TIL about DNA
finger printing footprinting. Transcription Factors (TFs) are proteins that bind to the DNA. Why? Because Natural Selection wanted to come up with regulators or switches to control the transcription of genes from DNA to RNA. You don’t wanna just convert all the DNA to RNA willy nilly. It would be silly to start transcribing genes for making an eye inside a liver. Better have specific triggers to start that process. Triggers based on right environment, right time or right external stimulus. Presence or absence of TFs on DNA can enlighten scientist about genes’ reactions to them. Does GeneA turns on when you add TF1? Or should you add TF1 + TF2 to turn it on? Or maybe add TF3 to turn GeneB off? Is TF3 a Transcription Friend or a Transcription Foe for GeneB?
But how do you find out whether a particular TF is bound or not? Use big ass microscopes to see them? Nah, we are not there yet. But worry not, we do have some indirect ways to figure it out. For instance, a protein called DNase can be used to cut the DNA into small fragments. If our TF is bound to the DNA, it will protect the DNA from this evil DNase to cut it into small pieces. Using a cool biotech method, we can observe a cleavage pattern with or without the TF. This cleavage pattern informs us whether TF in question was protecting the DNA or not. In other words, you try to infer the footprint of the protected DNA to then infer the TF’s binding site. One can even assess the strength of the bond between TF and the DNA site by regulating the concentration of the TF of interest.
More useful alternative seems to be in vivo footprinting because it captures TF and DNA interactions occuring inside the cell at a particular time point. If performed well, such footprinting experiments can capture the true state of a particular TF-DNA interaction. In this case though, your usual DNase cleaving method is prone to disturbing the cell as it can change the state of interactions inside the cell (unless you make cell memberance completely permeable). Therefore, UV irradiation is a more popular method because it can penetrate the cell membrane without disturbing the cell. But other than using a different cleavage agent, the idea remains the same.
With the advent of Next Generation Sequencing (NGS), large scale DNase-Seq and ATAC-seq experiments have been performed to profile binding sites of all known TFs across the whole genome. Since the scale is massive with these experiments, these NGS based methods are unhelpful by themselves and are usually followed by downstream bioinformatics analysis. Since I am in the process of learning one such bioinformatics software called CENTIPEDE, I will follow this TIL with a how to TIL for CENTIPEDE. How can we use it to analyze presence or absence of a particular TF?