Discovering and engineering new epigenome editing tools for better gene expression and epigenetic memory control across different contexts

Although all cell types in a multicellular organism contain the same genetic information, they are functionally and morphologically different. Such diversity can only be achieved by a highly regulated spatial and temporal control of gene expression by over 2000 transcription factors and chromatin regulators. The utilization of these transcriptional effector domains in gene regulation tools, as in CRISPRi/a and genetic circuits, have been instrumental in interrogating regulatory elements and gene function and holds promise for therapeutic gene or cell therapy applications. However, there is a need for new tools that do not rely on overexpressing effector domains and are small enough to deliver and combine for use in gene regulation tools. There is also the lack of systematic understanding of how effector domains regulate transcription across genomic and cell-type contexts and how their function depends on cellular cofactors. Characterizing the context of how these effectors function will help in the development of gene regulation tools that can perform across different contexts. First (in chapter 2), I develop a strategy for gene control using small single domain antibodies, called nanobodies, that bind and recruit endogenous chromatin regulators to a gene. I show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators and control gene expression, and that nanobodies against two different types of repressive chromatin regulators, HP1 and DNMT1, can silence a fluorescent reporter gene. Combining these nanobodies together or with other regulators, such as DNMT3A or KRAB, I observe enhanced silencing speed and epigenetic memory. Finally, I use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder. These results set the basis for using compact nanobodies against chromatin regulators for controlling gene expression and epigenetic memory. To better understand how the dynamics and mechanisms of chromatin-mediated gene control can vary in different cell types (chapter 3), I study the effect of two repressive histone methyltransferases, KRAB and EED, on gene expression and epigenetic memory at a fluorescent reporter gene in mouse embryonic stem cells (mESCs) and Chinese hamster ovary (CHO-K1) cells. I find that, when compared to CHO-K1 cells, KRAB had slower rates of silencing and that EED can only partially silence in mESCs. Moreover, silencing mediated by these effectors had shorter duration of epigenetic memory in mESCs, such that EED silencing was completely reversible. Overall, these results provide a preliminary look into the cell-type specific function of these regulators in stem cells and acts as a steppingstone for future experiments characterizing more transcriptional effectors across different contexts, such as cell-type, gene target, and DNA-binding domain. Lastly (in chapter 4), I used high-throughput recruitment (HT-recruit) to measure effector function for a pooled library of about 6,000 nuclear protein domains across 15 target gene, cell-type, and DNA-binding domain contexts. I discover context-dependent effectors (e.g., WW and HLH domains) and identify other context-independent effectors that can improve transcriptional perturbation tools (e.g., the tripartite activator NFZ and the ZNF705F KRAB repressor). Moreover, using CRISPR screens, we identify genetic dependencies for the context-dependent HLH repressors. Ultimately, these results reveal the context-dependence across human effectors, demonstrates how rare cell type- and target-specific effectors can be systematically characterized by combined high-throughput protein domain and genetic perturbation screens, and enables a new suite of transcriptional control tools for research and therapeutic applications. Collectively, this dissertation demonstrates my work in discovering and engineering epigenetic tools that allow for better and more potent transcriptional control across different genomic and cell-type contexts, in hopes of improving upon the existing gene regulation toolbox

Keywords: Biology.
Advisor: Bintu Lacramioara
Gozani Or Pinchas
Morrison Ashby J.
Wysocka Joanna