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Levodopa-induced dyskinesia (LID) is a persistent behavioral sensitization that develops after repeated levodopa (L-DOPA) exposure in Parkinson disease (PD) patients. we used reduced representation bisulfite sequencing to determine the methylation status of cytosines genome wide at base pair resolution following a parkinsonian-like lesion and LID development. Due to the enrichment of RRBS, we focused our analysis to cytosines in a CpG context and observed extensive locus-specific changes in DNA methylation, including a preponderance of demethylation, in the dorsal striatum following the development of dyskinetic behaviors in our animal model system. Changes in DNA methylation were concentrated in putative regulatory regions of many genes known to be aberrantly transcribed following L-DOPA exposure and enriched for genes relevant to mechanisms of synaptic plasticity. In the areas of the genome exhibiting the highest levels of effect, the magnitudes of change to methylation were strongly correlated with dyskinetic behaviors. Rats were given a unilateral dopaminergic lesion to the left medial forebrain bundle with 6-OHDA to destroy at least 90% of TH positive axons. Animals were allowed to recover for 3 weeks and then given daily injections of L-DOPA (6 mg/kg) for seven days to establish stable dyskinesia. Animals were sacked 3 hrs following the final L-DOPA injection and the dorsal striatum was immediately dissected and flash frozen. Striatal tissue samples were processed for nucleic acid isolation using the AllPrep DNA/RNA Mini Kit (Qiagen) following the manufacturer's instructions. One μg of gDNA sample was used for library construction. Bisulfite converted DNA libraries were produced and adaptor ligated, and single-end reads were sequenced on an Illumina HiSeq-2500 following library QC. Bisulfite conversion efficiency was greater that 98% for all of the samples and we obtained an average of 68 million reads per library. Quality control on raw reads was performed with FastQC (version 0.10.1, http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc), and adaptor trimming and removal of trimmed reads shorter than 20 bp was performed with Trim Galore (version 0.3.7, http://www.bioinformatics.babraham.ac.uk/projects/trim_galore). Trimmed reads were mapped to the UCSC Rattus norvegicus rn5 genome with the methylation-aware mapper bismark (version 0.13.0). Samtools (version 0.1.19–96b5f2294a) was used to sort SAM files produced by bismark and de-duplicate reads. SAM files were analyzed using MethylKit (version 0.9.2) in R (Akalin et al., 2012). Reads were filtered based on coverage, with a cutoff of at least ten reads per site, and normalized for each samples coverage before analysis. The genome was tiled at 250 bp and regions were counted if they contained at least 2 identified CpGs per tile. Differential methylation was defined as a sliding linear model correct p-value of <0.01 and, for highly dynamic regions, included at least a 5% change.

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