Applying super-resolution microscopy to investigate the regulatory structure of the genome

The three-dimensional (3D) organization of the genome is important for cellular function, such as gene expression and differentiation throughout development. Both the spatial and temporal expression of a gene are largely regulated by non-coding sequences in the genome. The genome is folded into compartments, topological associated domains (TADs), and loops, as determined by sequencing-based technology such as Hi-C. Many of the differences in cell type arise from specific interactions between distal enhancers and their target promoters, which are typically located thousands to hundreds of thousands of basepairs apart. Long-range enhancer and promoter activity and the specific of enhancer-promoter interactions are believed to arise from the cell-type specific genome folding. How this genome organization is established and regulated during development is not well understood. Hi-C and other sequencing-based assays lack information pertaining to the spatial organization of cells in tissues, and largely provide population-level information, not single cell, which makes it challenging to understand how genome folding might contribute to differences among cell types. Thus, there is a great need for approaches that provide a view of the chromatin organization and transcriptional activity in single cells. Here, I present my work developing and using a super-resolution technique to gain such an unprecedented view. Our novel super-resolution microscopy approached termed Optical Reconstruction of Chromatin Architecture (ORCA) to trace the DNA path in steps from 30 kb to 2 kb at the single-cell level. We discovered that single cells do have TAD-like structures that are heterogeneous across cells. However, the boundary positions of these single cell TADs do preferentially lie at insulator boundary protein CTCF and cohesin binding sites. Although depletion of cohesin is crucial for the presence of TADs at the population-level, we found that the TAD-like domains in single cells are not dependent on cohesin. Thus, my findings using ORCA in cultured cells (Chapter 2) shed important new light to genome organization in single cells. My interest in gene regulation led me to expand our microscopy approach by making ORCA compatible with multiplex RNA imaging to enable direct correlation between chromatin structure and gene expression on a cell-by-cell basis. Furthermore, I expanded our experimental system by applying ORCA to cryosectioned Drosophila embryos to investigate the role of 3D genome structure in loci, such as in the bithorax complex (BX-C), with well-studied enhancers. I discovered that cell-type specific 3D DNA folding of the BX-C correlates with BX-C expression patterns in different embryonic body segments. Using embryos with genetic perturbations allowed me to determine that the genetic elements at TAD boundaries drive proper cell-type specific enhancer-promoter contacts and gene expression. My results (Chapter 3) suggest that architectural proteins, such as CTCF and cohesin, at TAD boundaries are responsible for the establishment of 3D organization during development. Additionally, my results emphasize the need to study cell-type specific chromatin structures on a cell-by-cell and cell type basis, an area that is still largely unexplored. To facilitate such exploration, I worked towards making our approach accessible to other researchers that are interested in 3D genome architecture and transcriptional activity (Chapter 4). To determine the role of architectural proteins in genome organization (Chapter 5), I took advantage of Drosophila genetics and obtained null allele mutant embryos that lacked zygotic expression of architectural proteins such as Rad21, Wapl, CTCF, and CP190. Using ORCA, I found that these mutants have BX-C TADs that are similar to that of WT in mid to late staged embryos. However, as the maternal transcripts for these architectural proteins were present throughout embryogenesis, the maternally encoded proteins appeared to be sufficient to retain genome structure in the zygotic null mutants. I also observed BX-C TAD structure that looks similar to that of wild-type in the central nervous system (CNS) of mutant CTCF L3 larvae where maternal gene products were fully absent. My results raise the probability that other Drosophila insulator binding proteins, such as CP190, may play a redundant insulation function. To examine the role of various cis-acting insulator elements, I have begun preliminary studies in investigating how inserting insulators into the genome affects long-range cis-regulatory interactions (Chapter 6). Overall, the development of ORCA has enabled us to begin understanding the mechanisms underlying genome organization and their role in regulating transcription in a complex tissue. As our techniques improve and becomes more accessible to other researchers in the field, we are certain that the methods we have developed will play a role in un-covering the function of various chromatin components, such as transcription factors and epigenetic state, in establishing the 3D genome organization during development

Keywords: Developmental Biology.
Advisor: Boettiger Alistair 
Fuller Margaret T.
Villeneuve Anne
Wysocka Joanna