Studying Ascl1-Gsx2 interactions using a Luciferase reporter assay

1. Studying Ascl1-Gsx2 interactions using a Luciferase reporter assay - Juliana Madzia Mentor: KaushikRoychoudhury PI: Kenneth Campbell University of Cincinnati and Cincinnati Childrens Hospital Medical Center VZ SVZ Introduction: Mouse as a model system to study brain development Introduction: Transcription factors in brain development VZ - primary progenitors SVZ - intermediate progenitors • The mammalian brain is a complex organ composed of millions of neurons and glia • A class of protein molecules called transcription factors governs the expression of genes in the developing brain • Transcription factors bind to DNA and facilitate transcription of specific genes • During fetal development, transcription factors determine how neurons in the brain develop and mature. • Mutations and abnormalities in transcription factor expression leads to a multitude of syndromes ranging from mild to severe mental disabilities. • The house mouse (Musmusculus) is an excellent model to study brain development • Mice have significantly fewer neurons than humans (Millions as opposed to billions), making them easier to study • The ventral forebrain neural progenitors (neural stem cells) in mice give rise to the same types of neuron as in humans • Many types of genetic manipulations can be done in mice that facilitate understanding the mechanisms of embryonic development 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  A few transcription factors like Gsx2, Gsx1 and Ascl1 regulate progenitor maturation in the ventral telencephalon Luciferase reporter assay What is the mechanism of Gsx2–control of Ascl1? • Gsx2 protein directly interacts with Ascl1 protein • This binding is expected to block the DNA binding domain of Ascl1 • Gsx2 binding prevents Ascl1 form binding other transcription factors like E12 and E14 • Gsx2 inhibits Ascl1 binding to its target sequence of oligonucleotides • It is not clear yet if Gsx2 can inhibit transcription factor activity of Ascl1 • Hypothesis: • Gsx2 should be able to reduce Ascl1 mediated gene expression in cells • During development, Ventral telencephalic progenitors differentiate and give rise to neurons, oligodendriocytes 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 Ascl1 Ascl1 Luciferase Luciferase E-box on Delta Long E-box on Delta Long Source: Promega Light Gsx2 Light Source: Piercenet.com Methods Conclusions Results The luciferase reporter assay can be used in NIH3t3 cells to test Ascl1 mediated gene transcription Gsx2 inhibits Ascl1 mediated gene transcription Fold increase over control Luciferase Activity (Firefly/Renilla) 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 luminometerand quantified luminiscence Recorded firefly luciferase and renilla luciferase activity Future directions Repeat the same experiment with multiple replicates Study other target sequences of Ascl1 (other than Delta long E-box) and test whether Gsx2 inhibits transcription via those targets as well Investigate the regulators of Gsx2 using a yeast one hybrid system (Summer project) Ascl1 transfected (ng/well) Dose response of Delta enhancer long- luciferase construct to increasing Ascl1 The Delta enhancer showed a dose dependent increase in luciferase activity up to 100ng of Ascl1. Effect of Gsx2 on Ascl1 mediated Delta enhancer activation Gsx2 effectively repressed Ascl1 mediated luciferase reporter expression.