GDSC Graduate Student Brandon Park Passes his Qualifying Exam!
Thursday, August 26, 2021
Completing his 2nd year on a high note, GDSC Graduate Student Brandon Park, passed his Qualifying Exam. Brandon plans to focus his research on Nucleic Acid Biology, Epigenetics, and Development. His thesis proposal is titled “Using New Technologies to Investigate the Role of H2A.Z in Epigenetic Inheritance”. Brandon is pursuing his doctoral research under a co-mentorship with Dr. Mitch O’Connell, and Dr. Patrick J. Murphy.
Brandon Park’s thesis proposal may be read below.
Abstract: 5-methylcytosine (5mC) is a major form of DNA methylation that is often observed at the promoters and enhancers of repressed genes, but the mechanisms by which 5mC patterns are maintained in developing primordial germ cells (PGCs)is currently unknown. Nucleosomes that contain the histone variant H2A.Z function as “placeholders” in early embryos to deter DNA methylation following fertilization, and are thus located at hypomethylated regions, such as promoters for genes involved in early development. Therefore, I hypothesize that H2A.Z maintains this function during PGC development, allowing the DNA methylation patterns of stem cells to be maintained in PGCs. This mechanism would facilitate the trans-generational maintenance of 5mC patterns and might enable adaptive evolution if changes to these 5mC patterns are inherited. To date, technical limitations of Chromatin Immuno-Precipitation (ChIP) have made it difficult to assess the localization of chromatin modifications throughout PGC development and in mature oocytes. CUT&TAG is a recently developed alternative to ChIP-Seq that provides significantly improved signal to noise and can be done on just a few thousand cells. For this reason, I will use CUT&TAG to investigateH2A.Zpatterns in PGCs that have been purified via Fluorescence Assisted Cell Sorting (FACS), as well as whole WT sperm, oocytes, and early embryos. I will bioinformatically analyze the resulting H2A.Z landscapes to define regions where H2A.Z patterns change or are maintained in different cell types. These experiments will create a reference point for defining H2A.Z landscapes, and test our hypothesis that H2A.Z patterns are maintained throughout germ cell development. Functional studies of PGCs have also been limited by the availability of genetic tools in zebrafish, as well as difficulties generating tissue specific knockdown effects. Here I will create a tissue-specific CRISPR system to knockdown H2A.Zat various time points during PGC development using a heat shock inducible promoter to control gRNA expression. I will investigate the effects of these knockdowns by doing CUT&TAG for H2A.Z, locus specific bisulfite sequencing (through PCR), and monitoring for phenotypic changes in the mature germ cells and next generation embryos. Together, these experiments will combine CRISPR and CUT&TAG methods to establish several epigenomic profiles for various factors involved in regulating gene expression in developing zebrafish embryos and PGCs. These studies will also provide new insight into the role of H2A.Z in 5mCmaintenance and transgenerational epigenetic inheritance.