Job Dekker - US grants

PROJECT SUMMARY ? Additional Tool Development or Data Generation The 3D organization of chromosomes is central to gene regulation and to all aspects of chromosome dynamics. Recent 3C and Hi-C analyses have provided unprecedented detail about the 3D arrangement of chromosomes, and the Center will extend these analyses to provide 4D information. However, there are limitations to 3C/HiC data: first, it provides relative rather than absolute proximity information for gene loci. Second, 3C-based methods tend to have low signal-to-noise ratios, in part due to confounding non-specific contributions of sequence-based propinquity between genetically linked loci. Third, 3C by itself does not provide information about what other molecules (especially proteins and RNAs) are near genetic loci. Therefore, it is essential to develop new tools that provide additional 4D nucleosome organization data sets to both validate and complete the information provided by locus-juxtaposition maps. To do this we will introduce three new tools aimed at providing additional nucleome-mapping data. The first (Aim 1) will be pre- fragmentation of genomes followed by 3C analysis, which we expect to suppress non-specific contacts while maintaining long-range specific contacts. In addition to providing improvements in 3C signal:noise ratios, this approach promises to provide a cost-effective way to generate catalogs of specific looping interactions throughout the genome. The second approach (Aim 2) will be to develop the means to map nuclear components in a completely 3C-orthogonal manner by exploiting the RNA-guided genome binding capacity of nuclease-dead Cas9 (dCas9). In particular, we will adapt dCas9 for spatially restricted proximity labeling, allowing the biotinylation of both proteins and RNAs near specific types of nucleome structural elements. This has the potential to yield spatial position information as well as local protein/RNA enrichment near specific loci, providing insight into the factors that drive or respond to dynamic genome structure. Finally, we will develop new tools for direct, 3C-independent, microscopic visualization and physical study of chromatin in both its mitotic (Aim 3) and interphase (Aim 4) forms. Using micropipette-based microsurgery and microdissection, we will extract metaphase chromosomes and nuclei for combined enzymatic/mechanical study, using Cas9-fusion- protein technology to provide fluorescent labeling of specific chromatin loci. Using multiple labels we will directly test locus-juxtaposition data from Hi-C experiments, and we will be able to study the way that mechanical perturbation of chromosomes and genomes affects relative locus positioning to provide a test of the theoretical models developed in Component 3 of the overall proposal. Collectively, the novel tools that we develop and apply in this component will advance the field of nucleomics by connecting genome structure to the associated proteome and transcriptome, providing unique data on the mechanics of chromosomes and nuclei, and an initial glimpse of how the results of Hi-C and single-cell data correlate with each other.

Summary The three-dimensional organization of the genome is critical for regulation of gene expression, maintenance of genome stability and chromosome inheritance. Over the last several years there has been a tremendous increase in our knowledge of the spatial arrangements of chromosomes, and this is leading to insights into the molecular mechanisms that regulate genes, and how defects in genome folding can lead to human disease. We have developed powerful molecular and genomic technologies based on chromosome conformation capture (3C, 5C, Hi-C) to probe the three-dimensional structure of chromosomes. As cells go through the cell division cycle chromosomes alternate between two entirely different spatial conformations. We and others have used 3C-based assays to determine the structure of the human genome in interphase and in metaphase. In interphase the genome is composed of several different types of chromosomal domains, while within these domains genes are regulated by specific looping interactions between genes and their regulatory elements. A different structure is observed in mitotic cells, when chromosomes become highly compacted. We discovered that in mitosis chromosomes fold as linear arrays of consecutive chromatin loops. We have delineated a series of folding intermediates that show how the interphase conformation is converted into the metaphase state. These intermediates include extended linear loop arrays in prophase and more compacted helical arrays of nested loops in prometaphase. These studies lead to important new questions that we aim to address. First, it is not known in genomic detail how during prophase the interphase state is erased, chromosomes form initial loop arrays and sister chromatids become separated. Second, very little is known about the molecular machines that fold chromosomes. We propose innovative new strategies to identify new components of these machines that act during mitosis and interphase. Third, we hypothesize that these machines act through specific cis-elements that determine how and where they get loaded onto chromosomes, move to new sites and accumulate at yet other sites. We will identify and characterize these DNA elements that encode how the genome folds. Our proposed studies will uncover how the genome folds, unfolds and refolds.