Purification of Specific Chromatin Regions Using Oligonucleotides: Capture Hybridization Analysis of RNA Targets (CHART)

Davis, Connor; West, Jason A.

doi:10.1007/978-1-4939-2253-6_10

Cited by 17 publications

(13 citation statements)

References 5 publications

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“…To determine the molecular mechanism by which A-ROD activates DKK1 transcription we purified endogenous A-ROD RNA using antisense biotinylated oligos tiling the entire A-ROD transcript, modified from previously published methods (Chu et al, 2015; Davis & West, 2015; Vance et al, 2014). Using probes targeting the A-ROD RNA we obtain a specific recovery of A-ROD compared to both control probes and recovery of DKK1 or GAPDH mRNA transcripts (Figure 8A).…”

Section: Resultsmentioning

confidence: 99%

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Chromatin-release of the long ncRNA A-ROD is required for transcriptional activation of its target gene DKK1

Ntini

et al. 2017

Preprint

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Long non-coding RNAs (ncRNAs) are involved in both positive and negative regulation of transcription. Long ncRNAs are often enriched in the nucleus and at chromatin but whether chromatin-release plays a functional role is unknown. Here, we used epigenetic marks, expression level and strength of chromatin interactions to group long ncRNAs and find that those engaged in strong chromatin interactions are less enriched at chromatin in MCF-7 cells, suggesting a functional involvement of chromatin-release of long ncRNAs in transcriptional regulation. To study this further, we identify the long ncRNA A-ROD, an activating regulator of the Wnt signaling inhibitor DKK1. We show that A-ROD enhances transcription elongation of DKK1 in an RNA-dependent manner and that A-ROD recruits EBP1 to the DKK1 promoter. Our data suggest that the activating function depends on the release of A-ROD from chromatin, and further identify a functional regulatory interaction mediated by A-ROD in the transcription activation of DKK1. We propose that the release of a subset of long ncRNAs is important for their function, adding a new mechanistic perspective to the subcellular localization of long ncRNAs.

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Section: Resultsmentioning

confidence: 99%

“…Purification of target RNA and associated DNA and proteins was done based on the previously published methods ChIRP (Chu et al, 2015) and CHART (Davis & West, 2015) with modifications (see Supplementary Methods).…”

Section: Purification Of Target Rna and Associated Dna And Proteinsmentioning

confidence: 99%

Chromatin-release of the long ncRNA A-ROD is required for transcriptional activation of its target gene DKK1

Ntini

et al. 2017

Preprint

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“…Probes for CHART are empirically determined after RNase H assay to map the accessible regions on the RNA of interest. These regions will be targeted by specifically designed probes (Davis and West, 2015). On the other hand, both ChIRP and RAP do not require a priori knowledge of the lncRNA domains involved in the interaction since they tile oligonucleotides across the entire target RNA sequence trying to cover it as much as possible.…”

Section: Rna-centric Methodsmentioning

confidence: 99%

Probing Long Non-coding RNA-Protein Interactions

Barra

Leucci

2017

Front. Mol. Biosci.

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Non-coding RNA sequences outnumber the protein-coding genes in the human genome, however our knowledge of their functions is still limited. RNA-binding proteins follow the transcripts, including non-coding RNAs, throughout their life, regulating not only maturation, nuclear export, stability and eventually translation, but also RNA functions. Therefore, development of sophisticated methods to study RNA-protein interactions are key to the systematic characterization of lncRNAs. Although mostly applicable to RNA-protein interactions in general, many approaches, especially the computational ones, need adjustment to be adapted to the length and complexity of lncRNA transcripts. Here we critically review all the wet lab and computational methods to study lncRNA-protein interactions and their potential to clarify the dark side of the genome.

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“…Capture hybridization analysis of RNA targets (CHART) is a method to analyze RNA targets that are analogous to chromatin immunoprecipitation (ChIP) for proteins [ 51 ]. To identify the genomic targets of those large noncoding RNAs which can act on chromatin at sites distant from where they are transcribed, this endogenous RNA is enriched from cross-linked chromatin extracts using short biotinylated complementary oligodeoxyribonucleotides.…”

Section: Capture Hybridization Analysis Of Rna Targets (Chart)-seqmentioning

confidence: 99%

Methods to Study Long Noncoding RNA Biology in Cancer

Luo

2016

Advances in Experimental Medicine and Biology

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Thousands of long noncoding RNAs (lncRNAs) have been discovered in recent years. The functions of lncRNAs range broadly from regulating chromatin structure and gene expression in the nucleus to controlling messenger RNA (mRNA) processing, mRNA posttranscriptional regulation, cellular signaling, and protein activity in the cytoplasm. Experimental and computational techniques have been developed to characterize lncRNAs in high-throughput scale, to study the lncRNA function in vitro and in vivo, to map lncRNA binding sites on the genome, and to capture lncRNA-protein interactions with the identification of lncRNA-binding partners, binding sites, and interaction determinants. In this chapter, we will discuss these technologies and their applications in decoding the functions of lncRNAs. Understanding these techniques including their advantages and disadvantages and developing them in the future will be essential to elaborate the roles of lncRNAs in cancer and other diseases.

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Purification of Specific Chromatin Regions Using Oligonucleotides: Capture Hybridization Analysis of RNA Targets (CHART)

Cited by 17 publications

References 5 publications

Chromatin-release of the long ncRNA A-ROD is required for transcriptional activation of its target gene DKK1

Chromatin-release of the long ncRNA A-ROD is required for transcriptional activation of its target gene DKK1

Probing Long Non-coding RNA-Protein Interactions

Methods to Study Long Noncoding RNA Biology in Cancer

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