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Regulatory Networks in Arabidopsis

Arabidopsis thaliana Regulatory network (AtRegNet)

As more is discovered about how TFs function, it is increasingly recognized that they act as part of complex networks. Within these networks, TFs often regulate other TFs, forming branched networks. TFs also control the expression of other non-TF encoding genes more directly involved in cell differentiation and responses. A single TF may regulate, directly or indirectly, hundreds of genes. In order to understand the function of any TF, such complex networks must be recognized and visualized. AtRegNet is a tool used to display the regulatory networks of Arabidopsis thaliana transcription factors. It was developed to provide a fast and easy way for researchers to visualize the network of interactions specifically in Arabidopsis.

AtRegNet documents and visualizes networks formed by TFs and their direct target genes only. Indirect targets are not included, unless the intervening TF is known. This means that many interactions that have only been defined based on genetic data are not coded for in AtRegNet. As more data becomes available to support direct interactions, these interactions will be added to the database. Information presented in this database is taken from published data. We have defined two groups of direct targets: confirmed and unconfirmed. Unconfirmed direct targets have not been as extensively documented in the database as have direct targets. A confirmed direct target has been defined as a gene that responds to a given TF according to the following criteria. An unconfirmed direct target is one that has been demonstrated to be a target of a given TF by only one of those techniques.

1. The TF binds directly to the regulatory region of the target gene (as shown by electromobility shift assay (EMSA), yeast one-hybrid analysis, and/or chromatin immunoprecipitation (ChIP)).
2. The TF directly regulates the target gene, based on use of transgenic plants expressing an inducible TF-GR (glucocorticoid receptor) fusion protein. Fusion of TFs to the GR domain has been demonstrated to cause the fusion protein to be retained in the cytoplasm in the absence of steroid hormone. Upon application of the synthetic hormone dexamethasome (DEX), the protein moves into the nucleus and activates expression. Direct targets are those that are either induced or repressed by a given TF-GR upon DEX induction even in the presence of the translation inhibitor cycloheximide (CYC).
3. In vivo evidence of regulation: Expression of the target gene is affected by either loss of function mutations in the TF or ectopic expression of that TF in the plant.

Once it has been documented in the literature that a given gene is a direct target of a TF found in AtTFDB, it is entered into the AtRegNet database. If we have access to unpublished data on any TFs (either generated in-house by our group or communicated to us by others), these interactions will be put into the database as unconfirmed direct targets. Once the data has been published, the interactions will be updated.

Web interface:
AtRegNet can be accessed from the AGRIS website. In the title bar of all AGRIS pages is a link to Regulatory Networks, which will bring up a description of the AtRegNet database, and a blank workspace. A search bar for AtRegNet is provided in the interface. Another way to access AtRegNet is to search for a TF in AtTFDB and if regulatory network data is present on the TF, then a link is automatically provided to AtRegNet. When these links are used, the TF is immediately searched and the network is displayed when the AtRegNet page loads. 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. Non-TF targets (which can be any gene that encodes a product that does not act as a DNA-binding TF) are represented as rounded squares. TFs are provided with links to its interactions (if known) and both AtTFDB and AtcisDB. Non-TF targets are linked to AtcisDB. Clicking on the arrows linking the TF to the target can access links to papers documenting the interactions.

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