Browsing by Author "Carlson, Jennifer"
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Item Epigenetic regulation of chromatin structure by dKeap1 and CncC in Drosophila melanogaster(2019-08) Carlson, JenniferThe Nrf2-Keap1 pathway regulates transcriptional response to xenobiotic and oxidative stress, thereby reducing the adverse effects of these compounds on the health of an organism. According to the classic model, Keap1 interacts with Nrf2 in the cytoplasm and targets Nrf2 for ubiquitination and degradation. When xenobiotic compounds or reactive oxygen species are present, they disrupt the interaction between Keap1 and Nrf2, releasing Nrf2 to enter the nucleus and activate transcription of response genes. Although the short-term responses to xenobiotic factors are well understood, the mechanisms that mediate the effects of long-term exposure to xenobiotics on development remain unknown. It has been found that the Nrf2-Keap1 pathway can regulate normal development in Drosophila and mice. For example, Drosophila Nrf2 and Keap1 (CncC and dKeap1) regulate the synthesis of and response to ecdysone (an important hormone in metamorphosis). Elucidating other mechanisms by which Nrf2 and Keap1 can regulate development could help us understand how xenobiotics affect development and the complicated roles of Nrf2 and Keap1 in disease. Given the importance of epigenetic regulation in development, we investigated the role of CncC and dKeap1 in regulating chromatin package. A position effect variegation (PEV) assay uses a transcriptional reporter gene to assess heterochromatin formation. PEV assays revealed that knockdown of CncC and/or dKeap1 resulted in a reduction of gene silencing caused by pericentric heterochromatin. Knockdown of CncC or dKeap1 in embryos reduced the level of heterochromatin marker histone H3K9 dimethylation. However, knockdown of CncC or dKeap1 did not affect the expression of genes that encode heterochromatin components Su(var)3-9 and HP1 . These results indicate that CncC and Keap1 promote or maintain pericentric heterochromatin formation, likely at the post-transcriptional level. Thus, epigenetic regulation of chromatin may represent a novel mechanism by which the Kea[p1-Nrf2 xenobiotic response signaling controls development. Future studies will aim to determine the interacting partners of Nrf2 and Keap1 and establish whether Nrf2 and Keap1 also regulate euchromatin structure. Elucidating the roles of Nrf2 and Keap1 will help us understand how environmental toxins may impact epigenetics, development, and human health.Item Regulation of epigenetics and development by the xenobiotic/oxidative stress response factor Keap1 in Drosophila(2022-07) Carlson, JenniferEnvironmental toxins can cause diseases or developmental defects when not removed from cells, but the molecular mechanisms are not fully understood. The Keap1-Nrf2 complex is an important regulator of detoxification. Nrf2 is a transcription factor that activates expression of detoxification genes. Keap1 usually binds to Nrf2 in the cytoplasm and targets it for degradation. Toxins disrupt the Keap1-Nrf2 interaction, allowing Nrf2 to enter the nucleus and activate transcription. Interestingly, Keap1 and Nrf2 also bind to chromatin and regulate developmental genes. Thus, perhaps the developmental roles of Keap1-Nrf2 mediate the effects of toxins on development. To address this overall goal, this research used Drosophila to elucidate the developmental functions and molecular mechanisms of dKeap1 (Drosophila Keap1).Studying the developmental functions of dKeap1 is complicated because disrupting dKeap1 can cause mis-regulation of toxin response and lead to developmental defects. We found that a dKeap1 truncation with the C-terminal domain removed (dKeap1-ΔCTD) could still regulate expression of detoxifying genes but could no longer enter the nucleus to bind to chromatin. This allowed us to distinguish the developmental defects directly related to dKeap1 from the indirect effects of mis-regulated toxin response. We found that dKeap1-ΔCTD flies had defects in viability, fertility, and adipose tissue. These phenotypes likely resulted from mis-regulated developmental gene expression as we found that ecdysone biosynthetic/response genes and adipogenesis genes were down-regulated in specific tissues. We hypothesized that dKeap1 regulates transcription through controlling high-order chromatin structure. Here, we found that dKeap1 interacts with Lamin Dm0 proteins, which are intermediate filament proteins that form the nuclear lamina and organize the overall chromatin architecture. We also found that dKeap1 is required for the maintenance of a normal nuclear lamina, that dKeap1 overexpression redistributes the heterochromatin marker H3K9me2 along the chromosome arms and that dKeap1 and Lamin Dm0 function in the same genetic pathway. These results support a model where dKeap1 regulates chromatin structure and gene expression through interaction with Lamin proteins. Understanding the roles of dKeap1 in chromatin regulation and development can lead to a better understanding of the roles of Keap1-Nrf2 in disease and how environmental toxins influence epigenetics and development at the molecular level.