G9a influences neuronal subtype specification in striatum
ABSTRACT: 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:Goal of the experiment: Analysis of gene expression changes in the cortex, striatum, hippocampus, hypothalamus, Drd2-MSNs and Drd1-MSNs of mice with a postnatal, neuron-specific ablation of GLP or G9a as compared to control mice. Overall design: For microarray analysis, hippocampus, hypothalamus, cortex and striatum of Camk2a-Cre; GLPfl/fl, Camk2a-Cre; G9afl/fl and age (10-14 week old) and sex matched littermate controls were used for total RNA purification. Four biological replicates were performed for each experiment. Polyribosome associated mRNAs from five, age (10-14 week old) and sex matched Drd1-Cre; Drd1-bacTRAP; G9afl/fl, or Drd2-Cre; Drd2-bacTRAP; G9afl/fl and Drd1-bacTRAP; G9afl/fl or Drd2-bacTRAP; G9afl/fl control mice were used. Three biological replicates were performed for each experiment.
Project description:Goal of the experiment: Analysis of gene expression changes in the cortex, striatum, hippocampus, hypothalamus, Drd2-MSNs and Drd1-MSNs of mice with a postnatal, neuron-specific ablation of GLP or G9a as compared to control mice. For microarray analysis, hippocampus, hypothalamus, cortex and striatum of Camk2a-Cre; GLPfl/fl, Camk2a-Cre; G9afl/fl and age (10-14 week old) and sex matched littermate controls were used for total RNA purification. Four biological replicates were performed for each experiment. Polyribosome associated mRNAs from five, age (10-14 week old) and sex matched Drd1-Cre; Drd1-bacTRAP; G9afl/fl, or Drd2-Cre; Drd2-bacTRAP; G9afl/fl and Drd1-bacTRAP; G9afl/fl or Drd2-bacTRAP; G9afl/fl control mice were used. Three biological replicates were performed for each experiment.
Project description:Cocaine-induced alterations in gene expression cause changes in neuronal morphology and behavior that may underlie cocaine addiction. We identified an essential role for histone 3 lysine 9 (H3K9) dimethylation and the lysine dimethyltransferase G9a in cocaine-induced structural and behavioral plasticity. Repeated cocaine administration reduced global levels of H3K9 dimethylation in the nucleus accumbens. This reduction in histone methylation was mediated through the repression of G9a in this brain region. To identify whether changes in H3K9me2 correlated with genome-wide alterations in gene expression in the NAc, we employed microarray analyses to examine gene expression profiles induced by a challenge dose of cocaine in animals with or without a history of prior cocaine exposure. Animals that had received repeated cocaine displayed dramatically increased gene expression 1 hour after a cocaine challenge in comparison to acutely treated animals. This increased gene expression still occurred in response to a cocaine challenge given after 1 week of withdrawal from repeated cocaine. These data suggest that repeated, but not acute, cocaine exposure results in persistent sensitized genomic responses to a cocaine challenge, indicating that sensitized behavioral responses to repeated cocaine are likely the result of G9a-dependent alterations in global transcriptional responses to cocaine. Overall design: Four groups (3 independent biological replicates per group) were utilized for this microarray study, totaling 12 microarrays (see 'Treatment protocol' for cocaine treatment information). 1 hour following the last cocaine injection, animals were rapidly decapitated and brains were removed and placed on ice. Dissections of nucleus accumbens were taken using a 15-gauge needle punch and were quickly frozen on dry ice until RNA was extracted. Bilateral punches were pooled from four animals per replicate, totaling 12 mice per group. RNA isolation, microarray processing, and data analysis were performed as previously described. Briefly, RNA was isolated and purified and was checked for quality using Agilent’s Bioanalyzer. Reverse transcription, amplification, labeling and hybridization to Illumina MouseWG-6 v2.0 arrays were performed using standard procedures. Raw data were background subtracted and quantile normalized using Beadstudio software. Normalized data were analyzed using GeneSpring software and genelists were generated using significance criteria of a 1.3 fold change cutoff coupled with a non-stringent p-value cutoff of p < 0.05.
Project description:To understand the complexity of the brain, connectome and transcriptome maps of high resolution are being generated, but an equivalent catalogue of the brain proteome is lacking. To provide a starting point, we have performed an in-depth proteome analysis of the adult mouse brain, its major regions and cell types, which resulted in the so far largest collection of cell-type resolved protein expression data of the brain. Comparisons of the 12,934 identified proteins in oligodendrocytes, astrocytes, microglia and cortical neurons with deep sequencing data of the transcriptome indicated deep coverage of the proteome. We identified novel protein makers for different cell type and brain regions. These were validated either directly such as in case of cell types or by comparative analysis against Allen mouse brain atlas. The utility and the power of the resource were demonstrated by the identification of Lsamp, an adhesion molecule of the IgLON family, as a negative regulator of myelination in a subpopulation of neurons. Our in-depth proteome analysis of CNS cell types provides a framework towards a system-level understanding of cell type diversity in the CNS and serves as a rich resource to the neuroscience community for the better understanding of brain development and function.
Project description:mRNAseq for baseline transcriptional profiling of D1 and D2 receptor-expressing medium spiny neurons from 5-6 month BACHD mice, compared to respective WT littermates. BACHD iSPN and dSPN RNAseq data has been described in "Impaired TrkB receptor signaling underlies corticostriatal dysfunction in Huntington's disease"; Plotkin et al; 2014; PMID accession number 24991961. Overall design: Hemizygous BACHD (FVB) × Drd2 -EGFP BAC (FVB) and BACHD (FVB) × Drd1-EGFP BAC (FVB) mice were generated. Both D2BAC-EGFP (FVB) and D1BAC-EGFP (FVB) were obtained from Gensat/MMRRC. HD model BACHD (FVB) mice, a bacterial artificial chromosome (BAC)-mediated transgenic mouse model expressing full length -mHtt with 97 glutamine repeats under the control of endogenous Htt regulatory machinery on the BAC (Grey et al, 2008 PMID: 18550760) were obtained from CHDI Foundation (CHDI-81001010). Fluorescence activated cell sorting (FACS) for GFP was used to isolate BACHD and WT - indirect pathway medium spiny neurons (iMSNs) and direct pathway medium spiny neurons (dMSNs) from the D2BAC-EGFP or D1BAC-EGFP x HD model crosses, respectively. Cells were pooled from four animals (150,000 cells/animal) to create iSPN and dSPN pools for sequencing. RNA was extracted, and sequencing was performed by Expression Analysis on an Illumina Hi-seq 2000. Paired-end sequencing was performed, 4-plexed across lanes for a total of around 38 million 50mer paired reads per sample.