Control of insulator function and higher order genome organisation by the chromatin remodeling enzyme NURF

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The development of all cells in the body is determined by a set of instructions encoded in genes, found in DNA. All cells contain the same information. The immense variety of cell types in the human body, each with distinct functions is achieved by changing the way this information is read, or "expressed". In eukaryotes, the large amount of DNA required for correct development is compacted into manageable units by wrapping like thread around a protein spool to form structures called nucleosomes. In addition to compacting DNA, nucleosomes provide additional levels of so-called epigenetic information. By varying nucleosome position, access to gene control elements can be regulated and genes turned off or on, changing cell identity and function. Moreover, nucleosomes are the fundamental units in higher orders of genome organisation through which functional interactions between often-distant gene regulatory elements are stabilized, allowing correct patterns of gene expression. As such changes in nucleosome organisation can profoundly affect genome organisation and gene regulation. In our laboratory we study how nucleosome organisation is altered by chromatin remodeling enzymes. These large protein complexes use ATP, the cells energy transfer molecule, to change nucleosome position, allowing the wholesale reorganization ("remodeling") of genome architecture. We seek to understand how these complexes work as many human diseases are triggered by altered or disordered gene expression and global genome organization. By understanding the epigenetic mechanisms underlying genome organisation and gene regulation, new therapies to cure disease can be developed. In our research, we use the "model organism" Drosophila melanogaster (fruit flies). Although not immediately be apparent, Drosophila and humans have evolved from a common ancestor and thus share many design principles. A useful analogy is to compare a high-performance racing car and a child's go-kart. While one is more sophisticated, the basic elements of control - steering and brakes - are the same. Similarly, Drosophila uses many of the same mechanisms to control gene expression as humans. As such, we can use Drosophila as a stand-in for humans, a so-called "model organism". This is useful as it allows us to do experiments that are impossible or unethical in humans, for example deliberately deleting or altering genes to determine their role in development. In our work we use fly strains in which we can tag, alter or delete ("knock-out") the protein complexes that establish and regulate genome organisation to determine their function in gene regulation. In this study we will determine functions of a key chromatin remodeling enzyme called NURF in genome organisation. Drosophila provides an especially powerful system to characterize NURF, as NURF is only present in multicellular eukaryotes precluding studies in simpler model organisms like yeast. In our research we will determine how NURF regulates genome interactions critical for higher order chromatin organisation. To do this we will use variants of a technique called chromosome conformation capture (called ChIA-PET) to fish-out NURF complexes from nuclei. We can then co-purify regions of the genome brought into close physically proximity by NURF and identify these by determining their DNA sequence. Through genetic ablation of NURF and co-factor proteins at these sites we will determine the functional importance of NURF in establishing these regulatory genome interactions. This method will provide a high-resolution view of higher order regulatory genome interactions but requires many millions of cells to perform thus generating a snapshot of interactions averaged over many cells. To capture the dynamics of remodeler genome interactions in single cells over short time scales genome interactions we will also deploy advanced microscopy techniques that allow imaging of single remodeler and insulator molecules in live cells.