Question

it is a molecular biology question and requires designing an experiment and it is not missing information.

if we want to perform a ChiP-seq experiment in cell typeA to analyze genome-wide binding by favorite transcription activator (YFTA). I need to design an experiment that investigate if binding at one of the sites actually affects transcription. select one site that is located upstream of highly transcribed gene (HTGZ), which is expressed in cell typeA, but not expressed in cell type B. you hypothesize that YFTA is required for the high expression of HTGZ. Assume that you have the necessary reagents for your assays. in the answer, clearly state the experimental setup including controls . state the purpose of the controls. And be specific as possible when describing your reagents. state the expected results if your hypothesis is true.

( I am thinking about the reporter gene assay and deleting specific sequences ) ( It might be wrong )

Answer 1

We have the data from Chip-Seq experiment. Chip-Seq is done to predict the binding sites of a given transcription factor. We also know that only the cell type A transcribes the gene HTGZ. We also know that the binding site is in the upstream sequence element.

We can sequence the Upstream sequence element and screen for variations in the cell type A and cell type B. Of the sequences that are unique to A, we can screen for the predicted binding sites by Chip-Seq. The selected sequence(s) is (are) the predicted binding site(s) of the Transcription Factor YFTA.

1. To screen among the predicted binding sites and verify it experimentally, we first need to PCR amplify the sequence. This will require primers which can be designed based on the sequence of the predicted binding sites. The template DNA isolated from cell type A, primers for Predicted Binding Site (PBS), DNA polymerase, Mg2+ ions, Buffer for Polymerase and dNTPs can then be mixed together alongwith nuclease free water in a PCR mix. The PCR can be performed to amplify the PBS.

PBS can then be PCR purified or gel extracted based on the purity of the PCR product. This final product is our PBS.

2. The PBS then needs to be inserted into a plasmid backbone which has a reporter gene like GFP. The UPS is an upstream element, hence it can be cloned upstream to the promoter of the reporter gene. There are two steps that will clone the UPS into the plasmid backbone. Firstly, we need to find compatible restriction endonuclease sites within the backbone and the UPS. Then both of these are treated with the respective REs in appropriate buffers. They are then PCR purified and based on the concentration obtained, can be ligated using T4 ligase and ligation buffer. Thus, the UPS will be cloned upstream to a reporter gene.

These two steps are summarized in the figure below.

Upstream Sequence Element Promoter HTGZ Select Upstream Sequence unique to A and has the predicted binding site from Chip-Seq PCR amplify it and purify Predicted Binding Site Clone it upstream to a reporter Gene like GFP in a plasmid vector Predicted Binding Site Promoter Reporter Gene Plasmid Backbone

3. The plasmid thus obtained should have an antibiotic resistance marker. The cloned plasmid is then transformed into a host cell, which can be a bacterial cell like E.coli. The transformed cells can be selected in an antibiotic medium. The antibiotic resistance present in the plasmid will allow these cells to grow in the medium, whereas the untransformed cells will die.

4. The transformed cells will exhibit basal levels of transcription of the reporter gene which will then be translated to give basal levels of the protein product. However, when the cells are treated with the transcription factor YFTA and the PBS is indeed the binding site for this transcription factor, the PBS will induce increased levels of transcription of the reporter gene. The high levels of transcript will lead to higher levels of the protein product.

5. The reporter can be a fluorescent molecule like GFP. Thus, when the cells are excited at a particular wavelength, the GFP will get excited to emit fluorescent light that can be detected by a detector or by an eye. If the PBS is indeed the correct binding site, the GFP protein will be higher in the cells treated with PBS. Hence the fluorescence intensity should be higher in these cells.

These steps are summarized in the figure below.

PBS PBS P GFP PBS PBS HP HGFP GFP Protein Transform into a bacterial cell Check for fluorescence Provide the cell with TF PBS GFP PBS GFP GFP Protein

To conclude, if the hypothesis that the PBS is the binding site is true, we expect the GFP intensity in the YFTA cohort to be higher than that in the non-YFTA treated cohort. Thus the non-YFTA treated cohort acts as a control. However, to check if just the existence of PBS can have any effect on GFP intensity, we can perform the experiment wherein the plasmid is not cloned with the PBS and just has Promoter and GFP. If the YFTA is binding somewhere else on the plasmid, the GFP intensity should be high in this case too.