Comment[ArrayExpressAccession] E-GEOD-54443 MAGE-TAB Version 1.1 Public Release Date 2014-03-02 Investigation Title Mutant Huntingtin promotes neuronal death through cell autonomous microglial activation via myeloid lineage- determining factors Comment[Submitted Name] Mutant Huntingtin promotes neuronal death through cell autonomous microglial activation via myeloid lineage- determining factors Experiment Description Huntington's Disease (HD) is a fatal neurodegenerative disorder caused by an extended polyglutamine repeat in the N-terminus of the huntingtin (Htt) protein. Reactive microglia and elevated cytokine levels are observed in the brains of HD patients, but the extent to which neuroinflammation results from extrinsic or cell-autonomous mechanisms is unknown. Furthermore, the impact of microglia activation on the pathogenesis of HD remains to be established. Using genome-wide approaches, we show that expression of mutant Htt in microglia promotes cell-autonomous pro-inflammatory transcriptional activation within microglia by increasing the expression and transcriptional activities of the myeloid lineage-determining factors PU.1 and C/EBPs. Elevated levels of PU.1 and its target genes are observed in the brains of mouse models and HD individuals. Moreover, mutant Htt expressing microglia exhibit an increased capacity to induce neuronal death ex vivo and in vivo in the presence of sterile inflammation. These findings suggest that expression of mutant Htt in microglia may contribute to neuronal pathology in Huntingtin disease. RNA-Seq and ChIP-Seq for PU.1, C/EBP, and H3K4me2 in BV2 cells and RNA-Seq in primary microglia and macrophages Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Benner Crotti Benner Glass Person First Name Christopher Andrea Christopher Christopher Person Mid Initials K Person Email cbenner@salk.edu Person Affiliation Salk Institute Person Address Integrative Genomics and Bioinformatics Core, Salk Institute, 10010 North Torrey Pines Rd, La Jolla, CA, USA Person Roles submitter Protocol Name P-GSE54443-4 P-GSE54443-6 P-GSE54443-1 P-GSE54443-3 P-GSE54443-5 P-GSE54443-2 Protocol Description RNA-Seq: Reads were aligned to the mouse mm9 genome (NCBI Build 37) using STAR (v2.0.3e). RNA-Seq experiments were normalized and visualized by using HOMER (http://biowhat.ucsd.edu/homer/) to generate custom tracks for the UCSC Genome Browser (http://genome.ucsc.edu/). Gene expression values were generating for RefSeq annotated transcripts using HOMER and differential expression calculations were performed using edgeR. RNA-Seq RPKM tab-delimited text file is available on series record. Columes: 1: RefSeq ID, 2: chr, 3: gene start, 4: gene end, 5: strand, 6: mRNA length, 7: Copies of gene in genome, 8: Annotation information, 9-18: RPKM gene expression values Genome_build: mm9 ChIP-Seq: Reads were aligned to the mouse mm9 genome assembly (NCBI Build 37) using Bowtie allowing up to 2 mismatches. Only tags that mapped uniquely to the genome were considered for further analysis. ChIP-Seq experiments were normalized and visualized by using HOMER (http://biowhat.ucsd.edu/homer/) to generate custom tracks for the UCSC Genome Browser (http://genome.ucsc.edu/). Peak finding, motif finding, and peak annotation were performed using HOMER. Genome_build: mm9 pCDH, Nwt, and Nmu Constructs: N-terminus human wild-type and N-terminus mutant huntingtin have been cloned from pCAG-Htt19550-15Q and pCAG-Htt1955-128Q respectively into MCS of pCDH-CMV-MCS-EF1-Puro (System Bioscience) using EcoRI and NotI. Lentiviral production and BV2 cells transduction were performed according to the manufacturer's protocol. Control cell line was generated by transducing BV2 cells with Lentivirus obtained from pCDH-CMV-MCS-EF1-Puro (empty vector). Validation of plasmids used in this study was performed by Western blotting. RNA was purified using RNeasy Mini Kit (Qiagen) and enriched for Poly(A)-RNA with MicroPoly(A) Purist Kit (Ambion). Subsequently, RNA was treated with TURBO DNase (Ambion), fragmented with RNA Fragmentation Reagents (Ambion) and purified by a P-30 column (Biorad). Fragmented RNA was dephosphorylated with Antarctic phosphatase (New England Biolabs) heat inactivated and precipitated over-night. Poly(A)-tailing and cDNA synthesis was performed as previously described. For reverse transcription, oligos with custom barcodes (underlined) were used: 5M-bM-^@M-2-Phos-CA/TG/AC/GT GATCGTCGGACTGTAGAACTCT/idSp/CAAGCAGAAGACGGCATACGATTTT TTTTTTTTTTTTTTTTVN-3M-bM-^@M-2. Subsequently, exonuclease was used to remove the excess oligo. After heat-inactivation, RNA was hydrolyzed by alkaline treatment (100mM NaOH) and heat at 95M-0C for 25 min. The cDNA fragments of 50M-bM-^@M-^S150 nucleotides were purified on a denaturing Novex 10% polyacrylamide TBE-urea gel (Invitrogen). The recovered cDNA was circularized, linearized, amplified for 12 cycles, and gel purified as previously described. The library was sequenced on Illumina sequencers according to the manufacturer's instructions. 20x10^6 cells were crosslinked in Formaldehyde/PBS 1% for 10 min at RT. After quenching the reaction by adding 125mM glycine, cells were washed 2X with PBS and were centrifuged (8 min, 800g, 4M-0C). Cells were resuspended in swelling buffer (10mM HEPES/KOH pH 7.9, 85mM KCl, 1mM EDTA, 0.5% IGEPAL CA-630, 1X protease inhibitor cocktail (Roche), 1mM PMSF) for 5 min. Cells were spun down and resuspended in 500M-NM-