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Retinoid X receptor activation alters the chromatin landscape ot commit mesenchymal stem cells to the adipose lineage

ABSTRACT: We set out to study the effects of the endocrine disrupting chemical tributyltin (TBT) on early lineage commitment of primary bone marrow-derived mesenchymal stem cells (MSCs) from the long bones of C57BL/6 mice. We previously showed that prenatal exposure to nanomolar levels of TBT results in increased adiposity in mice. TBT is an agonist of two nuclear receptors, peroxisome proliferator-activated receptor gamma (PPARγ) and retinoid X receptor (RXR), master regulators of adipogenesis. To test if TBT could influence early MSC lineage commitment we treated these cells with 50 nM TBT, 100 nM Rosiglitazone (ROSI, a pure PPARγ agonist), 100 nM AGN194204 (4204, a pure RXR agonist), and vehicle control (0.1% DMSO) for 48 hours prior to differentiation with a standard adipogenic cocktail. This experiment revealed an RXR-dependent commitment to the adipose lineage. To better understand how TBT induces adipose commimtment, we sequenced RNA from MSCs treated with DMSO (0.1%), ROSI (100 nM), 4204 (100 nM), and TBT (50 nM) for 48 hrs, prior to any differentiation. Unbiased hierarchical clustering analysis showed a clear separation between DMSO and ROSI replicates from 4204 and TBT, strengthening our conclusion that the observed phentype is RXR-dependent. Pathway analysis of differentially expressed genes revealed that TBT and 4204 de-repress targets of the repressive histone modifier Enhancer of zeste 2 (EZH2), the catalytic member of the Polycomb repressive complex 2 (PRC2), which deposits H3K27me3 on histones. We there for preformed ChIP-Seq analysis on MSCs treated with DMSO (0.1%) or TBT (50 nM) for 48 hrs, hypothesizing that TBT would reduce H3K27me3 in proximity to genes that regulate adipose lineage commitment.

ORGANISM(S): Mus musculus

PROVIDER: GSE99565 | GEO | 2017/08/01

SECONDARY ACCESSION(S): PRJNA388939

REPOSITORIES: GEO

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Project description:We used mass spectrometry-based proteomics to unravel anaplastic lymphoma kinase (ALK) signaling in the ALK and MYCN amplified neuroblastoma cell line, NB1. We specifically measured the ALK interactome, phosphoproteome, phosphotyrosine interactome and proteome as outlined below. ALK Interactome and phosphoproteome: For each experiment, three SILAC conditions (‘Light’: Lys0,Arg0, ‘Medium’: Lys4,Arg6, ‘Heavy’: Lys8,Arg10) of NB1 cells were treated for 30 minutes with each of the following ALK-targeting small-molecule inhibitors: TAE684 (100 nM), crizotinib (500 nM), and LDK378 (250 nM). The experiments were performed as specified below: Experiment 1: Light: 500 nM crizotinib, Medium: 100 nM TAE684, Heavy: 0.1% DMSO Experiment 2: Light: 100 nM TAE684, Medium: 250 nM LDK378, Heavy: 0.1% DMSO Experiment 3: Light: 250 nM LDK378, Medium: 500 nM crizotinib, Heavy: 0.1% DMSO These three triple-SILAC experiments allowed the effect of each inhibitor to be measured in duplicates. ALK phosphotyrosine interactome: To identify phosphotyrosine-specific interaction partners of ALK we used a label-free mass spectrometry-based proteomics approach. Interacting proteins to the peptide sequences (phosphorylated and non-phosphorylated peptide) indicated in the table below were identified by a peptide pull-down assay and mass spectrometry. Peptide pull-downs were performed on NB1 cell lysate prepared from cells treated for 30 minutes with LDK378 (250 nM) or DMSO. Each peptide pull-down was performed on lysates from two biological replicates. Sample name (raw file) Gene Uniprot Site Peptide sequence A1 ALK Q9UM73 pY1078 ELQSPEpYKLSKLR A2 ALK Q9UM73 pY1092 RTIMTDpYNPNYSF A3 ALK Q9UM73 pY1096 TDYNPNpYSFAGKT A4 ALK Q9UM73 pY1131 GAFGEVpYEGQVSG A5 ALK Q9UM73 pY1278 GMARDIpYRASYYR A6 ALK Q9UM73 pY1282 DIYRASpYYRKGGS A7 ALK Q9UM73 pY1283 IYRASYpYRKGGSA A8 ALK Q9UM73 pY1359 RSPGPVpYRIMTQS A9 ALK Q9UM73 pY1507 RLWNPTpYGSWFTE A10 ALK Q9UM73 pY1584 RSGNVNpYGYQQQG A11 ALK Q9UM73 pY1604 APGAGHpYEDTILK A12 ALK Q9UM73 Y1078 ELQSPEYKLSKLR B1 ALK Q9UM73 Y1092 RTIMTDYNPNYSF B2 ALK Q9UM73 Y1096 TDYNPNYSFAGKT B3 ALK Q9UM73 Y1131 GAFGEVYEGQVSG B4 ALK Q9UM73 Y1278 GMARDIYRASYYR B5 ALK Q9UM73 Y1282 DIYRASYYRKGGS B6 ALK Q9UM73 Y1283 IYRASYYRKGGSA B7 ALK Q9UM73 Y1359 RSPGPVYRIMTQS B8 ALK Q9UM73 Y1507 RLWNPTYGSWFTE B9 ALK Q9UM73 Y1584 RSGNVNYGYQQQG B10 ALK Q9UM73 Y1604 APGAGHYEDTILK C3 IRS2 Q9Y4H2 pY675 SSRSDDpYMPMSPA C4 IRS2 Q9Y4H2 pY742 SPEDSGpYMRMWSG C5 IRS2 Q9Y4H2 pY978 RSPLSDpYMNLDFS C6 IRS2 Q9Y4H2 Y675 SSRSDDYMPMSPA C7 IRS2 Q9Y4H2 Y742 SPEDSGYMRMWSG C8 IRS2 Q9Y4H2 Y978 RSPLSDYMNLDFS Additional explanations to MS raw files: -1 extension indicates Control (DMSO lysate) replicate 1. -2 extension indicates LDK378 (treated lysate) replicate 1. -3 extension indicates Control (DMSO lysate) replicate 2. -4 extension indicates LDK378 (treated lysate) replicate 2. Proteome: The NB1 cell line proteome was measured by a label-free mass spectrometry-based approach. The analysis was performed in two biological replicates.

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Retinoid X receptor activation alters the chromatin landscape ot commit mesenchymal stem cells to the adipose lineage

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