Comment[ArrayExpressAccession] E-GEOD-37270 MAGE-TAB Version 1.1 Public Release Date 2013-09-20 Investigation Title BET bromodomain inhibition suppresses TH17-mediated pathology Comment[Submitted Name] BET bromodomain inhibition suppresses TH17-mediated pathology Experiment Description IL-17-producing T helper (TH17) cells have been selected through evolution for their ability to control fungal and bacterial infections. It is also firmly established that their aberrant generation and activation results in autoimmune conditions. Using a characterized potent and selective small molecule inhibitor, we show that the bromodomain and extra-terminal domain (BET) family of chromatin adaptors plays fundamental and selective roles in human and murine TH17 differentiation from naM-CM-/ve CD4+ T cells, as well as in the activation of previously differentiated TH17 cells. We provide evidence that BET controls TH17 differentiation in a bromodomain-dependent manner through a mechanism that includes the direct regulation of multiple effector TH17-associated cytokines, including IL17, IL21 and GMCSF. We also demonstrate that BET family members Brd2 and Brd4 associate with the Il17 locus in TH17 cells, and that this association requires bromodomains. We recapitulate the critical role of BET bromodomains in TH17 differentiation in vivo and show that therapeutic dosing of the BET inhibitor is efficacious in mouse models of autoimmunity. Our results identify the BET family of proteins as a fundamental link between chromatin signaling and TH17 biology, and support the notion of BET inhibition as a point of therapeutic intervention in autoimmune conditions. 4 samples were analyzed: two conditions in duplicate. Naive T cells were placed in conditions leading to TH17 differentiation, with and without BET inhibitor. RNA was collected at 48 hours. 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 Bryant Mele Salmeron Ghosh Huang Bryant Lora Person First Name Barbara Deanna Andres Srimoyee Hon-Ren Barbara Jose Person Mid Initials M A M M Person Email barbara.bryant@constellationpharma.com Person Affiliation Constellation Pharmaceuticals Person Phone 617-714-0561 Person Address Constellation Pharmaceuticals, 215 First Street, Cambridge, MA, USA Person Roles submitter Protocol Name P-GSE37270-1 P-GSE37270-5 P-GSE37270-6 P-GSE37270-2 P-GSE37270-3 P-GSE37270-4 P-GSE37270-7 Protocol Description CEL files were processed by the Affymetrix Expression Console software package using the gene-level RMA algorithm on the core set of probesets and the annotation file Huex-1_0-st-v2_na31_hg19 provided by Affymetrix. MoEx-1_0-st-v1.r2.pgf ID_REF = VALUE = Quantile normalized gene level expression values from RMA via Expression Console Total RNA was amplified using the NuGEN ApplauseM-bM-^DM-" WT Amp Plus ST Amplification System. First-strand synthesis of cDNA was performed using a unique first-strand DNA/RNA chimeric primer mix, resulting in cDNA/mRNA hybrid molecules. Following fragmentation of the mRNA component of the cDNA/mRNA molecules, second-strand synthesis was performed and double-stranded cDNA was formed with a unique DNA/RNA heteroduplex at one end. In the final amplification step, RNA within the heteroduplex was degraded using RNaseH, and replication of the resultant single-stranded cDNA was achieved through DNA/RNA chimeric primer binding and DNA polymerase enzymatic activity. A modification step targeting the amplified single-stranded cDNA resulted in the generation of sense target (ST) cDNA product compatible with the Affymetrix GeneChipM-. Exon arrays. The ST-cDNA was purified for accurate quantitation and to ensure optimal performance during the fragmentation and labeling process. The ST- cDNA was assessed using spectrophotometric methods in combination with the Agilent Bioanalyzer. The appropriate amount of amplified ST-cDNA was fragmented and labeled using the EncoreM-bM-^DM-" Biotin Module. The enzymatically and chemically fragmented product (50-100 nt) was labeled via the attachment of biotinylated nucleotides onto the 3'-end of the fragmented ST-cDNA. The fragmented and labelled ST-cDNA was added to the hybridization cocktail in accordance with the NuGENM-bM-^DM-" guidelines for hybridization onto Affymetrix GeneChipM-. arrays. Following the hybridization for 18 hours at 45M-0C in an Affymetrix GeneChipM-. Hybridization Oven 640, the array was washed and stained on the GeneChipM-. Fluidics Station 450 using the appropriate fluidics script. NaM-CM-/ve CD4+ T cells were treated with 150 nM JQ1 or DMSO under TH17 polarizing conditions for 48h. NaM-CM-/ve CD4 T cells were cultured in 24-well plates (1x106 cells/ml) and stimulated with anti-CD3/CD28 coated beads (Dynabeads 11452D; Invitrogen) for 4 days under TH17, polarizing conditions: 5 ng/ml TGF-M-NM-21 (100-B; R&D Biosystems), 20 ng/ml IL-6 (406-ML; R&D Biosystems), 20 ng/ml IL-23 (1887-ML-010; R&D Biosystems), 10 M-NM-