Mapping heterochromatin organization with high-throughput imaging

Oklahoma Medical Research Foundation, Oklahoma City OK

Investigators

Abstract

ABSTRACT Heterochromatin forms large-scale spatial domains within the nucleus through a process called self- association, and while this is evolutionarily conserved, developmentally regulated, and disrupted in disease, the mechanisms driving this organization and its functional ramifications remain unclear. The long-term goal is to understand how heterochromatin organization establishes and maintains chromatin state in human cells, which will require specifically altering organization and tracking its effects. The rationale for this project is that identifying the mechanisms that regulate heterochromatin self-association will not only provide significant insight into the basic regulation of the human genome, it will build the tools necessary to undertake functional studies by specifically altering genome organization. The objective of this application is therefore to understand what mechanisms determine self-association. The central hypothesis is that the mechanism by which heterochromatin self-associates in human cells is dependent upon chromatin type and mediated by the action of many chromatin- associated proteins and movement of the chromatin fiber. This hypothesis will be tested with three specific aims: 1) compare self-association across different heterochromatin domains marked by different histone modifications, 2) systematically identify proteins that are required for colocalization of heterochromatin loci, and 3) characterize the movements of heterochromatin loci in live cells. For the first aim, colocalizations and clustering between genomically distant heterochromatin loci in constitutive, facultative, and poised heterochromatin will be measured using a novel high-throughput imaging technique, and specific sequence-level or epigenetic features that characterize clustering will be determined using machine learning. For the second aim, a directed CRISPR mutagenesis screen will be undertaken to identify which chromatin-associated proteins are required for colocalization between genomically distant heterochromatin loci. For the third aim, individual heterochromatin loci will be tracked using catalytically dead and fluorescently tagged dCas9. The kinetics of these loci will determine whether the chromatin within a domain is a static scaffold or can freely diffuse. The proposed research is innovative as each aim leverages and develops novel techniques to yield new insights into heterochromatin organization. It is especially significant, as together these aims build towards a critically important study of how heterochromatin organization maintains or determines silencing. To ask this question, we must simultaneously track genome regions and transcription from those regions in live cells after specific disruption of heterochromatin self-association. In combination, these aims will set the groundwork for a well-developed, independent research plan addressing the functional relevance of genome architecture in gene regulation and human disease.