p53 is a central regulator driving neurodegeneration caused by C9orf72 poly(PR)

To analyze the epigenomic landscape of neurodegeneration caused by ALS-associated protein aggregation, we developed a modified version of ATAC-seq that works on primary neurons. We discovered that C9orf72-ALS/FTD associated poly(PR) activated a remarkably specific signature of chromatin accessibility, involving transcriptional targets of the tumor suppressor gene p53. Our findings reveal an unexpected role of p53 as a mediator of neurodegeneration elicited by poly(PR) and provide an example of how ATAC-seq can now be applied to neurons to define mechanisms of neurodegeneration. RNA and DNA were isolated from primary neurons treated for 20, 40 and 60h with either GFP, (PR)50, TDP-43 or untreated. Neurons were grown on 12 well plates each with 450,000 neurons and for each condition 4 biological replicates were pooled together and then split into 2 groups. To obtain RNA from the same neurons that were used for ATAC-seq, we performed sequential lysis. Neurons were first lysed in ATAC-seq resuspension RSB buffer, followed by sequential RNA extraction and isolation in RLT buffer of the QIAGEN RNeasy Plus Micro Kit (QIAGEN), according to the manufacturer’s protocol. In brief, cells were lysed, homogenized, and loaded on a genomic DNA (gDNA) eliminator column. After removal of genomic DNA, RNA was purified using RNeasy spin columns. RNA quantity and purity were determined by optical density measurements of OD260/280  and OD230/260  using a NanoDrop spectrophotometer. Structural integrity of the total RNA was assessed using a 2200 TapeStation Instrument with RNA ScreenTapes (Agilent Technologies, Santa Clara, CA). mRNA libraries were prepared using a SureSelect Strand-Specific RNA Library Preparation kit for Illumina (G9691B) on an Agilent Bravo Automated Liquid Handling Platform, following the manufacturer’s protocol. Libraries were sequenced on an Illumina HiSeq 4000. For the ATACseq Primary cortical neurons treated for 20, 40 and 60h with either GFP, (PR)50, TDP-43 or untreated, were pretreated with 200 U/ml DNase (Worthington) for 30 min at 37°C to remove free-floating DNA and to digest DNA from dead cells. This medium was then washed out, and the cells were washed in cold PBS four times. To avoid neuronal cell death that is associated with trypsinization, neurons were directly lysed in 1ml of cold ATAC-seq resuspension buffer (RSB; 10mM Tris-HCl pH 7.4, 10mM NaCl, and 3mM MgCl2 in water, containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin) on ice for 10 min. After lysis cells nuclei were collected and counted, 50,000 viable cells were centrifuged at 500xg for 5 min in a pre-chilled (4°C) fixed-angle centrifuge. After centrifugation, supernatant was carefully aspirated, with two pipetting steps; The remaining 100μl of supernatant was carefully aspirated by pipetting with a P200 pipette tip to avoid the cell pellet. Cell pellets were then resuspended in 1 ml of ATAC-seq RSB containing 0.1% Tween-20 (without NP40 or digitonin), and the tubes were inverted to mix. Nuclei were then centrifuged for 10 min at 500 RCF in a pre-chilled (4°C) fixed-angle centrifuge. Supernatant was carefully removed with two pipetting steps, as described before, and nuclei were resuspended in 50μl of transposition mix (25μl 2× TD buffer (20 mM Tris-HCl pH 7.6, 10 mM MgCl2, 20% Dimethyl Formamide), 2.5μl transposase (100 nM final), 16.5μl PBS, 0.5μl 1% digitonin, 0.5μl 10% Tween-20, and 5μl water) by pipetting up and down six times. Transposition reactions were incubated at 37°C for 30 min in a thermomixer with shaking at 1,000 r.p.m. Reactions were cleaned up with Zymo DNA Clean and Concentrator columns. The complete eluate underwent PCR, as follows. After initial extension, 5 cycles of pre-amplification using indexed primers (Buenrostro et al., 2015) and NEB Next High-Fidelity 2X PCR Master Mix (NEB) were conducted, before the number of additional cycles was assessed by quantitative PCR using SYBR Green. Typically, 7-9 additional cycles were run. The final library was purified using a MinElute PCR kit (QIAGEN) and quantified using a Qubit dsDNA HS Assay kit (Invitrogen) and a High Sensitivity DNA chip run on a Bioanalyzer 2100 system (Agilent). All libraries were sequenced using 150bp Nextseq High Output Cartridge kits and a Nextseq 500 sequencer (Illumina).