ReIN: An interactive tool to create and visualize regulatory networks in Arabidopsis thaliana

ReIN is a web-based software tool for creating, visualizing and identifying regulatory networks in Arabidopsis thaliana. Regulatory motifs represent the simplest units of the overall regulatory network of an organism. A simple graphical interface tool like ReIN will facilitate biologists a fast and easy way to visualize networks of interactions in Arabidopsis with the potential to be extended to other organisms.

A significant and unique feature of ReIN is that it displays different colours, shapes and variations for each object (TFs, non-TFs, targets and its regulation type) in the network. TFs are represented as circles with lines connecting them to their targets. Lines with blue arrows indicate that the TF positively regulates the target, while lines with red arrows indicated the TF represses the target. By clicking on the arrows, the user can link to the corresponding papers in PubMed that document the interactions. Non-TF targets (which can be any gene that encodes a product that does not act as a DNA-binding TF) are represented as squares and are always the end of a given network motif. TFs and non-TFs are provided with dynamic links to its interactions. The users can save their work in graphical format, like a TXT file, or like a XGMML file to be displayed at Cytoscape

TUTORIAL

CONTENT

1. Go to ReIN

2. Search a Gene Regulatory Network (GRN)

3. Explore and modify the network displayed

4. Saving network results

4.1 To export an image file

4.2 To export an Text file

4.3 To export an XGMML file

5. Importing a plain files for network visualization

6. Adding individual new node to a network

1. Go to ReIN

Go to http://arabidopsis.med.ohio-state.edu/REIN/ This address can be accessed doing click in the link to Regulatory Networks that is in the title bar of all AGRIS pages.There you will find a page, which contains an Applet that requires a Java plugin to be used.

2. Search a Gene Regulatory Network (GRN)

Go to the text box and use a keyword to retrieve information from the AGRIS databases.

The first step to search a network is to write a word query in the text box, this query word could be Transcription Factor name (i.e AP3), Target name (i.e CDKB1;1), Transcription Factor Locus id (i.e At3g54340) or Target Locus id (i.e At3g54180). Notice that Locus id are TAIR identifiers.

Click on the button and ReIN will display a graph that shows nodes connecting between them with edges to represent one gene regulatory motif.

In a ReIN graph a node can be simple or complex, and in the graph it could be representing a source node, a target node or both.

Source nodes are always representing transcriptions factors (TFs) and they are displayed like circles, target nodes could be representing TFs (circle) or any gene that encodes a product that does not act as DNA-binding it is a non-TFs this last one are displayed like rounded squares.

Interactions are represented as edges between nodes, each edge has always one source and one target, according to the interaction type the graph displays:

• Blue arrow represent a positive interaction (target is activated by TF)
• Red arrow represent a negative interaction (target is repressed by TF) and
• A black circle represent an interaction with as yet not established consequences on gene expression.

Some edges are displayed as a dotted line and others as a solid line. This is because some interactions are considers unconfirmed (dotted line) and others confirmed (solid line). One interaction is defined confirmed or unconfirmed according to the kind of experimental evidence that has been published which supports that a TF directly binds to a direct target. To know more details Click here .

There are in databases some large regulatory motifs, for example because many direct targets have been identified for a particular TF. For performance, ReIN filters networks with more than 200 nodes and just show confirmed interactions. To understand the criteria employed in defining a confirmed interaction, please Click here .

Troubleshooting:

If after clicking on you get 'No results! Try another query' it likely be because there is no information for this particular query or that the information is not under that queried word.

For example, if you use APETALA 3 or T12E18.30 instead AP3 you are not going to obtain the regulatory network, but if you use locus id At3g54340 you always are going to obtain AP3 (APETALA 3, T12E18.30) gene regulatory motif.

3. Explore and modify the network displayed

In the bottom panel there are and buttons for  zooming in and zooming out.

Using the bottom panel, you can change the mode for the mouse work on the network panel. With the you can move all the network on the network panel and with the mouse mode you can  click with the left mouse button on a node or select several nodes by holding down and dragging the left mouse to define a rectangular area and drag it for change its position.

Move the mouse over nodes or over arrows on the edge and the system shows tooltips with more information.

For any node you can display the locus id just by moving the mouse over it e.g., 'At3g543401/At5g20240 Complex'.
For any edge you can display a small label that summarizes the edge information e.g., 'CAULIFLOWER is activated by At5g03790 (Unconfirmed)'.

Select any simple node by clicking it with the right mouse button and pop up a menu with more information about the node. Simple node pop up menu contains locus id, Protein-DNA interactions, More info from AtcisDB, More info from AtTFDB and More info from TAIR. The followings actions are possible on pop up menu for simple nodes: 

• If you click on 'Protein-DNA interactions', the system searches more interactions in the database related to this node, and if found more interactions this are added to the previous regulatory motif.
• If you click on 'More info from AtcisDB' the browser displays a small area below applet with a message like this: 'Click here for more information about ... in AtcisDB', which links to information in AtcisDB about the specific node that you have consulted.
• If you click on 'More info from AtTFDB' the browser displays a small area below applet with a message like this 'Click here for more information about ... in AtTFDB', which links to AtTFDB information  about the specific node that you have consulted.
• If you click on 'More info from TAIR' the browser displays a small area below applet with a message like this 'Click here for the TAIR reference', which links to TAIR information about the specific node that you have consulted.

Select any node that is represents a complex by clicking it with the right mouse button and pop up a menu with more information about the node. A complex pop out menu contains Protein-Protein interactions, Expand node, More info from AtcisDB, More info from AtTFDB and More info from TAIR.

The followings actions are possible on pop up menu for complex: 

If you click on 'Protein-Protein interactions', a small windows gives the option to choose one protein for the complex. After you have chosen the protein, the system searches for more interactions in the database related to this node, and if found, they are added to the old regulatory motif.

For complex node you can select the option 'Expand node'  to look that interactions correspond for the complex, and which interactions correspond for each individual TF. If you want to restore the complex view, click with the right mouse button on the little black circle in the middle of both TFs, display the pop up menu and choose the option 'Contract node' .

• If you click on 'More info from AtcisDB', a small windows gives the option to choose one protein for the complex. After you have chosen the protein, the browser displays a small area below the applet with a message like this 'Click here for more information about ... in AtcisDB', which links to information in AtcisDB about the specific protein that you have consulted.
• If you click on 'More info from AtTFDB', a small windows gives the option to choose one protein for the complex. After you have chosen the protein, the browser displays a small area below the applet with a message like this 'Click here for more information about ...in AtTFDB', which links to AtTFDB information  about the specific protein that you have consulted.
• If you click on 'More info from TAIR', a small windows gives the option to choose one protein for the complex. After you have chosen the protein, the browser displays a small area below the applet with a message like this 'Click here for the TAIR reference', which links to TAIR information about the specific protein that you have consulted.

Select any edges by clicking, them with the right mouse button and pop up a menu with more information about the edge. Pop up menu contains 'PMID' and 'Click for more info from PubMed'.

The followings actions are possible on the pop up menu for the edges:  

• If you click on 'Click for more info from PubMed', your browser will display a small area below applet with a message like this: 'Click here for the PubMed reference', which links to the  PubMed's abstract page with the reference most relevant to support the interaction that you have consulted.

Troubleshooting:

If the edges are not linked to any paper, it is probably an error from our side and we would appreciate it you please send an e-mail to our curator about it.

If the node that has been selected is not a TF when you click on  'Click here for more information about ... in AtTFDB' the page shows  'No matches'

4. Saving network results

In the bottom panel you have the following options and . With those you can export a text file, which contains columns separated by tabulations, or an eXtensible Graph Markup Language (XGMML or .xgmml) file or create an image file of your network data.

To export an image file:

Click on Notice that the system displays the following message 'Your file is available for viewing. Please click on the link below'.

After you press OK, the browser displays a small area below the applet with a message like this: 'Click here to see the image'.

Do click with the left mouse button and a new browser windows will show the image.

Do click with the right mouse button on the image and select the option 'Save as image'

To export a text file:

Click on Notice that the system displays the following message 'Your file is available. Please click on the link  with the mouse right button to download it'.

After you press OK, the browser displays a small area below the applet with a message like this 'Click here to download the file'.

Do click with the right mouse button and select the option 'Save link as'.

The result file stores information on the network connectivity and it could be open in any text editor or spreadsheet

To export an XGMML file:

Click on Notice that the system displays the following message 'Your file is available. Please click on the link  with the mouse right button to download it'.

After you press OK, the browser displays a small area below the applet with a message like this 'Click here to download the file'.

Do click with the right mouse button and select the option 'Save link as'.

The resulting file is non amenable to (most) human editing. However, it stores information on the network connectivity and it is in a format readable by the network visualizator Cytoscape.

In order to use the XGMML file you need to download Cytoscape. For this, go to http://www.cystoscape.org/ and follow the instructions.

When you have Cytoscape in your desktop go to the menu and via File --> Import -->Network (multiple file types)... or Ctrl + l

Cytoscape displays a small panel where you can select options to Import Network file. There select local, press select button, choose the .xgmml that previously you downloaded from ReIN and press the 'import' option.

Now you can use Cytoscape tool and manipulate the network.

via Layouts --> yFiles --> Hierarchich

via Layouts --> Cytoscape layouts --> Group atributes layout --> TF

via JGraph Layouts --> Circle layouts

Experiment with Cytoscape's tools or follow a tutorial to learn more about many others options to manipulate network information that ReIN gives you.

5. Importing a plain files for network visualization.

ReIN not only display network after query in AGRIS databases,  but you can also upload your own network in plain text files. So, you need create a text file, columns separate by spaces where each row describes one relation between any two nodes, Click here for a sample file .

TF Locus Id	TF Name	Target Locus Id	Target Name	Interaction Type	Is a TF	Experimentally Confirmed or UnConfirmed	PubMed Id
At3g23130	SUP	At3g54340	AP3	Repression	Yes	Confirmed	15634696
At1g69120	AP1	At3g54340	AP3	Activation	Yes	Confirmed	NA

After localizing the file in your system, click on

Then the network contained in your file is displayed and you can use all tools of ReIN.

6. Adding individual new node to a network.

Another capability of ReIN is to display an assistant to submit information in order to draw any two connected nodes.

After clicking on the ,the system displays a window where you should give the following information:  TF Locus ID, TF Label, Target Locus ID, Target Label and choose the Interaction Type, by default is Activation, but you can choose between Activation, Repression or Unknown.

Finally you must submit the information by clicking on option.

Then the network area will show two nodes connected and you can use all tools of ReIN.