scholarly journals MAPPING OF LONG-RANGE CHROMATIN INTERACTIONS BY PROXIMITY LIGATION ASSISTED CHIP-SEQ

2016 ◽  
Author(s):  
Rongxin Fang ◽  
Miao Yu ◽  
Guoqiang Li ◽  
Sora Chee ◽  
Tristin Liu ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Embryonic Stem ◽  
Cost Effective ◽  
Eukaryotic Cells ◽  
Proximity Ligation ◽  
Chromatin Interactions ◽  
Genome Wide ◽  
Cost Effective Method ◽  
Highly Sensitive ◽  
State Of Art

AbstractWe report a highly sensitive and cost-effective method for genome-wide identification of chromatin interactions in eukaryotic cells. Combining proximity ligation with chromatin immunoprecipitation and sequencing, the method outperforms the state of art approach in sensitivity, accuracy and ease of operation. Application of the method to mouse embryonic stem cells improves mapping of enhancer-promoter interactions.

scholarly journals RedChIP identifies noncoding RNAs associated with genomic sites occupied by Polycomb and CTCF proteins

2021 ◽  
Vol 119 (1) ◽  
pp. e2116222119
Author(s):  
Alexey A. Gavrilov ◽  
Rinat I. Sultanov ◽  
Mikhail D. Magnitov ◽  
Aleksandra A. Galitsyna ◽  
Erdem B. Dashinimaev ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Wide Spectrum ◽  
Noncoding Rnas ◽  
Ctcf Binding ◽  
Polycomb Repressive Complex 2 ◽  
Proximity Ligation ◽  
Chromatin Interactions ◽  
Genome Wide ◽  
Architectural Protein

Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. The progress in studying nuclear ncRNAs depends on the ability to identify the genome-wide spectrum of contacts of ncRNAs with chromatin. To address this question, a panel of RNA–DNA proximity ligation techniques has been developed. However, neither of these techniques examines proteins involved in RNA–chromatin interactions. Here, we introduce RedChIP, a technique combining RNA–DNA proximity ligation and chromatin immunoprecipitation for identifying RNA–chromatin interactions mediated by a particular protein. Using antibodies against architectural protein CTCF and the EZH2 subunit of the Polycomb repressive complex 2, we identify a spectrum of cis- and trans-acting ncRNAs enriched at Polycomb- and CTCF-binding sites in human cells, which may be involved in Polycomb-mediated gene repression and CTCF-dependent chromatin looping. By providing a protein-centric view of RNA–DNA interactions, RedChIP represents an important tool for studies of nuclear ncRNAs.

scholarly journals Pluripotency Transcription Factor Oct4 Mediates Stepwise Nucleosome Demethylation and Depletion

2015 ◽  
Vol 35 (6) ◽  
pp. 1014-1025 ◽  
Author(s):  
Arvind Shakya ◽  
Catherine Callister ◽  
Alon Goren ◽  
Nir Yosef ◽  
Neha Garg ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Chromatin Immunoprecipitation ◽  
Time Course ◽  
Embryonic Stem ◽  
Assay System ◽  
Histone Demethylation ◽  
Oct4 Expression ◽  
Genome Wide ◽  
Histone Lysine Demethylase ◽  
Lysine Demethylase

The mechanisms whereby the crucial pluripotency transcription factor Oct4 regulates target gene expression are incompletely understood. Using an assay system based on partially differentiated embryonic stem cells, we show that Oct4 opposes the accumulation of local H3K9me2 and subsequent Dnmt3a-mediated DNA methylation. Upon binding DNA, Oct4 recruits the histone lysine demethylase Jmjd1c. Chromatin immunoprecipitation (ChIP) time course experiments identify a stepwise Oct4 mechanism involving Jmjd1c recruitment and H3K9me2 demethylation, transient FACT ( fa cilitates c hromatin t ransactions) complex recruitment, and nucleosome depletion. Genome-wide and targeted ChIP confirms binding of newly synthesized Oct4, together with Jmjd1c and FACT, to the Pou5f1 enhancer and a small number of other Oct4 targets, including the Nanog promoter. Histone demethylation is required for both FACT recruitment and H3 depletion. Jmjd1c is required to induce endogenous Oct4 expression and fully reprogram fibroblasts to pluripotency, indicating that the assay system identifies functional Oct4 cofactors. These findings indicate that Oct4 sequentially recruits activities that catalyze histone demethylation and depletion.

scholarly journals Genome-wide screen of Saccharomyces cerevisiae null allele strains identifies genes involved in selenomethionine resistance

2008 ◽  
Vol 105 (46) ◽  
pp. 17682-17687 ◽  
Author(s):  
Jessica Bockhorn ◽  
Bharvi Balar ◽  
Dongming He ◽  
Eden Seitomer ◽  
Paul R. Copeland ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Null Allele ◽  
Cost Effective ◽  
Anomalous Dispersion ◽  
Gene Encoding ◽  
X Ray ◽  
X Ray Crystallography ◽  
Genome Wide ◽  
Cost Effective Method ◽  
Strain Collection

Selenomethionine (SeMet) is a potentially toxic amino acid, and yet it is a valuable tool in the preparation of labeled proteins for multiwavelength anomalous dispersion or single-wavelength anomalous dispersion phasing in X-ray crystallography. The mechanism by which high levels of SeMet exhibits its toxic effects in eukaryotic cells is not fully understood. Attempts to use Saccharomyces cerevisiae for the preparation of fully substituted SeMet proteins for X-ray crystallography have been limited. A screen of the viable S. cerevisiae haploid null allele strain collection for resistance to SeMet was performed. Deletion of the CYS3 gene encoding cystathionine gamma-lyase resulted in the highest resistance to SeMet. In addition, deletion of SSN2 resulted in both increased resistance to SeMet as well as reduced levels of Cys3p. A methionine auxotrophic strain lacking CYS3 was able to grow in media with SeMet as the only source of Met, achieving essentially 100% occupancy in total proteins. The CYS3 deletion strain provides advantages for an easy and cost-effective method to prepare SeMet-substituted protein in yeast and perhaps other eukaryotic systems.

scholarly journals Multi-omics analysis of chromatin accessibility and interactions with transcriptome by HiCAR

2020 ◽  
Author(s):  
Xiaolin Wei ◽  
Yu Xiang ◽  
Ruocheng Shan ◽  
Derek T Peters ◽  
Tongyu Sun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Long Range ◽  
Spatial Interaction ◽  
Embryonic Stem ◽  
Cost Effective ◽  
Chromatin Accessibility ◽  
Open Chromatin ◽  
Low Input ◽  
Chromatin Interactions

The long-range interactions of cis-regulatory elements (cREs) play a central role in regulating the spatial-temporal gene expression program of multi-cellular organism. cREs are characterized by the presence of accessible (or open) chromatin, which can be identified at genome-wide scale with assays such as ATAC-seq, DHS-seq, and FAIRE-seq. However, it remains technically challenging to comprehensively identify the long-range physical interactions that occur between cREs, especially in a cost effective manner using low-input samples. Here, we report HiCAR (High-throughput Chromosome conformation capture on Accessible DNA with mRNA-seq co-assay), a method that enables simultaneous assessment of cis-regulatory chromatin interactions and chromatin accessibility, as well as evaluation of the transcriptome, which represents the functional output of chromatin structure and accessibility. Unlike immunoprecipitation-based methods such as HiChIP, PLAC-seq, and ChIA-PET, HiCAR does not require target-specific antibodies and thus can comprehensively capture the cis-regulatory chromatin contacts anchored at accessible regulatory DNA regions and associated with diverse epigenetic modifications and transcription factor binding. Compared to Trac-looping, another method designed to capture interactions between accessible chromatin regions, HiCAR produced a 17-fold greater yield of informative long-range cis- reads at a similar sequencing depth and required 1,000-fold fewer cells as input. Applying HiCAR to H1 human embryonic stem cells (hESCs) revealed 46,792 cis-regulatory chromatin interactions at 5kb resolution. Interestingly, we found that epigenetically poised, bivalent, and repressed cREs exhibit comparable spatial interaction activity to those transcriptionally activated cREs. Using machine learning approaches, we predicated 22 epigenome features that are potentially important for the spatial interaction activity of cREs in H1 hESC. Lastly, we also identified long-range cis-regulatory chromatin interactions in GM12878 and mouse embryonic stem cells with HiCAR. Our results demonstrate that HiCAR is a robust and cost-effective multi-omics assay, which is broadly applicable for simultaneous analysis of genome architecture, chromatin accessibility, and the transcriptome using low-input samples.

scholarly journals Optimization of Cost-Effective and Reproducible Flexible Humidity Sensors Based on Metal-Organic Frameworks

Sensors ◽  
2020 ◽  
Vol 20 (23) ◽  
pp. 6981
Author(s):  
Victor Toral ◽  
Florin C. Loghin ◽  
Antonio Rodríguez-Diéguez ◽  
Alejandro Lapresta-Fernández ◽  
Diego P. Morales ◽  
...  
Keyword(s):  
Metal Organic Framework ◽  
Metal Organic Frameworks ◽  
Three Dimensional ◽  
Cost Effective ◽  
Film Deposition ◽  
Flexible Substrates ◽  
Humidity Sensors ◽  
Cost Effective Method ◽  
Highly Sensitive ◽  
Metal Organic

In this letter, we present the extension of a previous work on a cost-effective method for fabricating highly sensitive humidity sensors on flexible substrates with a reversible response, allowing precise monitoring of the humidity threshold. In that work we demonstrated the use of three-dimensional metal-organic framework (MOF) film deposition based on the perylene-3,4,9,10-tetracarboxylate linker, potassium as metallic center and the interspacing of silver interdigitated electrodes (IDEs) as humidity sensors. In this work, we study one of the most important issues in efficient and reproducible mass production, which is to optimize the most important processes’ parameters in their fabrication, such as controlling the thickness of the sensor’s layers. We demonstrate this method not only allows for the creation of humidity sensors, but it also is possible to change the humidity value that changes the actuator state.

scholarly journals Quantitative occupancy of myriad transcription factors from one DNase experiment enables efficient comparisons across conditions

2020 ◽  
Author(s):  
Kaixuan Luo ◽  
Jianling Zhong ◽  
Alexias Safi ◽  
Linda K. Hong ◽  
Alok K. Tewari ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Chromatin Immunoprecipitation ◽  
Cost Effective ◽  
Cell Types ◽  
Hierarchical Regression ◽  
Bayesian Hierarchical ◽  
Genome Wide ◽  
Regression Framework ◽  
Quantitative Changes ◽  
Using Data

AbstractOver a thousand different transcription factors (TFs) bind with varying occupancy across the human genome. Chromatin immunoprecipitation (ChIP) can assay occupancy genome-wide, but only one TF at a time, limiting our ability to comprehensively observe the TF occupancy landscape, let alone quantify how it changes across conditions. We developed TOP, a Bayesian hierarchical regression framework, to profile genome-wide quantitative occupancy of numerous TFs using data from a single DNase-seq experiment. TOP is supervised, and its hierarchical structure allows it to predict the occupancy of any sequence-specific TF, even those never assayed with ChIP. We used TOP to profile the quantitative occupancy of nearly 1500 human TF motifs, and examined how their occupancies changed genome-wide in multiple contexts: across 178 cell types, over 12 hours of exposure to different hormones, and across the genetic backgrounds of 70 individuals. TOP enables cost-effective exploration of quantitative changes in the landscape of TF binding.

scholarly journals Genome-Wide Technologies to Study RNA–Chromatin Interactions

2020 ◽  
Vol 6 (2) ◽  
pp. 20 ◽  
Author(s):  
Masaki Kato ◽  
Piero Carninci
Keyword(s):  
Nuclear Organization ◽  
Oligonucleotide Probes ◽  
Hybridization Capture ◽  
Proximity Ligation ◽  
Advantages And Disadvantages ◽  
Chromatin Interactions ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
Non Coding Rnas

An increasing number of studies have revealed that long non-coding RNAs (lncRNAs) play important roles in gene regulation and nuclear organization. Although the mechanisms are still largely unknown, many lncRNAs have been shown to interact with chromatin. Thus, one approach to understanding the function of these lncRNAs is to identify their sites of genomic interaction. Hybridization capture methods using oligonucleotide probes have been used for years to study chromatin-associated RNA. Recently, several groups have developed novel methods based on proximity ligation to investigate RNA–chromatin interactions at a genome-wide scale. This review discusses these technologies and highlights their advantages and disadvantages for the consideration of potential users.

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