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Analysis of the transcriptome of undifferentiated SH-SY5Y neuroblastoma cells following AS3MT knockout with CRISPR/Cas9.

ABSTRACT: Aims/Hypothesis: AS3MT is a risk gene for schizoprhenia and the role in neuronal cell lines has not been established. Using CRISPR/Cas9 generated two AS3MT knockout lines (B6/C6) and age matched wild type controls (D2/D3) in SH-SY5Y neuroblastoma cells and characterised the transcriptional response following knockout. We carried out two independent sequencing runs, including independent cell culturing, RNA extraction, library prep, and sequencing and identify considerable overlap of differentially expressed genes identifying a robust transcriptional response following AS3MT knockout. We identified a number of genes involved in cellular adhesions, ribosomal function, cell-cell contact, and neuronal development, as well as a significant enrichment of previously associated schizophrenia genes, including ZNF80a and DRD2.

ORGANISM(S): Homo sapiens

PROVIDER: GSE182157 | GEO | 2021/10/04

REPOSITORIES: GEO

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Project description:Cocaine-mediated repression of the histone methyltransferase (HMT) G9a has recently been implicated in transcriptional, morphological, and behavioral responses to chronic cocaine administration. Here, using a ribosomal affinity purification approach, we find that G9a repression by cocaine occurs in both Drd1 (striatonigral)- and Drd2 (striatopallidal)-expressing medium spiny neurons (MSNs). Conditional knockout and overexpression of G9a within these distinct cell types, however, reveals divergent behavioral phenotypes in response to repeated cocaine treatment. Our studies further indicate that such developmental deletion of G9a selectively in Drd2 neurons results in the unsilencing of transcriptional programs normally specific to striatonigral neurons, and the acquisition of Drd1-associated projection and electrophysiological properties. This partial striatopallidal to striatonigral ‘switching’ phenotype in mice indicates a novel role for G9a in contributing to neuronal subtype identity, and suggests a critical function for cell-type specific histone methylation patterns in the regulation of behavioral responses to environmental stimuli. Polyribosome associated mRNAs from 2-5 month old, age and sex matched Drd1-Cre; Drd1-TRAP; G9afl/fl and Drd1-TRAP; G9afl/fl, Drd2-Cre; Drd2-TRAP; G9afl/fl and Drd2-TRAP; G9afl/fl mice (n = 2-4 mice/genotype/drug treatment, 2 hours after the last of eight repeated cocaine injections of 20 mg/kg/day) were obtained as previously described. EGFP labeled ribosomes and associated mRNAs were immunoprecipitated using a mix of two monoclonal anti-GFP antibodies (50 μg of clones #19C8 and #19F7 for each IP, available at Sloan-Kettering Monoclonal Antibody Facility). Purified mRNA was amplified and processed for microarray and qPCR analysis using the Affymetrix two-cycle cDNA Synthesis kit (Affymetrix) as previously described. Affymetrix Mouse Genome 430 2.0 arrays were used in all experiments. Information regarding the array design and features can be found at www.affymetrix.com. Mouse Genome 430 2.0 arrays were scanned using the GeneChip Scanner 3000 (Affymetrix) and globally scaled to 150 using the Affymetrix GeneChip Operating Software (GCOS v1.4).

Project description:Background: Chronic exposure to inorganic arsenic (iAs) has been associated with type 2 diabetes (T2D). However, potential sex divergence and the underlying mechanisms remain understudied. iAs is not metabolized uniformly across species, which is a limitation of typical exposure studies in rodent models. The development of a new “humanized” mouse model overcomes this limitation. In this study, we leverage this model to study sex differences in the context of iAs exposure. Objectives: The aim of this study iwas to determine if males and females exhibit different liver and adipose molecular profiles and metabolic phenotypes in the context of iAs exposure. Methods: Our study was performed on wild-type (WT) 129S6/SvEvTac and humanized arsenic +3 methyl transferase (human AS3MT) 129S6/SvEvTac mice treated with 400 ppb of iAs via drinking water ad libitum. After 1 month, mice were sacrificed and the liver and epididymales gonadal adipose depot were harvested for iAs quantification as well as sequencing-based microRNA and gene expression analysis. Serum blood was collected for fasting blood glucose, fasting plasma insulin, and HOMA-IR. Results: We detected sex divergence in liver and adipose markers of diabetes (e.g., insulin signaling pathways, fasting blood glucose, fasting plasma insulin, and HOMA-IR) only in humanized (not WT) male mice. In humanized female mice, numerous genes that promote insulin sensitivity and glucose tolerance in both the liver and adipose are elevated compared to humanized male mice. We also identified Klf11 as a putative master regulator of the sex divergence in gene expression in humanized mice. Discussion: Our study underscoreds the importance of future studies leveraging the humanized mouse model to study iAs-associated metabolic disease. The findings also suggest suggested that humanized females are protected from metabolic dysfunction relative to humanized males in the context of iAs exposure. Future investigations should focus on the detailed mechanisms that underlie the sex divergence, including the potential role of miR-34a and/or Klf11.

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Analysis of the transcriptome of undifferentiated SH-SY5Y neuroblastoma cells following AS3MT knockout with CRISPR/Cas9.

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