# Kara M Dismuke Week 12 Journal

## Procedure

### Using YEASTRACT to Infer which Transcription Factors Regulate a Cluster of Genes

In the previous analysis using STEM, we found a number of gene expression profiles (aka clusters) which grouped genes based on similarity of gene expression changes over time. The implication is that these genes share the same expression pattern because they are regulated by the same (or the same set) of transcription factors. We will explore this using the YEASTRACT database.

1. In Excel, open the gene list the profile/cluster that you analyzed for the Week 11 Assignment. For me, this was Profile 45.
• Copy the list of gene IDs.
2. Launch a web browser and go to the YEASTRACT database.
• On the left panel of the window, click on the link to Rank by TF.
• Paste your list of genes from your cluster into the box labeled ORFs/Genes.
• Check the box for Check for all TFs.
• Accept the defaults for the Regulations Filter.
• Rank genes by TF using: The % of genes in the list and in YEASTRACT regulated by each TF.
• Click the Search button.
• In the results window that appears, the p values colors represents the following
• pink: "not significant"
• yellow: "borderline significant"
• green: "significant"
• How many transcription factors are green or "significant"?'
• List the "significant" transcription factors on your wiki page, along with the corresponding "% in user set", "% in YEASTRACT", and "p value".
• Are CIN5, GLN3, HMO1, and ZAP1 on the list?
4. For the mathematical model that we will build in class, we need to define a gene regulatory network of transcription factors (with the help of YEASTRACT) that regulate other transcription factors. We want to generate a network with approximately 15-30 transcription factors in it.
• Click on the link: Generate Regulation Matrix.
• Copy and paste the list of transcription factors you identified (plus CIN5, GLN3, HMO1, and ZAP1) into both the "Transcription factors" field and the "Target ORF/Genes" field.
• We are going to generate several regulation matrices, with different "Regulations Filter" options.
• (1): "Documented", "DNA binding plus expression evidence"
• In the results window that appears, click on the link to the "Regulation matrix (Semicolon Separated Values (CSV) file)" that appears and save it. Rename this file with a meaningful name so that you will be able to distinguish it from the other files you will generate.
• (2) Repeat to generate a matrix that applies the Regulations Filter: "Documented", "Only DNA binding evidence".
• (3) Repeat to generate a matrix that applies the Regulations Filter: "Documented", DNA binding and expression evidence".

### Analyzing and Visualizing Your Gene Regulatory Networks

We will analyze the regulatory matrix files you generated above in Microsoft Excel and visualize them using GRNsight to determine which one will be appropriate to pursue further in the modeling.

1. First we need to properly format the output files from YEASTRACT. You will repeat these steps for each of the three files you generated above.
• Open the file in Excel. Select the entire Column A. Then go to the "Data" tab and select "Text to columns". In the Wizard that appears, select "Delimited" and click "Next". In the next window, select "Semicolon", and click "Next". In the next window, leave the data format at "General", and click "Finish". What is generated is called an "adjacency matrix." If there is a "1" in the cell, that means there is a connection between the trancription factor in that row with that column.
• Save this file in Microsoft Excel workbook format (.xlsx).
• Check to see that all of the transcription factors in the matrix are connected to at least one of the other transcription factors by making sure that there is at least one "1" in a row or column for that transcription factor. If a factor is not connected to any other factor, delete its row and column from the matrix.
• Make sure that you still have somewhere between 15 and 30 transcription factors in your network after this pruning.
• We need to transpose the matrix so we can use it in the GRNmap (the modeling software) and GRNsight (the visualization software), Insert a new worksheet into your Excel file and name it "network". Go back to the previous sheet and select the entire matrix and copy it. Go to you new worksheet and "Paste special" from the "Home" tab in cell "A1". In the window that appears, check the box for "Transpose". This will paste your data with the columns transposed to rows and vice versa.
• The labels for the genes in the columns and rows need to match. Thus, delete the "p" from each of the gene names in the columns. Adjust the case of the labels to make them all upper case.
• In cell A1, copy and paste the text "rows genes affected/cols genes controlling".
2. Now we will look at some of the network properties. Again, repeat these steps for each of the three gene regulatory matrices you generated above. See this file for an example of how to do the following instructions.
• Create a new worksheet and call it "degree". Copy and paste your adjacency matrix from the "network" sheet into this new worksheet.
• In the first empty cell in column A, type "Out-degree". In the cell to the right of that in Column B, type the equation =SUM( and select the range of cells in column B that has 1's and 0's in it, close the parentheses, and press Enter. This quantity is the number of genes that the transcription factor in that column is controlling, or the out-degree. Copy and paste that equation across all of the columns.
• In Cell 1 of the first empty column to the right of the adjacency matrix, type "In-degree". In Cell 2 of this column, type the equation =SUM( and select the entire row of 1's and 0's, close the parentheses, and press Enter. This quantity is the number of transcription factors that regulate the gene in that row, or the in-degree. Copy and paste the equation down the entire column, including the row that contains the out-degree sums.
• The number in the lower right-hand corner, the sum of sums, is the total number of edges in the adjacency matrix. We would like to see about 50 (40-60 or so) edges in the matrix. If the matrix is too dense, it will slow down the modeling program because it will be difficult to estimate the parameters in the model.
• We want to plot the degree distributions for each of your gene regulatory networks. In the "degree" worksheet, create three columns to the right called "Frequency", "In-degree total", and "Out-degree total". In the "Frequency" column, number sequentially from 1 to the largest degree number in your calculations above. In the "In-degree total" column, type the number of genes with that in-degree for each of the frequencies. In the "Out-degree total" column, type the number of genes with that out-degree for each of the frequencies.
• Select the "Frequency", "In-degree total", and "Out-degree total" columns. Go to the "Insert" tab and select the column chart type to insert a plot of the degree distribution. Copy and paste the charts for each gene regulatory matrix into your PowerPoint presentation.
3. Now we will visualize what these gene regulatory networks look like with the GRNsight software.
• Go to the GRNsight home page (you can either use the version on the home page or the beta version, which has slightly different visualization properties).
• Select the menu item File > Open and select one of the regulation matrix .xlsx file that has the "network" worksheet in it that you formatted above. If the file has been formatted properly, GRNsight should automatically create a graph of your network. Move the nodes (genes) around until you get a layout that you like and take a screenshot of the results. Paste it into your PowerPoint presentation. Repeat with the other two regulation matrix files. You will want to arrange the genes in the same order for each screenshot so that the graphs can be easily compared.
4. Write a paragraph discussing and explaining the results of each aspect of today's work.
• Determining candidate transcription factors that regulate a cluster of genes from your dataset.
• Creating three candidate gene regulatory networks.
• Determining the total number of edges and degree distribution of your three gene regulatory networks.
• Visualizing the networks.
• Choosing a particular gene regulatory network to pursue for the modeling.

## Results and Discussion

### YEASTRACT Results and Discussion

Profile 45

• How many transcription factors are green or "significant"?
• 26
• List the "significant" transcription factors on your wiki page, along with the corresponding "% in user set", "% in YEASTRACT", and "p value".
• Are CIN5, GLN3, HMO1, and ZAP1 on the list?
• No, they are not.
• How many of the transcription factors appear in both your list and your partner's list (aftter comparison)?
• We had 21 shared transcription factors. Sfp1p, Yhp1p, Yox1p, Fkh2p, Cyc8p, YLR278C, Rif1p, ACE2p, Msn2p, Cse2p, Stb5p, Ndt80p, Asg1p, Snf2p, Swi5p, Mig2p,Spt20p,Snf6p, Pdr1p, Dcr2p, Gat3p.
• We decided to include all of our shared transcription factors and the four additional ones that we were required to add (CIN5, GLN3, HMO1, ZAP1) since we had a total amount that fell within the suggested range.

### Analyzing and Visulaizing Results and Discussion

#### Pruning: TFs

• After the pruning process (deleting rows/columns for transcription factors without at least one "1" in it's row/column), we observe that we have the following number of transcription factors:
• DNA binding PLUS expression evidence: 29 TFs
• deleted:
• rows: Cyc8p, Asg1p, Gat3p
• columns: none
• ONLY DNA binding evidence: 10 TFs
• deleted:
• rows: Cyc8p, Rif1p, Ace2p, Cse2p, Stb5p, Asg1p, Snf2p, Spt20p, Pdr1p, Gcr2p, Gat3p, Gln3p
• columns: CSE2, STB5, SNF2, MIG2, SPT20, PDR1, GCR2, GAT3, GLN3, ZAP1
• DNA binding AND expression evidence: 2 TFs
• deleted all rows and columns except for row Fkh2p and column ACE2
• Note: after pruning, it is clear that some of these data sets may not be able to be used in the future because they do not have the required 15-30 remaining transcription factors.

#### Pruning: Edges

• After finding the number of edges (the sum of sums of the adjacency matrix), we observe the following number of edges:
• DNA binding PLUS expression evidence: 141 edges
• ONLY DNA binding evidence: 35 edges
• DNA binding AND expression evidence: 5 edges
• Note: after pruning, it is clear that some of these data sets may not be able to be used in the future because they do not have the required 40-60 edges in the matrix.