nucleosomes on DNA and the stability of the underlying histone–DNA interactions affect the access of regulatory proteins to DNA. Both stability and position are regulated through DNA sequence, histone post-translational modifications, histone variants, chromatin remodelers, and transcription factors. Here, we explored the functional implications of nucleosome properties on gene expression and development in Caenorhabditis elegans embryos. We performed a time-course of micrococcal nuclease (MNase) digestion and measured the relative sensitivity or resistance of nucleosomes throughout the genome. Fragile nucleosomes were defined by nucleosomal DNA fragments that were recovered preferentially in early MNase-digestion time points. Nucleosome fragility was strongly and positively correlated with the AT content of the underlying DNA sequence. There was no correlation between promoter nucleosome fragility and the levels of histone modifications or histone variants. Genes with fragile nucleosomes in their promoters tended to be lowly expressed and expressed in a context-specific way, operating in neuronal response, the immune system, and stress response. In addition to DNA-encoded nucleosome fragility, we also found fragile nucleosomes at locations where we expected to find destabilized nucleosomes, for example, at transcription factor binding sites where nucleosomes compete with DNA-binding factors. Our data suggest that in C. elegans promoters, nucleosome fragility is in large part DNA-encoded and that it poises genes for future context-specific activation in response to environmental stress and developmental cues.

Efficient Preparation of High-Complexity ChIP-Seq Profiles from Early Xenopus Embryos.

Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) has become a powerful tool to acquire a precise and genome-wide snapshot of many chromatin features in vivo. These chromatin profiles are obtained by immunoprecipitation of cross-linked chromatin fragments to enrich the feature of interest. Sequencing and aligning the underlying DNA sequences to the genome make it possible to virtually reconstruct the global distribution of most chromatin features. We present here recent improvements to the ChIP-seq protocol by means of Xenopus embryos to prepare high-complexity DNA libraries from small amounts of biological material. This approach allows researchers to explore the landscape of chromatin regulators and states in early vertebrate embryos or in any biological entity with small numbers of cells.