Epigenetic gene regulation is the process by which external factors regulate the genome. This research studies Polycomb Group (PcG) proteins which function as epigenetic agents that work together in complexes to maintain gene silencing for multiple cellular generations. Drosophila melanogaster PcG proteins can be organized into three canonical complexes: Pho-RC, PRC1, and PRC2. Though there are multiple proposed models for the order of recruitment, it is generally accepted that PhoRC, PRC1, and PRC2 interact with each other to stably recruit to a target gene. Since these proteins are highly conserved, this project studies PcG proteins in the model organism, Drosophila melanogaster. In Drosophila, these proteins are key regulators of developmental genes, such as giant. In previous work conducted by this lab, it has been shown that a PcG protein Pho, a core component of Pho-RC, binds to giant at two different Polycomb Response Elements (PREs). Through Chromatin Immunoprecipitation experiments, the timing of PcG proteins’ de novo recruitment to giant PREs has been established.
To further our understanding of PcG recruitment, my project looks at the roles of individual PcG proteins. Studies have elucidated the activity and function of PcG complexes, but not the behavior and activity of each PcG protein. Through the depletion of one PcG protein at a time, we were able to independently assay the effect of Pho and Sfmbt on recruitment and assay the effect on other PcG proteins.
This project follows two experimental aims. The first aim is to assay the effect of the depletion of Pho and Sfmbt, respectively, to observe the impact on components of PRC1 and PRC2. Through ChIP analyses at three distinct developmental time points, a pattern emerges when analyzing the presence of PcG proteins at gt PRE1 and PRE2. The data suggest that Sfmbt may play a role in the recruitment of PhoRC and potentially interacts independently of Pho. In addition, the divergence of dRING from Pc suggests its involvement with non-canonical PcG complexes through early developmental time stages. Further research needs to be conducted to see if there is a reciprocal effect of other PcG protein knockdowns on Pho and Sfmbt.
The second aim is to assay the effectiveness of a novel transgene to reduce early embryonic expression of Pcl to determine if this method would allow for effective knockdown of Pcl for future experimentation. Zygotic gene expression occurs post-Midblastula transition (MBT) in embryos, but this vector was designed to induce pre-MBT expression to more effectively deplete zygotically expressed Pcl. Future studies would selectively knockdown Pcl to assay the effect on other PcG recruitment.
Genetics, Molecular Biology
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Kum, Anni, "The Roles of Individual Proteins in De Novo Recruitment of PcG Repressive Complexes to Giant" (2022). Biological Sciences Theses and Dissertations. 14.