The role of chromatin remodeling in Parkinson’s Disease

Advisor: Dr. Scott Ryan, Molecular Cell Biology

Familial Parkinson’s Disease (PD) can be caused by an Alanine 53 to Threonine (A53T) amino acid substitution in the protein α-Synuclein (α-syn). This causes increased redox-stress in dopaminergic neurons, which has been suggested to lead to chromatin remodeling. These chromatin changes are predicted to affect the regulation of several genes needed for neuronal function, and thus chromatin dysregulation may result in the death of dopaminergic neurons, as seen in familial PD. To investigate changes in chromatin structure in PD, Chromatin Immunoprecipitation with deep sequencing (ChIP-seq) was used to isolate H3K9me and sequence regions of open chromatin from stem cell-derived neurons harboring the A53T substitution. For this investigation, four different cell-lines were used: human induced pluripotent stem cells differentiated into dopaminergic neurons from a patient with familial PD harboring the A53T mutation (hiPSC_A53T) that will be compared against a genome corrected control cell line (hiPSC_Corr), or wild type human embryonic stem cells that were differentiated into dopaminergic neurons (hESC_WT), that will be compared against hESCs in which the A53T mutation was introduced by genome editing (hESC-A53T). We postulate that there is a significant shift in chromatin structure in dopaminergic neurons due to the presence of the A53T α-syn mutation and subsequent redox stress.

Objective: Develop a bioinformatic pipeline to process the raw ChIP-seq reads, identify areas of open chromatin, and investigate these areas for the presence of different regulatory element and DNA binding motifs. If time permits, analyze the results from all 4 cell lines to identifying changes in chromatin structure that are found in both mutant cell lines but are absent in the control cell lines. The processing pipeline will consist of six main steps. First, quality control of the raw reads will be assessed. Second, the reads will be aligned to the human reference genome GRCh38. Third, reads will be filtered for unique alignment, and then duplicates will be removed. At this point, the data will be visualized to ensure quality alignment. Step five will consist of peak calling and finally, the identified peaks will be annotated to the nearest gene. In addition, regulatory elements and DNA binding motifs that are significantly present in each sample will be found.