Developing a Dual Luciferase Assay to Validate VSX2 Binding Sites Identified by Chromatin Immunoprecipitation Followed By High Through-Put Sequencing (ChIP-seq)
Mentor 1
Dr. David Gamm
Mentor 2
Dr. Elizabeth Capowski
Location
Union Wisconsin Room
Start Date
24-4-2015 2:30 PM
End Date
24-4-2015 3:45 PM
Description
During optic vesicle formation, intrinsic and extrinsic factors initiate the activation of cell fate promoters. These factors differentiate multipotent cells into neural retina (NR) and retinal pigment epithelium (RPE). Two such intrinsic factors are Microplthalmia Transcript Factor (MITF) and Visual Homeobox System 2 (VSX2). MITF expression drives RPE cell fate while VSX2 promotes NR cell fate, in part by downregulating MITF transcription. A previously performed unbiased search for VSX2 binding sites identified three sites in MITF introns. The goal of this experiment was to develop a dual luciferase reporter assay to determine whether VSX2 can bind the three identified sites in vitro. Once the correct ratio of Renilla to luciferase was determined, the assay system was used to test whether VSX2 bound to identified target sites, whether it repressed transcription of the linked reporter gene, and whether fusing the strong VP16 activator domain to VSX2 converted its activity from repression to activation. VSX2 did not repress luciferase activity but results are inconclusive because activity was not high enough to begin with. Now, there is a better understanding of the strengths and weaknesses of the dual luciferase assay system as a method to test potential transcription factor binding sites. Future experiments will resolve the technical issues and pinpoint the relationship between VSX2 and the three binding sites in MITF introns.
Developing a Dual Luciferase Assay to Validate VSX2 Binding Sites Identified by Chromatin Immunoprecipitation Followed By High Through-Put Sequencing (ChIP-seq)
Union Wisconsin Room
During optic vesicle formation, intrinsic and extrinsic factors initiate the activation of cell fate promoters. These factors differentiate multipotent cells into neural retina (NR) and retinal pigment epithelium (RPE). Two such intrinsic factors are Microplthalmia Transcript Factor (MITF) and Visual Homeobox System 2 (VSX2). MITF expression drives RPE cell fate while VSX2 promotes NR cell fate, in part by downregulating MITF transcription. A previously performed unbiased search for VSX2 binding sites identified three sites in MITF introns. The goal of this experiment was to develop a dual luciferase reporter assay to determine whether VSX2 can bind the three identified sites in vitro. Once the correct ratio of Renilla to luciferase was determined, the assay system was used to test whether VSX2 bound to identified target sites, whether it repressed transcription of the linked reporter gene, and whether fusing the strong VP16 activator domain to VSX2 converted its activity from repression to activation. VSX2 did not repress luciferase activity but results are inconclusive because activity was not high enough to begin with. Now, there is a better understanding of the strengths and weaknesses of the dual luciferase assay system as a method to test potential transcription factor binding sites. Future experiments will resolve the technical issues and pinpoint the relationship between VSX2 and the three binding sites in MITF introns.
