DNA Methylation Remodeling Research Articles

Genome-Wide Analysis of DNA Methylation and Gene Expression Changes in Two Arabidopsis Ecotypes and Their Reciprocal Hybrids

Heterosis is a fundamental biological phenomenon characterized by the superior performance of a hybrid over its parents in many traits, but the underlying molecular basis remains elusive. To investigate whether DNA methylation plays a role in heterosis, we compared at single-base-pair resolution the DNA methylomes of Arabidopsis thaliana Landsberg erecta and C24 parental lines and their reciprocal F1 hybrids that exhibited heterosis. Both hybrids displayed increased DNA methylation across their entire genomes, especially in transposable elements. Interestingly, increased methylation of the hybrid genomes predominantly occurred in regions that were differentially methylated in the two parents and covered by small RNAs, implying that the RNA-directed DNA methylation (RdDM) pathway may direct DNA methylation in hybrids. In addition, we found that 77 genes sensitive to methylome remodeling were transcriptionally repressed in both reciprocal hybrids, including genes involved in flavonoid biosynthesis and two circadian oscillator genes circadian clock associated1 and late elongated hypocotyl. Moreover, growth vigor of F1 hybrids was compromised by treatment with an agent that demethylates DNA and by abolishing production of functional small RNAs due to mutations in Arabidopsis RNA methyltransferase HUA enhancer1. Together, our data suggest that genome-wide remodeling of DNA methylation directed by the RdDM pathway may play a role in heterosis.

Big (sequencing) future of non-coding RNA research for the understanding of cocaine.

Big (sequencing) future of non-coding RNA research for the understanding of cocaine.

Molecular dissection of an adaptive epigenetic memory mechanism in norway spruce

Molecular dissection of an adaptive epigenetic memory mechanism in norway spruce

There Goes the (Gene Expression) Neighbourhood Theory

The production of proteins from their respective genes is a tightly regulated process that helps cells conserve energy while ensuring they have the raw materials needed to perform basic life processes. The expression of any particular gene can be controlled by various mechanisms, including the actions of transcription factors and repressors, and epigenetic factors such as DNA methylation or chromatin remodelling. In addition, many eukaryotic genomes, from plants to humans, appear to have another regulatory trick at their disposal: ‘‘gene expression neighbourhoods’’—areas in which genes that are close together tend to have similar expression profiles. Many researchers have proposed that the organization of these neighbourhoods might be important for the coordinated expression of the genes that reside within them. In a new study, Meadows et al. have tested this theory in the fruitfly Drosophila by disrupting several gene expression neighbourhoods that contain testis-specific genes. Their results undercut the notion that gene expression neighbourhoods may play a regulatory role. Several gene expression neighbourhoods have been shown to be evolutionarily conserved among species, raising the possibility that there is some selective advantage to keeping these genes together in the genome. In Drosophila, at least 20% of all genes seem to be clustered into neighbourhoods, and one study concluded that around 45% of genes that are expressed exclusively in the testes were found in testis-specific gene expression neighbourhoods. Might this clustering into neighbourhoods somehow facilitate the coordinated expression of genes that have similar expression profiles? To find out, Meadows et al. generated targeted chromosomal inversions in the fly genome to split up specific gene expression neighbourhoods populated by testis-specific genes. They then used microarray analysis to compare the expression of the genes found in the affected neighbourhoods in flies carrying the inversions with otherwise genetically identical flies that did not carry the inversions. Perhaps surprisingly, the authors found no significant differences in gene expression between the flies with the inversions and those without. Even though the testisspecific genes no longer occupied the same neighbourhood (and thus were no longer clustered in the same way as in the wildtype genome), their expression patterns were normal. This finding was consistent whether the authors measured gene expression in the whole flies or in just the testes. They also generated flies with an inversion in an embryo expressed gene neighbourhood and again found no significant effect on gene expression, suggesting that the lack of effect of the inversions was not confined to sexually dimorphic genes or to the testis-specific genes themselves. One effect of gene clustering might be to ensure that genes are physically close together in the nucleus, perhaps bringing them under the influence of common factors that might affect their transcription. To investigate whether the clustered genes were physically separated in the nucleus by the chromosomal inversions, the authors used fluorescent markers to measure the distance between the genes. Genes that came from the same neighbourhood, but which were split apart by an inversion, were significantly further apart in the nucleus than the same genes in wild-type flies, which suggests that colocalization in the nucleus did not overcome the effects of the inversions. Meadows et al. have shown that, at least in Drosophila and at least for certain clusters of genes, the existence of gene expression neighbourhoods is not necessary for the correct and coordinated expression of genes that have the same expression profiles. These findings suggest that one shouldn’t assume that genes inhabit the same neighbourhood for a reason. Why these clusters have weathered the passage of evolutionary eons, however, remains an open question. It may well be, the authors suggest, that the evolutionary advantage of neighbourhoods might lie in more subtle effects, which await the creation of more powerful tools to reveal their purpose.

Targeting Epigenetic Modifications for the Treatment and Prevention of Breast Cancer

DNA methylation, histone modification, and nucleosome remodeling are the three intercalated events that result in epigenetic modification, which in turn alters gene expression. Aberrant epigenetic regulation resulting in altered gene expression has been clearly implicated in the initiation and progression of breast cancer. Our understanding of the landscape of these changes in breast cancer has increased tremendously over the past decade. Significant advancement has been made in the preclinical arena to identify targets that are epigenetically altered in breast cancer and to modulate these targets with epigenetic therapies to improve tumor response. Clinical translation of these concepts is currently ongoing and shows promise in improving outcomes in breast cancer. This article provides a comprehensive review of the completed and ongoing clinical trials of epigenetic therapy in the management and prevention of breast cancer and the rationale leading to the design of these trials.

Epigenetics and parental effects

Parental effects are a major source of phenotypic plasticity and may influence offspring phenotype in concert with environmental demands. Studies of "environmental epigenetics" suggest that (1) DNA methylation states are variable and that both demethylation and remethylation occur in post-mitotic cells, and (2) that remodeling of DNA methylation can occur in response to environmentally driven intracellular signaling pathways. Studies of mother-offspring interactions in rodents suggest that parental signals influence the DNA methylation, leading to stable changes in gene expression. If parental effects do indeed enhance the "match" between prevailing environmental demands and offspring phenotype, then the potential for variation in environmental conditions over time would suggest a mechanism that requires active maintenance across generations through parental signaling. We suggest that parental regulation of DNA methylation states is thus an ideal candidate mechanism for parental effects on phenotypic variation.

Aberrant DNA methylation in porcine in vitro-, parthenogenetic-, and somatic cell nuclear transfer-produced blastocysts.

Early embryonic development in the pig requires DNA methylation remodeling of the maternal and paternal genomes. Aberrant remodeling, which can be exasperated by in vitro technologies, is detrimental to development and can result in physiological and anatomic abnormalities in the developing fetus and offspring. Here, we developed and validated a microarray based approach to characterize on a global scale the CpG methylation profiles of porcine gametes and blastocyst stage embryos. The relative methylation in the gamete and blastocyst samples showed that 18.5% (921/4,992) of the DNA clones were found to be significantly different (P < 0.01) in at least one of the samples. Furthermore, for the different blastocyst groups, the methylation profile of the in vitro-produced blastocysts was less similar to the in vivo-produced blastocysts as compared to the parthenogenetic- and somatic cell nuclear transfer (SCNT)-produced blastocysts. The microarray results were validated by using bisulfite sequencing for 12 of the genomic regions in liver, sperm, and in vivo-produced blastocysts. These results suggest that a generalized change in global methylation is not responsible for the low developmental potential of blastocysts produced by using in vitro techniques. Instead, the appropriate methylation of a relatively small number of genomic regions in the early embryo may enable early development to occur.

Epigenetic processes and cancer risk assessment

The U.S. Environmental Protection Agency's Guidelines for Carcinogen Risk Assessment encourages the use of mechanistic data in the assessment of human cancer risk at low (environmental) exposure levels. The key events that define a particular mode of action for tumor formation have been concentrated to date more on mutational responses that are broadly the result of induced DNA damage and enhanced cell proliferation. While it is clear that these processes are important in terms of tumor induction, other modes that fall under the umbrella of epigenetic responses are increasingly being considered to play an important role in susceptibility to tumor induction by environmental chemicals and as significant modifiers of tumor responses. Alterations in gene expression, DNA repair, cell cycle control, genome stability and genome reprogramming could be the result of modification of DNA methylation and chromatin remodeling patterns as a consequence of exposure to environmental chemicals. These concepts are described and discussed.

Research Advances on the Relationship of PI3-Kinase/Akt/mTOR Pathway and Epigenetic Modification

The mammalian target of rapamycin (mTOR) has been shown to link growth factor signaling and posttranscriptional control of protein translation through activation of the PI3K/Akt pathway, which is frequently involved in cell cycle progression. The inhibition of mTOR has promising potential in anticancer and immunosuppressive therapies, and additional phase II clinical trials are ongoing. Epigenetic modification, which involves DNA methylation, histone modification and chromatin remodeling, as well as the recently described RNAi mechanism, can initiate the formation of silenced chromatin. Persistent activation or inhibition of the mTOR pathway may affect epigenetic modification. In this paper, we reviewed the research advances in the relationship between PI3K/Akt/mTOR and epigenetic modification.

86. Transcriptional Gene Silencing in Human Cells

In mammalian cells, small interfering RNA (siRNA) is known to produce post-transcriptional gene silencing (PTGS) in the cytoplasm. We recently reported transcriptional gene silencing (TGS) mediated by siRNAs when targeted to the EF1alpha promoter and delivered to the nucleus of human cells 1. We have expanded our initial observation to address the mechanism of siRNA mediated TGS. We screened the binding potential of DNMT1, 3A, 3A2, 3B1, 3B2, and HP1-alpha, beta, and gamma to the EF52 siRNA. Interestingly, DNMT3A, 3A2, and 3B2 bound EF52 with DNMT3A displaying the most robust binding. Moreover, the observed binding appears to be strand-specific. These data suggest a link between siRNA mediated targeting of genomic regions (promoters), DNA methylation, and chromatin remodeling complexes in mammalian cells. One possible model is that the siRNA binds DNMT3A and specifically targets the promoter. Once targeted to the promoter DNMT3A could then have a secondary interaction with histone deacetylase and/or the histone methyltransferase SUV39H1 2. These interactions would then be expected to ultimately result in histone 3 lysine 9 methylation, gene silencing, and eventually if reinforced, DNA methylation of the targeted promoter.

401. Engineering Retroviral Vectors for Stem Cell Therapies - Overcoming the Effect of Gene Silencing

The combined application of gene therapy and stem cell engineering for tissue regeneration have has drawn immense recent interest. In recent clinical trials with several patients who suffered from different forms of severe combined immunodeficiency syndrome (SCID), corrective genes were introduced into the patients' hematopoeitic stem cells via Moloney Murine Leukemia (MoMLV) viral vectors. This marked the first successful reports of gene therapy clinical trials. However, improvements to the vectors are needed to improve gene expression and safety of retroviral gene delivery to stem cells. For example, MoMLV vectors suffer from transcriptional silencing in many stem cell systems in vitro and in vivo, and enhancing gene expression after vector delivery could improve therapeutic efficacy, as well as improve safety by lowering the necessary dosage. The mechanism by which stem cells shutdown expression from an integrated retrovirus is not completely understood, but it is thought to be achieved via a combination of DNA methylation, histone modification, and chromatin remodeling. So far, five different regions within the retroviral genome have been identified as recognized targets that promote silencing, most of which lie within the U3 region of the viral Long Terminal Repeat (LTR). The effect of silencing can be reduced by modification and/or deletions of these regions; however, complete relief has never been achieved. Furthemore, improvements in long-term expression by these LTR modifications usually come at the cost of significant reductions in viral titer.

Profiling methyl-CpG specific determinants on transcriptionally silent chromatin.

Transcriptional activity is closely associated with DNA methylation and chromatin remodelling. Evidence is emerging that a family of methylation specific (methyl-CpG binding domain, MBD) proteins have the capacity to bind to methylated sequences and repress transcription. Recent advances in this area reveal that many of the MBD proteins are associated with histone deacetylase (HDAC) dependant repression. The capacity of MBD association to repress transcription would largely be defined by promoter structure and this is best explained by the position and density of DNA methylation. The mechanism of specific targeting of MBD family members to methylated sequences remains largely unknown. In order to understand the mechanistic details of silencing the current challenge is to identify and map these molecular determinants assembled on native chromatin in model systems of human development and disease. Downstream targets such as the methylated Fragile X Mental Retardation gene 1 (FMR1) gene and tumour suppressor genes are likely candidates. In this article, we describe a powerful strategy that involves the immunoprecipitation of in vivo formaldehyde fixed chromatin to identify MBD binding complexes directly isolated from the natural chromosomal environment. We demonstrate the methylated human Multidrug Resistance gene 1 (MDR1) is enriched with transcriptional repressors that belong to the MBD family and this would account for transcriptional silencing.