THE ROLE OF THE MTOR PATHWAY IN DEVELOPMENTAL REPROGRAMMING OF HEPATIC LIPID METABOLISM AND THE HEPATIC TRANSCRIPTOME AFTER EXPOSURE TO 2,2',4,4'- TETRABROMODIPHENYL ETHER (BDE-47) An Honors Thesis Presented By JOSEPH PAUL MCGAUNN Approved as to style and content by: ________________________________________________________** Alexander Suvorov 05/18/20 10:40 ** Chair ________________________________________________________** Laura V Danai 05/18/20 10:51 ** Committee Member ________________________________________________________** Scott C Garman 05/18/20 10:57 ** Honors Program Director ABSTRACT An emerging hypothesis links the epidemic of metabolic diseases, such as non-alcoholic fatty liver disease (NAFLD) and diabetes with chemical exposures during development. Evidence from our lab and others suggests that developmental exposure to environmentally prevalent flame-retardant BDE47 may permanently reprogram hepatic lipid metabolism, resulting in an NAFLD-like phenotype. Additionally, we have demonstrated that BDE-47 alters the activity of both mTOR complexes (mTORC1 and 2) in hepatocytes. The mTOR pathway integrates environmental information from different signaling pathways, and regulates key cellular functions such as lipid metabolism, innate immunity, and ribosome biogenesis. Thus, we hypothesized that the developmental effects of BDE-47 on liver lipid metabolism are mTOR-dependent. To assess this, we generated mice with liver-specific deletions of mTORC1 or mTORC2 and exposed these mice and their respective controls perinatally to BDE-47. We found that developmental exposure to BDE-47 permanently reprograms gene expression related to hepatic lipid metabolism, innate immunity, and other key cellular functions in an mTORC1- and 2-dependent manner. Our results also provide a hypothetical model of gene- environment interaction in which early-life BDE-47 exposure triggers life-long reprogramming of liver lipid metabolism and other key cellular functions in an mTOR-dependent manner, and indicate that modulation of the mTOR pathway by environmental chemicals such as BDE-47 may lead to long-lasting changes in liver disease susceptibility. 1 Acknowledgements I would like to thank Dr. Alexander Suvorov for his incredible mentorship over the last three years, his dedication to helping me grow as a scientist, his willingness to always take time out of his busy day to answer questions and hypotheses, and his passion for science that made working under him so inspiring and worthwhile. I would like to thank Dr. Laura V. Danai for her guidance over the last three years as well. Dr. Danai taught me much of the biochemistry and molecular biology that I used for this work, and her willingness to encourage me to grow as a scientist in the lab, in the classroom, and in our discussions has proven invaluable. I would also like to thank other members of the Suvorov Lab who made critical contributions to this project. Anthony Poluyanoff also conducted RNA-seq and western blotting for all samples and conducted analysis for mTORC1 and 2 knockout data in tandem with me. Victoria Salemme conducted analysis of triglyceride levels in blood and liver samples. Ahmed Khalil provided guidance regarding western blotting for Anthony and me, and Menna Teffera assisted in bioinformatic analysis of gene expression data using Ingenuity Pathway Analysis (IPA). I would also like to thank the laboratory of Dr. R. Thomas Zeller for assistance with western blot imaging. Lastly, I would like to thank all my family, friends, and other mentors who have supported me throughout this project and over the course of my development as a scientist. Funding for this project was provided by the Research Enhancement Award from the Dean of the School of Public Health and Health Sciences to Dr. Alexander Suvorov and The Commonwealth Honors College Honors Research Grant. 2 Table of Contents I. Experimental goals .......................................................................................................................................................... 9 II. Introduction ..................................................................................................................................................................... 10 III. Methodology .............................................................................................................................................................. 21 IV. Results part 1: the comparative roles of mTORC1 and 2 in liver gene expression ............................. 27 V. Results part 2: The role of mTORC1 in mediating the long-term effects of BDE-47 on the liver ...... 58 VI. Results part 3: the role of mTORC2 in mediating long-term effects of BDE-47 on the liver .......... 81 VII. Discussion ................................................................................................................................................................ 106 VIII. Conclusions .............................................................................................................................................................. 148 IX. References ................................................................................................................................................................ 154 X. Appendix ....................................................................................................................................................................... 165 3 List of tables and figures Figures: ❖ Figure 1. A Visual Representation of our Exposure Paradigm. ❖ Figure 2. A Visual Representation of the Groups of Animals Used for this Series of Experiments. ❖ Figure 3. Western Blots to Characterize Phosphorylation Activity in Mice with a Liver- Specific Deletion of mTORC1 or 2. ❖ Figure 4. Metascape Reveals Ontology Terms Regulated by mTORC1 or 2 ❖ Figure 5. Comparison Analysis Reveals Similar and Differential Regulatory Roles for mTORC1 and 2 at the Transcriptional Level. ❖ Figure 6. Metascape Results for Genes Dependent on mTORC1 Only. ❖ Figure 7. Metascape Results for Genes Dependent on mTORC2 Only. ❖ Figure 8. Metascape Results for Genes Inversely Dependent on mTORC1 and 2. ❖ Figure 9. Metascape Results for Genes Similarly Altered in Expression by mTORC1 and 2 Knockouts. ❖ Figure 10. Overlap of Genes whose Expression was Altered by mTORC1 and mTORC2 Knockouts Filtered by Dependency Analysis. ❖ Figure 11. Comparison of Significantly Enriched GSEA Gene Sets for mTORC1 and 2 Knockouts. ❖ Figure 12. IPA-Generated Networks of Transcription Regulators Downstream of mTORC1 and 2. 4 ❖ Figure 13. IPA Disease and Biological Functions for mTORC1 Knockout (Left) and mTORC2 Knockout (Right). ❖ Figure 14. IPA Toxicological Functions for mTORC1 Knockout (Left) and mTORC2 Knockout (Right). ❖ Figure 15. Western Blots to Characterize the Relationship Between Early-Life BDE-47 Exposure and mTORC1 Activity. ❖ Figure 16. Metascape Reveals Ontology Terms Enriched Due to BDE-47 Exposure in the Presence and Absence of mTORC1 in the mTORC1 Experiment. ❖ Figure 17. Comparison Analysis Reveals mTORC1 Dependent and Independent Effects of Early-Life BDE-47 Exposure in the mTORC1 Experiment. ❖ Figure 18. Metascape Results for Genes for which Deletion of mTORC1 Abolished Effects of BDE-47 Exposure in the mTORC1 Experiment. ❖ Figure 19. Metascape Results for Genes for which Deletion of mTORC1 Permitted an Effect of BDE-47 Exposure mTORC1 Experiment. ❖ Figure 20. Metascape Results for Genes for which Effects of BDE-47 Exposure are Independent of mTORC1. ❖ Figure 21. Comparison of Significantly Enriched GSEA Gene Sets In BDE-47 Exposed mTORC1 Control and Knockout Mice. ❖ Figure 22. IPA Disease and Biological Functions for BDE-47 Exposed Control (Left) and mTORC1 Knockout (Right) mice in the mTORC1 Experiment. ❖ Figure 23. IPA Toxicological Functions for BDE-47 Exposed Control (Left) and mTORC1 Knockout (Right) mice in the mTORC1 Experiment. 5 ❖ Figure 24. Western Blots to Characterize the Relationship Between Early-Life BDE-47 Exposure and mTORC2 Activity. ❖ Figure 25. BDE-47 Exposure Significantly Increases Serum Triglycerides in the mTORC2 experiment and is mTORC2 dependent. ❖ Figure 26. Metascape Reveals Ontology Terms Enriched Due to BDE-47 Exposure in the Presence and Absence of mTORC2 in the mTORC2 Experiment. ❖ Figure 27. Comparison Analysis Reveals mTORC2 Dependent and Independent Effects of Early-Life BDE-47 Exposure in the mTORC2 Experiment. ❖ Figure 28. Metascape Results for Genes for which Deletion of mTORC2 Abolished Effects of BDE-47 Exposure in the mTORC2 Experiment. ❖ Figure 29. Metascape Results for Genes for which Effects of BDE-47 Exposure are Independent of mTORC2. ❖ Figure 30. Comparison of Significantly Enriched GSEA Gene Sets In BDE-47 Exposed mTORC2 Control and Knockout Mice. ❖ Figure 31. IPA-Generated Network of Transcription Factors Downstream of mTORC1 and 2 that are Activated and Inhibited due to BDE-47 Exposure in mTORC2 control mice. ❖ Figure 32. IPA Disease and Biological Functions for BDE-47 Exposed Control (Left) and mTORC2 Knockout (Right) mice in the mTORC2 Experiment. ❖ Figure 33. IPA Toxicological Functions for BDE-47 Exposed Control (Left) and mTORC2 Knockout (Right) mice in the mTORC2 Experiment. ❖ Figure 34. A Visual Representation of the Gene-Environment Interaction that Modulates Expression of a Hypothetical Gene in the mTORC1 and mTORC2 experiments. 6 ❖ Figure 35. A Theoretical Model of Modulation of Gene Expression by BDE-47 via mTORC1 and 2. Tables: ❖ Table 1. Selected