Project Abstract: Faithful epigenetic maintenance of repression is essential to developmental processes and is frequently disrupted in diseases like cancer. Repression is maintained, in part, by chromatin-associated proteins, like HP1 and Polycomb (Pc) groups that biochemically alter chromatin by depositing histone marks and spatially reorganize chromatin by compaction and/or phase separation mechanisms. As such, a physical mechanism of repression through stable compaction or phase separation of chromatin has been proposed as the function of heterochromatin organization, resulting in discrete open (active) and closed (repressed) chromatin states. My prior studies challenge this dogma by revealing that while Pc-repressed regions are compact and separated on average, at the single-locus level there exists a continuum of repressed chromatin conformations. I propose that instead of providing a physical mechanism of transcriptional repression, heterochromatin indirectly represses chromatin by regulating epigenetic memory. A mechanism of spatial feedback, through which dynamic chromatin folding permits distal loci to reinforce the deposition and maintenance of histone marks, serves as an epigenetic memory regulator without the need for a stably compact or phase-separated organization. In this model, the rates of interaction frequencies, facilitated by cell type- or locus-specific HP1/Polycomb proteins, refresh epigenetic marks in the face of nucleosome turnover and cell division. This dynamic chromatin organization can regulate stability of epigenetic memory such that a balance between maintenance of the existing epigenetic state and reprogrammability scales with cell plasticity. In this proposal I will rigorously investigate this functional feedback between 3D genome organization and the repressive epigenetic memory that underlies developmental gene regulation and cell plasticity. In Aim 1, I will evaluate how dynamic chromatin organization regulates epigenetic memory during development through a highly multiplexed epigenetic state and chromatin imaging methodology. This will enable me to analyze single-locus epigenetic states and chromatin folding in an organoid model, unveiling how spatial feedback shapes cellular reprogramming and fate commitment. In Aim 2, I will perform live imaging of heterochromatin dynamics which will allow me to define the motion of HP1 and Pc-bound chromatin and measure how chromatin motion influences epigenetic memory. Finally, I will develop a super-resolution imaging methodology to quantify protein-DNA interactions and chromatin folding to better understand the role of Pc associated proteins’ ability to alter chromatin organization and create different levels of chromatin spatial feedback.

The mechanistic relationship between 3D genome folding, chromatin dynamics, and stability of epigenetic memory is unclear due to challenges of simultaneously measuring these parameters in single cells. As such, we have a limited understanding of how chromatin folding impacts epigenetic stability and how this effects development processes and disease. Drawing on recent advances in multiplexed microscopy and super resolution imaging, I aim to develop new imaging technologies to study the dynamic feedback between chromatin folding and epigenetic memory across time scales.