Genome architecture is associated with many essential cellular processes from transcriptional regulation to chromosome segregation. Recent technological innovations have enabled detailed characterization of long- range chromosome conformations. Long-range chromosome compaction appears at repressive regions collectively referred to as heterochromatin. These genomic regions are vital for proper cell type-dependent gene expression patterns, and their architecture also helps to protect against genomic instability by controlling the expression of parasitic transposons and by regulating the chromatin structure near centromeres, telomeres, and other DNA repeats. Despite advances in understanding long-range chromatin compaction, few methods exist that measure the spatial organization of DNA at sub-nucleosome resolution, which is the length scale relevant to transcription and other critical DNA processes. Furthermore, many heterochromatic structures contain DNA repeats, which are difficult to study due to their inability to be mapped to a single genomic locus. I seek to determine the short-range compaction states of heterochromatin, using a recently developed method, RICC-seq, which can measure 3D DNA contacts at sub-nucleosome resolution. I will create new RICC-seq- based methods using Nanopore long-read sequencing to enable measurements of DNA repeats. I will also genetically manipulate histone modification pathways that regulate heterochromatin to determine their effects on short-range chromatin structure. Histone deacetylation and methylation are two major epigenetic pathways that dynamically regulate heterochromatin. In addition to these modifications, multiple isoforms of the conserved heterochromatin protein 1 (HP1) help regulate heterochromatin structures. I will determine the respective in vivo contributions that these epigenetic factors have on chromatin compaction and transcriptional silencing. In addition to defining the basic rules governing heterochromatin organization and function, I also propose to investigate the compaction states of phase-separated condensates. Phase separation is thought to regulate heterochromatin dynamics and transcription, however, how it affects short-range chromatin organization has yet to be addressed. I will determine the 3D DNA folding conformations of in vitro phase- separated chromatin, connecting phenomena observed in vitro with measurements of chromatin compaction in cells. This proposed work will tease out fundamental principles of genomic organization at nanoscale resolution and provide a structural foundation for understanding heterochromatin regulation and the possible impacts of its disruption in disease states.
Repetitive sequences of DNA fold into structures and compartments that protect against disease-associated genomic instability. Due to the inability to map a DNA repeat to a single genomic location, it is difficult to determine the spatial organization of repeats and to understand the mechanisms that prevent their mutagenesis. This proposal combines cutting-edge DNA sequencing technology with genetic approaches in the fission yeast model organism to determine how DNA repeats, and other critical regions of the genome, are regulated by the three-dimensional architecture of their chromosomes.
