Using yeast two-hybrid to check for specific binders reveals many non-specific

A straightforward method of determining if a prey protein interacts specifically with Isl1 is to screen the prey against other potential binding partners. Yeast two-hybrid assays were used for screening all potential interactors against Isl2, the close homolog of Isl1, and additional other proteins as explained below.

3.7.3.1 Non-specific interactions in the Isl1LIM prey pool

A set of 54 prey constructs representing each Isl1LIM-interacting protein identified were tested

for specificity of binding to Isl1LIM by screening against LIM domains from other proteins,

including Isl2, LIM domain only protein 4 (Lmo4, another LIM domain transcription factor), and LIM kinase 2 (Limk2, a cytoplasmic LIM protein). Hits that interacted with Lmo4LIM

may represent biologically relevant, but less specific, binding partners. Hits that interact with Limk2LIM are more likely to represent non-specific interactions that are not biologically

relevant to the function of Isl1.

Of the pool tested, 42 Isl1LIM-interacting prey constructs showed strong interactions with

Limk2LIM, with many also showing strong interactions with Lmo4LIM (Table 3.7). All prey

constructs except two showed a strong interaction with Isl2LIM. Those were Meprin A subunit

beat (Mep1b) and Muskelin (Mkln1), which only showed evidence of weak interactions with any of the tested non-Isl1 LIM domains, highlighting them as potentially specific Isl1LIM

interaction partners.

Table 3.7: Screening putative interactors for interaction specificity.

Breakdown of putative interactors by Isl1 construct, showing strengths of interactions with Isl1-like proteins.

Limk2 interaction strength

Strong Moderate Weak No interaction

L m o4 in te rac tion str en gt h Strong 15 0 0 3 Moderate 5 0 0 2 Weak 20 0 0 7 No interaction 2 0 0 0

Overall, nine of the Isl1LIM-interacting pool showed strongest binding to Isl1LIM, with

minimal binding to the other LIM domains screened. These were: Ddx20, Dfna5, Lace1, Mep1b, Mkln1, Nup50, Rps18, Sparcl1, and Zfand1.

3.7.3.2 Checking the interaction interface of Ldb1LID/Isl1LIM-interacting prey

A set of six hits from the original Ldb1LID/Isl1LIM screen were tested for specificity:

Isoaspartyl peptidase (Asrgl1), Coatomer subunit beta (Copb1), COP9 signalosome complex subunit 5 (Cops5), Mkln1, Transient receptor potential channel 1 (Trpc1), and Ubiquitin D (Ubd). Of these, Asrgl1, Cops5, and Mkln1 had also been isolated in the Isl1LIM screening.

This was taken as an indication that these proteins may not be interacting with the Ldb1LID/Isl1LIM complex as a whole, but might compete with Ldb1LID for binding of Isl1LIM.

Accordingly, Ldb1LID/Isl1LIM-interacting hits were screened against Isl1LIM, Isl2LID, and

Lmo4LIM, to check both for specificity of binding to Ldb1LID/Isl1LIM, and for which part of

the Ldb1LID/Isl1LIM construct was facilitating the interaction.

Asrgl1, Cops5, and Trpc1 were found to interact strongly with Isl1LIM and Lmo4LIM,

indicating that they most likely interact with LIM domains, but not specifically with Isl1 (Figure 3.9). Therefore, they were not pursued further. Copb1, Mkln1, and Ubd were found to interact weakly with Lmo4, but showed varied interaction strengths with Isl1LIM. Copb1

showed weak binding, Ubd showed moderate binding, and Mkln1 showed strong binding to Isl1LIM.

Figure 3.9: Yeast two-hybrid spot test validations for specificity of Ldb1L ID/Isl1L IM interacting proteins. Yeast were co-transformed with one pGBT9

plasmid and one pGAD plasmid and grown on a range of selective media to screen for interactions. pGBT9 plasmids used were: empty pGBT9 (E), Isl1LIM, Lmo4LIM, and Isl2LID.

pGAD plasmids used were: empty pGAD10 (E), and pGADT7-RecAB plasmids encoding the prey proteins Ubd, Trpc1, Mkln1, Cops5, Copb1, and Asrgl1.

Whereas Mkln1 did appear in Isl1LIM screening, Copb1 and Ubd did not, although they

showed an interaction with Isl1LIM in the above specificity validations. This absence may be

explained by the fact that the interactions of these two proteins with Isl1LIM are relatively

weak, and so would not have been isolated in the Isl1LIM screening process, which focussed

on the strongest interactions detected. As Copb1 interacted equally strongly with Ldb1LID/Isl1LIM, Isl1LIM, and Lmo4LIM, it was not pursued further. This leaves Mkln1 and Ubd

as the remaining likely Ldb1LID/Isl1LIM-interacting proteins, although it appears likely that

they are binding only to Isl1LIM.

3.7.3.3 Determining Isl1∆LIM prey interaction specificity

Isl1∆LIM-interacting hits were screened against full length Isl2 and Isl2LID (Figure 3.10). As

the C-terminus of Isl1(other than the LID) has an unknown domain structure, further screening against homologous domains could not be conducted. None of the 17 proteins tested showed an interaction with Isl2LID. Most of the Isl1∆LIM-interacting proteins showed

similarly strong interactions with full length Isl1 and Isl2, indicating no preference for interacting with a particular Islet protein.

Figure 3.10: Representative yeast two-hybrid spot test validations for specificity of Ldb1L I D/Isl1L IM interacting proteins. Yeast were co-transformed with

one pGBT9 plasmid and one pGAD plasmid, and grown on a range of selective media to screen for interactions. pGBT9 plasmids used were: empty pGBT9, Isl1FL, Isl2FL, and Isl2LID.

pGAD plasmids used were: empty pGAD10, and pGADT7-RecAB plasmids encoding the prey proteins Rnf167, Scpep1, Spata7, Tigd2, Usp8, and Zdhhc20.

3.7.3.4 Fhl1

Screening with Isl1LIM resulted in a total of 51 hits encoding 3 different truncations of the

protein Four and a half LIM domains protein 1 (Fhl1), making it the most represented protein. In fact, Fhl1 represented 14% of the total pool of Isl1LIM hits, and 23% of the total

pool may indicate a particularly strong interaction, or simply that Fhl1 is an abundant transcript in the library.

Unlike other protein hits, the different Fhl1 fragments identified varied by less than 5 residues (or 15 bp). The shortest construct of Fhl1 found was 19 amino acids in length, and the longest was 23, both containing the very C-terminal portion of the Fhl1 protein. Specificity validation experiments showed that Fhl1 interacted very strongly with both Isl2LIM and Limk2LIM, suggesting that it is a non-specific LIM-binding sequence (further

discussed in Section 3.8.2).