Unknown,Transcriptomics,Genomics,Proteomics

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Activation of the Integrated Stress Response by a Jumonji Histone Demethylase Inhibitor Induces Pro-Apoptotic Metabolic Reprogramming in Multiple Myeloma

ABSTRACT: Regulation of gene expression by chromatin modification through methylation of histone lysine residues is a dynamic, reversible process that when deregulated is associated with cancer development. In multiple myeloma, combined inhibition of the histone demethylases JARID1B, UTX and JmjD3 by the small molecule GSK-J4 prevents cellular glutamine utilization leading to amino acids deprivation, activates the integrated stress response via GCN2-dependent ATF4 activation, and induces apoptosis. This response is associated with a profound upregulation of metallothionein genes. Combined with clinical data demonstrating that overexpression of JARID1B is associated with shorter survival in multiple myeloma patients, this study highlights histone demethylases as epigenetic drug targets and places this demethylase inhibitor chemotype as having unique potential relative to established anti-myeloma treatment options. In total there are 7 different samples analyzed and one input control. Treatments are carried out with the demethylase inhibitor (or DMSO as negative control) at 6h and 48h, or with LNA targeting demethylases (or scrambled LNA) at 7 days. A negative control at 0h is included.

ORGANISM(S): Homo sapiens

SUBMITTER: Laurynas Pliuskys

PROVIDER: E-GEOD-60626 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Identification and characterization of HP1BP3 (a human histone H1 homologue) as a novel chromatin retention factor essential for the co-transcriptional processing of pri-miRNA. We generated BAC transgenic cells at 80% confluency (~1x107) were cross-linked with 1% formaldehyde for 10 minutes at 37Â°C, and quenched with 125 mM glycine at room temperature for 5 minutes. The fixed cells were washed twice with cold PBS, scraped, and transferred into 1 ml PBS containing protease inhibitors (Roche). After centrifugation at 700 g for 4 minutes at 4Â°C, the cell pellets were resuspended in 100 Î¼l ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4Â°C with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 15 minutes). The sheared chromatin with a fragment length of ~200 â 600 bp) was centrifuged at 20,000 g for 15 minutes at 4Â°C). 100 Î¼l of the supernatant was used for ChIP or as input. A 1:10 dilution of the solubilized chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl 16.7 mM Tris-HCl [pH 8.1]) was incubated at 4Â°C overnight with 6 Î¼g/ml of a goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP). Immunoprecipitation was carried out by incubating with 40 Î¼l pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4Â°C, followed by five washes for 10 minutes with 1ml of the following buffers: Buffer I: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl; Buffer II: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl; Buffer III: 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]; twice with TE buffer [pH 8.0]. Elution from the beads was performed twice with 100 Î¼l ChIP elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature (RT) for 15 minutes. Protein-DNA complexes were de-crosslinked by heating at 65Â°C in 192 mM NaCl for 16 hours. DNA fragments were purified using QiaQuick PCR Purification kit (QIAGEN) and eluted into 30 Î¼l H2O according to the manufacturerâs protocol after treatment with RNase A and Proteinase K.

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Activation of the Integrated Stress Response by a Jumonji Histone Demethylase Inhibitor Induces Pro-Apoptotic Metabolic Reprogramming in Multiple Myeloma

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