Dose-dependent nuclear delivery and transcriptional repression with a cell-penetrant MeCP2

Abstract Methyl-CpG-binding-protein 2 (MeCP2) is a nuclear protein expressed in all cell types, especially neurons 1 . Mutations in the MECP2 gene cause Rett syndrome (RTT), an incurable neurological disorder that disproportionately affects young girls 2 . Strategies to restore MeCP2 expression phenotypically reverse RTT-like symptoms in male and female MeCP2-deficient mice 3–5 , suggesting that direct nuclear delivery of functional MeCP2 could restore MeCP2 activity. We report that ZF- t MeCP2, a conjugate of MeCP2(Δaa13-71, 313-484) 6 and the cell-permeant mini-protein ZF5.3 7–11 , both binds DNA in a methylation-dependent manner and reaches the nucleus of model cell lines intact at concentrations above 700 nM. When delivered to live cells, ZF- t MeCP2 engages the NCoR/SMRT co-repressor complex and selectively represses transcription from methylated promoters. Efficient nuclear delivery of ZF- t MeCP2 relies on a unique endosomal escape portal provided by HOPS-dependent endosomal fusion. The Tat conjugate of MeCP2 (Tat- t MeCP2), evaluated for comparison, is degraded within the nucleus, is not selective for methylated promoters, and trafficks in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini-protein ZF5.3. Such a strategy could broaden the impact of multiple families of protein-derived therapeutics.