Encoding memory in 3D : how polycomb-mediated genome folding facilitates epigenetic memory through dynamic chromatin architecture

The genome is non-randomly packaged and organized within a nucleus such that interactions between regulatory elements are either preferentially enriched or prevented. The growing challenge in the genome organization field is understanding how a highly heterogeneous chromatin organization is able to promote the precise gene expression programs necessary for development. In order to address this challenge, it is necessary to confidently measure genome organization at single-locus resolution. My thesis focuses on this challenge by building tools to accurately sample the distributions of single-locus organization and proposes a model for how heterogeneous organization can be leveraged to stabilize epigenetic memory. Chapter 1 briefly introduces the concept of epigenetic memory and its relationship to genome organization. It concludes by discussing some future perspectives and how this work may be extended for further investigations. In chapter 2, I discuss the development of our novel imaging technology, Optical Reconstruction of Chromatin Architecture (ORCA) which allows for simultaneous measurement of genome organization and gene expression in single-cells. We first applied this technology to Drosophila embryos at the Bithorax (BX-C) locus. At the BX-C, Polycomb repressed regions progressively retract as the locus is activated. The organization of polycomb-bound regions of the locus adopted an inverted structure where long-range interactions were closer than expected and short-range interactions were further than expected. This curious organization led me to investigate the biophysical properties of polycomb-bound chromatin and ask how the organization was functional for the Polycomb system. This investigation of Polycomb-mediated genome organization is chapter 3. Here, we examined the 3D-organization of the mammalian Hoxa locus using ORCA. Prevailing models of Polycomb-mediated repression suggested that compaction of a locus or phase-separation were physical mechanisms of repression, making the locus inaccessible to activating factors. However, our ability to confidently examine heterogeneity of Hoxa folding demonstrated that the organization was not stable enough to result in physical repression. Instead, we proposed a model, supported by biophysical polymer simulations, that the heterogeneous structure we observe across cell-types and tissues may act as a spatial feedback mechanism to retain epigenetic memory of repressed loci. In chapter 4, I describe an organoid system called gastruloids that capture early developmental axial patterning. I adapted the gastruloid system for compatibility with ORCA imaging and outline the protocol so that it can be used for future experimentation addressing questions of spatial feedback during development. It is my hope that leveraging a model that captures the spatiotemporal pattering of Hox loci will provide ample opportunity for more dynamic measurements of genome organization and corresponding epigenetic state. Finally, in chapter 5, I discuss a different aspect of my PhD that I hold in equal value to my scientific training--work in the diversity, equity, and inclusion space. It is a goal of mine to make academia a more inclusive space and I highlight some of the lessons I've learned and best-practices to improve representation in the biosciences.

Keywords: Medicine.
Genetics.
Advisor: Boettiger Alistair
Bintu Lacramioara
Fire Andrew
Villeneuve Anne