Interactions between MAGE and EID Family Proteins Table 2. MAGE and EID proteins used in this study. S. pombe protein Human proteins Nse3 Nse4 MAGEG1 MAGEF1 MAGED4b Necdin MAGEA1 EID1 EID2 EID2b EID3/NSE4b NSE4a Type II MAGE – ortholog of Nse3 Type II MAGE - closely related to MAGEG1 Type II MAGE Type I MAGE Type II MAGE – expressed in postmitotic neurons Identified as transcriptional repressor Identified by sequence similarity to EID1. Also shown to be transcriptional repressor Identified by sequence similarity to EID2. Also shown to be transcriptional repressor Identified by sequence similarity to EID1. Testis-specific transcriptional repressor. Ortholog of Nse4 in testis. Ortholog of Nse4 in somatic cells doi:10.1371/journal.pone.0017270.t002 unpublished data). We showed previously that MAGEG1 is the only MAGE protein detected in the SMC5-6 complex, and that MAGEF1 could not be integrated into the complex [18]. This is consistent with our finding that MAGEF1 does not interact with NSE4a or b (Figure 7E). Instead MAGEF1 protein can form complexes with EID proteins (which lack the C-terminal WHD domain essential for binding to the SMC5 head domain). Remarkably we found that most of the MAGE proteins that we examined interacted with both NSE4a and NSE4b (Figure 9A). However, with the exception of the MAGEG1 interactions, the MAGE-NSE4 interactions do not take place in the context of the SMC5-6 complex, since neither NSE1 nor SMC6 is found in the immunoprecipitates (Fig. 7B, data not shown). Consistent with our results, Doyle et al. found that most of the MAGE proteins that they examined were unable to interact with NSE1 [14]. We have shown that Nse1 stabilizes the interaction between S. pombe Nse4 and Nse3 (Figure 4A, [8]), and the same is probably the case for the human orthologs. Without NSE1, it is likely that the MAGE-NSE4 subcomplexes are not able to bind to the SMC6- SMC5-NSE2 subcomplex (Fig. 9B). Furthermore, we previously showed that not only Nse4 but also Nse3 (as well as Nse5 and Nse6 in S. pombe) bound to the head domain of Smc6 ([13]; K. Bednarova unpublished data). We speculate that the MAGE proteins (other than MAGEG1), have lost their ability to bind to the SMC6 head domain and to NSE1. The evolutionary diversification of such a binding surface(s) then resulted in a gain of new binding partners and the formation of novel MAGE complexes with RING-finger proteins (Figure 9B) ([14]; our unpublished data). Remarkably, the EID family shows a similar pattern of evolutionary diversification to the MAGE family, albeit to a less dramatic extent, namely a single member (Nse4) in most eukaryotes up to non-placental mammals (although there are two in the plant Arabidopsis thaliana; [24]) and four members in placental mammals. The fifth member, EID2b, is found only in rodents and primates. Our finding that, of the pairs that we examined, most MAGE proteins interacted with most of the EID proteins (Figure 9A) suggests that the diversification of these two protein families may be connected. Interestingly, two tumour-related mutations in MAGE proteins were described recently. In MAGEA1 Glu217 (corresponding to Phe235 in yeast Nse3, Table 1) was mutated to Lys in a melanoma sample [25]. We speculate that this change could disturb the MAGEA1 binding to NSE4/EID partners. Similarly in MAGEC1 Ile1001 (corresponding to Met214 in yeast Nse3, Table 1) was mutated to Phe in glioblastoma multiforme cells [26]. Although this change is less severe, it could change the affinity and/or specificity of the binding of MAGEC1 to its putative NSE4/EID partner. The physical interaction between the MAGE and EID proteins raises the question of their functional significance. In contrast to the broadly similar physical interactions between members of the two families, their effects in the transcriptional activation reporter system were quite different. In the EID family, only EID1 repressed transcription in the Gal4-SF1 system in HEK293 cells. Of the MAGE proteins examined, MAGEA1 and D4b were strong transcription co-activators, whereas several other MAGE proteins had little effect. There are various reports in the literature on the effects of MAGE proteins on transcription systems. MAGEA1 represses transcription mediated by Ski interacting protein [27], whereas Wilson and co-workers reported that MAGEA11 increased the transcriptional activity of the androgen receptor [28] via an interaction with p300 [29]. MAGED1 was shown to be a co-activator of the RORa and RORc proteins, but this co-activation did not require the MHD of MAGED1 [30]. We found that, when EID and MAGE proteins were co-expressed, EID1 reversed the co-activation mediated by MAGEA1 and MAGED4b, whereas it had no effect on the much lower activation in the presence of necdin. The latter result agrees with the finding of Bush and Wevrick [21]. Our results suggest a relatively specific functional interplay between MAGE and EID proteins which contrasts with the general physical interactions that we have observed. It is evident that other proteins interacting with these partners may influence the transcription level. Much more detailed studies need to be carried out in future work in order to unravel the nature of these complex interactions and to understand the functions of these two protein families in their normal cellular contexts. In conclusion, we have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydropho- bic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Materials and Methods Plasmids All pTriEx4 plasmids described in this study were generated by PCR and ligase-independent cloning (Merck). The primers used PLoS ONE | www.plosone.org 10 February 2011 | Volume 6 | Issue 2 | e17270