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Find video protocols related to scientific articles indexed in Pubmed.
Decoding the role of chromatin architecture in development: coming closer to the end of the tunnel.
Front Plant Sci
PUBLISHED: 08-21-2014
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Form and function in biology are intimately related aspects that are often difficult to untangle. While the structural aspects of chromatin organization were apparent from early cytological observations long before the molecular details of chromatin functions were deciphered, the extent to which genome architecture may impact its output remains unclear. A major roadblock to resolve this issue is the divergent scales, both temporal and spatial, of the experimental approaches for examining these facets of chromatin biology. Recent advances in high-throughput sequencing and informatics to model and monitor genome-wide chromatin contact sites provide the much-needed platform to close this gap. This mini-review will focus on discussing recent efforts applying new technologies to elucidate the roles of genome architecture in coordinating global gene expression output. Our discussion will emphasize the potential roles of differential genome 3-D structure as a driver for cell fate specification of multicellular organisms. An integrated approach that combines multiple new methodologies may finally have the necessary temporal and spatial resolution to provide clarity on the roles of chromatin architecture during development.
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The chromatin landscape of the moss Physcomitrella patens and its dynamics during development and drought stress.
Plant J.
PUBLISHED: 04-14-2014
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The moss Physcomitrella patens is an important model organism for evo-devo studies. Here, we determined the genome-wide chromatin landscape of five important histone three (H3) modifications (H3K4me3, H3K27me3, H3K27Ac, H3K9Ac and H3K9me2) and describe the changes to these histone marks in two contrasted situations, developmental transition and abiotic (drought) stress. Integrative analysis of these histone H3 modifications revealed their preferential association into 15 chromatin states (CS) in genic regions of the P. patens genome. Synergistic relationships that influence expression levels were revealed for the three activating marks H3K4me3, H3K27Ac and H3K9Ac, while an antagonistic relationship was found between CS containing the H3K27me3 and H3K27Ac marks, suggesting that H3K27 is a key indexing residue regarding transcriptional output. Concerning the alteration of histone marks in response to developmental transition (juvenile to adult) and drought stress, the three activating marks H3K4me3, H3K27Ac and H3K9Ac show significant changes in both situations. However, changes to H3K27me3 are central only for genes differentially expressed during development. Interestingly, genes induced during drought stress show significant histone mark toggling during developmental transition. This situation suggests that drought induced adult (gametophore expressed) genes are primed to respond to this stress during the juvenile to adult transition.
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Quantitatively profiling genome-wide patterns of histone modifications in Arabidopsis thaliana using ChIP-seq.
Methods Mol. Biol.
PUBLISHED: 01-31-2014
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Genome-wide quantitative profiling of chromatin modifications is a critical experimental approach to study epigenetic and transcriptional control mechanisms. Since first being reported in 2007, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) has soon became a popular method-of-choice for profiling chromatin modifications and transcription factor-binding sites in eukaryote genomes. ChIP-seq has the advantage over the earlier ChIP-chip approach in multiple aspects including the lower amount of input DNA required, an expanded dynamic range and compatibility with sample multiplexing. Here we describe a detailed protocol for profiling histone modification in the Arabidopsis thaliana genome with ChIP-seq using the SOLiD™ 2.0 high-throughput sequencing platform. As read length and sequencing depth are two critical factors determining data quality and cost, we have developed bioinformatics approach to evaluate the effect of read length and sequencing depth on the alignment accuracy and the generated chromatin profile, respectively. Our analyses suggest that 2-3 million high quality sequencing tags with a read length of 35 nucleotides would be sufficient to profile the majority of histone modifications in this popular model plant species.
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Chromatin beacons: global sampling of chromatin physical properties using chromatin charting lines.
Methods Mol. Biol.
PUBLISHED: 04-13-2011
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The extent to which physical properties and intranuclear locations of chromatin can influence transcription output remains unclear and poorly quantified. Because the scale and resolution at which structural parameters can be queried are usually so different from the scale that transcription outputs are measured, the integration of these data is often indirect. To overcome this limitation in quantifying chromatin structural parameters at different locations in the genome, a Chromatin Charting collection with 277 transposon-tagged Arabidopsis lines has been established in order to discover correlations between gene expression and the physical properties of chromatin loci within the nuclei. In these lines, dispersed loci in the Arabidopsis genome are tagged with an identical transgene cassette containing a luciferase gene reporter, which permits the quantification of gene expressions in real time, and an ?2 kb LacO repeat that acts as a "chromatin beacon" to facilitate the visual tracking of a tagged locus in living plants via the expression of LacI-GFP fusion proteins in trans. In this chapter, we describe the methods for visualizing and tracking these insertion loci in vivo and illustrate the potential of using this approach to correlate chromatin mobility with gene expression in living plants.
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ANCORP: a high-resolution approach that generates distinct chromatin state models from multiple genome-wide datasets.
Plant J.
PUBLISHED: 04-26-2010
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Chromatin components can be extensively modified and dynamically regulated by a plethora of catalytic complexes. The numerous modifications may form a type of molecular pattern that defines particular local and global chromatin states through extensive cross-talk. Analyses that can integrate multiple genome-wide datasets are essential to determine the interactions and biological function of chromatin modifications in various contexts. Through a combination of hierarchical clustering and pattern visualization, we categorized all annotated Arabidopsis genes into 16 chromatin state clusters using combinations of four chromatin marks (H3K4me3, H3K36me2, H3K27me3 and cytosine methylation) using publicly available data. Our results suggest that gene length may be an important factor in shaping chromatin states across transcription units. By analysis of two rare chromatin states, we found that the enrichment of H3K36me2 around the transcription start site is negatively correlated with transcriptional activities. High-resolution association analyses in the context of chromatin states have identified inter-correlations between chromatin modifications. H3K4me3 were found to be under-represented in actively transcribed regions that are modified by DNA methylation and the H3K36me2 mark, concomitant with increased nucleosome occupancy in these regions. Lastly, quantitative data from transcriptome analyses and gene ontology partitioning were integrated to determine the possible functional relevance of the corresponding chromatin states. We show that modelling the plant epigenome in terms of chromatin states and combining correlative visualization methods can be a productive approach to unravel complex relationships between epigenomic features and the functional output of the genome.
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Chromatin charting: global mapping of epigenetic effects.
Methods Mol. Biol.
PUBLISHED: 07-10-2009
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To tackle the question of how chromatin organization is involved in global regulation of genome-related processes such as transcription, we have recently created a collection of 277 transposon-tagged Arabidopsis lines comprised of a single insert with a common luciferase reporter cassette and a LacO repeat array for visual tracking of the tagged region via fluorescent protein fusion technology. Using this collection of plants, one can begin to map transgene position effects as well as global epigenetic control in response to developmental or externally applied cues. In this chapter, we will outline the approach and methods for deploying this novel resource for the study of global gene control, using Arabidopsis as a convenient model system.
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Defining the functional network of epigenetic regulators in Arabidopsis thaliana.
Mol Plant
PUBLISHED: 04-03-2009
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Development of ChIP-chip and ChIP-seq technologies has allowed genome-wide high-resolution profiling of chromatin-associated marks and binding sites for epigenetic regulators. However, signals for directing epigenetic modifiers to their target sites are not understood. In this paper, we tested the hypothesis that genome location can affect the involvement of epigenetic regulators using Chromatin Charting (CC) Lines, which have an identical transgene construct inserted at different locations in the Arabidopsis genome. Four CC lines that showed evidence for epigenetic silencing of the luciferase reporter gene were transformed with RNAi vectors individually targeting epigenetic regulators LHP1, MOM1, CMT3, DRD1, DRM2, SUVH2, CLF, and HD1. Involvement of a particular epigenetic regulator in silencing the transgene locus in a CC line was determined by significant alterations in luciferase expression after suppression of the regulators expression. Our results suggest that the targeting of epigenetic regulators can be influenced by genome location as well as sequence context. In addition, the relative importance of an epigenetic regulator can be influenced by tissue identity. We also report a novel approach to predict interactions between epigenetic regulators through clustering analysis of the regulators using alterations in gene expression of putative downstream targets, including endogenous loci and transgenes, in epigenetic mutants or RNAi lines. Our data support the existence of a complex and dynamic network of epigenetic regulators that serves to coordinate and control global gene expression in higher plants.
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Charting functional and physical properties of chromatin in living cells.
Curr. Opin. Genet. Dev.
PUBLISHED: 01-14-2009
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With the vast transcriptome database now available, global patterns of gene expression have been mapped in various species to reveal higher order structures in the genome. Location-dependent control of gene expression has also been studied in human cell cultures and in Arabidopsis plants using well-characterized insertion and transposition cell line collections. With the added genome-wide mapping of chromatin features at a high resolution, via advanced microarray and sequencing technologies, comprehensive analysis of structure-function relationships deduced from chromatin organization and gene expression data is now feasible. This has begun to reveal micro-heterogeneity in the genome with respect to structural and functional segmentations.
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Integrative analysis of chromatin states in Arabidopsis identified potential regulatory mechanisms for natural antisense transcript production.
Plant J.
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Genome-wide analyses of epigenomic and transcriptomic profiles provide extensive resources for discovering epigenetic regulatory mechanisms. However, the construction of functionally relevant hypotheses from correlative patterns and the rigorous testing of these hypotheses may be challenging. We combined bioinformatics-driven hypothesis building with mutant analyses to identify potential epigenetic mechanisms using the model plant Arabidopsis thaliana. Genome-wide maps of nine histone modifications produced by ChIP-seq were used together with a strand-specific RNA-seq dataset to profile the epigenome and transcriptome of Arabidopsis. Combinatorial chromatin patterns were described by 42 major chromatin states with selected states validated using the re-ChIP assay. The functional relevance of chromatin modifications was analyzed using the ANchored CORrelative Pattern (ANCORP) method and a newly developed state-specific effects analysis (SSEA) method, which interrogates individual chromatin marks in the context of combinatorial chromatin states. Based on results from these approaches, we propose the hypothesis that cytosine methylation (5mC) and histone methylation H3K36me may synergistically repress production of natural antisense transcripts (NATs) in the context of actively expressed genes. Mutant analyses supported this proposed model at a significant proportion of the tested loci. We further identified polymerase-associated factor as a potential repressor for NAT abundance. Although the majority of tested NATs were found to localize to the nucleus, we also found evidence for cytoplasmically partitioned NATs. The significance of the subcellular localization of NATs and their biological functions remain to be defined.
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Analysis of the epigenetic status of telomeres by using ChIP-seq data.
Nucleic Acids Res.
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The chromatin structure of eukaryotic telomeres plays an essential role in telomere functions. However, their study might be impaired by the presence of interstitial telomeric sequences (ITSs), which have a widespread distribution in different model systems. We have developed a simple approach to study the chromatin structure of Arabidopsis telomeres independently of ITSs by analyzing ChIP-seq data. This approach could be used to study the chromatin structure of telomeres in some other eukaryotes. The analysis of ChIP-seq experiments revealed that Arabidopsis telomeres have higher density of histone H3 than centromeres, which might reflects their short nucleosomal organization. These experiments also revealed that Arabidopsis telomeres have lower levels of heterochromatic marks than centromeres (H3K9(Me2) and H3K27(Me)), higher levels of some euchromatic marks (H3K4(Me2) and H3K9Ac) and similar or lower levels of other euchromatic marks (H3K4(Me3), H3K36(Me2), H3K36(Me3) and H3K18Ac). Interestingly, the ChIP-seq experiments also revealed that Arabidopsis telomeres exhibit high levels of H3K27(Me3), a repressive mark that associates with many euchromatic genes. The epigenetic profile of Arabidopsis telomeres is closely related to the previously defined chromatin state 2. This chromatin state is found in 23% of Arabidopsis genes, many of which are repressed or lowly expressed. At least, in part, this scenario is similar in rice.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.