Our laboratory studies the spatial organization of the genome, with implications for gene regulation, genome integrity, and diseases such as cancer, aging, and neurodegenerative disorders. We use Drosophila and mammals in combination with cellular, molecular, genetic, and computational tools to elucidate how chromosomes are functionally organized in 3-D space and time. We also develop and utilize new technologies that use fluorescent in situ hybridization (FISH) to interrogate chromosome positioning at single-cell resolution. These include a pipeline for high-throughput FISH (Hi-FISH), and a new type of probe, called Oligopaints, which reduces the cost and increases the resolution of FISH.
CHROMOSOME TERRITORIES AND GENOME INTEGRITY
The major feature that stands out in the nucleus of all metazoan genomes are the chromosome territories (CTs), which have nonrandom radial positions that are conserved through evolution. We are using our custom Oligopaint FISH probes to identify signatures of chromosome arrangement across different cell cycle stages, tissues, and developmental time points in both Drosophila and mammalian cells. Our experiments allow us to integrate levels of chromatin compaction, chromosome intermingling, and the epigenetic state of large domains in a single-cell and genome-wide manner. Using this approach, we are dissecting the contributions of putative architectural proteins to the radial positioning and folding of chromosomes as well as their function in genome integrity.
HIGH-THROUGHPUT FISH-BASED SCREENING
Although chromosome interactions are a fundamental aspect of nuclear organization little is known about how they are established, regulated, and inherited across cell divisions. To address this gap, we developed a fully automated FISH-based imaging pipeline, called Hi-FISH, to quantitatively determine the position and interaction frequency of multiple loci in the nucleus. This technique enables us to image thousands of cells and use special statistical methods to determine whether and to what extent a variable (RNAi, CRISPR, drug, etc) alters chromosome organization and function. Recently, we have shown the potential of Hi-FISH by conducting the first FISH-based whole-genome RNAi screen for somatic pairing factors in Drosophila, using their well-established capacity to form these interactions as a model for chromosome interactions in general. Collectively, we isolated 105 cellular factors, many of which not previously implicated in genome organization. We are currently characterizing the function of these candidates and are also further enhancing our Hi-FISH method to screen for additional features of nuclear organization.
SPATIAL ORGANIZATION OF THE EPIGENOME
The emerging view is that interactions between regulatory elements and promoters are insulated within extensive self-interacting units termed topologically associated domains (TADs), which are further positioned in the nucleus to spatially segregate active and silent chromatin. Proper targeting of sequences to their respective chromatin environment is necessary for genome stability and gene regulation. We have expanded our high-resolution chromosome paints to better visualize epigenome organization across individual chromosomes and are using this tool to study the regulation of architectural proteins and their effects on nuclear, cellular, and physiological phenotypes.