Short linear motifs and disordered context of Arabidopsis transcription factors in coregulator binding

Transcription depends on complex protein networks in order to ensure proper gene expression of the right genes in different cellular responses. Transcription factors (TFs) activate target genes by interaction with the DNA through DNA-binding domains and with the general transcriptional machinery often through large intrinsically disordered regions (IDRs) mediated by activation domains (ADs). The plasticity and motif features in the IDRs are essential in the regulation of TFs. Due to the diverse transcriptional responses of different TFs, this is a complex system that is still not fully understood.

This thesis aimed to investigate how short linear motifs (SLiMs) in IDRs of TFs are influenced by their disordered context in the interaction with coregulators in the regulation of the transcriptional response. The Arabidopsis model system of TFs binding to the folded domain, RST, of the repressor Radical-Induced Cell Death1 (RCD1) was central to the investigation. The TFs in this system share a common SLIM which is located within ADs to facilitate the binding to RCD1-RST.

From an evolutionary-guided approach to design substitutions, we were able to show substitutions in the TF DREB2A tuned the degree of residual helix in its unbound state and the SLiM-mediated interaction with RCD1-RST. Kinetic analysis showed that the complex lifetime was affected by the substitutions and the association of the complex is not affected by preformed structure.
The ability of D-peptides to bind in a continuum of disorder-based interactions was investigated and demonstrated that the three TFs ANAC013, ANAC046, and DREB2A interacting with RCD1-RST showed a scale of disorder in the complex with RCD1-RST based on their responsiveness to stereochemistry. Lastly, bioinformatic analysis of SLiMs in the IDRs of a TF clade was correlated with AD activity to study the TF ANAC046 in more detail. ADs overlapping with SLiMs in the ANAC046 IDR interacted with multiple coregulator domains with a preference for the C-terminal AD suggestive of a possible model for regulation of the transcriptional activity of ANAC046.

In summary, the work presented in this thesis highlights the complexity found both in the near context regarding SLiMs and ADs and the larger context spanning the entire IDR for the regulation of TFs through coregulator binding.