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| Status |
Public on Mar 18, 2019 |
| Title |
A Chemical Toolbox for the Study of Bromodomains and Epigenetic Signaling |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by array
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| Summary |
Bromodomains (BRDs) are evolutionary conserved epigenetic protein interaction modules which recognize (“read”) acetyl-lysine, however their role(s) in regulating cellular states and their potential as targets for the development of targeted treatment strategies is poorly understood. Here we present a set of 25 chemical probes, selective tool small molecule inhibitors, covering 29 human bromodomain targets. We comprehensively evaluate the selectivity of this probe-set using BROMOscan® and demonstrate the utility of the set in studies of muscle cell differentiation and triple negative breast cancer (TNBC). We identified cross talk between histone acetylation and the glycolytic pathway resulting in a vulnerability of TNBC cell lines to inhibition of BRPF2/3 BRDs under conditions of glucose deprivation or GLUT1 inhibition. This chemical probe set will serve as a resource for future applications in the discovery of new physiological roles of bromodomain proteins in normal and disease states and as a toolset for bromodomain target validation.
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| Overall design |
C2C12 mouse myoblast cells were grown in DMEM containing 20% FBS. For differentiation experiments in the presence and absence of mentioned drugs, 0.3x106 cells were seeded and allowed to grow for 36 hrs before switching to differentiation media (DMEM containing 2% horse serum, 10 µg/ml insulin and 10 µg/ml Transferrin). Indicated drugs were added at the time of differentiation at 0.5 μM (JQ1, LP99) or 2 μM (GSK2801, BAZ2ICR, BSP) for 12h before they were harvested for extraction of total RNA. Total RNA was extracted using PureLinkTM RNA kit as per the manufacturer’s protocol.
Raw CEL data were processed in R (v.3.5.1) using Bioconductor (v.3.7) and the oligo package (v.1.44). Quality controls were carried out using the arrayQualityMetrics package (v.3.36.0) taking into account array intensity distributions, distance between arrays and variance mean-dependence. Principal component analysis was used to decide which arrays to process together. On this basis one of the three arrays treated with BSP was considered to be an outlier and was discarded (sample 'BSP_rep3', file 'BSP_rep3.CEL'), with the rest of the analysis performed without taking it into account. Background correction and normalization were carried out employing the Robust Multichip Array (RMA) method implemented in the oligo package. Data were filtered using the genefilter package (v.1.62.0) employing the nsFilter function and an interquartile range (IQR) cut-off of 0.28 (or >log(1.2), determined by the midpoint of the shortest interval containing half of the data. Probe sets that did not have an Entrez gene identifier were also removed. From the 29129 features available on the Clariom S Mouse HT chip, 14538 remained after filtering.
A linear model was applied employing the limma package (v.3.36.2) followed by empirical Bayesian analysis to determine differential expression between not-treated and treated samples. Genes were considered differentially expressed if the adjusted P-value, calculated using the Benjamini–Hochberg method in order to minimize false discovery rate, was less than 0.05 and the mean level of expression was greater than 1.5-fold. Data were annotated using the clariomsmousehttranscriptcluster.db annotation data (https://www.bioconductor.org/packages/release/data/annotation/html/clariomsmousehttranscriptcluster.db.html).
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| Contributor(s) |
Nakka K, Dilworth J, Filippakopoulos P |
| Citation(s) |
31015424 |
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| Submission date |
Jul 24, 2018 |
| Last update date |
May 02, 2019 |
| Contact name |
Panagis Filippakopoulos |
| E-mail(s) |
panagis.filippakopoulos@gmail.com
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| Organization name |
Oxford University
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| Department |
Nuffield Department of Medicine
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| Lab |
Structural Genomics Consortium
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| Street address |
ORCRB - Roosevelt Drive
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| City |
Oxford |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX3 7DQ |
| Country |
United Kingdom |
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| Platforms (1) |
| GPL24242 |
[Clariom_S_Mouse_HT] Affymetrix Clariom S Assay HT, Mouse (Includes Pico Assay) |
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| Samples (17)
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| Relations |
| BioProject |
PRJNA482645 |
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