1. Studying Ascl1-GSX2 interactions
Juliana Madzia
Mentor: Kaushik Roychoudhury
PI: Kenny Campbell

2. Transcription Factor Regulation of Progenitor Maturation
During development, Ventral telencephalic progenitors differentiate and give rise to neurons, oligodendrocytes, and astrocytes.
The early progenitors divide to form more progenitors or give rise to intermediate progenitors that mature into neurons or glia
Presence of GSX2 in the LGE progenitors keep them from differentiating
Presence of GSX1 and Ascl1 in the progenitors help them differentiate
GSX2 appears to be upstream of Ascl1 in LGE progenitors

3. Background
While performing a variety of candidate protein-protein interaction tests, we found that in the yeast, Ascl1 (Mash1) binds directly to GSX2, but not GSX1
We could co-precipitate Mash1 while pulling down GSX2, both from in vitro synthesized protein mixture as well as from E12.5 embryo telencephalon.

4. Question #1
Can GSX2 neutralize the ability of Mash to bind to its DNA target sequence and reduce Mash1 mediated transcription?

5. Luciferase assay Basic Construct Promoter
Binding site
Reporter (Firefly luciferase)

6. Luciferase Assay Mash Only Promoter Reporter (Firefly luciferase)
Binding site
Reporter (Firefly luciferase)

7. Luciferase Assay Mash and GSX2 Promoter Mash1
Binding site
Reporter (Firefly luciferase)

8. Kaushik’s old data on high affinity e-box (In HEK 293T cells)
Fold increase over control
Increasing GSX2
12.5ng Mash

9. Kaushik’s old data on Delta N E-box (Low affinity E-box) in P-19 cells

10. Experimental Procedure
Plated 2x104 NIH3t3 fibroblast cell line in wells of 48 well plates
Incubated in a 5% CO2 incubator at 37°C for 12 hours
Transfected cells with different combinations of Delta enhancer Luciferase construct, Ascl1, Gsx2, Beta actin promoter driven renilla luciferase and empty pCDNA6V5 plasmid.
Incubated another 48 hours in 5% CO2, 37°C
Lysed cells, transferred lysate to luminometer and quantified luminiscence
Recorded firefly luciferase and renilla luciferase activity

11. Brian made some low copy High and low affinity e-boxes
High Affinity E-Box
Firefly/Renilla
Low Affinity E- Box
Mash (Ng)

12. Delta M Long
Firefly/renilla
Firefly/renilla
Ng of Mash
Ng of Mash
Firefly/renilla
Ng Mash

13. Error bar = 1 Standard Deviation of three independent cultures
Results
16ng Mash1
64ng Mash1
14 fold
6.5 fold
Error bar = 1 Standard Deviation of three independent cultures

14. Question #2
Do Mash1 and GSX2 physically interact in mammalian cells?

15. Proximity Ligation Assay
A pair of oligonucleotide labeled secondary antibodies (PLA probes) generates a signal only when the two PLA probes have bound in close proximity
The signal from each detected pair of PLA probes is visualized as an individual fluorescent spot
PLA signals can be quantified (counted) and assigned to a specific subcellular location based on microscopy images

16. 1st Principle
The samples are incubated with primary antibodies that bind to the protein(s) to be detected.

17. 2nd Principle
Secondary antibodies conjugated with oligonucleotides (PLA probe MINUS and PLA probe PLUS) are added to the reaction and incubated.

18. 3rd Principle
The Ligation solution, consisting of two oligonucleotides and Ligase, is added and the oligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity.

19. 4th Principle
The Amplification solution is added together with Polymerase. The oligonucleotide arm of one of the PLA probes acts as a primer for a rolling-circle amplification reaction using the ligated circle as a template, generating a concatemeric (repeated sequence) product. The fluorescently labeled oligonucleotides will hybridize to the RCA product. The signal is easily visible as a distinct fluorescent spot and analyzed by fluorescence microscopy.

20. Protocol Summary Add blocking solution to each sample.
Add two diluted primary antibodies.
Dilute the two PLA probes with your chosen buffer and add to the samples.
Add Ligation-Ligase solution.
Add Amplification-Polymerase solution.
Carry out a final wash and leave to dry in the dark.
Mount slides with DAPI and perform microscopy with fluorescence or confocal microscope.

21. Typical Results One antibody, positive and negative control
Two antibodies, positive and negative control

22. In our assay we used: Cells: MNS-70 cells developed by Masato Nakafuku
These cells have high Ascl1 and GSX2 levels.
Antibodies: 1- Goat Anti ASH1 (Mash1/Ascl1)
2- Rabbit Anti GSX1/2
PLA Probes: anti goat (plus) ; anti rabbit (minus)
Block: 10% Normal Donkey Serum in Tris Buffered Saline with 0.1% Tween 20
Detection reagent: Orange- (Cy3 spectra)

23. Microscopy Result
Blue: DAPI Purple: Areas where GSX2 and Ascl1 are in close proximity

24. Results
There were many cells that did not have any of the desired proteins in close proximity (25 nm) to one another, but there were some that did show to be in close proximity.
All of the regions in which Ascl1 and GSX2 were shown to be within close proximity to each other were found in the nuclear regions of the cells.