AID	Name	Type	Comment
602436	Broad Institute Screen for Small Molecule Inhibitors of ATP Dependent Chromatin Remodeling Inhibitor Probe Project	summary

Keywords:
Mouse embryonic stem cells, ATP-dependent chromatin-remodeling complex, esBAF, Bmi1, cell-based reporter assay, inhibitor screen

Assay Overview:
Recent data indicate that the SWI/SNF-like, ATP-dependent chromatin-remodeling complex (Brg/Brahma Associated Factors complex, referred to as BAF) is both necessary and, in certain contexts, sufficient for the induction of pluripotency. The ATPase, Brg1 or Brm, is a central component of the complex. Mutations in these genes have been linked to human cancer. Recent genome-wide studies from a conditional Brg knockout ES cell line indicate that the esBAF complex can both activate and repress transcription and it frequently regulates promoters at a distance from its binding sites. This mode of regulation is significantly different from the regulation of SWI/SNF complexes in Saccharomyces cerevisiae, which exclusively activate genes. A major group of genes directly repressed by esBAF are members of the Polycomb repressive complex, including Bmi1, Cbx7, Ring1, Phc1, Phc2, and Suz12. We selected Bmi1 as a reporter for esBAF activity and have developed a murine embryonic stem cell (mESC) line with a firefly luciferase reporter knocked into the Bmi1 locus. Bmi1 is a particularly interesting target due to its essential role in the maintenance and self-renewal of hematopoietic and neural stem cells. These modified mESCs were treated with compounds for 24 hours and Bmi-1 expression is measured with the Steady Glo luciferase reagnet (Promega). The identified inhibitors from the screen will provide the precision and temporal control needed to tease out the precise functions and mechanisms of the BAF complex. These compounds will prove useful to researchers studying stem cells, chromatin remodeling, and cancer. Another important reason for selecting Bmi1 is that compounds regulating the expression of Bmi1 will be interesting independent of their effect on the BAF complex.

Expected Outcome:
The esBAF complex represses the transcription of Bmi1 and firefly luciferase is knocked into the Bmi1 locus. Therefore, an inhibitor of the esBAF complex will cause an increase in signal (i.e. increase firefly luciferase activity).

Luciferase Reporter Assay Protocol
Materials
Corning CellBIND Surface T175 Flasks (Cat# 3292) and Corning HYPERFlasks (Cat# 10024)
Corning customized 384-well white CellBIND surface assay plates

ES media:
150 mL ES FBS (Applied Stem Cell, ASM-5007)
10 ml HEPES (GIbco 15630)
10 mL Sodium Pyruvate (Gibco 11360)
10 mL Pen/Strep/Glutamine (Invitrogen 10378-016)
10 mL Non essential amino acids (Gibco 1140)
1 mL Beta-mercaptoethanol (Gibco 21985-023)
2 mL LIF conditioned media (from Assay Provider)
800 mL DMEM (Gibco 11960) for Propagation media or
800 mL DMEM no Phenol Red (Gibco 31053) for Assay media

Cell line Development
A stable mESC line with a luciferase reporter knocked into the Bmi1 locus was developed. The cell line was confirmed by Southern blot and the neomycin cassette was deleted using transfected Cre recombinase. The parental cell line requires mouse embryonic fibroblast (MEF) feeder cells for growth and maintenance of pluripotency. A feeder-free cell line, BLR FF, was established for high throughput screening through multiple passage selection on gelatin surfaced tissue culture vessels with uninpaired growth and a mESC phenotype.

Culturing cells
1.#Thaw 3 million BLR FF mouse embryonic stem cells in 40mL Propagation media to a T175 CellBIND Flask.
2.#Culture the cells in a 37 degrees C, 5% CO2 incubator.
3.#Change media daily and split the cells after 3 days with 0.25% Trypsin-EDTA (Gibco, 25200-056).
4.#Add 20 million cells per HYPERFlask filled with Propagation media; Change media daily.
5.#Trypsinize the cells from HYPERFlask after 3 days.
6.#Count the cells and plate 10,000 cells in 30uL of Assay media per well to a 384-well white CellBIND surface plate.
7.#Culture the plates in a 37 degrees C, 5% CO2 incubator overnight.

Compound Treatments
1.#Pin transfer 100nL/well of 3.75mM compound library along with 100nL/well of 0.75mM of Positive control (Pubchem SID: 85814977) to the assay plates based on plate map on GS.
2.#Return the assay plates to the incubator for 24 hours compound treatment.

Reporter level detection
1.#Take the assay plates from incubator and equilibrate for 10 minutes to room temperature
2.#Dispense 10uL/well of reconstituted Promega SteadyGlo solution to each well of the assay plates
3.#Shake the assay plates at 1000 rpm for 15 second at room temperature
4.#Incubate the assay plates for another 10 minutes at room temperature
5.#Read on a Perkin Elmer Envision in Ultra Sensitive Luminescence mode

PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.

PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 20.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

Grant Number: R03 DA032469-01