MECP2 directly interacts with RNA polymerase II to modulate transcription in human neurons

ABSTRACT: This dataset comprises 15 raw AP-MS files, each with an associated peak list, captured using a Vanquish Neo nanoLC system in tandem with an Orbitrap Eclipse mass spectrometer. To generate MECP2 hESC-reporter lines for wild type (WT) and various mutations of MECP2, including R133C, R168X, and R270X, we first used CRISPR/Cas9 to create MECP2 alleles carrying the green fluorescent protein (GFP) sequences in the endogenous gene. The R133C mutation was then introduced into the WT MECP2-GFP reporter line. Mutations R133C, R168X, and R270X are recognized as loss-of-function variants in MECP2 and are also identified as primary Rett syndrome-causing mutations. For efficient neuronal differentiation, a doxycycline (DOX)-responsive NGN2 construct was incorporated at their AAVS1 safe harbor locus. Upon the addition of DOX, homogenous populations of neurons were generated within three weeks from those four MECP2 hESC-reporter lines. Subsequently, GFP-pull down assay and AP-MS were performed using these WT MECP2-GFP neurons along with R133C-, R168X-, and R270X-mutant MECP2-GFP reporter neurons. Neurons expressing only the GFP tag served as a negative control. AP-MS analysis identified proteins interacting differently between WT and mutant MECP2 within human neurons.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens

SUBMITTER: Fabian Schulte  

PROVIDER: MSV000093210 | MassIVE | Fri Oct 27 13:54:00 BST 2023

REPOSITORIES: MassIVE