qHTS Assay for Epigenetic Modulators
The Locus Derepression assay detects the derepression of a GFP reporter that is stably integrated in a region of the genome of murine c127i mammary cells that is presumably silenced. GFP transcription in this construct is controlled by a CMV promoter, which normally is strong and constitutively active. However, this line was selected for lack of constitutive expression of the GFP protein. GFP more ..
BioActive Compounds: 57
Depositor Specified Assays
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]
MLSCN Grant: 1 X01 MH079860-01
Assay Provider: Elisabeth D. Martinez, University of Texas SW Medical Center
NCGC Assay Overview:
The Locus Derepression assay detects the derepression of a GFP reporter that is stably integrated in a region of the genome of murine c127i mammary cells that is presumably silenced. GFP transcription in this construct is controlled by a CMV promoter, which normally is strong and constitutively active. However, this line was selected for lack of constitutive expression of the GFP protein. GFP production can be induced by incubating the cells with histone deacetylase or DNA methyltransferase inhibitors. Compounds that cause derepression of the locus to express GFP are identified by enumerating GFP positive cells using a laser-scanning microplate cytometer.
For further details of this screen and follow-up characterization, see R.L. Johnson, W. Huang, A. Jadhav, C.P. Austin, J. Inglese, E.D. Martinez, A Quantitative High-Throughput Screen Identifies Potential Epigenetic Modulators of Gene Expression, Analytical Biochemistry (2008) 375(2):237-48.
NCGC Assay Protocol Summary:
Two hundred and fifty cells in 5 uL per well were dispensed into black, clear-bottom 1536-well plates. Twenty-three nL compound was transferred to the assay plate and cells were incubated 30 hours at 37 C. Medium was removed, cells were washed two times with PBS, and GFP-positive cells were enumerated as fluorescent objects between the sizes of 20 to 120 um width and depth using an Acumen Explorer.
Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, qHTS, NCGC, qHTS, HDAC, DNMT, HAT, DNA methyltransferases, histone acetyl transferases, histone deacetylases
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Classification". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Data Table (Concise)