About MECP2 NeuroAtlas:
The MECP2 NeuroAtlas provides a comprehensive integration of multi-omics MECP2 datasets, encompassing CUT&Tag, AP-MS, RNA-Seq, WGBS, and ATAC-Seq. To develop MECP2 human embryonic stem cell (hESC)-reporter lines for wild type (WT) and R168X, and R270X mutations of MECP2, we used CRISPR/Cas9 to create MECP2 alleles carrying the green fluorescent protein (GFP) sequences in the endogenous gene. Additionally, the R133C mutation was introduced into the WT MECP2-GFP reporter line. This GFP tag enables targeted comparison between WT and mutant MECP2 across genomics and proteomics profiling. The hESC line (WIBR1) used in this study was developed by the Jaensich lab (NIHhESC-10-0074), and its genetic stability was verified via karyotype analysis. To efficiently induce neuronal differentiation, we introduced a doxycycline (DOX)-inducible NGN2 construct into the safe harbor AAVS1. Upon the addition of DOX, homogenous populations of neurons were generated within three weeks from those MECP2 hESC-reporter lines. Mutations R133C, R168X, and R270X are recognized as loss-of-function variants in MECP2 and are also identified as primary Rett syndrome-causing mutations (referencing the database from Rettsyndrome.org). Given this context, our NGN2-reporter neurons present a relevant human model to study MECP2’s role in cellular physiology and disease development. Therefore, we aim for the MECP2 NeuroAtlas to offer researchers streamlined access to explore these MECP2 datasets with the broad spectrum of genomics, epigenetics, and proteomics, paving the way to study the multifaceted functions of MECP2 in gene regulation and its molecular impact on human brain disorders.
Portal Data Summary:
The MECP2 NeuroAtlas features 16 CUT&Tag datasets, 2 ATAC-Seq datasets, 1 WGBS dataset, 12 RNA-Seq datasets, and 15 AP-MS datasets, all derived from the aforementioned MECP2-GFP reporter neurons. Raw and processed data files from these datasets have been deposited in GEO (GSE230716). Detailed information for each data category is provided below.
- CUT&Tag MECP2-GFP, RNA Pol II, H3K4me3, and H3K9me3:
CUT&Tag experiments were conducted on WT MECP2-GFP, R168X, R270X, and R133C mutant CRISPR-Cas9 edited reporter neurons. We utilized an anti-GFP antibody (Abcam ab290), RNA polymerase II antibody (RNA Pol II; Abcam ab290), H3K4me3 antibody (Active Motif 39160), and H3K9me3 antibody (Active Motif 39162) for these assays. CUT&Tag using Rabbit IgG (Cell Signaling #2729) in WT MECP2-GFP reporter neurons served as a negative control. The genomic tracks display pooled data from individual bigWig files originating from three distinct biological replicates. Below is a summary of the labels for the genome tracks presented:
- The assay for transposase-accessible chromatin with sequencing (ATAC-Seq):
ATAC-Seq was performed on WT MECP2-GFP neurons. The genomic track displays data from individual bigWig files combined from two distinct biological replicates.
- Whole Genome Bisulfite Sequencing (WGBS):
WGBS was carried out on WT MECP2-GFP neurons using three biological replicates that achieved a minimum of 30x coverage. Cytosines were partitioned into CpG and mCA contexts, and any sites with read coverage less than five were removed. The genomic tracks show the methylation fraction in CpG and CA contexts of WT MECP2-GFP reporter neurons, representing pooled data from individual bigWig files derived from three biological replicates.
- RNA sequencing (RNA-Seq):
RNA-Seq was conducted on WT MECP2-GFP, R168X, R133C mutant, and MECP2 knockout (KO) CRISPR-Cas9 edited reporter neurons. The box plots and bar graphs represent the expression levels of a gene based on data from three biological replicates.
- Affinity-Purification /mass spectrometry (AP-MS):
The GFP pull-down assay was performed with WT MECP2-GFP, R168X, R270X, and R133C mutant reporter neurons. Neurons stably expressing GFP-empty were used as a negative control. The captured proteins were further identified by a Vanquish Neo nanoLC system coupled with an Orbitrap Eclipse mass spectrometer. The analysis of proteome data was conducted using the DIA-NN 1.8.1 software platform, and the raw intensity values (protein peak areas) from three biological replicates were normalized by median normalization and then adjusted by subtracting the AP-MS results from the negative control (GFP-empty). This normalized binding intensity was presented in the box plots and bar graphs to indicate the differential interaction of a protein with WT and mutant MECP2 in human neurons.
Frequently Asked Questions
- How can I navigate MECP2 NeuroAtlas?
As an exploratory portal, users can input individual genes to generate a variety of visualizations that showcase their genomic, transcriptomic, and proteomic behaviors in MECP2 WT and mutant human neurons.
- What format is required for my input genes?
Entries should be provided in gene symbol format.
- How can I provide feedback or reach out to support?
Please contact Yi Liu at yiliu@wi.mit.edu
- Website citation:
Please cite this article in press as: Liu et al., MECP2 directly interacts with RNA polymerase II to modulate transcription in human neurons, Neuron (2024), https://doi.org/10.1016/j.neuron.2024.04.007