LAUSR

Dear Editor, Methyl-CpG-binding protein 2 (MeCP2) is a ubiquitously expressed nuclear protein originally identified as a methylated DNA binding protein, which is particularly abundant in mature neurons. Deficiency or excess of MeCP2 causes severe neurological problems. Mutations in MeCP2 account for 95% of the dominant X-linked neurological disorder Rett syndrome. MeCP2 have two key functional domains: the methyl-DNA binding domain (MBD) and the transcriptional repressor domain (TRD). Almost all of missense Rett mutations are clustered in these two domains, such as R133C, F155S, T158M in MBD, and R306H in TRD. The mechanism of the mutations leading to Rett syndrome is still not well understood. Here, we reveal that MeCP2 can drive the liquid–liquid phase separation (LLPS) in complex with DNA. Interestingly, this ability is compromised in the presence of mutations found in Rett syndrome patients, suggesting a potential common mechanism by disrupting LLPS of MeCP2 droplets underlying Rett syndrome. MeCP2 forms sharp condensed foci which highly overlap with DNA dense compartments in neuronal nuclei. Recently, LLPS has been recognized as an important mechanism to condensate molecules to form membraneless compartments within a cell. As both the MBD and TRD of MeCP2 bind to DNA, we hypothesized that the sharp puncta of MeCP2 in the nuclei were phase separated liquid droplets mediated by multivalent interactions between MeCP2 and DNA. To test this hypothesis, we purified full-length recombinant His-MBPMeCP2 (mouse MeCP2-e2 if not specified) and released the His-MBP tag by cutting with TEV protease (Supplementary Fig. S1). CpG methylated DNA was prepared by methylating a 1.1 kb DNA fragment with the M.SssI methyltransferase and verified by digesting with a methylation sensitive endonuclease, BsiWI (Supplementary Fig. S2). Phase separation was assayed using different concentration of a 1.1 kb unmodified or methylated DNA with MeCP2 in a buffer containing 150 mM NaCl and a low concentration of DAPI for visualizing DNA. Puncta formation was observed both in the presence of DNA or methylated DNA (Fig. 1a). In contrast, MeCP2 protein alone and DNA alone could not form the puncta (Supplementary Fig. S3a, b). In addition, His-MBP in mixture with mEGFP protein with or without DNA could not form puncta either (Supplementary Fig. S3c), confirming that MeCP2 together with DNA or methylated DNA drives the puncta formation. Next, we monitored the property of the puncta in more details. The MeCP2/DNA droplets can fuse together upon contact (Fig. 1b). Moreover, we observed quick recovery of EGFP fluorescence after photo-bleaching of the middle of MeCP2-mEGFP/DNA droplets (Fig. 1c). These evidences support that the puncta formed by MeCP2 and DNA are LLPS droplets. To compare the ability of DNA and methylated DNA in promoting the phase separation of MeCP2, we calculated ‘Occupancy Rate’ of the droplets formed by MeCP2 with methylated DNA over unmethylated DNA. We found that about 66% values were positive (stronger) and 25% values were negative (weaker), indicating that CpG methylation could moderately promote LLPS of MeCP2 in complex with DNA (Fig. 1d). Taken together, these data reveal that MeCP2 can undergo LLPS with DNA which can be moderately enhanced by methylation at CpG sites. Rett mutations have been found both within and outside the MBD domain. Therefore, the loss of the binding to methylated CpG is not the sole mechanism of MeCP2