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SRX7175047: GSM4173701: Crispr TFAM ChIPseq rep1; Rattus norvegicus; ChIP-Seq
1 ILLUMINA (HiSeq X Ten) run: 31M spots, 4.6G bases, 1.6Gb downloads

Submitted by: NCBI (GEO)
Study: Mitochondrial Dysfunction Induces Epigenetic Dysregulation by H3K27 Hyperacetylation to Perturb Active Enhancers in Parkinson's Disease Models
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We aim to explore the predominant mitochondrial dysfunctions in PD, with a focus on how altering the histone code contributes to PD pathogenesis. We employ a multidisciplinary approach to convincingly demonstrate that neurotoxicant exposure- and genetic mutation-driven mitochondrial dysfunction share a common mechanism of epigenetic dysregulation. Under both scenarios, lysine 27 acetylation of likely variant H3.2 (H3.2K27ac) increased in dopaminergic neuronal models of PD, thereby opening that region to active enhancer activity via H3K27 hyperacetylation. These vulnerable epigenomic loci represent potential transcription factor motifs for PD pathogenesis. Our results reveal an exciting axis of 'exposure/mutation-mitochondrial dysfunction-metabolism-H3K27ac-transcriptome' for PD pathogenesis. Collectively, the novel mechanistic insights presented here interlinks mitochondrial dysfunction to epigenetic transcriptional regulation in dopaminergic degeneration as well as offer potential new epigenetic intervention strategies for PD. Overall design: We performed genome-wide ChIP-Seq characterization of H3K27ac as well as transcriptome profiling in dopaminergic neurons whose mitochondria had been impaired either by exposure to a neurotoxic pesticide or by genetic modifications.
Sample: Crispr TFAM ChIPseq rep1
SAMN13318881 • SRS5681832 • All experiments • All runs
Instrument: HiSeq X Ten
Strategy: ChIP-Seq
Selection: ChIP
Layout: SINGLE
Construction protocol: Total RNAs from treatment and control groups were isolated and quality controlled according to the Illumina protocols. A total of 2 μg RNA per sample was used as initial material for library construction. Chromatin immunoprecipitation (ChIP) was performed as described previously (Wang et al., 2⁠ 009b; Wang et al., 2⁠ 008). In brief, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and the reaction was quenched with 125 mM glycine. After chromatin fragmented to 100 to 1000bp by sonication, chromatin templates from 20 million cells were used for ChIP experiment. Samples were immunoprecipitated with 2- 4μg of H3K27ac antibody overnight at 4 °C followed by washing steps. For RNAseq library construction, Poly-A containing mRNA molecules was purified by using poly-T oligo-attached magnetic beads and fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity is achieved by replacing dTTP with dUTP, followed by second strand cDNA synthesis. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are then purified and enriched with PCR to create the final cDNA library. For ChIPseq library construction, after reverse cross-linking, ChIP-DNA fragments were purified and constructed into libraries by using the ThruPLEX DNA-seq Kit (Rubicon Genomics, Takara, Japan). Amplified libraries around 300 – 500 bp were isolated from agarose gel prior to sequencing.
Experiment attributes:
GEO Accession: GSM4173701
Runs: 1 run, 31M spots, 4.6G bases, 1.6Gb
Run# of Spots# of BasesSizePublished


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