Drawing and annotating pathways in PathVisio

In this tutorial you will create a pathway step by step in PathVisio. We are using the circadian clock pathway as an example.
We provide screenshots and short videos to guide you through the process of creating a new biological pathway.

The circadian clock pathway
The circadian clock is a molecular mechanism that maintains the cell’s 24-hour molecular rhythm by driving the rhythmic expression of a wide range of genes, involved in metabolism, physiology and behavior. In this tutorial, you will draw a simplified version of the pathway, restricting to the mPer/mCry feedback loop.


Step 1: Download and open PathVisio


Go to pathvisio.org and click at Downloads -> Download PathVisio

In section 1 you can download the Java Webstart version of PathVisio.
Download PathVisio 1

After downloading PathVisio the following will be displayed

Download PathVisio


Step 2: Download the mouse gene databases and open in PathVisio


Go to pathvisio.org and click at Downloads -> Download mapping databases

Click at Mus musculus under database, download the zipped file and unzip the Mm_Derby_20130701.zip. (Please note that you need a program like Winrar or Winzip to unzip this file).

Mouse_gene_database

Open mouse ID mapping database in PathVisio. Go to ‘data’ and ‘Select Gene Database’.
Now select the Mm_Derby_20130701.bridge file.

Gene_database


Step 3: Create new pathway


Click at File and thereafter New .

Add_new_pathway

Click at the left corner on Title: New pathway and change pathway title in “Circadian clock” and select “Mus musculus” under organism.

Add_title_pathway

You can now start drawing your pathway on the drawing area.


Step 4: The proteins of the circadian clock pathway


The circadian clock pathway is formed by interactions between the following proteins:

proteins_circadian_clock

A good way to start drawing the pathway is to add the proteins. On a  pathway in PathVisio, all biological entities with an annotation are stored as a DataNode. A DataNode can have different types, in this case, we choose the type gene-product. To add a gene-product click the  gene-product button, followed by clicking on the drawing area on the location that you want the element to be added. Repeat this to add 6 gene-products. Your pathway now looks something like this:

Adding a gene product


Annotating DataNodes

Now you are going to annotate the DataNodes and set a proper text label. DataNodes can be annotated using an identifier from one of the available datasources. A pathway with annotated DataNodes has several advantages:

  • Detailed information about the entity is directly available in the backpage panel
  • The pathway page provides direct links to extended info at the annotation datasource website (e.g. Ensembl)
  • The pathway can be used for computational analysis, e.g. visualization and statistical analysis with PathVisio, or in network analysis with Cytoscape.

To annotate a DataNode, go to the annotations tab in the properties dialog, by double-clicking the DataNode or choosing ‘properties’ from the right-click menu. Below is a screenshot of the annotation dialog:

Dialog_gene_product

Annotating gene product

In the dialog, you need to set the identifier, datasource and a text label that will be displayed on the pathway. You can manually fill in these values or search in the identifier mapping database. To search for an annotation, fill in the search term and press the search button. If there are results you can select the identifier/datasource that you want to use that will automatically be filled in the manual entry fields. The text you type in the text label field will be displayed on the pathway and doesn’t affect the annotation.

After annotating a DataNode, you can click on the DataNode to see whether it’s correctly annotated. If the identifier can be found in the database, the annotation info will show up in the Backpage tab of the side panel:

Backpage

Note: If you try to search for Bmal1, you’ll notice that it is not found. This is because this particular gene has two synonymous names (Bmal1 and Arntl1), but only the latter is stored in the internal database. However, since we have a valid Ensembl ID we can still annotate this gene manually. Just fill out the Ensembl ID and Bmal1 in the right fields. You can check the backpage to confirm that the manual entry is cross-referenced.


Step 5: Illustrate the E-box enhancer

Target genes of the circadian clock pathway contain the E-box cis-regulatory enhancer elements. The Bmal1/Clock heterodimer directly activates expression of these genes by binding to the E-box sequence. In this section we are going to draw an illustration of this process. It will become a purely visual element of the pathway, so it’s not annotated to any biological database and will be added for illustration purpose only.

Drawing the E-box element

The video shows how to draw the E-box element, which in the end will look like this:
Ebox

Below is a step-by-step description of how to create such an element.

1. Draw a horizontal line

Start with a horizontal line, by clicking the Basic shapes drop-down menu and selecting the line button. Since this will be a purely graphical line, we are using the shapes object line as opposed to an interaction line. Next, move to the location on the drawing where you want the line to start. Press down the mouse button, drag the mouse to the right (while holding the button down!) and release the button at the location where you want the line to end. You can modify the location of the start and end points by dragging the handles (yellow squares). You can modify the location of the whole line by clicking on the line itself and dragging it.

Note: You can easily make a perfectly horizontal or vertical line by holding the Shift button on you keyboard while moving a line end. This will snap the line fixed angles.

2. Copy/paste the line

We now want a second horizontal line of the same length and parallel to the first line. Therefore, we are going to copy the line from the previous step. Select the line by clicking on it, the line will turn red and the two handles will be visible. Now click the copy button. The line is now copied to your system’s clipboard. You can paste a copy of the line by pressing the paste button. A copy of the line now appears a little below the original line.

3. Align the two lines vertically

To put the lines exactly below each other, select both lines and press the align horizontal center button. You can select multiple objects by pressing the mouse on an empty part of the drawing, hold the mouse button down and drag the mouse to include or exclude elements from the selection. You can also select or deselect multiple objects by clicking them while holding the Shift button on your keyboard.

4. Draw a label

Now we have two parallel horizontal lines that represent a piece of DNA. We can denote the E-box region by placing a label over the lines. Add a label using the label button.

5. Set the label text to ‘E-box’ and apply the ‘square’ outline

Label properties can be edited in the Properties table. Set the property Text Label to ‘E-box’ by double clicking on the cell in the Value column:
label

In the same way, you can set a rectangular outline, by selecting the ‘Rectangle’ for the Shape type property. Finally, use the handles to resize the label so that it exactly fits between the two horizontal lines.

6. Copy/paste the label and change text to ‘Gene’

Downstream of the E-box element is the gene that will be activated. We can denote this with a label too. Copy/paste the E-box label and change the text to ‘Genes’, since we use a single element to depict multiple genes that will be activated. Drag the label to put it at the right position.

7. Draw a transcription start symbol

To make it clear that the genes downstream of the E-box element are transcribed after activation, the following symbol is used:
TSS

This symbol is made out of a vertical line and horizontal arrow. Draw the line and arrow by using the graphical line object and setting the end line style to arrow in the Properties panel. Correctly place them by dragging the handles.

8. Group all lines and labels

To make it more easy to handle the E-box illustration, you can group it. This way the elements will be selected together, so you can move the group as a whole, instead of having to move each element separately. To create the group, select all lines and labels you created in the previous steps, right-click on one of the lines and select Group->Group from the pop-up menu, or press CTRL-G on the keyboard. You can ungroup the grouped elements by right-clicking the group and selecting Group->Ungroup, or pressing CTRL-G again.


Step 6: Clock and Bmal1 dimerize and bind to the E-box element

To activate the target genes of the Circadian clock pathway, the Clock and Bmal1 proteins dimerize and the resulting heterodimer will bind to the E-box elements of the target genes to activate transcription. We are now going to draw this process in our pathway, making it look like this:

Clock_di-1

Drawing the dimerization and binding to the E-box element

1. Draw the Clock/Bmal1 in separate and dimerized state

Drag the Clock and Bmal1 gene-products to position them above the previously drawn E-box illustration. Use the align horizontal center button to align the two gene-products horizontally. Now copy and paste the two elements and place them below each other, like in the image above. The bottom Clock/Bmal1 refers to the heterodimer and this is illustrated by stacking the two gene-product boxes horizontally. You can easily stack multiple elements by selecting them and clicking the stack horizontally button. Finally, you can specify that the two proteins are in dimerized state. To do this, select both gene-product boxes and select ‘Create complex’ in the right-click menu, or pressing Ctrl-P. You will now see a gray bounding box around the two gene-product boxes.

2. Draw the dimerization event

The actual event of dimerization is drawn by two curved arrows that point from the individual elements representing the separate states and the complex element representing the dimerized state. First draw two straight interactions, choosing arrow as the end line style, pointing to the Clock/Bmal1 complex, one from the separate Bmal1 element and one from the separate Clock element. You will notice that while you are moving a start or end point of a line near another element, little bulls eyes appear:

Clock_2

These objects are called link anchors and can be used to connect line ends to elements on the pathway. When you move the line end near a link anchor, it will snap on to it and the line will be connected to the corresponding shape. This means that the line will stick to the shape when you move it. You can release the connection by moving the line end away from the link anchor. Notice that when you move over a group or complex, the group boundaries will also show link anchors. For example, if you move the point over the Clock/Bmal1 complex, you will see connectors for the individual gene-product boxes and the complex:

Clock_3

In this case we want to connect the end point of the lines to the complex, since the line represents the transition from the separate protein to the dimer.

Note: It’s good habit to connect the lines in your pathway wherever you can. It makes it easier to change the layout of the pathway in the future, because when you move an object, the connected lines will stick to it. Another advantage of defining connections is that it enables conversion to a graph. When the interactions between the entities in the pathway are explicitly defined, this information can be used for computational purposes. For example, the pathway can be converted to a Cytoscape network to perform various graph analysis algorithms (see http://www.cytoscape.org).

To make the two arrows more visually appealing, you can change their type. Here we will change the line type to ‘curved’, to create a connector that smoothly curves from the start to the end object. Change the line type of both lines by right clicking on the line and choosing Line type->curved. You will see that the line will now curve towards the Clock/Bmal1 complex

Clock_4

3. The Clock/Bmal1 dimer binds to the E-box element

Finally we are going to draw how the Clock/Bmal1 binds to the E-box element. Copy the Clock/Bmal1 dimer and place the copy just above the E-box element. Now draw a line from the top dimer to the bottom dimer and connect the start and end points to the dimers.


Step 7: Target genes are activated by the E-box element

After the Clock/Bmal1 dimer binds to the E-box element, transcription of several target genes are activated. This simplified version of the Circadian clock pathway, will restrict to the mPer/mCry genes, which will act as a negative feedback loop. As long as Per1 or Per2 exist as monomers, they will be phosphorylated by casein kinase 1 delta or epsilon, followed by degradation. In the pathway drawing we simplify it a bit by leaving out the phosphorylation event, resulting in the following representation:

Clock_5

Drawing the transcription of mPer/mCry and degradation of mPer

1. Stack the mCry/mPer DataNodes

To illustrate that the mCry/mPer genes are activated by the E-box element, we can stack them below the ‘Gene’ label. Place the mPer/mCry DataNodes below the ‘Gene’ label and stack them in two vertical pairs, using the stack vertically button. Group each stacked pair.

2. Copy the gene-products

The genes will be translated into proteins, to show this on the pathway, copy the stacked genes and move them to the right of the stacked DataNodes.

3. Draw a line from the genes to the gene-products

To illustrate that the genes are translated into proteins, we draw a dashed arrow from each pair of genes to the copied pair. Connect the start and end points to the groups.

4. Create a degradation shape

As long as Per1 or Per2 is not dimerized with Cry1 or Cry2, it will be degraded. We can indicate degradation by using the degradation shape:
degradation

You can find this shape in under Basic Shapes in the Objects Tab.

5. Draw an arrow from the mPer DataNodes to the degradation shape

To show that the mPer proteins are degraded, draw an dashed arrow from the two mPer DataNodes to the degradation shape. Connect the start of this line to group 2.1 to specify that only the mPer proteins are degraded.


Step 8: The negative feedback loop

The final event we are going to add to the pathway is the negative feedback loop formed by the mPer/mCry dimer. In order to inhibit the expression of the Circadian clock genes, the mPer and mCry proteins need to dimerize and translocate to the nucleus. After dimerization, mPer is protected from degradation (see step 7). Once the dimer is in the nucleus, it will inhibit the activation of the E-box element by Clock/Bmal1, thereby acting as a negative feedback loop. After this step, the pathway will look like this:

Clock6

Drawing the negative feedback loop

1. Copy the mCry and mPer DataNodes

The negative feedback loop will need two additional variations on the mCry and mPer proteins:

The mCry/mPer dimer outside the nucleus
The mCry/mPer dimer inside the nucleus

To add these DataNodes, you can copy the 4 mCry and mPer DataNodes under the Gene label you added in step 7.1 twice and move them to right location (see image above).

2. Draw the dimerization of mPer and mCry

Drawing this is similar to drawing the dimerization of Clock and Bmal1 in step 5.2. First, copy the 4 mCry and mPer DataNodes under the Gene label you added in step 6.1. Place the copied DataNodes above/right of the separate mPer and mCry DataNodes, stack them and create a complex (select all four DataNodes and press CTRL-P). Connect two arrows from the individual mPer and mCry elements to the newly created mPer/mCry complex and set their line type to ‘curved’.

3. Draw the translocation of mPer/mCry

After dimerization, the mPer/mCry dimer translocates to the nucleus. There is no information about cellular location on the pathway yet, this is going to happen in step 8, but we can already draw the translocation. Copy the mCry/mPer complex and move the copy next to the Clock/Bmal DataNodes. Draw and connect a dashed arrow between the original and copied dimer. You can set the line style of the dashed arrow to ‘elbow’.

4. Draw the inhibition of Clock/Bmal1 function by mPer/mCry

The mPer/mCry complex located in the nucleus, inhibits the activation of the E-box element by Clock/Bmal1. Inhibition can be represented by the T-bar line:

T-line

Draw and connect a T-bar line from the mPer/mCry complex in the nucleus to the Clock/Bmal1 dimer bound to the E-box using the newtbar.gif T-bar button on the toolbar.

5. Add colors to the degradation and dimerization events

To emphasize the two routes mPer can go (either degradation or dimerization with mCry), you can give each route a different color. Change the color of the arrow and degradation shape to red. Select the arrow and degradation shape and click on the Color property in the properties table. A dialog will appear where you can choose the color. Repeat the same steps for the arrows that represent the dimerization, but now set the color to blue.


Step 9: Draw cellular locations

In this step, we will add information about cellular localization to the pathway, to show which event happen in the nucleus. The Clock and Bmal1 dimerization, E-box activation, transcription of target genes and inhibition of Clock/Bmal1 by mPer/mCry all happen in the nucleus. We can show this by drawing a rectangle that defines the boundaries of the nucleus and place the above events within that boundary. After this step, the pathway will look like this:

Clock_7

Drawing a nucleus

Draw a rectangle to represent the nucleus

To indicate the boundaries of the nucleus, draw a rectangle that is big enough to contain the events that take place in the nucleus.

Add a label

To show that the rectangle represents the nucleus, add a label and set the text to ‘Nucleus’. You can increase the font size to make the label stand out more, by changing the Font Size property to 200 and enabling the Bold property in the properties table.


Step 10: Adding bibliography

In this step, we are going to add references to literature to the pathway. Providing your modifications to a pathway with evidence from peer-reviewed publications is important to guarantee quality of the pathway. It also enables other users to get more detailed information or background about the process you are describing in the pathway, e.g. by adding references to review articles. Here we are going to add a reference to the following review article:

Ramsey KM, Marcheva B, Kohsaka A, and Bass J. The clockwork of metabolism. Annu Rev Nutr 2007; 27 219-40. doi:10.1146/annurev.nutr.27.061406.093546 pmid:17430084.

Add a literature reference to the pathway

To add a reference to the pathway as a whole, right click on the infobox and select Literature->add literature reference.

Reference_1

In the dialog that appears, you can fill in the reference information manually or by querying the information from PubMed by filling in a PubMed id. In this case, the reference is available in PubMed under the id 17430084. Fill in this id in the PubMed ID field and click the Query Pubmed button. The title, year, source and author fields are now automatically filled in.

Reference_2

Click Ok to save the information.

Note: You can also add references to individual elements or groups, by right-clicking on the selected element(s) instead of the infobox


Step 11: Saving pathway in PathVisio

We are now done with editing the pathway, so it can be saved in PathVisio.

Save pathway as pgml

Go to File -> Save and save the pathway in gpml format. This is the PathVisio specific format.

Save_gpml

Save pathway as image

If you want to transform your pathway into a static image and use it in for example a paper or powerpoint presentation go to File -> Export and save the pathway in the format you want. You can save the pathway as png, tiff, svg and pdf.

Save_image