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Assays for Detecting Protein-DNA Interactions
- 1. Assays for determining Protein DNA interaction Amaan shaikh MSc part I
- 2. • Proteins interact with DNA at many points for critical functions to maintain the overall integrity of the cell. • This interaction may be specific or non specific. • Some assays for determining protein-DNA interactions – Chromatin immunoprecipitation Yeast one hybrid assay Immunosorbant assay DPI ELISA In silico tools SELEX Protein binding assays Xray crystallography DAP-seq DNA footprinting
- 4. Cell Fixation of bound proteins with DNA using formaldehyde Cells are later lysed
- 5. DNA to which proteins may or may not be bound are fragmented using sonication
- 6. Immunoprecipitation of Protein-DNA complexes using antibodies and washed Fixating agent is removed to get free DNA and proteins DNA and proteins are isolated and processed for ChIPseq, ChIPMS, ChIPchip
- 7. Strengths and Limitations • Allows observation of highly dynamic events. • Can be combined with sequencing, PCR, cloning, footprinting, microarray, etc. • Only proteins for which antibodies are available can be studied. • Conformational changes on binding may prevent antibody binding.
- 8. Yeast one hybrid system Y1H
- 9. • Construct 1/Bait plasmid: Bait+ Promoter + Reporter gene BAIT REPORTER GENE Bait is the sequence of our interest along with promoter Transformed into yeast cells Reporter gene will be expressed Pro • Construct 2/Prey plasmid: Prey + AD + Marker Prey is the sequence encoding for TF of interest Yeast activating domain [GAL4 usually] Selection marker Transformed into pretransformed yeast cells
- 10. BAIT REPORTER GENE Pro BAIT REPORTER GENE Pro Case 1: If the protein recognizes and binds to SOI, transcription will result in expression of reporter gene Case 2: No transcription of reporter gene will happen if protein can’t recognize SOI
- 11. Strengths and Limitations • Can detect interactions between non transcription proteins and DNA. • Compatible with many existing libraries. • Rates of false positive are high. • Improper protein folding.
- 12. In Silico tools
- 13. • Laboratory methods are very time consuming and very expensive. • Low cost and efficient computational tools provide a way to study protein-DNA complexes. • There are many tools, software or web based, freely available for such interactive studies. • These tools uses structural information, model simulations, thermodynamic parameters, experimental conditions, biochemical properties, evolutionary info, scoring functions to predict protein- DNA binding affinity quantitively.
- 14. • TRANSFAC (TRANScription FACtor database) – DB of eukaryotic TFS, their binding sites, DNA binding profiles; helps in identifying potential TFBSs and in transcriptional regulation. • DISPLAR (DNA-Interaction Site Prediction from a List of Adjacent residues) – Predicts the residues of a protein which interact with DNA. • YEASTRACT (Yeast Search for Transcriptional Regulators And Consensus Tracking) – DB of TFs and target genes of yeast, helps in determining potential TFBSs. • Some web based tools for DNA-binding protein interaction studies – DP-Bind, HMMBinder, PiDNA, iDNA-Prot, PreDBA.
- 15. References • Cozzolino, F., Iacobucci, I., Monaco, V., & Monti, M. (2021). Protein-DNA/RNA interactions: An overview of investigation methods in the -omics era. Journal of Proteome Research, 20(6), 3018– 3030. https://doi.org/10.1021/acs.jproteome.1c00074 • Das, P. M., Ramachandran, K., vanWert, J., & Singal, R. (2004). Chromatin immunoprecipitation assay. BioTechniques, 37(6), 961–969. https://doi.org/10.2144/04376RV01 • Dey, B., Thukral, S., Krishnan, S., Chakrobarty, M., Gupta, S., Manghani, C., & Rani, V. (2012). DNA- protein interactions: methods for detection and analysis. Molecular and Cellular Biochemistry, 365(1–2), 279–299. https://doi.org/10.1007/s11010-012-1269-z • Emamjomeh, A., Choobineh, D., Hajieghrari, B., MahdiNezhad, N., & Khodavirdipour, A. (2019). DNA- protein interaction: identification, prediction and data analysis. Molecular Biology Reports, 46(3), 3571–3596. https://doi.org/10.1007/s11033-019-04763-1 • Ferraz, R. A. C., Lopes, A. L. G., da Silva, J. A. F., Moreira, D. F. V., Ferreira, M. J. N., & de Almeida Coimbra, S. V. (2021). DNA-protein interaction studies: a historical and comparative analysis. Plant Methods, 17(1), 82. https://doi.org/10.1186/s13007-021-00780-z • Pandey, P., Hasnain, S., & Ahmad, S. (2019). Protein-DNA Interactions. In Encyclopedia of Bioinformatics and Computational Biology (pp. 142–154). Elsevier. https://doi.org/10.1016/b978-0- 12-809633-8.20217-3 • Watson, J. D., Baker, T. A., Bell, S. P., Gann, A., Levine, M., & Losick, R. (2014). Molecular biology of the gene (7th ed.). Pearson.
- 16. THANK YOU
Editor's Notes
- Good morning sir/ma’am, Myself Amaan Shaikh and my topic is assays for determining protein DNA interactions
- Replication, transcription, translation, repair, regulation, etc….This interaction can be specific or non specific depending upon the functions.. As someone rightly said, life is a relation between molecules, not a property of any one molecule. So by learning proteins DNA interactions, humans will be a lil bit more closer to understand life, to understand different processes that happen within the cell. And this understanding will help in designing novel/modified proteins with different specificity, affinities n functions for various purposes. These are some assays for determining protein DNA interactions. I am going to explain couple of em. for example, DNA binding proteins in with different specificity, affinity, helping in transcriptional regulation, gene therapy functional genomics cellular functions including gene transcription, DNA replication and recombination, repair, segregation, chromosomal stability, cell cycle progression, and epigenetic silencing. The 3-dimensional structure of chromatin is maintained by the binding of histones and other regulatory proteins to the DNA. It is vital to know how DNA-binding proteins affect the functioning of any particular gene and to identify which particular protein binds to a specific DNA sequence in vivo https://www.scripps.edu/barbas/pdf/Segal00COCB.pdf
- R2 Chromatin immunoprecipitation (ChIP) has become a very widely used technique for determining the in vivo location of binding sites of various transcription factors. As we all know, chromatin is basically complex of DNA and protein complexes….in a lil bit, we will see what immunoprecipitation means here
- So we have a DNA here, and some DBPs. They can bind and unbind to DNA but here, we require proteins DNA complexes. And thus, we use formaldehyde [or Methylene blue, acridine orange, UV, laser] as a cross linking agent to fix proteins on DNA after which cells are lysed, using ionic salts [Nacl] or buffering salts [tris HCL]…. ref5
- R5- Next important step is fragmentation of DNA to 100-500bps fragments to pinpoint the location of the DNA sequence of interest. This can be done by sonication, RE digestion or by Nuclease In sonication, sound energy (>20khz) is applied resulting in agitation, bubble formation, bubble growth, bubble break resulting in shearing forces
- Here comes the immunoprecipitation part….we use Protein A/G coated beads specific to protein of interest to isolate and elute out protein-DNA complexes. Later unlinking of crosslinking agent [that is aldehyde] is done by incubating the protein-DNA complexes at higher temperature [68 degree Celsius accordingly] to get free DNA and protein. In post immunoprecipitation, proteins and DNA can be processed for sequencing in chipseq, chip Mass spec or chip micro array chips. Then proteins are can be either digested or used in post immunoprecipitation analyses.
- These are some strengths n limitations of this technique. It allows observation of minute-by-minute rapid changes at a single promoter. This technique can be combined with various other techniques to broaden the applications. One of the disadvantage here is antibodies against protein of interest should be available. Also, as the protein binds to the DNA, some conformational change may hide the epitope and we may get false negative results. Difficult to adapt for high throughput screening
- This is powerful method to identify proteins [any proteins and not just transcription factors] that can interact with a DNA sequence of interest, is a modified version of yeast two hybrid system which is used as a protein protein interaction assays
- This system is kinda like fishing,,,, here we are providing a bait and prey may or may not take that bait. First construct consist of a bait which is a sequence of interest, promoter and a reporter gene..umm, let’s say…LacZ…this will help in detecting whether the bait is taken by prey or not. Bait construct is ligated with the integrative vector which is later transformed into a suitable yeasts cells. 2nd construct here is our prey [that is, protein of interest], yeast activating domain that brings polymerase, normally Gal4 promoter, and a selection marker for example HIS3. This 2nd construct is then transformed into yeast cells which were pretransformed with bait construct.
- After culturing and selecting using a minimal medium without leucine, 2 cases are possible. Case 1: So if the prey protein is capable of recognizing and binding to the bait, it will bind to it, activating domain will bring promoter and other transcription factors ultimately leading to the expression of reporter gene…lacZ in our case which can be assayed using the galactosidase assay. Case 2: If prey can’t bind to the bait, there will be no expression of reporter gene so no signals in galactosidase assay. Leu2 leucine, ura3 for uracil C3, c4, cam pathway
- So, by fusing protein(s) of interest to a strong activation domain allows Y1H to detect a variety of DNA-binding proteins, including those that do not directly function in transcription, e.g. replication proteins, DNA repair proteins, and repressor proteins. Most Y1H experiments can use hybrid prey libraries [that is, prey with activating domains] that have been constructed for Y2H applications, e.g. Gal4p- or LexA-based protein libraries can also be used for screening against various DNA baits in Y1H. Polymerase may bind to promoter leading to false positive results…and fusion with activating domain may result in conformation change so prey might not bind to the bait sequences, which might be capable of binding originally. https://bitesizebio.com/25900/an-overview-of-the-yeast-one-hybrid-assay/
- Instead of going for laboratory methods which are expensive and time consuming, we can opt for computational tools. These tools are low cost plus are an efficient way to study protein-DNA complexes in short time. There are many tools freely available for such purposes. All these tools uses structural information, biochemical and thermodynamical properties, model simulations, etc to score the sequence of interest using some scoring matrices and thus predicting where a particular protein is capable of binding on our sequence of interest. https://www.nature.com/articles/s41598-020-57778-1
- These are some of the tools that are available. TRANSFAC helps in identifying eukaryotic Transcription factor binding sites…similarly for yeasts, YEASTRACT, 3rd one.. is available…then there is Displar- which when provided with structure of protein known to bind DNA, can predict amino residues that t=interact with DNA with accuracy over 80%.....and lastly, some web based tools that help in protein-DNA interaction studies. So, as the database and techniques increases and by using combinations of different techniques, along with modifications, protein DNA interactions can be studied efficiently. r6
- This are the references that I have used.
- With this, I end my presentation. I hope I was clear with the basic principle at least. Thank you very much. Myelin gene promoters – TFs, 10.1007/978-1-4939-9554-7_37 Whole TFs, then methylation dependent - https://pubmed.ncbi.nlm.nih.gov/14706630/ In silico DNA binding proteins generally