Mutations in methyl-CpG binding protein 2 (MeCP2) have been attributed as the cause of Rett syndrome (RTT). RTT is an X-linked neurological disorder affecting approximately 1/10000 females. MeCP2 is a ubiquitously expressed protein and its main function is to act as a transcriptional repressor via association with NCoR/SMRT complex. However, MeCP2 has various reported functions and interacting partners. For this reason, the exact role of MeCP2 in RTT, its function and acting mechanism are not yet fully known.
In order to address this, the aim of this thesis was to shed light on the protein interactions of MeCP2 in a novel way, using proximity labelling (PL). Here, two promiscuous ligases, TurboID and miniTurbo, were fused to the C-Terminus of MeCP2 to enable biotinylation of proximal proteins. Mouse fibroblast cells were transfected with MeCP2-TurboID and MeCP2-miniTurbo and exposed to biotin. The high affinity of biotin to streptavidin allows biotinylated proteins to be isolated and identified.
Firstly, I have shown that fusion in the C-Terminus of MeCP2 does not affect its ability to localise to the nucleus. Additionally, supplementation with biotin allowed the visualisation of biotinylated proteins in mouse fibroblast cells. I have confirmed that protein biotinylation using the named ligases can occur in as little as 10 minutes. More exogenous biotinylation occurred in TurboID transfected cells than miniTurbo transfected cells. A cell line has been produced of MeCP2- TurboID. Overall, PL appears to be a promising avenue of research for identifying protein partners of MeCP2. In the future, this will aid more detailed insights into molecular mechanisms of MeCP2 and, indeed, implications on RTT.
