Maintenance Of Epigenetic States Research Articles

An immunochemistry-based screen for chemical inhibitors of DNA-protein interactions and its application to human CGGBP1

BackgroundInhibition of DNA-binding of proteins by small-molecule chemicals holds immense potential in manipulating the activities of DNA-binding proteins. Such a chemical inhibition of DNA-binding of proteins can be used to modulate processes such as replication, transcription, DNA repair and maintenance of epigenetic states. This prospect is currently challenged with the absence of robust and generic protocols to identify DNA-protein interactions. Additionally, much of the current approaches to designing inhibitors requires structural information of the target proteins.MethodsWe have developed a simple dot blot and immunodetection-based assay to screen chemical libraries for inhibitors of DNA-protein interactions. The assay has been applied to a library of 1685 FDA-approved chemicals to discover inhibitors of CGGBP1, a multifunctional DNA-binding protein with no known structure. Additional in vitro and in cellulo assays have been performed to verify and supplement the findings of the screen.ResultsOur primary screen has identified multiple inhibitors of direct or indirect interactions between CGGBP1 and genomic DNA. Of these, one inhibitor, Givinostat, was found to inhibit direct DNA-binding of CGGBP1 in the secondary screen using purified recombinant protein as the target. DNA and chromatin immunoprecipitation assays reinforced the findings of the screen that Givinostat inhibits CGGBP1-DNA binding.ConclusionsThe assay we have described successfully identifies verifiable inhibitors of DNA-binding of protein; in this example, the human CGGBP1. This assay is customizable for a wide range of targets for which primary antibodies are available. It works with different sources of the target protein, cell lysates or purified recombinant preparations and does not require special equipment, DNA modifications or protein structural data. This assay is scalable and highly adaptable with the potential to discover inhibitors of transcription factors with implications in cancer biology.

The Arabidopsis epigenetic regulator ICU11 as an accessory protein of Polycomb Repressive Complex 2

Molecular mechanisms enabling the switching and maintenance of epigenetic states are not fully understood. Distinct histone modifications are often associated with ON/OFF epigenetic states, but how these states are stably maintained through DNA replication, yet in certain situations switch from one to another remains unclear. Here, we address this problem through identification of Arabidopsis INCURVATA11 (ICU11) as a Polycomb Repressive Complex 2 accessory protein. ICU11 robustly immunoprecipitated in vivo with PRC2 core components and the accessory proteins, EMBRYONIC FLOWER 1 (EMF1), LIKE HETEROCHROMATIN PROTEIN1 (LHP1), and TELOMERE_REPEAT_BINDING FACTORS (TRBs). ICU11 encodes a 2-oxoglutarate-dependent dioxygenase, an activity associated with histone demethylation in other organisms, and mutant plants show defects in multiple aspects of the Arabidopsis epigenome. To investigate its primary molecular function we identified the Arabidopsis FLOWERING LOCUS C (FLC) as a direct target and found icu11 disrupted the cold-induced, Polycomb-mediated silencing underlying vernalization. icu11 prevented reduction in H3K36me3 levels normally seen during the early cold phase, supporting a role for ICU11 in H3K36me3 demethylation. This was coincident with an attenuation of H3K27me3 at the internal nucleation site in FLC, and reduction in H3K27me3 levels across the body of the gene after plants were returned to the warm. Thus, ICU11 is required for the cold-induced epigenetic switching between the mutually exclusive chromatin states at FLC, from the active H3K36me3 state to the silenced H3K27me3 state. These data support the importance of physical coupling of histone modification activities to promote epigenetic switching between opposing chromatin states.

Yeast epigenetics: the inheritance of histone modification states.

Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast) are two of the most recognised and well-studied model systems for epigenetic regulation and the inheritance of chromatin states. Their silent loci serve as a proxy for heterochromatic chromatin in higher eukaryotes, and as such both species have provided a wealth of information on the mechanisms behind the establishment and maintenance of epigenetic states, not only in yeast, but in higher eukaryotes. This review focuses specifically on the role of histone modifications in governing telomeric silencing in S. cerevisiae and centromeric silencing in S. pombe as examples of genetic loci that exemplify epigenetic inheritance. We discuss the recent advancements that for the first time provide a mechanistic understanding of how heterochromatin, dictated by histone modifications specifically, is preserved during S-phase. We also discuss the current state of our understanding of yeast nucleosome dynamics during DNA replication, an essential component in delineating the contribution of histone modifications to epigenetic inheritance.

Nucleosome dynamics and maintenance of epigenetic states of CpG islands.

Methylation of mammalian DNA occurs primarily at CG dinucleotides. These CpG sites are located nonrandomly in the genome, tending to occur within high density clusters of CpGs (islands) or within large regions of low CpG density. Cluster methylation tends to be bimodal, being dominantly unmethylated or mostly methylated. For CpG clusters near promoters, low methylation is associated with transcriptional activity, while high methylation is associated with gene silencing. Alternative CpG methylation states are thought to be stable and heritable, conferring localized epigenetic memory that allows transient signals to create long-lived gene expression states. Positive feedback where methylated CpG sites recruit enzymes that methylate nearby CpGs, can produce heritable bistability but does not easily explain that as clusters increase in size or density they change from being primarily methylated to primarily unmethylated. Here, we show that an interaction between the methylation state of a cluster and its occupancy by nucleosomes provides a mechanism to generate these features and explain genome wide systematics of CpG islands.

Exact Computation of Velocity Field and Probability Flux of Time-Evolving Probability Landscape of Stochastic Networks

Stochastic networks play important roles in cellular processes such as gene regulation, maintenance of epigenetic states, and decision of cellular fate. The discrete chemical master equation (dCME) provides a general framework for studying stochasticity in cellular mesoscopic systems. The time-evolving probabilistic landscape governed by dCME gives a comprehensive picture of the stochastic behavior of the network. Probability flux and entropy production of stochastic network have been previously studied using Fokker-Plank equation based on Gaussian processes. Here we formulate the time-dependent evolution of the probability landscape as a general diffusion process [1], and construct the velocity field and the probability flux based on exactly computed time-evolving probabilistic landscape. We dispense with Gaussian approximations and the truncation of the jump operator necessary when using the Fokker-Plank equation. We computed the dynamics of probability fluxes and vector fields over the full state space across different time scale. We showed details of the time-evolving probability fluxes and vector fields for the ubiquitous birth-death process, the bistable Schlogl process, the oscillatory Schnakenberg model, and the toggle switch model at different time points using the exact ACME algorithm [2]. Our results identify important cellular states, characterize high probability paths and trajectories along which the probability mass of the network state evolve. Furthermore, we provide exact assessment of dissipative entropy production associated with the complex diffusion process.[1] Eisenberg, Bob et al. The Journal of Chemical Physics 133.10 (2010): 104104.[2] Cao, Youfang et al. BMC Systems Biology 2.1 (2008): 30.

Consistent and heritable alterations of DNA methylation are induced by tissue culture in maize.

Plants regenerated from tissue culture and their progenies are expected to be identical clones, but often display heritable molecular and phenotypic variation. We characterized DNA methylation patterns in callus, primary regenerants, and regenerant-derived progenies of maize using immunoprecipitation of methylated DNA (meDIP) to assess the genome-wide frequency, pattern, and heritability of DNA methylation changes. Although genome-wide DNA methylation levels remained similar following tissue culture, numerous regions exhibited altered DNA methylation levels. Hypomethylation events were observed more frequently than hypermethylation following tissue culture. Many of the hypomethylation events occur at the same genomic sites across independent regenerants and cell lines. The DNA methylation changes were often heritable in progenies produced from self-pollination of primary regenerants. Methylation changes were enriched in regions upstream of genes and loss of DNA methylation at promoters was associated with altered expression at a subset of loci. Differentially methylated regions (DMRs) found in tissue culture regenerants overlap with the position of naturally occurring DMRs more often than expected by chance with 8% of tissue culture hypomethylated DMRs overlapping with DMRs identified by profiling natural variation, consistent with the hypotheses that genomic stresses similar to those causing somaclonal variation may also occur in nature, and that certain loci are particularly susceptible to epigenetic change in response to these stresses. The consistency of methylation changes across regenerants from independent cultures suggests a mechanistic response to the culture environment as opposed to an overall loss of fidelity in the maintenance of epigenetic states.

Molecular versatility: the many faces and functions of noncoding RNA

Molecular versatility: the many faces and functions of noncoding RNA

The Maintenance of Epigenetic States by p53: The Guardian of the Epigenome

// Arnold J. Levine 1 and Benjamin Greenbaum 1 1 Institute for Advanced Study, Einstein Road, Princeton, N.J. Correspondence: Arnold J. Levine , email: // Keywords : TP53, virus, cancer, cell death, epigenetic stability Received : December 08, 2012, Accepted : December 10, 2012, Published : December 11, 2012 Commentary on: Leonova et al. p53 cooperates with DNA methylation and a suicidal interferon response to maintain epigenetic silencing of repeats and noncoding RNAs. PNAS, USA, 2012; in press.

A Polycomb-based switch underlying quantitative epigenetic memory

The conserved Polycomb repressive complex 2 (PRC2) generates trimethylation of histone 3 lysine 27 (H3K27me3), a modification associated with stable epigenetic silencing. Much is known about PRC2-induced silencing but key questions remain concerning its nucleation and stability. Vernalization, the perception and memory of winter in plants, is a classic epigenetic process that, in Arabidopsis, involves PRC2-based silencing of the floral repressor FLC. The slow dynamics of vernalization, taking place over weeks in the cold, generate a level of stable silencing of FLC in the subsequent warm that depends quantitatively on the length of the prior cold. These features make vernalization an ideal experimental system to investigate both the maintenance of epigenetic states and the switching between them. Here, using mathematical modelling, chromatin immunoprecipitation and an FLC:GUS reporter assay, we show that the quantitative nature of vernalization is generated by H3K27me3-mediated FLC silencing in the warm in a subpopulation of cells whose number depends on the length of the prior cold. During the cold, H3K27me3 levels progressively increase at a tightly localized nucleation region within FLC. At the end of the cold, numerical simulations predict that such a nucleation region is capable of switching the bistable epigenetic state of an individual locus, with the probability of overall FLC coverage by silencing H3K27me3 marks depending on the length of cold exposure. Thus, the model predicts a bistable pattern of FLC gene expression in individual cells, a prediction we verify using the FLC:GUS reporter system. Our proposed switching mechanism, involving the local nucleation of an opposing histone modification, is likely to be widely relevant in epigenetic reprogramming.

Multiple Functions for Drosophila Mcm10 Suggested Through Analysis of Two Mcm10 Mutant Alleles

DNA replication and the correct packaging of DNA into different states of chromatin are both essential processes in all eukaryotic cells. High-fidelity replication of DNA is essential for the transmission of genetic material to cells. Likewise the maintenance of the epigenetic chromatin states is essential to the faithful reproduction of the transcriptional state of the cell. It is becoming more apparent that these two processes are linked through interactions between DNA replication proteins and chromatin-associated proteins. In addition, more proteins are being discovered that have dual roles in both DNA replication and the maintenance of epigenetic states. We present an analysis of two Drosophila mutants in the conserved DNA replication protein Mcm10. A hypomorphic mutant demonstrates that Mcm10 has a role in heterochromatic silencing and chromosome condensation, while the analysis of a novel C-terminal truncation allele of Mcm10 suggests that an interaction with Mcm2 is not required for chromosome condensation and heterochromatic silencing but is important for DNA replication.

Paramutation: the tip of an epigenetic iceberg?

Paramutation describes the transfer of an acquired epigenetic state to an unlinked homologous locus, resulting in a meiotically heritable alteration in gene expression. Early investigations of paramutation characterized a mode of change and inheritance distinct from mendelian genetics, catalyzing the concept of the epigenome. Numerous examples of paramutation and paramutation-like phenomena have now emerged, with evidence that implicates small RNAs in the transfer and maintenance of epigenetic states. In animals Piwi-interacting RNA (piRNA)-mediated retrotransposon suppression seems to drive a vast system of epigenetic inheritance with paramutation-like characteristics. The classic examples of paramutation might be merely informative aberrations of pervasive and broadly conserved mechanisms that use RNA to sense homology and target epigenetic modification. When viewed in this context, paramutation is only one aspect of a common and broadly distributed form of inheritance based on epigenetic states.

Characterization of a novel obesity phenotype caused by interspecific hybridization

Interspecific hybridization in mammals causes hybrid dysgenesis effects, such as sterility and abnormal placentation. Here, we describe a novel obesity syndrome caused by interspecific hybridization in the genus Mus and show that this obesity, appearing sporadically in F1 littermates derived from inbred strains, has an epigenetic basis. Mus hybrids from various strains of M. musculus and M. spretus were generated and the sporadic obese phenotype was confirmed through assessment of physiological and biochemical parameters in littermates. To understand the underlying mechanisms, large-scale and candidate gene expression assays, global DNA methylation assays and allelic expression analysis were performed. Studies showed that obese hybrids are similar to other known models of obesity. While increased axial growth indicated a defect in POMC pathway, comparison of global gene expression patterns in brain of obese F1 and obese Pomc mutant mice showed little similarity. In F1 obese mice many genes involved in the maintenance of epigenetic states, as well as several imprinted genes, were differentially expressed. Global DNA methylation analysis in brain showed that increased methylation levels were associated with obesity. The imprinted gene Gnasxl, known to be important in lipid homeostasis, was found over expressed in the obese hybrids. Allelic expression and methylation analysis of Gnasxl showed that alterations of epigenetic marks underlying F1 obesity are probably many and multi-factorial. Conclusions: This model of obesity, which is both spontaneous and epigenetic, may be a useful tool to address the epigenetic aspects of clinical obesity.

Physiological functions of plant DNA methyltransferases

Epigenetic regulation is defined as mechanisms that control gene expression without altering base sequences. Cytosine methylation, chromatin remodeling, and modifications at the N-termini of core histones are key factors in this regard. Epigenetic modifications are found throughout the eukaryotes, suggesting that they developed at an early stage in biological evolution, although actual molecular mechanisms show considerable variation among species. In particular, plants are unique in establishment and maintenance of epigenetic states, as exemplified by species-specific enzymes that catalyze DNA methylation. Since the function and diversity of DNA methyltransferases in individual species are not fully understood, I here summarize recent findings in plant epigenetics, focusing on DNA methyltransferases classified into three major groups. Their possible biological functions are also discussed with reference to histone modification and chromatin remodeling.

Epigenetic variability and the evolution of human cancer

Although the leading dogma for the origin of the diversity in cancer cell subpopulations is based on a stepwise selection and accumulation of genetic changes that allow uncontrollable malignant growth, there is an emerging understanding that the variability of heritable phenotypes in cancer and cancer-prone cells may also involve epigenetic mechanisms. We discuss here experimental data that allow us to postulate that the genome is organized into epigenetic territories with lineage-specific differences in the stringencies of the active and inactive states. Low-stringency epigenetic states are predicted to be closer to mosaicism, or chaos, than high-stringency states. In pathological situations, the result is an epigenetic variability upon which selection mechanisms can act during tumor progression. This view may have significant implications on clinical assessment and prognosis, and also suggests that major factors involved in the resetting and/or maintenance of epigenetic states may serve as new attractive targets for therapeutic interventions.

Plant chromatin: development and gene control.

It is increasingly clear that chromatin is not just a device for packing DNA within the nucleus but also a dynamic material that changes as cellular environments alter. The precise control of chromatin modification in response to developmental and environmental cues determines the correct spatial and temporal expression of genes. Here, we review exciting discoveries that reveal chromatin participation in many facets of plant development. These include: chromatin modification from embryonic and meristematic development to flowering and seed formation, the involvement of DNA methylation and chromatin in controlling invasive DNA and in maintenance of epigenetic states, and the function of chromatin modifying and remodeling complexes such as SWI/SNF and histone acetylases and deacetylases in gene control. Given the role chromatin structure plays in every facet of plant development, chromatin research will undoubtedly be integral in both basic and applied plant biology.