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Advanced applications
[edit]In vivo footprinting
[edit]In vivo footprinting is a technique used to analyze the protein-DNA interaction occurring within a cell at a given time point.[1] This method helps identify regions of DNA occupied by proteins, revealing insights into in vivo gene regulation with the cell.
Cleavage agents are used to degrade the unbound DNA while preserving protein-bound DNA. DNase I is commonly used as a cleavage agent when the cellular membrane has been permeabilized, making it more porous to allow better penetration of external substances.[2] However, the most common cleavage agent used is UV-irradiation, because it penetrates the cell membrane without disrupting the cell and can thus capture interactions that are sensitive to cellular changes. However, this comes with the drawback that DNase I provides higher specificity and accuracy. DNase I is capable of cleaving unprotected DNA regions, leaving a footprint where proteins are bound. This allows for precise identification of protein-DNA interactions, while UV radiation induces widespread damage, such that, it can be difficult to find the exact binding sites.[3]
Once the DNA has been cleaved or damaged by UV, the cells can be lysed and DNA purified for analysis of a region of interest. Ligation-mediated PCR is an alternative method to footprint in vivo. Following DNA cleavage and isolation, linker proteins are attached at the breakpoints. A region of interest is amplified between the linker and a gene-specific primer, and when run on a polyacrylamide gel, will have a footprint, a gap, where a protein was bound.[4]
In vivo footprinting can be combined with immunoprecipitation to assess protein specificity at particular locations throughout the genome.[5] This assay involves either using chemical crosslinkers or UV light to cross-link DNA to its associated proteins. After digesting unbound DNA, the DNA-protein complexes will remain. The protein of interest can be then selectively immunoprecipitated when detected by a complimentary antibody. Once detected, the immunoprecipitated DNA can be purified, released from crosslink and analyzed using DNA footprinting techniques like PCR or sequencing the region of interest.[6] The DNA bound to a protein of interest can be immunoprecipitated with an antibody to that protein, and then specific region binding can be assessed using the DNA footprinting technique.
Quantitative footprinting
[edit]The DNA footprinting technique can be modified to assess the binding strength of a protein to specific regions of DNA.[7] By using a range of protein concentrations in the footprinting experiment, the intensity of banding and the footprint can be tracked. This concentration-dependent technique investigates the affinity between DNA and the protein of interest by testing it under a range of protein concentrations. After DNase I treatment, the resulting fragments will be visualized on a PAGE gel and computationally analyzed. The intensity of banding and presence of the footprint will reflect the binding affinity between the protein of interest and a specific region of DNA. It is expected that with lower protein concentration, the gaps that signify the protein-DNA interactions will disappear, because fewer proteins are bound to the DNA, leading to more random cleavage.[8][9]
Detection by capillary electrophoresis
[edit]To adapt the footprinting technique to updated detection methods, the labelled DNA fragments are detected by a capillary electrophoresis device instead of being run on a polyacrylamide gel.[10] If the DNA fragment to be analyzed is produced by polymerase chain reaction (PCR), it is straightforward to couple a fluorescent molecule such as carboxyfluorescein (FAM) to the primers. This way, the fragments produced by DNase I digestion will contain FAM, and will be detectable by the capillary electrophoresis machine. Typically, carboxytetramethyl-rhodamine (ROX)-labelled size standards are also added to the mixture of fragments to be analyzed. Binding sites of transcription factors have been successfully identified this way.[11]
Capillary electrophoresis can be used to detect length differences of DNA fragments of interest within a sample.[12] Additionally, this technique offers high resolution, allowing for the detection of even minor variations in fragment length. The automation and high-throughput capabilities of capillary electrophoresis make it a valuable tool for large-scale studies and applications where rapid and accurate results are required. Furthermore, the use of fluorescent labelling enhances sensitivity and allows for multiplexing, enabling the simultaneous analysis of multiple samples or target regions within a single run.[13]
Cell-free fragmentomics
[edit]Cell-free (Cf) DNA fragmentomics analyzes the fragmentation patterns of cfDNA.[14] DNA footprinting is applied to cfDNA to study the binding sites of DNA-binding proteins. This allows researchers to identify and analyze protein-DNA interactions non-invasively. This is specifically used for early cancer detection by assessing disease-associated fragmentation patterns. This is done by extracting DNA from a body fluid sample, undergo sequencing and analysis, where specific features like fragment size, end motifs, and fragment distribution across different genomic regions to detect disease in a non-invasive manner.[15][16]
Genome-wide assays
[edit]Next-generation sequencing has enabled a genome-wide approach to identify DNA footprints. Open chromatin assays such as DNase-Seq[17] and FAIRE-Seq[18] have proven to provide a robust regulatory landscape for many cell types.[19] However, these assays require some downstream bioinformatics analyses in order to provide genome-wide DNA footprints. The computational tools proposed can be categorized in two classes: segmentation-based and site-centric approaches.
Segmentation-based methods are based on the application of Hidden Markov models or sliding window methods to segment the genome into open/closed chromatin region. Examples of such methods are: HINT,[20] Boyle method and Neph method[21]. Site-centric methods, on the other hand, find footprints given the open chromatin profile around motif-predicted binding sites, i.e., regulatory regions predicted using DNA-protein sequence information (encoded in structures such as position weight matrix). Examples of these methods are CENTIPEDE and Cuellar-Partida method.[22][23]
- ^ Becker, Michael M.; Wang, James C. (1984-06). "Use of light for footprinting DNA in vivo". Nature. 309 (5970): 682–687. doi:10.1038/309682a0. ISSN 0028-0836.
{{cite journal}}: Check date values in:|date=(help) - ^ Dellweg, H.; Wacker, A. (1966-01). "THE ENZYMATIC DEGRADATION OF ULTRAVIOLET‐IRRADIATED DEOXYRIBONUCLEIC ACID*". Photochemistry and Photobiology. 5 (1): 119–126. doi:10.1111/j.1751-1097.1966.tb05766.x. ISSN 0031-8655.
{{cite journal}}: Check date values in:|date=(help) - ^ Pfeifer, Gerd P.; Tornaletti, Silvia (1997-02). "Footprinting with UV Irradiation and LMPCR". Methods. 11 (2): 189–196. doi:10.1006/meth.1996.0405.
{{cite journal}}: Check date values in:|date=(help) - ^ Dai, Shu-Mei; Chen, Hsiu-Hua; Chang, Cheng; Riggs, Arthur D.; Flanagan, Steven D. (2000-10). "Ligation-mediated PCR for quantitative in vivo footprinting". Nature Biotechnology. 18 (10): 1108–1111. doi:10.1038/80323. ISSN 1087-0156.
{{cite journal}}: Check date values in:|date=(help) - ^ Dai, Shu-Mei; Chen, Hsiu-Hua; Chang, Cheng; Riggs, Arthur D.; Flanagan, Steven D. (2000-10). "Ligation-mediated PCR for quantitative in vivo footprinting". Nature Biotechnology. 18 (10): 1108–1111. doi:10.1038/80323. ISSN 1087-0156.
{{cite journal}}: Check date values in:|date=(help) - ^ Dey, Bipasha; Thukral, Sameer; Krishnan, Shruti; Chakrobarty, Mainak; Gupta, Sahil; Manghani, Chanchal; Rani, Vibha (2012-06). "DNA–protein interactions: methods for detection and analysis". Molecular and Cellular Biochemistry. 365 (1–2): 279–299. doi:10.1007/s11010-012-1269-z. ISSN 0300-8177.
{{cite journal}}: Check date values in:|date=(help) - ^ Mir, Rakeeb Ahmad; Mansoor, Sheikh; Zargar, Sajad Majeed (2023). Principles of genomics and proteomics. Amsterdam, Netherlands Oxford, United Kingdom Cambridge MA: Elsevier. ISBN 978-0-323-99045-5.
- ^ Hampshire, A; Rusling, D; Broughtonhead, V; Fox, K (2007-06). "Footprinting: A method for determining the sequence selectivity, affinity and kinetics of DNA-binding ligands". Methods. 42 (2): 128–140. doi:10.1016/j.ymeth.2007.01.002.
{{cite journal}}: Check date values in:|date=(help) - ^ Shubsda, Michael; Kishikawa, Hiroko; Goodisman, Jerry; Dabrowiak, James (1994-06). "Quantitative footprinting analysis". Journal of Molecular Recognition. 7 (2): 133–139. doi:10.1002/jmr.300070210. ISSN 0952-3499.
{{cite journal}}: Check date values in:|date=(help) - ^ Sivapragasam, Smitha; Pande, Anuja; Grove, Anne (2015-07). "A recommended workflow for DNase I footprinting using a capillary electrophoresis genetic analyzer". Analytical Biochemistry. 481: 1–3. doi:10.1016/j.ab.2015.04.013.
{{cite journal}}: Check date values in:|date=(help) - ^ Kovács, Krisztián A.; Steinmann, Myriam; Magistretti, Pierre J.; Halfon, Olivier; Cardinaux, Jean‐René (2006-09). "C/EBPβ couples dopamine signalling to substance P precursor gene expression in striatal neurones". Journal of Neurochemistry. 98 (5): 1390–1399. doi:10.1111/j.1471-4159.2006.03957.x. ISSN 0022-3042.
{{cite journal}}: Check date values in:|date=(help) - ^ Wilson, Douglas O.; Johnson, Peter; McCord, Bruce R. (2001-06). "Nonradiochemical DNase I footprinting by capillary electrophoresis". ELECTROPHORESIS. 22 (10): 1979–1986. doi:10.1002/1522-2683(200106)22:10<1979::AID-ELPS1979>3.0.CO;2-A. ISSN 0173-0835.
{{cite journal}}: Check date values in:|date=(help) - ^ Ferraz, Ricardo André Campos; Lopes, Ana Lúcia Gonçalves; da Silva, Jessy Ariana Faria; Moreira, Diana Filipa Viana; Ferreira, Maria João Nogueira; de Almeida Coimbra, Sílvia Vieira (2021-12). "DNA–protein interaction studies: a historical and comparative analysis". Plant Methods. 17 (1). doi:10.1186/s13007-021-00780-z. ISSN 1746-4811.
{{cite journal}}: Check date values in:|date=(help)CS1 maint: unflagged free DOI (link) - ^ Kim, Jaeryuk; Hong, Seung-Pyo; Lee, Seyoon; Lee, Woochan; Lee, Dakyung; Kim, Rokhyun; Park, Young Jun; Moon, Sungji; Park, Kyunghyuk; Cha, Bukyoung; Kim, Jong-Il (2023-10-28). "Multidimensional fragmentomic profiling of cell-free DNA released from patient-derived organoids". Human Genomics. 17 (1). doi:10.1186/s40246-023-00533-0. ISSN 1479-7364.
{{cite journal}}: CS1 maint: unflagged free DOI (link) - ^ Chiu, Rossa W K; Heitzer, Ellen; Lo, Y M Dennis; Mouliere, Florent; Tsui, Dana W Y (2020-12-01). "Cell-Free DNA Fragmentomics: The New "Omics" on the Block". Clinical Chemistry. 66 (12): 1480–1484. doi:10.1093/clinchem/hvaa258. ISSN 0009-9147.
- ^ Bruhm, Daniel C.; Vulpescu, Nicholas A.; Foda, Zachariah H.; Phallen, Jillian; Scharpf, Robert B.; Velculescu, Victor E. (2025-03-04). "Genomic and fragmentomic landscapes of cell-free DNA for early cancer detection". Nature Reviews Cancer. doi:10.1038/s41568-025-00795-x. ISSN 1474-175X.
- ^ Song, Lingyun; Crawford, Gregory E. (2010-02). "DNase-seq: A High-Resolution Technique for Mapping Active Gene Regulatory Elements across the Genome from Mammalian Cells". Cold Spring Harbor Protocols. 2010 (2): pdb.prot5384. doi:10.1101/pdb.prot5384. ISSN 1940-3402.
{{cite journal}}: Check date values in:|date=(help) - ^ Giresi, Paul G.; Kim, Jonghwan; McDaniell, Ryan M.; Iyer, Vishwanath R.; Lieb, Jason D. (2007-06). "FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin". Genome Research. 17 (6): 877–885. doi:10.1101/gr.5533506. ISSN 1088-9051.
{{cite journal}}: Check date values in:|date=(help) - ^ Thurman, Robert E.; Rynes, Eric; Humbert, Richard; Vierstra, Jeff; Maurano, Matthew T.; Haugen, Eric; Sheffield, Nathan C.; Stergachis, Andrew B.; Wang, Hao; Vernot, Benjamin; Garg, Kavita; John, Sam; Sandstrom, Richard; Bates, Daniel; Boatman, Lisa (2012-09). "The accessible chromatin landscape of the human genome". Nature. 489 (7414): 75–82. doi:10.1038/nature11232. ISSN 0028-0836.
{{cite journal}}: Check date values in:|date=(help) - ^ Gusmao, Eduardo G.; Dieterich, Christoph; Zenke, Martin; Costa, Ivan G. (2014-11-15). "Detection of active transcription factor binding sites with the combination of DNase hypersensitivity and histone modifications". Bioinformatics. 30 (22): 3143–3151. doi:10.1093/bioinformatics/btu519. ISSN 1367-4811.
- ^ Boyle, Alan P.; Song, Lingyun; Lee, Bum-Kyu; London, Darin; Keefe, Damian; Birney, Ewan; Iyer, Vishwanath R.; Crawford, Gregory E.; Furey, Terrence S. (2011-03). "High-resolution genome-wide in vivo footprinting of diverse transcription factors in human cells". Genome Research. 21 (3): 456–464. doi:10.1101/gr.112656.110. ISSN 1088-9051.
{{cite journal}}: Check date values in:|date=(help) - ^ Pique-Regi, Roger; Degner, Jacob F.; Pai, Athma A.; Gaffney, Daniel J.; Gilad, Yoav; Pritchard, Jonathan K. (2011-03). "Accurate inference of transcription factor binding from DNA sequence and chromatin accessibility data". Genome Research. 21 (3): 447–455. doi:10.1101/gr.112623.110. ISSN 1088-9051.
{{cite journal}}: Check date values in:|date=(help) - ^ Cuellar-Partida, Gabriel; Buske, Fabian A.; McLeay, Robert C.; Whitington, Tom; Noble, William Stafford; Bailey, Timothy L. (2012-01-01). "Epigenetic priors for identifying active transcription factor binding sites". Bioinformatics. 28 (1): 56–62. doi:10.1093/bioinformatics/btr614. ISSN 1367-4811.