ChiRP-Seq

Last updated September 10, 2024

ChiRP-Seq (Chromatin Isolation by RNA purification) is a high-throughput sequencing method to discover regions of the genome which are bound by a specific RNA (or by a ribonucleoprotein containing the RNA of interest). Recent studies have shown that a significant proportion of some genomes (including mouse and human genomes) synthesize RNA that apparently do not code for proteins. The function of most of these non-coding RNA still has to be ascertained. Various genomic methods are being developed to map the functional association of these novel RNA to distinct regions of the genome to gain a better understanding of their function. ChiRP-Seq is one of these new methods which uses the massively parallel sequencing capability of 2nd generation sequencers to catalog the binding sites of these novel RNA molecules on a genome.

Although many have believed that RNAs mainly encode for proteins a very large portion of the eukaryotic genome is composed of RNAs that do not. These RNAs were originally considered junk until new advancements lead to the realization that they may indeed have a biological purpose. Over the last few years lncRNAs have been the least explored and functionally characterized emerging regulatory molecules, especially in comparison to their short counterparts, small ncRNAs.^[1] ChiRP-Seq is a new technique that has allowed us to map long RNA occupancy across the genome at a higher resolution than ever before. ChiRP-Seq works via affinity capture of a target complex of lncRNA and chromatin by tiling antisense-oligos.^[2] This technique will allow scientists to generate a map of genomic binding sites of several hundred bases very accurately due to high sensitivity and low background.

Overview of method

Tens of oligonucleotide probes are designed to be complementary to the RNA of interest. These oligos are labeled with biotin. Cells are cross-linked by UV or formalin and nuclei are isolated from these treated cells. The isolated nuclei were lysed and the released chromatin was fragmented by sonication to produce approximately 100-500 bp sized fragments. These chromatin fragments were hybridized to the biotinylated probe set. Complexes containing biotin-probe + RNA of interest + DNA fragment are captured by magnetic beads labeled with streptavidin.

Overview of a next generation sequencing method to characterize RNA binding sites to chromatin. Chromatin isolation by RNA purification. ChiRP-Seq.png — Overview of a next generation sequencing method to characterize RNA binding sites to chromatin. Chromatin isolation by RNA purification.

DNA is isolated from an aliquot of the bound complex by treatment with RNAse (or proteinase followed by RNAse) to digest associated protein and RNA. RNA may also be isolated from an additional aliquot of the bound complex to detect other RNA molecules associated with the RNA of interest. The purified DNA is then used to prepare a sequencing library and the library is sequenced on a next generation DNA sequencing system. The sequencing reads are then mapped to the genome. A pile-up of reads at specific locations on the genome indicates that the RNA of interest had bound to that region of the genome. This helps delineate specific genomic regions that interact with RNA. For example, genomic targets of enhancer RNA which act at a distance from their site of synthesis can be easily evaluated by ChiRP-Seq.^[3]^[4]

Related Research Articles

<span class="mw-page-title-main">Complementary DNA</span> DNA reverse transcribed from RNA

In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed from an RNA. cDNA exists in both single-stranded and double-stranded forms and in both natural and engineered forms.

Functional genomics is a field of molecular biology that attempts to describe gene functions and interactions. Functional genomics make use of the vast data generated by genomic and transcriptomic projects. Functional genomics focuses on the dynamic aspects such as gene transcription, translation, regulation of gene expression and protein–protein interactions, as opposed to the static aspects of the genomic information such as DNA sequence or structures. A key characteristic of functional genomics studies is their genome-wide approach to these questions, generally involving high-throughput methods rather than a more traditional "candidate-gene" approach.

ChIP-on-chip is a technology that combines chromatin immunoprecipitation ('ChIP') with DNA microarray ("chip"). Like regular ChIP, ChIP-on-chip is used to investigate interactions between proteins and DNA in vivo. Specifically, it allows the identification of the cistrome, the sum of binding sites, for DNA-binding proteins on a genome-wide basis. Whole-genome analysis can be performed to determine the locations of binding sites for almost any protein of interest. As the name of the technique suggests, such proteins are generally those operating in the context of chromatin. The most prominent representatives of this class are transcription factors, replication-related proteins, like origin recognition complex protein (ORC), histones, their variants, and histone modifications.

ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global binding sites precisely for any protein of interest. Previously, ChIP-on-chip was the most common technique utilized to study these protein–DNA relations.

DNA adenine methyltransferase identification, often abbreviated DamID, is a molecular biology protocol used to map the binding sites of DNA- and chromatin-binding proteins in eukaryotes. DamID identifies binding sites by expressing the proposed DNA-binding protein as a fusion protein with DNA methyltransferase. Binding of the protein of interest to DNA localizes the methyltransferase in the region of the binding site. Adenine methylation does not occur naturally in eukaryotes and therefore adenine methylation in any region can be concluded to have been caused by the fusion protein, implying the region is located near a binding site. DamID is an alternate method to ChIP-on-chip or ChIP-seq.

RIP-chip is a molecular biology technique which combines RNA immunoprecipitation with a microarray. The purpose of this technique is to identify which RNA sequences interact with a particular RNA binding protein of interest in vivo. It can also be used to determine relative levels of gene expression, to identify subsets of RNAs which may be co-regulated, or to identify RNAs that may have related functions. This technique provides insight into the post-transcriptional gene regulation which occurs between RNA and RNA binding proteins.

Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence. Epigenomic maintenance is a continuous process and plays an important role in stability of eukaryotic genomes by taking part in crucial biological mechanisms like DNA repair. Plant flavones are said to be inhibiting epigenomic marks that cause cancers. Two of the most characterized epigenetic modifications are DNA methylation and histone modification. Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis. The study of epigenetics on a global level has been made possible only recently through the adaptation of genomic high-throughput assays.

Chromatin Interaction Analysis by Paired-End Tag Sequencing is a technique that incorporates chromatin immunoprecipitation (ChIP)-based enrichment, chromatin proximity ligation, Paired-End Tags, and High-throughput sequencing to determine de novo long-range chromatin interactions genome-wide.

Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or other DNA binding sites, and possibly define cistromes. ChIP also aims to determine the specific location in the genome that various histone modifications are associated with, indicating the target of the histone modifiers. ChIP is crucial for the advancements in the field of epigenomics and learning more about epigenetic phenomena.

ChIP-exo is a chromatin immunoprecipitation based method for mapping the locations at which a protein of interest binds to the genome. It is a modification of the ChIP-seq protocol, improving the resolution of binding sites from hundreds of base pairs to almost one base pair. It employs the use of exonucleases to degrade strands of the protein-bound DNA in the 5'-3' direction to within a small number of nucleotides of the protein binding site. The nucleotides of the exonuclease-treated ends are determined using some combination of DNA sequencing, microarrays, and PCR. These sequences are then mapped to the genome to identify the locations on the genome at which the protein binds.

Chem-seq is a technique that is used to map genome-wide interactions between small molecules and their protein targets in the chromatin of eukaryotic cell nuclei. The method employs chemical affinity capture coupled with massively parallel DNA sequencing to identify genomic sites where small molecules interact with their target proteins or DNA. It was first described by Lars Anders et al. in the January, 2014 issue of "Nature Biotechnology".

CRISPR-Display (CRISP-Disp) is a modification of the CRISPR/Cas9 system for genome editing. The CRISPR/Cas9 system uses a short guide RNA (sgRNA) sequence to direct a Streptococcus pyogenes Cas9 nuclease, acting as a programmable DNA binding protein, to cleave DNA at a site of interest.

CUT&RUN sequencing, also known as cleavage under targets and release using nuclease, is a method used to analyze protein interactions with DNA. CUT&RUN sequencing combines antibody-targeted controlled cleavage by micrococcal nuclease with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that CUT&RUN sequencing does not.

Spatial transcriptomics is a method for assigning cell types to their locations in the histological sections. Recent work demonstrated that the subcellular localization of mRNA molecules, for example, in the nucleus can also be studied.

BLESS, also known as breaks labeling, enrichment on streptavidin and next-generation sequencing, is a method used to detect genome-wide double-strand DNA damage. In contrast to chromatin immunoprecipitation (ChIP)-based methods of identifying DNA double-strand breaks (DSBs) by labeling DNA repair proteins, BLESS utilizes biotinylated DNA linkers to directly label genomic DNA in situ which allows for high-specificity enrichment of samples on streptavidin beads and the subsequent sequencing-based DSB mapping to nucleotide resolution.

CUT&Tag-sequencing, also known as cleavage under targets and tagmentation, is a method used to analyze protein interactions with DNA. CUT&Tag-sequencing combines antibody-targeted controlled cleavage by a protein A-Tn5 fusion with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that CUT&RUN and CUT&Tag sequencing do not. CUT&Tag sequencing is an improvement over CUT&RUN because it does not require cells to be lysed or chromatin to be fractionated. CUT&RUN is not suitable for single-cell platforms so CUT&Tag is advantageous for these.

ChIL sequencing (ChIL-seq), also known as Chromatin Integration Labeling sequencing, is a method used to analyze protein interactions with DNA. ChIL-sequencing combines antibody-targeted controlled cleavage by Tn5 transposase with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that ChIL-Sequencing does not. ChIL-Seq is a precise technique that reduces sample loss could be applied to single-cells.

H3R17me2 is an epigenetic modification to the DNA packaging protein histone H3. It is a mark that indicates the di-methylation at the 17th arginine residue of the histone H3 protein. In epigenetics, arginine methylation of histones H3 and H4 is associated with a more accessible chromatin structure and thus higher levels of transcription. The existence of arginine demethylases that could reverse arginine methylation is controversial.

Ribose-seq is a mapping technique used in genetics research to determine the full profile of embedded ribonucleotides, specifically ribonucleoside monophosphates (rNMPs), in genomic DNA. Embedded ribonucleotides are thought to be the most common alteration to DNA in cells, and their presence in genomic DNA can affect genome stability. As recent studies have suggested that ribonucleotides in mouse DNA may affect disease pathology, ribonucleotide incorporation in genomic DNA has become an important target of medical genetics research. Ribose-seq allows scientists to determine the precise location and type of ribonucleotides that have been incorporated into eukaryotic or prokaryotic DNA.

Proximity ligation-assisted chromatin immunoprecipitation sequencing (PLAC-seq) is a chromatin conformation capture(3C)-based technique to detect and quantify genomic chromatin structure from a protein-centric approach. PLAC-seq combines in situ Hi-C and chromatin immunoprecipitation (ChIP), which allows for the identification of long-range chromatin interactions at a high resolution with low sequencing costs. Mapping long-range 3-dimensional(3D) chromatin interactions is important in identifying transcription enhancers and non-coding variants that can be linked to human diseases.

References

↑ Chu, Quinn, J., & Chang, H. Y. (2012). Chromatin isolation by RNA purification (ChIRP). Journal of Visualized Experiments, 61. https://doi.org/10.3791/3912
↑ Jathar, Kumar, V., Srivastava, J., & Tripathi, V. (2017). Technological Developments in lncRNA Biology. Long Non Coding RNA Biology, 283–323. https://doi.org/10.1007/978-981-10-5203-3_10
↑ Chu, Ci; Qu, Kun; Zhong, Franklin L.; Artandi, Steven E.; Chang, Howard Y. (31 August 2011). "Genomic Maps of Long Noncoding RNA Occupancy Reveal Principles of RNA-Chromatin Interactions". Molecular Cell. 44 (4): 667–678. doi:10.1016/j.molcel.2011.08.027. PMC 3249421 . PMID 21963238.
↑ Li, W.; Notani, D.; Ma, Q.; Tanasa, B.; Nunez, E.; et al. (2013). "Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation". Nature. 498 (7455): 516–520. Bibcode:2013Natur.498..516L. doi:10.1038/nature12210. PMC 3718886 . PMID 23728302.

This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.

[1] Chu, Quinn, J., & Chang, H. Y. (2012). Chromatin isolation by RNA purification (ChIRP). Journal of Visualized Experiments, 61. https://doi.org/10.3791/3912

[2] Jathar, Kumar, V., Srivastava, J., & Tripathi, V. (2017). Technological Developments in lncRNA Biology. Long Non Coding RNA Biology, 283–323. https://doi.org/10.1007/978-981-10-5203-3_10

[3] Chu, Ci; Qu, Kun; Zhong, Franklin L.; Artandi, Steven E.; Chang, Howard Y. (31 August 2011). "Genomic Maps of Long Noncoding RNA Occupancy Reveal Principles of RNA-Chromatin Interactions". Molecular Cell. 44 (4): 667–678. doi:10.1016/j.molcel.2011.08.027. PMC 3249421 . PMID 21963238.

[4] Li, W.; Notani, D.; Ma, Q.; Tanasa, B.; Nunez, E.; et al. (2013). "Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation". Nature. 498 (7455): 516–520. Bibcode:2013Natur.498..516L. doi:10.1038/nature12210. PMC 3718886 . PMID 23728302.

[1]

[2]

[3]

[4]