Amanda Fitzgerald
OURS Undergraduate Research Fellow, Fall 08-Spring 09
Determining the role of individual domains of Williams Syndrome Transcription Factor in Xenopus laevis embryos
Williams Syndrome is a developmental disorder exhibiting visual/spatial defects and mental retardation in affected patients. Individuals with Williams Syndrome are missing the Williams Syndrome Transcription Factor (WSTF), a gene that together with Imitation Switch (ISWI) forms the WICH complex. This complex is a member of a family of ATP-dependent chromatin remodelers which utilize the energy from the hydrolysis of ATP to alter the nucleosomal arrangement of DNA. This is done to allow the precise expression of specific genes throughout an organism's development. WSTF expression can be blocked in Xenopus laevis embryos by injecting them with anti-WSTF morpholino, resulting in abnormalities in neural and eye development. Xenopus injected with anti-WSTF morpholino should exhibit restoration of normal developmental function of WSTF if they are also injected with normal WSTF, this is known as a “rescue” experiment. Mutants of WSTF can be constructed with deletions of specific domains, including the bromodomain and the PHD finger. These mutants will also possess a mutation in the region that the anti-WSTF morpholino recognizes, ensuring that the morpholino's presence will not affect the expression of the mutant WSTF. Site-directed mutagenesis will be used to create these domain-lacking WSTF mutants. I will use these mutants in a series of rescue experiments, by injecting them into Xenopus embryos that are WSTF- depleted. These embryos will be monitored for their ability to rescue phenotypes including those in brain development and eye formation. Relatively little is known about the role of these individual domains in the functioning of WSTF, and this study will determine whether the bromodomain and/or PHD finger of WSTF are required for the rescue of developmentally functioning WSTF.
Williams Syndrome is a developmental disorder exhibiting visual/spatial defects and mental retardation in affected patients. Individuals with Williams Syndrome are missing the Williams Syndrome Transcription Factor (WSTF), a gene that together with Imitation Switch (ISWI) forms the WICH complex. This complex is a member of a family of ATP-dependent chromatin remodelers which utilize the energy from the hydrolysis of ATP to alter the nucleosomal arrangement of DNA. This is done to allow the precise expression of specific genes throughout an organism's development. WSTF expression can be blocked in Xenopus laevis embryos by injecting them with anti-WSTF morpholino, resulting in abnormalities in neural and eye development. Xenopus injected with anti-WSTF morpholino should exhibit restoration of normal developmental function of WSTF if they are also injected with normal WSTF, this is known as a “rescue” experiment. Mutants of WSTF can be constructed with deletions of specific domains, including the bromodomain and the PHD finger. These mutants will also possess a mutation in the region that the anti-WSTF morpholino recognizes, ensuring that the morpholino's presence will not affect the expression of the mutant WSTF. Site-directed mutagenesis will be used to create these domain-lacking WSTF mutants. I will use these mutants in a series of rescue experiments, by injecting them into Xenopus embryos that are WSTF- depleted. These embryos will be monitored for their ability to rescue phenotypes including those in brain development and eye formation. Relatively little is known about the role of these individual domains in the functioning of WSTF, and this study will determine whether the bromodomain and/or PHD finger of WSTF are required for the rescue of developmentally functioning WSTF.