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Characterization of human mosaic Rett syndrome brain tissue by single-nucleus RNA sequencing (MeCP2 ChIP-seq)

ABSTRACT: In females with X-linked genetic disorders, wild-type and mutant cells coexist within brain tissue because of random X-chromosome inactivation. This cellular mosaicism leads to phenotypic variability and poses significant challenges for interpreting the effects of X-linked mutant alleles on gene expression. We present a single-nucleus RNA sequencing approach that resolves mosaicism by using single nucleotide polymorphisms in genes that are expressed in cis with the X-linked mutation to determine whether individual nuclei express the wild-type or mutant allele even when the mutant gene is not directly detected. This approach enables genome-wide comparisons of gene expression between mutant and wild-type cells within the same individual and eliminates the variability introduced by comparisons to controls with different genetic backgrounds. We apply this approach to mosaic female mouse models and humans with Rett syndrome, an X-linked neurodevelopmental disorder caused by mutations in the methyl-DNA-binding protein MECP2, and reveal that cell-type-specific DNA methylation patterns largely predict the degree of gene upregulation by MECP2 in specific neuronal subtypes. The approach described here can be broadly applied to the characterization of gene expression in additional mosaic X-linked conditions.

ORGANISM(S): Mus musculus

PROVIDER: GSE113903 | GEO | 2018/11/15

REPOSITORIES: GEO

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Project description:The goal of this study was to characterize a novel Mecp2 allele in the laboratory rat, a distinct rodent species from the laboratory mouse with unique features. The allele was created by zinc finger-nuclease (ZFN) targeting (SAGE/Horizon) of the X-linked gene, Methyl-CpG-Binding Protein 2 (Mecp2), resulting in normal Mecp2 RNA abundance, but absent protein in male rats as expected due to the presence of only one copy of mutant Mecp2, and an approximate 50% reduction in female rats as expected from animals that harbor one wild-type copy and one mutant copy of Mecp2. Behavioral characterization of female rats with the Mecp2 ZFN allele and wild-type littermates was conducted, and Mecp2 ZFN/+ female rats showed behavioral phenotypes that were consistent with disease-like features present during the early stages of disease onset in the neurological disorder Rett syndrome. The goal of conducting RNAseq studies was to compare existing gene expression alterations in the Mecp2 rat with one of the most widely studied Mecp2 mouse model. Hypothalami were obtained from males with complete loss of MeCP2 (ZFN/y) and wild-type littermate male rats for RNA-seq studies. Common and unique gene expression alterations among the Mecp2 rodents that were then tested in human Rett and control postmortem brain revealed the benefit of combining findings from both models, suggesting the predictive validity of this approach for future studies for the identification of potential preclinical outcome measures. QPCR validation in an independent set of rats was conducted with a subset of genes from the Mecp2 ZFN rat RNA-seq data as an additional control measure. Taken together, these findings demonstrate that the Mecp2 rat model is a complementary tool with unique features for the study of RTT and highlight the potential benefit of cross-species analyses in identifying potential disease-relevant preclinical outcome measures. Transcriptional profiles of hypothalamic samples obtained from male rats haboring a novel zinc-finger nuclease Mecp2 loss-of-function allele and corresponding wild-type littermate rats were generated using RNA-seq

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Characterization of human mosaic Rett syndrome brain tissue by single-nucleus RNA sequencing (MeCP2 ChIP-seq)

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