Showing papers in "Epigenetics & Chromatin in 2015"

De novo identification of differentially methylated regions in the human genome

Abstract: The identification and characterisation of differentially methylated regions (DMRs) between phenotypes in the human genome is of prime interest in epigenetics. We present a novel method, DMRcate, that fits replicated methylation measurements from the Illumina HM450K BeadChip (or 450K array) spatially across the genome using a Gaussian kernel. DMRcate identifies and ranks the most differentially methylated regions across the genome based on tunable kernel smoothing of the differential methylation (DM) signal. The method is agnostic to both genomic annotation and local change in the direction of the DM signal, removes the bias incurred from irregularly spaced methylation sites, and assigns significance to each DMR called via comparison to a null model. We show that, for both simulated and real data, the predictive performance of DMRcate is superior to those of Bumphunter and Probe Lasso, and commensurate with that of comb-p. For the real data, we validate all array-derived DMRs from the candidate methods on a suite of DMRs derived from whole-genome bisulfite sequencing called from the same DNA samples, using two separate phenotype comparisons. The agglomeration of genomically localised individual methylation sites into discrete DMRs is currently best served by a combination of DM-signal smoothing and subsequent threshold specification. The findings also suggest the design of the 450K array shows preference for CpG sites that are more likely to be differentially methylated, but its overall coverage does not adequately reflect the depth and complexity of methylation signatures afforded by sequencing. For the convenience of the research community we have created a user-friendly R software package called DMRcate, downloadable from Bioconductor and compatible with existing preprocessing packages, which allows others to apply the same DMR-finding method on 450K array data.

Constitutive heterochromatin formation and transcription in mammals

Abstract: Constitutive heterochromatin, mainly formed at the gene-poor regions of pericentromeres, is believed to ensure a condensed and transcriptionally inert chromatin conformation. Pericentromeres consist of repetitive tandem satellite repeats and are crucial chromosomal elements that are responsible for accurate chromosome segregation in mitosis. The repeat sequences are not conserved and can greatly vary between different organisms, suggesting that pericentromeric functions might be controlled epigenetically. In this review, we will discuss how constitutive heterochromatin is formed and maintained at pericentromeres in order to ensure their integrity. We will describe the biogenesis and the function of main epigenetic pathways that are involved and how they are interconnected. Interestingly, recent findings suggest that alternative pathways could substitute for well-established pathways when disrupted, suggesting that constitutive heterochromatin harbors much more plasticity than previously assumed. In addition, despite of the heterochromatic nature of pericentromeres, there is increasing evidence for active and regulated transcription at these loci, in a multitude of organisms and under various biological contexts. Thus, in the second part of this review, we will address this relatively new aspect and discuss putative functions of pericentromeric expression.

Making sense of GWAS: using epigenomics and genome engineering to understand the functional relevance of SNPs in non-coding regions of the human genome.

Abstract: Considerable progress towards an understanding of complex diseases has been made in recent years due to the development of high-throughput genotyping technologies. Using microarrays that contain millions of single-nucleotide polymorphisms (SNPs), Genome Wide Association Studies (GWASs) have identified SNPs that are associated with many complex diseases or traits. For example, as of February 2015, 2111 association studies have identified 15,396 SNPs for various diseases and traits, with the number of identified SNP-disease/trait associations increasing rapidly in recent years. However, it has been difficult for researchers to understand disease risk from GWAS results. This is because most GWAS-identified SNPs are located in non-coding regions of the genome. It is important to consider that the GWAS-identified SNPs serve only as representatives for all SNPs in the same haplotype block, and it is equally likely that other SNPs in high linkage disequilibrium (LD) with the array-identified SNPs are causal for the disease. Because it was hoped that disease-associated coding variants would be identified if the true casual SNPs were known, investigators have expanded their analyses using LD calculation and fine-mapping. However, such analyses also identified risk-associated SNPs located in non-coding regions. Thus, the GWAS field has been left with the conundrum as to how a single-nucleotide change in a non-coding region could confer increased risk for a specific disease. One possible answer to this puzzle is that the variant SNPs cause changes in gene expression levels rather than causing changes in protein function. This review provides a description of (1) advances in genomic and epigenomic approaches that incorporate functional annotation of regulatory elements to prioritize the disease risk-associated SNPs that are located in non-coding regions of the genome for follow-up studies, (2) various computational tools that aid in identifying gene expression changes caused by the non-coding disease-associated SNPs, and (3) experimental approaches to identify target genes of, and study the biological phenotypes conferred by, non-coding disease-associated SNPs.

Epigenetic regulatory functions of DNA modifications: 5-methylcytosine and beyond

Abstract: The chemical modification of DNA bases plays a key role in epigenetic gene regulation. While much attention has been focused on the classical epigenetic mark, 5-methylcytosine, the field garnered increased interest through the recent discovery of additional modifications. In this review, we focus on the epigenetic regulatory roles of DNA modifications in animals. We present the symmetric modification of 5-methylcytosine on CpG dinucleotide as a key feature, because it permits the inheritance of methylation patterns through DNA replication. However, the distribution patterns of cytosine methylation are not conserved in animals and independent molecular functions will likely be identified. Furthermore, the discovery of enzymes that catalyse the hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine not only identified an active demethylation pathway, but also a candidate for a new epigenetic mark associated with activated transcription. Most recently, N6-methyladenine was described as an additional eukaryotic DNA modification with epigenetic regulatory potential. Interestingly, this modification is also present in genomes that lack canonical cytosine methylation patterns, suggesting independent functions. This newfound diversity of DNA modifications and their potential for combinatorial interactions indicates that the epigenetic DNA code is substantially more complex than previously thought.

Selective impairment of methylation maintenance is the major cause of DNA methylation reprogramming in the early embryo

Abstract: Background DNA methylomes are extensively reprogrammed during mouse pre-implantation and early germ cell development. The main feature of this reprogramming is a genome-wide decrease in 5-methylcytosine (5mC). Standard high-resolution single-stranded bisulfite sequencing techniques do not allow discrimination of the underlying passive (replication-dependent) or active enzymatic mechanisms of 5mC loss. We approached this problem by generating high-resolution deep hairpin bisulfite sequencing (DHBS) maps, allowing us to follow the patterns of symmetric DNA methylation at CpGs dyads on both DNA strands over single replications.

Characterization of whole-genome autosomal differences of DNA methylation between men and women

Abstract: Disease risk and incidence between males and females reveal differences, and sex is an important component of any investigation of the determinants of phenotypes or disease etiology. Further striking differences between men and women are known, for instance, at the metabolic level. The extent to which men and women vary at the level of the epigenome, however, is not well documented. DNA methylation is the best known epigenetic mechanism to date. In order to shed light on epigenetic differences, we compared autosomal DNA methylation levels between men and women in blood in a large prospective European cohort of 1799 subjects, and replicated our findings in three independent European cohorts. We identified and validated 1184 CpG sites to be differentially methylated between men and women and observed that these CpG sites were distributed across all autosomes. We showed that some of the differentially methylated loci also exhibit differential gene expression between men and women. Finally, we found that the differentially methylated loci are enriched among imprinted genes, and that their genomic location in the genome is concentrated in CpG island shores. Our epigenome-wide association study indicates that differences between men and women are so substantial that they should be considered in design and analyses of future studies.

Wanderer, an interactive viewer to explore DNA methylation and gene expression data in human cancer

Abstract: Background The Cancer Genome Atlas (TCGA) offers a multilayered view of genomics and epigenomics data of many human cancer types However, the retrieval of expression and methylation data from TCGA is a cumbersome and time-consuming task

Concordant and discordant DNA methylation signatures of aging in human blood and brain

Abstract: DNA methylation is an epigenetic mark that balances plasticity with stability. While DNA methylation exhibits tissue specificity, it can also vary with age and potentially environmental exposures. In studies of DNA methylation, samples from specific tissues, especially brain, are frequently limited and so surrogate tissues are often used. As yet, we do not fully understand how DNA methylation profiles of these surrogate tissues relate to the profiles of the central tissue of interest. We have adapted principal component analysis to analyze data from the Illumina 450K Human Methylation array using a set of 17 individuals with 3 brain regions and whole blood. All of the top five principal components in our analysis were associated with a variable of interest: principal component 1 (PC1) differentiated brain from blood, PCs 2 and 3 were representative of tissue composition within brain and blood, respectively, and PCs 4 and 5 were associated with age of the individual (PC4 in brain and PC5 in both brain and blood). We validated our age-related PCs in four independent sample sets, including additional brain and blood samples and liver and buccal cells. Gene ontology analysis of all five PCs showed enrichment for processes that inform on the functions of each PC. Principal component analysis (PCA) allows simultaneous and independent analysis of tissue composition and other phenotypes of interest. We discovered an epigenetic signature of age that is not associated with cell type composition and required no correction for cellular heterogeneity.

Histone modifications rather than the novel regional centromeres of Zymoseptoria tritici distinguish core and accessory chromosomes

Abstract: Supernumerary chromosomes have been found in many organisms. In fungi, these “accessory” or “dispensable” chromosomes are present at different frequencies in populations and are usually characterized by higher repetitive DNA content and lower gene density when compared to the core chromosomes. In the reference strain of the wheat pathogen, Zymoseptoria tritici, eight discrete accessory chromosomes have been found. So far, no functional role has been assigned to these chromosomes; however, they have existed as separate entities in the karyotypes of Zymoseptoria species over evolutionary time. In this study, we addressed what—if anything—distinguishes the chromatin of accessory chromosomes from core chromosomes. We used chromatin immunoprecipitation combined with high-throughput sequencing (“ChIP-seq”) of DNA associated with the centromere-specific histone H3, CENP-A (CenH3), to identify centromeric DNA, and ChIP-seq with antibodies against dimethylated H3K4, trimethylated H3K9 and trimethylated H3K27 to determine the relative distribution and proportion of euchromatin, obligate and facultative heterochromatin, respectively. Centromeres of the eight accessory chromosomes have the same sequence composition and structure as centromeres of the 13 core chromosomes and they are of similar length. Unlike those of most other fungi, Z. tritici centromeres are not composed entirely of repetitive DNA; some centromeres contain only unique DNA sequences, and bona fide expressed genes are located in regions enriched with CenH3. By fluorescence microscopy, we showed that centromeres of Z. tritici do not cluster into a single chromocenter during interphase. We found dramatically higher enrichment of H3K9me3 and H3K27me3 on the accessory chromosomes, consistent with the twofold higher proportion of repetitive DNA and poorly transcribed genes. In contrast, no single histone modification tested here correlated with the distribution of centromeric nucleosomes. All centromeres are similar in length and composed of a mixture of unique and repeat DNA, and most contain actively transcribed genes. Centromeres, subtelomeric regions or telomere repeat length cannot account for the differences in transfer fidelity between core and accessory chromosomes, but accessory chromosomes are greatly enriched in nucleosomes with H3K27 trimethylation. Genes on accessory chromosomes appear to be silenced by trimethylation of H3K9 and H3K27.

RNA:DNA hybrids in the human genome have distinctive nucleotide characteristics, chromatin composition, and transcriptional relationships

Abstract: RNA:DNA hybrids represent a non-canonical nucleic acid structure that has been associated with a range of human diseases and potential transcriptional regulatory functions. Mapping of RNA:DNA hybrids in human cells reveals them to have a number of characteristics that give insights into their functions. We find RNA:DNA hybrids to occupy millions of base pairs in the human genome. A directional sequencing approach shows the RNA component of the RNA:DNA hybrid to be purine-rich, indicating a thermodynamic contribution to their in vivo stability. The RNA:DNA hybrids are enriched at loci with decreased DNA methylation and increased DNase hypersensitivity, and within larger domains with characteristics of heterochromatin formation, indicating potential transcriptional regulatory properties. Mass spectrometry studies of chromatin at RNA:DNA hybrids shows the presence of the ILF2 and ILF3 transcription factors, supporting a model of certain transcription factors binding preferentially to the RNA:DNA conformation. Overall, there is little to indicate a dependence for RNA:DNA hybrids forming co-transcriptionally, with results from the ribosomal DNA repeat unit instead supporting the intriguing model of RNA generating these structures in trans. The results of the study indicate heterogeneous functions of these genomic elements and new insights into their formation and stability in vivo.

CENP-A nucleosomes localize to transcription factor hotspots and subtelomeric sites in human cancer cells

Abstract: Background: The histone H3 variant CENP-A is normally tightly regulated to ensure only one centromere exists per chromosome. Native CENP-A is often found overexpressed in human cancer cells and a range of human tumors. Consequently, CENP-A misregulation is thought to contribute to genome instability in human cancers. However, the consequences of such overexpression have not been directly elucidated in human cancer cells. Results: To investigate native CENP-A overexpression, we sought to uncover CENP-A-associated defects in human cells. We confirm that CENP-A is innately overexpressed in several colorectal cancer cell lines. In such cells, we report that a subset of structurally distinct CENP-A-containing nucleosomes associate with canonical histone H3, and with the transcription-coupled chaperones ATRX and DAXX. Furthermore, such hybrid CENP-A nucleosomes localize to DNase I hypersensitive and transcription factor binding sites, including at promoters of genes across the human genome. A distinct class of CENP-A hotspots also accumulates at subtelomeric chromosomal locations, including at the 8q24/Myc region long-associated with genomic instability. We show this 8q24 accumulation of CENP-A can also be seen in early stage primary colorectal tumors. Conclusions: Our data demonstrate that excess CENP-A accumulates at noncentromeric locations in the human cancer genome. These findings suggest that ectopic CENP-A nucleosomes could alter the state of the chromatin fiber, potentially impacting gene regulation and chromosome fragility.

Occupancy by key transcription factors is a more accurate predictor of enhancer activity than histone modifications or chromatin accessibility

Abstract: Regulated gene expression controls organismal development, and variation in regulatory patterns has been implicated in complex traits. Thus accurate prediction of enhancers is important for further understanding of these processes. Genome-wide measurement of epigenetic features, such as histone modifications and occupancy by transcription factors, is improving enhancer predictions, but the contribution of these features to prediction accuracy is not known. Given the importance of the hematopoietic transcription factor TAL1 for erythroid gene activation, we predicted candidate enhancers based on genomic occupancy by TAL1 and measured their activity. Contributions of multiple features to enhancer prediction were evaluated based on the results of these and other studies. TAL1-bound DNA segments were active enhancers at a high rate both in transient transfections of cultured cells (39 of 79, or 56%) and transgenic mice (43 of 66, or 65%). The level of binding signal for TAL1 or GATA1 did not help distinguish TAL1-bound DNA segments as active versus inactive enhancers, nor did the density of regulation-related histone modifications. A meta-analysis of results from this and other studies (273 tested predicted enhancers) showed that the presence of TAL1, GATA1, EP300, SMAD1, H3K4 methylation, H3K27ac, and CAGE tags at DNase hypersensitive sites gave the most accurate predictors of enhancer activity, with a success rate over 80% and a median threefold increase in activity. Chromatin accessibility assays and the histone modifications H3K4me1 and H3K27ac were sensitive for finding enhancers, but they have high false positive rates unless transcription factor occupancy is also included. Occupancy by key transcription factors such as TAL1, GATA1, SMAD1, and EP300, along with evidence of transcription, improves the accuracy of enhancer predictions based on epigenetic features.

Affinity -seq detects genome-wide PRDM9 binding sites and reveals the impact of prior chromatin modifications on mammalian recombination hotspot usage

Abstract: Genetic recombination plays an important role in evolution, facilitating the creation of new, favorable combinations of alleles and the removal of deleterious mutations by unlinking them from surrounding sequences. In most mammals, the placement of genetic crossovers is determined by the binding of PRDM9, a highly polymorphic protein with a long zinc finger array, to its cognate binding sites. It is one of over 800 genes encoding proteins with zinc finger domains in the human genome. We report a novel technique, Affinity-seq, that for the first time identifies both the genome-wide binding sites of DNA-binding proteins and quantitates their relative affinities. We have applied this in vitro technique to PRDM9, the zinc-finger protein that activates genetic recombination, obtaining new information on the regulation of hotspots, whose locations and activities determine the recombination landscape. We identified 31,770 binding sites in the mouse genome for the PRDM9Dom2 variant. Comparing these results with hotspot usage in vivo, we find that less than half of potential PRDM9 binding sites are utilized in vivo. We show that hotspot usage is increased in actively transcribed genes and decreased in genomic regions containing H3K9me2/3 histone marks or bound to the nuclear lamina. These results show that a major factor determining whether a binding site will become an active hotspot and what its activity will be are constraints imposed by prior chromatin modifications on the ability of PRDM9 to bind to DNA in vivo. These constraints lead to the presence of long genomic regions depleted of recombination.

Targeted epigenome editing of an endogenous locus with chromatin modifiers is not stably maintained.

Abstract: DNA methylation and histone 3 lysine 9 (H3K9) methylation are considered as epigenetic marks that can be inherited through cell divisions. To explore the functional consequences and stability of these modifications, we employed targeted installment of DNA methylation and H3K9 methylation in the vascular endothelial growth factor A (VEGF-A) promoter using catalytic domains of DNA or H3K9 methyltransferases that are fused to a zinc finger protein which binds a site in the VEGF-A promoter. Expression of the targeted DNA and H3K9 methyltransferases caused dense deposition of DNA methylation or H3K9 di- and trimethylation in the promoter of VEGF-A and downregulation of VEGF-A gene expression. We did not observe positive feedback between DNA methylation and H3K9 methylation. Upon loss of the targeted methyltransferases from the cells, the epigenetic marks, chromatin environment, and gene expression levels returned to their original state, indicating that both methylation marks were not stably propagated after their installment. The clear anti-correlation between DNA or H3K9 methylation and gene expression suggests a direct role of these marks in transcriptional control. The lack of maintenance of the transiently induced silenced chromatin state suggests that the stability of epigenetic signaling is based on an epigenetic network consisting of several molecular marks. Therefore, for stable reprogramming, either multivalent deposition of functionally related epigenetic marks or longer-lasting trigger stimuli might be necessary.

DNase I hypersensitivity analysis of the mouse brain and retina identifies region-specific regulatory elements

Abstract: Background: The brain, spinal cord, and neural retina comprise the central nervous system (CNS) of vertebrates. Understanding the regulatory mechanisms that underlie the enormous cell-type diversity of the CNS is a significant challenge. Whole-genome mapping of DNase I-hypersensitive sites (DHSs) has been used to identify cis-regulatory elements in many tissues. We have applied this approach to the mouse CNS, including developing and mature neural retina, whole brain, and two well-characterized brain regions, the cerebellum and the cerebral cortex. Results: For the various regions and developmental stages of the CNS that we analyzed, there were approximately the same number of DHSs; however, there were many DHSs unique to each CNS region and developmental stage. Many of the DHSs are likely to mark enhancers that are specific to the specific CNS region and developmental stage. We validated the DNase I mapping approach for identification of CNS enhancers using the existing VISTA Browser database and with in vivo and in vitro electroporation of the retina. Analysis of transcription factor consensus sites within the DHSs shows distinct region-specific profiles of transcriptional regulators particular to each region. Clustering developmentally dynamic DHSs in the retina revealed enrichment of developmental stagespecific transcriptional regulators. Additionally, we found reporter gene activity in the retina driven from several previously uncharacterized regulatory elements surrounding the neurodevelopmental gene Otx2. Identification of DHSs shared between mouse and human showed region-specific differences in the evolution of cis-regulatory elements. Conclusions: Overall, our results demonstrate the potential of genome-wide DNase I mapping to cis-regulatory questions regarding the regional diversity within the CNS. These data represent an extensive catalogue of potential cis-regulatory elements within the CNS that display region and temporal specificity, as well as a set of DHSs common to CNS tissues. Further examination of evolutionary conservation of DHSs between CNS regions and different species may reveal important cis-regulatory elements in the evolution of the mammalian CNS.

Dose-dependent alcohol-induced alterations in chromatin structure persist beyond the window of exposure and correlate with fetal alcohol syndrome birth defects.

Abstract: In recent years, we have come to recognize that a multitude of in utero exposures have the capacity to induce the development of congenital and metabolic defects. As most of these encounters manifest their effects beyond the window of exposure, deciphering the mechanisms of teratogenesis is incredibly difficult. For many agents, altered epigenetic programming has become suspect in transmitting the lasting signature of exposure leading to dysgenesis. However, while several chemicals can perturb chromatin structure acutely, for many agents (particularly alcohol) it remains unclear if these modifications represent transient responses to exposure or heritable lesions leading to pathology. Here, we report that mice encountering an acute exposure to alcohol on gestational Day-7 exhibit significant alterations in chromatin structure (histone 3 lysine 9 dimethylation, lysine 9 acetylation, and lysine 27 trimethylation) at Day-17, and that these changes strongly correlate with the development of craniofacial and central nervous system defects. Using a neural cortical stem cell model, we find that the epigenetic changes arising as a consequence of alcohol exposure are heavily dependent on the gene under investigation, the dose of alcohol encountered, and that the signatures arising acutely differ significantly from those observed after a 4-day recovery period. Importantly, the changes observed post-recovery are consistent with those modeled in vivo, and associate with alterations in transcripts encoding multiple homeobox genes directing neurogenesis. Unexpectedly, we do not observe a correlation between alcohol-induced changes in chromatin structure and alterations in transcription. Interestingly, the majority of epigenetic changes observed occur in marks associated with repressive chromatin structure, and we identify correlative disruptions in transcripts encoding Dnmt1, Eed, Ehmt2 (G9a), EzH2, Kdm1a, Kdm4c, Setdb1, Sod3, Tet1 and Uhrf1. These observations suggest that the immediate and long-term impacts of alcohol exposure on chromatin structure are distinct, and hint at the existence of a possible coordinated epigenetic response to ethanol during development. Collectively, our results indicate that alcohol-induced modifications to chromatin structure persist beyond the window of exposure, and likely contribute to the development of fetal alcohol syndrome-associated congenital abnormalities.

Genetic and epigenetic variation among inbred mouse littermates: identification of inter-individual differentially methylated regions

Abstract: Phenotypic variability among inbred littermates reared in controlled environments remains poorly understood. Metastable epialleles refer to loci that intrinsically behave in this way and a few examples have been described. They display differential methylation in association with differential expression. For example, inbred mice carrying the agouti viable yellow (A vy ) allele show a range of coat colours associated with different DNA methylation states at the locus. The availability of next-generation sequencing, in particular whole genome sequencing of bisulphite converted DNA, allows us, for the first time, to search for metastable epialleles at base pair resolution. Using whole genome bisulphite sequencing of DNA from the livers of five mice from the A vy colony, we searched for sites at which DNA methylation differed among the mice. A small number of loci, 356, were detected and we call these inter-individual Differentially Methylated Regions, iiDMRs, 55 of which overlap with endogenous retroviral elements (ERVs). Whole genome resequencing of two mice from the colony identified very few differences and these did not occur at or near the iiDMRs. Further work suggested that the majority of ERV iiDMRs are metastable epialleles; the level of methylation was maintained in tissue from other germ layers and the level of mRNA from the neighbouring gene inversely correlated with methylation state. Most iiDMRs that were not overlapping ERV insertions occurred at tissue-specific DMRs and it cannot be ruled out that these are driven by changes in the ratio of cell types in the tissues analysed. Using the most thorough genome-wide profiling technologies for differentially methylated regions, we find very few intrinsically epigenetically variable regions that we term iiDMRs. The most robust of these are at retroviral elements and appear to be metastable epialleles. The non-ERV iiDMRs cannot be described as metastable epialleles at this stage but provide a novel class of variably methylated elements for further study.

Prenatal ethanol exposure alters adult hippocampal VGLUT2 expression with concomitant changes in promoter DNA methylation, H3K4 trimethylation and miR-467b-5p levels

Abstract: Maternal consumption of alcohol during pregnancy is associated with a range of physical, cognitive and behavioural outcomes in the offspring which are collectively called foetal alcohol spectrum disorders. We and others have proposed that epigenetic modifications, such as DNA methylation and post-translational histone modifications, mediate the effects of prenatal alcohol exposure on gene expression and, ultimately, phenotype. Here we use an inbred C57BL/6J mouse model of early gestational ethanol exposure equivalent, developmentally, to the first 3–4 weeks of pregnancy in humans to examine the long-term effects on gene expression and epigenetic state in the hippocampus. Gene expression analysis in the hippocampus revealed sex- and age-specific up-regulation of solute carrier family 17 member 6 (Slc17a6), which encodes vesicular glutamate transporter 2 (VGLUT2). Transcriptional up-regulation correlated with decreased DNA methylation and enrichment of histone H3 lysine 4 trimethylation, an active chromatin mark, at the Slc17a6 promoter. In contrast to Slc17a6 mRNA levels, hippocampal VGLUT2 protein levels were significantly decreased in adult ethanol-exposed offspring, suggesting an additional level of post-transcriptional control. MicroRNA expression profiling in the hippocampus identified four ethanol-sensitive microRNAs, of which miR-467b-5p was predicted to target Slc17a6. In vitro reporter assays showed that miR-467b-5p specifically interacted with the 3′UTR of Slc17a6, suggesting that it contributes to the reduction of hippocampal VGLUT2 in vivo. A significant correlation between microRNA expression in the hippocampus and serum of ethanol-exposed offspring was also observed. Prenatal ethanol exposure has complex transcriptional and post-transcriptional effects on Slc17a6 (VGLUT2) expression in the mouse hippocampus. These effects are observed following a relatively moderate exposure that is restricted to early pregnancy, modelling human consumption of alcohol before pregnancy is confirmed, and are only apparent in male offspring in adulthood. Our findings are consistent with the idea that altered epigenetic and/or microRNA-mediated regulation of glutamate neurotransmission in the hippocampus contributes to the cognitive and behavioural phenotypes observed in foetal alcohol spectrum disorders. Although further work is needed in both mice and humans, the results also suggest that circulating microRNAs could be used as biomarkers of early gestational ethanol exposure and hippocampal dysfunction.

A comprehensive epigenome map of Plasmodium falciparum reveals unique mechanisms of transcriptional regulation and identifies H3K36me2 as a global mark of gene suppression

Abstract: Role of epigenetic mechanisms towards regulation of the complex life cycle/pathogenesis of Plasmodium falciparum, the causative agent of malaria, has been poorly understood. To elucidate stage-specific epigenetic regulation, we performed genome-wide mapping of multiple histone modifications of P. falciparum. Further to understand the differences in transcription regulation in P. falciparum and its host, human, we compared their histone modification profiles. Our comprehensive comparative analysis suggests distinct mode of transcriptional regulation in malaria parasite by virtue of poised genes and differential histone modifications. Furthermore, analysis of histone modification profiles predicted 562 genes producing anti-sense RNAs and 335 genes having bidirectional promoter activity, which raises the intriguing possibility of RNA-mediated regulation of transcription in P. falciparum. Interestingly, we found that H3K36me2 acts as a global repressive mark and gene regulation is fine tuned by the ratio of activation marks to H3K36me2 in P. falciparum. This novel mechanism of gene regulation is supported by the fact that knockout of SET genes (responsible for H3K36 methylation) leads to up-regulation of genes with highest occupancy of H3K36me2 in wild-type P. falciparum. Moreover, virulence (var) genes are mostly poised and marked by a unique set of activation (H4ac) and repression (H3K9me3) marks, which are mutually exclusive to other Plasmodium housekeeping genes. Our study reveals unique plasticity in the epigenetic regulation in P. falciparum which can influence parasite virulence and pathogenicity. The observed differences in the histone code and transcriptional regulation in P. falciparum and its host will open new avenues for epigenetic drug development against malaria parasite.

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

Abstract: 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.

Ethnicity-specific epigenetic variation in naïve CD4+ T cells and the susceptibility to autoimmunity

Abstract: Background Genetic and epigenetic variability contributes to the susceptibility and pathogenesis of autoimmune diseases. T cells play an important role in several autoimmune conditions, including lupus, which is more common and more severe in people of African descent. To investigate inherent epigenetic differences in T cells between ethnicities, we characterized genome-wide DNA methylation patterns in naive CD4+ T cells in healthy African-Americans and European-Americans, and then confirmed our findings in lupus patients.

Tip60 complex binds to active Pol II promoters and a subset of enhancers and co-regulates the c-Myc network in mouse embryonic stem cells.

Abstract: Tip60 (KAT5) is the histone acetyltransferase (HAT) of the mammalian Tip60/NuA4 complex. While Tip60 is important for early mouse development and mouse embryonic stem cell (mESC) pluripotency, the function of Tip60 as reflected in a genome-wide context is not yet well understood. Gel filtration of nuclear mESCs extracts indicate incorporation of Tip60 into large molecular complexes and exclude the existence of large quantities of “free” Tip60 within the nuclei of ESCs. Thus, monitoring of Tip60 binding to the genome should reflect the behaviour of Tip60-containing complexes. The genome-wide mapping of Tip60 binding in mESCs by chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) shows that the Tip60 complex is present at promoter regions of predominantly active genes that are bound by RNA polymerase II (Pol II) and contain the H3K4me3 histone mark. The coactivator HAT complexes, Tip60- and Mof (KAT8)-containing (NSL and MSL), show a global overlap at promoters, whereas distinct binding profiles at enhancers suggest different regulatory functions of each essential HAT complex. Interestingly, Tip60 enrichment peaks at about 200 bp downstream of the transcription start sites suggesting a function for the Tip60 complexes in addition to histone acetylation. The comparison of genome-wide binding profiles of Tip60 and c-Myc, a somatic cell reprogramming factor that binds predominantly to active genes in mESCs, demonstrate that Tip60 and c-Myc co-bind at 50–60 % of their binding sites. We also show that the Tip60 complex binds to a subset of bivalent developmental genes and defines a set of mESC-specific enhancer as well as super-enhancer regions. Our study suggests that the Tip60 complex functions as a global transcriptional co-activator at most active Pol II promoters, co-regulates the ESC-specific c-Myc network, important for ESC self-renewal and cell metabolism and acts at a subset of active distal regulatory elements, or super enhancers, in mESCs.

Pan-cancer stratification of solid human epithelial tumors and cancer cell lines reveals commonalities and tissue-specific features of the CpG island methylator phenotype

Abstract: The term CpG island methylator phenotype (CIMP) has been used to describe widespread DNA hypermethylation at CpG-rich genomic regions affecting clinically distinct subsets of cancer patients. Even though there have been numerous studies of CIMP in individual cancer types, a uniform analysis across tissues is still lacking. We analyze genome-wide patterns of CpG island hypermethylation in 5,253 solid epithelial tumors from 15 cancer types from TCGA and 23 cancer cell lines from ENCODE. We identify differentially methylated loci that define CIMP+ and CIMP− samples, and we use unsupervised clustering to provide a robust molecular stratification of tumor methylomes for 12 cancer types and all cancer cell lines. With a minimal set of 89 discriminative loci, we demonstrate accurate pan-cancer separation of the 12 CIMP+/− subpopulations, based on their average levels of methylation. Tumor samples in different CIMP subclasses show distinctive correlations with gene expression profiles and recurrence of somatic mutations, copy number variations, and epigenetic silencing. Enrichment analyses indicate shared canonical pathways and upstream regulators for CIMP-targeted regions across cancer types. Furthermore, genomic alterations showing consistent associations with CIMP+/− status include genes involved in DNA repair, chromatin remodeling genes, and several histone methyltransferases. Associations of CIMP status with specific clinical features, including overall survival in several cancer types, highlight the importance of the CIMP+/− designation for individual tumor evaluation and personalized medicine. We present a comprehensive computational study of CIMP that reveals pan-cancer commonalities and tissue-specific differences underlying concurrent hypermethylation of CpG islands across tumors. Our stratification of solid tumors and cancer cell lines based on CIMP status is data-driven and agnostic to tumor type by design, which protects against known biases that have hindered classic methods previously used to define CIMP. The results that we provide can be used to refine existing molecular subtypes of cancer into more homogeneously behaving subgroups, potentially leading to more uniform responses in clinical trials.

Transcription-coupled recruitment of human CHD1 and CHD2 influences chromatin accessibility and histone H3 and H3.3 occupancy at active chromatin regions.

Abstract: CHD1 and CHD2 chromatin remodeling enzymes play important roles in development, cancer and differentiation. At a molecular level, the mechanisms are not fully understood but include transcriptional regulation, nucleosome organization and turnover. Here we show human CHD1 and CHD2 enzymes co-occupy active chromatin regions associated with transcription start sites (TSS), enhancer like regions and active tRNA genes. We demonstrate that their recruitment is transcription-coupled. CHD1 and CHD2 show distinct binding profiles across active TSS regions. Depletion of CHD1 influences chromatin accessibility at TSS and enhancer-like chromatin regions. CHD2 depletion causes increased histone H3 and reduced histone variant H3.3 occupancy. We conclude that transcription-coupled recruitment of CHD1 and CHD2 occurs at transcribed gene TSSs and at intragenic and intergenic enhancer-like sites. The recruitment of CHD1 and CHD2 regulates the architecture of active chromatin regions through chromatin accessibility and nucleosome disassembly.

Role of Ccr4-Not complex in heterochromatin formation at meiotic genes and subtelomeres in fission yeast

Abstract: Heterochromatin is essential for chromosome segregation, gene silencing and genome integrity. The fission yeast Schizosaccharomyces pombe contains heterochromatin at centromeres, subtelomeres, and mating type genes, as well as at small islands of meiotic genes dispersed across the genome. This heterochromatin is generated by partially redundant mechanisms, including the production of small interfering RNAs (siRNAs) that are incorporated into the RITS protein complex (RNAi-Induced Transcriptional Silencing). The assembly of heterochromatin islands requires the function of the RNA-binding protein Mmi1, which recruits RITS to its mRNA targets and to heterochromatin islands. In addition, Mmi1 directs its targets to an exosome-dependent RNA elimination pathway. Ccr4-Not is a conserved multiprotein complex that regulates gene expression at multiple levels, including RNA degradation and translation. We show here that Ccr4-Not is recruited by Mmi1 to its RNA targets. Surprisingly, Ccr4 and Caf1 (the mRNA deadenylase catalytic subunits of the Ccr4-Not complex) are not necessary for the degradation or translation of Mmi1 RNA targets, but are essential for heterochromatin integrity at Mmi1-dependent islands and, independently of Mmi1, at subtelomeric regions. Both roles require the deadenylase activity of Ccr4 and the Mot2/Not4 protein, a ubiquitin ligase that is also part of the complex. Genetic evidence shows that Ccr4-mediated silencing is essential for normal cell growth, indicating that this novel regulation is physiologically relevant. Moreover, Ccr4 interacts with components of the RITS complex in a Mmi1-independent manner. Taken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.

Cells adapt to the epigenomic disruption caused by histone deacetylase inhibitors through a coordinated, chromatin-mediated transcriptional response.

Abstract: The genome-wide hyperacetylation of chromatin caused by histone deacetylase inhibitors (HDACi) is surprisingly well tolerated by most eukaryotic cells. The homeostatic mechanisms that underlie this tolerance are unknown. Here we identify the transcriptional and epigenomic changes that constitute the earliest response of human lymphoblastoid cells to two HDACi, valproic acid and suberoylanilide hydroxamic acid (Vorinostat), both in widespread clinical use. Dynamic changes in transcript levels over the first 2 h of exposure to HDACi were assayed on High Density microarrays. There was a consistent response to the two different inhibitors at several concentrations. Strikingly, components of all known lysine acetyltransferase (KAT) complexes were down-regulated, as were genes required for growth and maintenance of the lymphoid phenotype. Up-regulated gene clusters were enriched in regulators of transcription, development and phenotypic change. In untreated cells, HDACi-responsive genes, whether up- or down-regulated, were packaged in highly acetylated chromatin. This was essentially unaffected by HDACi. In contrast, HDACi induced a strong increase in H3K27me3 at transcription start sites, irrespective of their transcriptional response. Inhibition of the H3K27 methylating enzymes, EZH1/2, altered the transcriptional response to HDACi, confirming the functional significance of H3K27 methylation for specific genes. We propose that the observed transcriptional changes constitute an inbuilt adaptive response to HDACi that promotes cell survival by minimising protein hyperacetylation, slowing growth and re-balancing patterns of gene expression. The transcriptional response to HDACi is mediated by a precisely timed increase in H3K27me3 at transcription start sites. In contrast, histone acetylation, at least at the three lysine residues tested, seems to play no direct role. Instead, it may provide a stable chromatin environment that allows transcriptional change to be induced by other factors, possibly acetylated non-histone proteins.

Tissue-specific expression of histone H3 variants diversified after species separation

Abstract: Background The selective incorporation of appropriate histone variants into chromatin is critical for the regulation of genome function. Although many histone variants have been identified, a complete list has not been compiled.

DNA demethylation by 5-aza-2′-deoxycytidine is imprinted, targeted to euchromatin, and has limited transcriptional consequences

Abstract: Background DNA methylation can be abnormally regulated in human disease and associated with effects on gene transcription that appear to be causally related to pathogenesis. The potential to use pharmacological agents that reverse this dysregulation is therefore an attractive possibility. To test how 5-aza-2′-deoxycytidine (5-aza-CdR) influences the genome therapeutically, we exposed non-malignant cells in culture to the agent and used genome-wide assays to assess the cellular response.

The BisPCR 2 method for targeted bisulfite sequencing

Abstract: DNA methylation has emerged as an important regulator of development and disease, necessitating the design of more efficient and cost-effective methods for detecting and quantifying this epigenetic modification. Next-generation sequencing (NGS) techniques offer single base resolution of CpG methylation levels with high statistical significance, but are also high cost if performed genome-wide. Here, we describe a simplified targeted bisulfite sequencing approach in which DNA sequencing libraries are prepared following sodium bisulfite conversion and two rounds of PCR for target enrichment and sample barcoding, termed BisPCR2. We have applied the BisPCR2 technique to validate differential methylation at several type 2 diabetes risk loci identified in genome-wide studies of human islets. We confirmed some previous findings while not others, in addition to identifying novel differentially methylated CpGs at these genes of interest, due to the much higher depth of sequencing coverage in BisPCR2 compared to prior array-based approaches. This study presents a robust, efficient, and cost-effective technique for targeted bisulfite NGS, and illustrates its utility by reanalysis of prior findings from genome-wide studies.

A high-throughput screen of inactive X chromosome reactivation identifies the enhancement of DNA demethylation by 5-aza-2′-dC upon inhibition of ribonucleotide reductase

Abstract: Background DNA methylation is important for the maintenance of the silent state of genes on the inactive X chromosome (Xi). Here, we screened for siRNAs and chemicals that reactivate an Xi-linked reporter in the presence of 5-aza-2′-deoxycytidine (5-aza-2′-dC), an inhibitor of DNA methyltransferase 1, at a concentration that, on its own, is not sufficient for Xi-reactivation.