Aside from its role in the motor neuron complex, the action of the Isl1 homeodomain is also relevant to the other roles of the Isl1 protein. The data presented here using Isl1/Lhx3 fusion constructs provides hints about how Isl1 can play a role in many different contexts.
As Isl1 has been shown to play a role regulating gene expression in many tissues (Section 1.4), it may be that weak DNA binding is essential to its function. Making a smaller contribution to DNA binding affinity could allow Isl1 to participate in a wider range of protein-DNA complexes, targeting a broader range of DNA sequences than would be possible if it bound DNA with a higher affinity or increased specificity. It is also possible that the combination of a weakly DNA-binding Isl1 in complex with another protein that binds DNA more strongly allows more rapid identification and binding of target sequences, which can be crucial in a developmental context, where cell fate decisions are made with precise timing [403, 404]. This ability to use non-specific binding to search for specific gene targets is a known feature of homeodomains [405].
The influence of Isl1 on the DNA-binding of partner proteins could be easily investigated through the production and study of other fusion constructs containing Isl1HD and another
DNA-binding domain. Studying the DNA-binding behaviour of such fusion constructs would allow a broader insight into how the Isl1 homeodomain influences the DNA-binding preferences of other proteins. As Isl1 is thought to cooperate with a wide variety of transcription factors in vivo, it is likely that it influences the binding specificities of at least some other protein binding partners in a similar manner to that seen in the case of the motor neuron complex.
It is plausible that the primary role of Isl1 is as a protein-protein adaptor, bringing together other transcription factors so that they may bind DNA. In support of this statement, Isl1 and Isl2 are the only LIM-homeodomain proteins to have two known protein-protein interaction interfaces (LIM domains and LIDs), and so are the only proteins in the LIM-HD transcription factor family that can form higher order complexes through those two interfaces. However, all the LIM-HD proteins have uncharacterised C-termini, so dual protein-binding sites may not be exclusive to the Islet proteins. Moreover, the Islet proteins may also contain additional interaction domains that have not yet been identified.
It must be noted that LIM domains themselves can bind to multiple binding partners concurrently. Examples of this include the case of the hematopoietic transcriptional complex that contains Lmo2 [406, 407], and the cytoskeletal complex that contains another LIM- domain containing protein, Testin [408, 409]. In situations such as this, binding partners are in very close proximity, meaning they have the opportunity to interact with each other, as well as with the LIM domain protein. The LIDs appear to be more limited in their ability to interact with other partners. To date they appear to bind only one protein at any instance, and whereas Ldb1LID can interact with multiple partners [51], Isl1LID preferentially binds only
Lhx3 and Lhx4, with apparently lower affinity binding to Lmx1b [54]. The LIDs from Isl1 and Isl2 have another proposed role in shielding the Isl1 LIM domains from non-specific or off-target interactions [54]. However, it may be possible for the intramolecular LIM:LID interaction to co-exist with another LIM domain based protein-protein interaction. This may act as a mechanism by which higher order complexes are formed: initial binding of a protein to the Isl1LIM domains, before displacement of the Isl1LID and formation of further protein-
protein interactions.
From the data presented here, it is clear that the study of both protein-protein and protein- DNA interactions are important in examining the function of Isl1. Data from Chapter 5 suggests that Isl1 may not be able to function as a DNA-binding transcription factor without the presence of additional protein binding partners. Given that the specificity of the Lhx3:Isl1 complex is different to that of an independent Lhx3, it appears that Isl1 can influence the DNA-binding specificity of its protein binding partners. In vivo, this is potentially a mechanism by which Lhx3:Isl1 complexes can target different areas of the genome than Lhx3-only containing complexes. This behaviour extends to other transcriptional complexes containing Isl1. For example, Isl1 has been found to influence the DNA-binding preferences of the transcription factor Phox2a during cranial motor neuron development, in a similar fashion to its behaviour with Lhx3 [105]. Further work may identify other instances of Isl1 modulating the DNA-binding specificity of its protein-protein interaction partners.
It may be that the weak binding of Isl1 to DNA is an advantage in the context of multi- protein complexes binding to DNA. Assuming that this property helps Isl1-containing complexes find their targets more quickly (see above), Isl1 may refine the in vivo DNA- targeting properties of any transcriptional complex it is a part of, and allow that complex to find its genomic targets efficiently. As Isl1 can bind multiple partners, it could influence the
DNA-binding properties of many different transcriptional complexes, and thereby direct development in a broad range of contexts.
Further study is needed to determine whether this model of the action of Isl1 is correct. Investigation of the DNA-binding behaviour of Isl1 in the context of different protein binding partners will generate a more complete picture of the role of Isl1 in transcriptional regulation of gene expression, giving new insight into how the interplay of protein-protein and protein- DNA interactions can influence gene regulation.