Aims
The human genome exists as a folded, physical structure within the nucleus of each cell. Whereas some aspects of the genome’s architecture are likely to be relatively stable, many spatial features are dynamic, representing both reflections of, and determinants of, a cell’s state. Although the past few years have seen incredible progress, there is an acute need to improve the reliability and resolution of experimental methods for characterizing genome architecture, for advancing computational modeling of the nucleome in space and time, and for integrating this information with key cellular functions. Today we know almost nothing about how the configuration of the 4D nucleome influences health and disease.
The overall aims of the University of Washington Center for Nuclear Organization and Function (UW-CNOF) are as follows:
The overall aims of the University of Washington Center for Nuclear Organization and Function (UW-CNOF) are as follows:
Aim 1To develop and validate robust methodologies for the mapping of 4D nucleomes with high resolution and minimal bias, including from large numbers of single cells. We recently developed a new protocol, termed DNase Hi-C, that has considerably higher resolution and lower input requirements than the conventional restriction enzyme-based Hi-C protocol. We will optimize DNase Hi-C for robustness, scalability and exportability, as well as to further reduce its input requirements. We will also reduce to practice and optimize a novel combinatorial indexing strategy for generating DNase Hi-C data on very large numbers of single cells. Finally, we will develop methods for concurrently measuring genome architecture (DNase Hi-C) and transcriptional output (RNA-seq) within each of many single cells.
Aim 3To biologically validate experimental maps and computational models of the 4D nucleome. We will validate our maps and models by high resolution FISH and by functional studies in several systems: in vivo tissues and cell lines from interspecific F1 hybrid mice, to distinguish alleles; directed differentiation of mouse ESCs or skeletal myoblasts; and human cell lines including K562 (ENCODE Tier 1) and Hap1 (haploid). We will also test specific predictions of the models in response to targeted (Crispr/Cas9-based genome editing) or large-scale (Xist-mediated silencing of entire chromosomes) perturbations. Aspects of the maps and models that will be validated include predicted intra- and inter-chromosomal interactions, perturbations of sequence determinants of nuclear architecture, and analyses of the dynamic relationship between nuclear architecture and cellular processes including the cell cycle and differentiation.
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Aim 2To build models for visualizing and quantifying 4D nucleomes in space and time. We will develop innovative computational methodologies in three areas. First, we will advance our existing statistical inference procedure for estimating 3D architecture to explicitly model diploidy and to probabilistically represent heterogeneity within populations of cells. Second, we will adapt methods that we developed for pseudotemporal analysis of single cell RNA-seq data to single cell DNase Hi-C data, to enable modeling of the dynamics of nuclear architecture over the course of the cell cycle or a process of differentiation. Finally, we will build predictive models that integrate the 4D nucleome with existing 1D genomic datasets, thereby linking nuclear architecture to DNA replication, transcription factor binding, and gene expression.
Aim 4To demonstrate that maps and models of the 4D nucleome are useful for advancing our understanding of fundamental biology as well as of disease states. We will develop models and generate data for three systems to validate the utility of understanding the 4D nucleome. First, we will define the dynamics of nuclear architecture in bulk populations and in single cells during the directed differentiation of naïve human embryonic stem cells (ELF1) into cardiomyocytes and endothelial cells. Second, we will test the hypothesis that cardiomyopathy-inducing mutations in the nuclear scaffolding protein, lamin A, are associated with derangements in cardiomyocyte nuclear architecture. Finally, we will determine the changes in human cardiomyocyte nuclear architecture induced by trisomy 21 (Down Syndrome).
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