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Intrinsically disordered regions are highly represented among mammalian transcription factors, where they often contribute to the formation of multiprotein complexes that regulate gene expression. An example of this occurs with LIM-homeodomain (LIM-HD) proteins in the developing spinal cord. The LIM-HD protein LHX3 and the LIM-HD cofactor LDB1 form a binary complex that gives rise to interneurons, whereas in adjacent cell populations, LHX3 and LDB1 form a rearranged ternary complex with the LIM-HD protein ISL1, resulting in motor neurons. The protein-protein interactions within these complexes are mediated by ordered LIM domains in the LIM-HD proteins and intrinsically disordered LIM interaction domains (LIDs) in LDB1 and ISL1; however, little is known about how the strength or rates of binding contribute to complex assemblies. We have measured the interactions of LIM:LID complexes using FRET-based protein-protein interaction studies and EMSAs and used these data to model population distributions of complexes. The protein-protein interactions within the ternary complexes are much weaker than those in the binary complex, yet surprisingly slow LDB1:ISL1 dissociation kinetics and a substantial increase in DNA binding affinity promote formation of the ternary complex over the binary complex in motor neurons. We have used mutational and protein engineering approaches to show that allostery and modular binding by tandem LIM domains contribute to the LDB1 binding kinetics. The data indicate that a single intrinsically disordered region can achieve highly disparate binding kinetics, which may provide a mechanism to regulate the timing of transcriptional complex assembly.

Transcription is an essential process in biology whereby gene-specific transcription factors target sites on DNA to recruit the basal transcription machinery that will produce messenger RNA (mRNA). It is a highly regulated multi-step process that involves many proteins and protein complexes. Transcription factors, the proteins that mark genes for activation, and other transcriptional regulators are highly enriched in low-complexity disordered regions, which are strongly linked to multivalent binding and phase separation. These disordered regions can form multivalent dynamic complexes that are essential for many aspects of transcription. Many of these proteins can phase separate in vitro and show evidence of phase separation in vivo. Whether these interactions represent biologically relevant phase separation in vivo is controversial. However, what these events do demonstrate is that many transcriptional proteins co-cluster with other factors in vivo, forming multivalent dynamic clusters that contribute to transcriptional events. We review some of these recently investigated events and consider how they contribute to our understanding of transcription.

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