Different cell types in a given animal, such as heart cells and brain cells display different behaviors because they express different sets of genes. Yet, they all have DNA with essentially the same sequence and thus the same set of genes. How is it that the same DNA is used to generate different cell types? Which genes are on and which genes are off is controlled by how their underlying DNA sequences are packaged. DNA is packaged by wrapping it around specific proteins called histones to generate bead-like structures called nucleosomes. Strings of nucleosomes are then further folded to condense the underlying DNA and make it less accessible. Structures called heterochromatin are thought to be particularly effective at compacting strings of nucleosomes and turning off the underlying genes. A few years ago it was discovered that proteins named HP1 proteins, which are core components of heterochromatin, can sequester DNA into droplets that are separated from the surrounding solution in a different phase. This discovery provides a novel way to think about DNA packaging while also raising new fundamental questions such as: how are these droplet-based DNA compartments regulated by cellular signals and; how do changes in droplet mediated DNA organization impact biology at the level of a whole animal? To address these questions the PIs have assembled a multi-disciplinary team that brings together expertise in mouse biology, cutting-edge imaging technology and advanced biophysical methods. Another key goal of the project is to provide middle school students from underrepresented communities hands-on experience in carrying out experiments with packaged DNA. The research will: (i) shed light on how small collections of molecules can drive heritable changes at the level of a whole animal and; (ii) introduce middle-school students to the wonders of scientific discovery.

A major form of heritable gene regulation is driven by heterochromatin, which silences specific subsets of genes and is essential for cellular differentiation, environmental adaptation and organismal physiology. The discovery that heterochromatin can form by phase-separation based mechanisms have led to a new paradigm for imagining genome organization, in which phase-separation enables genome sequestration. Given the novelty of the findings many fundamental questions remain unanswered such as: (i) what are the physico-chemical rules underlying phase-separation by heterochromatin; (ii) what types of emergent properties are conferred by phase-separation and; (iii) what are the physiological consequences? Addressing these questions requires working at the intersection of multiple disciplines. Therefore this project is organized within the physiological context of mouse olfactory receptor regulation as studied by Dr. Lomvardas and uses new imaging technologies pioneered by Dr. Larabell and biophysical tools developed by Dr. Narlikar. The project integrates experimental enquiry across multiple scales, from atomic-level studies of HP1 behavior to assessment of whole mouse phenotypes. Specifically the PIs will study olfactory receptor (OR) expression in mice, which is controlled by specialized heterochromatic compartments (ORH). ORH is spatially distinct from heterochromatin formed near centromeres (PH), strongly indicative of two different phase-separated states. ORH and PH are enriched for different HP1 paralogs, HP1 and HP1, respectively. Using a combination of mouse genetics, soft-Xray tomography and quantitative phase-separation methods the PIs will investigate (i) whether ORH and PH have different physico-chemical properties that prevent mixing and enable distinct physiological functions and, (ii) whether these different properties may arise from differences in the substructures formed within HP1 vs. HP1 phases. The PIs anticipate these studies will illuminate how atomic scale differences in sequence between HP1; and HP1; result in meso-scale differences in droplet structure, which in turn have a defined physiological impact. A key goal of the project is also to provide middle school students a hands-on experience in heterochromatin based phase-separation experiments through an annual summer workshop.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

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J.D. Swanson
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Columbia University
New York
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