ChIL-sequencing

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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. [1]

Contents

Uses

ChIL-sequencing can be used to examine gene regulation or to analyze transcription factor and other chromatin-associated protein binding. Protein-DNA interactions regulate gene expression and are responsible for many biological processes and disease states. This epigenetic information is complementary to genotype and expression analysis. ChIL-Seq is an alternative to the current standard of ChIP-seq. ChIP-Seq suffers from limitations due to the cross linking step in ChIP-Seq protocols that can promote epitope masking and generate false-positive binding sites. [2] [3] As well, ChIP-seq suffers from suboptimal signal-to-noise ratios and poor resolution. [4] ChIL-sequencing has the advantage of being a simpler technique suitable for low sample input due to the high signal-to-noise ratio, requiring less depth in sequencing for higher sensitivity. [5]

Specific DNA sites in direct physical interaction with proteins such as transcription factors can be isolated by Protein-A (pA) conjugated Tn5 bound to a protein of interest. Tn5 mediated cleavage produces a library of target DNA sites bound to a protein of interest in situ . Sequencing of prepared DNA libraries and comparison to whole-genome sequence databases allows researchers to analyze the interactions between target proteins and DNA, as well as differences in epigenetic chromatin modifications. Therefore, the ChIL-Seq method may be applied to proteins and modifications, including transcription factors, polymerases, structural proteins, protein modifications, and DNA modifications.

Protocols

There are detailed ChIL-Seq workflows available in an open-access methods repository. [6]

Limitations

The primary limitation of ChIL-seq is the likelihood of over-digestion of DNA due to inappropriate timing of the Magnesium-dependent Tn5 reaction. This is biased towards open chromatin like ATAC-Seq and similar techniques. [5] A similar limitation exists for contemporary ChIP-Seq protocols where enzymatic or sonicated DNA shearing must be optimized. As with ChIP-Seq, a good quality antibody targeting the protein of interest is required. As with other techniques using Tn5, the library preparation has a strong GC bias and has poor sensitivity in low GC regions or genomes with high variance in GC content. [7] [8] [9]


ChIL-Seq requires numerous laboratory steps and takes longer than other techniques such as CUT&RUN or CUT&Tag. It is still a broadly applicable technique which avoids sample loss suitable for small numbers of cells. However, the consumables cost of ChIL-Seq is substantially lower allowing more samples to be processed. [10]

Similar methods

See also

Related Research Articles

ChIP-on-chip Molecular biology method

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.

RNA immunoprecipitation chip

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

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 defining 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-exo

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.

ATAC-seq is a technique used in molecular biology to assess genome-wide chromatin accessibility. In 2013, the technique was first described as an alternative advanced method for MNase-seq, FAIRE-Seq and DNase-Seq. ATAC-seq is a faster and more sensitive analysis of the epigenome than DNase-seq or MNase-seq.

Single cell epigenomics Study of epigenomics in individual cells by single cell sequencing

Single cell epigenomics is the study of epigenomics in individual cells by single cell sequencing. Since 2013, methods have been created including whole-genome single-cell bisulfite sequencing to measure DNA methylation, whole-genome ChIP-sequencing to measure histone modifications, whole-genome ATAC-seq to measure chromatin accessibility and chromosome conformation capture.

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.

H3K9me3 is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the tri-methylation at the 9th lysine residue of the histone H3 protein and is often associated with heterochromatin.

H3K79me2 is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the di-methylation at the 79th lysine residue of the histone H3 protein. H3K79me2 is detected in the transcribed regions of active genes.

H3K14ac is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the acetylation at the 14th lysine residue of the histone H3 protein.

H3K36ac is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the acetylation at the 36th lysine residue of the histone H3 protein.

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.

H3K36me2 is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the di-methylation at the 36th lysine residue of the histone H3 protein.

H3K36me is an epigenetic modification to the DNA packaging protein Histone H3, specifically, the mono-methylation at the 36th lysine residue of the histone H3 protein.

H3R42me is an epigenetic modification to the DNA packaging protein histone H3. It is a mark that indicates the mono-methylation at the 42nd 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.

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.

References

  1. ↑ "When less is more: A promising approach for low-cell-number epigenomic profiling". Science Daily. 11 December 2018. Retrieved 24 December 2019.
  2. ↑ Meyer CA, Liu XS (November 2014). "Identifying and mitigating bias in next-generation sequencing methods for chromatin biology". Nature Reviews. Genetics. 15 (11): 709–21. doi:10.1038/nrg3788. PMC   4473780 . PMID   25223782.
  3. ↑ Baranello L, Kouzine F, Sanford S, Levens D (May 2016). "ChIP bias as a function of cross-linking time". Chromosome Research. 24 (2): 175–81. doi:10.1007/s10577-015-9509-1. PMC   4860130 . PMID   26685864.
  4. ↑ He C, Bonasio R (February 2017). "A cut above". eLife. 6. doi:10.7554/eLife.25000. PMC   5310838 . PMID   28199181.
  5. 1 2 Harada A, Maehara K, Handa T, Arimura Y, Nogami J, Hayashi-Takanawa Y, Shirahige K, Kurumizaka H, Kimura H, Ohkawa Y (December 2018). "A chromatin integration labelling method enables epigenomic profiling with lower input". Nature Cell Biology. 21 (2): 287–296. doi:10.1038/s41556-018-0248-3. PMID   30532068. S2CID   54463772.
  6. ↑ Ohkawa Y, Kimura H, Handa T, Harada A, Maehara K (20 Dec 2018). "Detailed protocol ─ Chromatin Integration labeling". Protocol Exchange. doi: 10.1038/protex.2018.122 .
  7. ↑ Lan, James H.; Yin, Yuxin; Reed, Elaine F.; Moua, Kevin; Thomas, Kimberly; Zhang, Qiuheng (March 2015). "Impact of three Illumina library construction methods on GC bias and HLA genotype calling". Human Immunology. 76 (2–3): 166–175. doi:10.1016/j.humimm.2014.12.016. PMC   5089167 . PMID   25543015.
  8. ↑ Sato, Mitsuhiko P; Ogura, Yoshitoshi; Nakamura, Keiji; Nishida, Ruriko; Gotoh, Yasuhiro; Hayashi, Masahiro; Hisatsune, Junzo; Sugai, Motoyuki; Takehiko, Itoh; Hayashi, Tetsuya (1 October 2019). "Comparison of the sequencing bias of currently available library preparation kits for Illumina sequencing of bacterial genomes and metagenomes". DNA Research. 26 (5): 391–398. doi: 10.1093/dnares/dsz017 . PMC   6796507 . PMID   31364694.
  9. ↑ Chen, Yen-Chun; Liu, Tsunglin; Yu, Chun-Hui; Chiang, Tzen-Yuh; Hwang, Chi-Chuan (29 April 2013). "Effects of GC Bias in Next-Generation-Sequencing Data on De Novo Genome Assembly". PLOS ONE. 8 (4): e62856. Bibcode:2013PLoSO...862856C. doi: 10.1371/journal.pone.0062856 . PMC   3639258 . PMID   23638157.
  10. ↑ Handa, Tetsuya; Harada, Akihito; Maehara, Kazumitsu; Sato, Shoko; Nakao, Masaru; Goto, Naoki; Kurumizaka, Hitoshi; Ohkawa, Yasuyuki; Kimura, Hiroshi (October 2020). "Chromatin integration labeling for mapping DNA-binding proteins and modifications with low input". Nature Protocols. 15 (10): 3334–3360. doi:10.1038/s41596-020-0375-8. PMID   32807906. S2CID   221145784 . Retrieved 3 December 2020.
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