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Studying Ascl1-Gsx2 interactions using a Luciferase reporter assay
Juliana Madzia
Mentor: Kaushik Roychoudhury
PI: Kenneth Campbell
University of Cincinnati and Cincinnati Childrens Hospital Medical Center
Introduction:
Mouse as a model system to study brain
development
Introduction:
Transcription factors in brain development
• The mammalian brain is a complex organ composed of millions of
neurons and glia
•
•
Mice have significantly fewer neurons than humans (Millions as
opposed to billions), making them easier to study
• Transcription factors bind to DNA and facilitate transcription of
specific genes
•
The ventral forebrain neural progenitors (neural stem cells) in
mice give rise to the same types of neuron as in humans
• During fetal development, transcription factors determine how
neurons in the brain develop and mature.
•
Many types of genetic manipulations can be done in mice that
facilitate understanding the mechanisms of embryonic
development
A few transcription factors like Gsx2, Gsx1 and Ascl1
regulate progenitor maturation in the ventral
telencephalon
VZ
SVZ
SVZ - intermediate progenitors
The house mouse (Mus musculus) is an excellent model to study
brain development
• A class of protein molecules called transcription factors governs the
expression of genes in the developing brain
• Mutations and abnormalities in transcription factor expression leads
to a multitude of syndromes ranging from mild to severe mental
disabilities.
VZ - primary progenitors
-
Neural progenitors in the ventral
telencephalon give rise to neurons that
then migrate to the cortex, striatum, or
olfactory bulb.
The VZ houses the primary (early)
progenitors while the SVZ houses more
mature intermediate progenitors. LGE
progenitors give rise to neurons.
Goulburn et al. 2012. Stem Cell Res. 8:412
Luciferase reporter assay
What is the mechanism of Gsx2–control of Ascl1?
• Gsx2 protein directly interacts with Ascl1 protein
• During development, Ventral telencephalic progenitors differentiate and give rise to
neurons, oligodendriocytes and astrocytes.
• This binding is expected to block the DNA binding domain of Ascl1
• The early progenitors divide to form more progenitors or give rise to intermediate
progenitors that mature into neurons or glia
• Gsx2 binding prevents Ascl1 form binding other transcription factors like E12
and E14
Source: Promega
Light
Ascl1
• Gsx2 inhibits Ascl1 binding to its target sequence of oligonucleotides
• Presence of Gsx2 in the LGE progenitors keep them from differentiating
E-box on Delta Long
Luciferase
• It is not clear yet if Gsx2 can inhibit transcription factor activity of Ascl1
• Presence of Gsx1 and Ascl1 in the progenitors help them differentiate
• Gsx2 appears to be upstream of Ascl1 in LGE progenitors
Hypothesis:
Light
Gsx2 should be able to reduce Ascl1 mediated gene expression in cells
Source: Piercenet.com
E-box on Delta Long
Methods
Luciferase
Conclusions
Results
35
NIH3t3 fibroblast cell line in wells of 48 well plates
3. Transfected cells with different combinations of Delta enhancer
Luciferase construct, Ascl1, Gsx2, Beta actin promoter driven renilla
luciferase and empty pCDNA6V5 plasmid.
1.6
25
1.4
Fold increase over control
2. Incubated in a 5% CO2 incubator at 37°C for 12 hours
1.2
1
0.8
0.6
0.4
0.2
0
0
40
60
80
100
500
Ascl1 transfected (ng/well)
4. Incubated another 48 hours in 5% CO2, 37°C
5. Lysed cells, transferred lysate to luminometer and quantified
luminiscence
6. Recorded firefly luciferase and renilla luciferase activity
1. The luciferase reporter assay can be used in NIH3t3 cells to test Ascl1 mediated
gene transcription
30
1.8
Luciferase Activity (Firefly/Renilla)
1. Plated
2x104
Dose response of Delta enhancer longluciferase construct to increasing Ascl1
20
2. Gsx2 inhibits Ascl1 mediated gene transcription
15
10
5
0
0ng Ascl1
100ng Ascl1
100 Ascl1+100ng
Gsx2
100ng Gsx2
Effect of Gsx2 on Ascl1 mediated Delta
enhancer activation
The Delta enhancer showed a dose
Gsx2 effectively repressed Ascl1 mediated
dependent increase in luciferase activity up luciferase reporter expression.
to 100ng of Ascl1.
Future directions
1. Repeat the same experiment with multiple replicates
2. Study other target sequences of Ascl1 (other than Delta long E-box) and test
whether Gsx2 inhibits transcription via those targets as well
3. Investigate the regulators of Gsx2 using a yeast one hybrid system (Summer
project)