Regulation of the transcription cycle by co-ordinate interaction of ATP-dependent chromatin remodelling and histone post-translational modifications.

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The development and identity of all cells in the body is determined by a set of instructions encoded in genes, found in DNA in the cell nucleus. 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, such as humans, DNA is folded and compacted into manageable units by wrapping like thread around a spool composed of proteins called histones, to form chromatin. These manageable units, called nucleosomes, offer an additional level of information to the cell as the histone proteins at their core can be selectively modified by the incorporation of acetate, phosphate, methyl or other chemical groups. These histone post-translational modifications (HPTMs) can act as so-called epigenetic "marks" to encode additional information in the genome. These marks can function to tether or recruit enzyme complexes that either silence or allow gene expression. By varying the distribution of histone post-translational modifications, and their ability to be read or decoded, genes can be selectively turned off or on in cells, controlling the development and function of cells. We seek to understand how this occurs as many human diseases, such as cancers and lymphomas, are triggered by altered or disordered gene expression. By understanding these epigenetic mechanisms of gene regulation, new therapies to cure disease can be developed. In our research, we use both human cell lines and the "model organism" Drosophila melanogaster (fruit flies). Although at first glance it may not seem so, Drosophila and humans have evolved from a common ancestor and thus share many design principals. A useful analogy is to compare a high-performance racing car and a child's go-kart. Although one is more sophisticated, the basic elements of control - steering and brakes - are the same. In the same way, 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 interpret epigenetic marks to determine their function in gene regulation. In this study we will determine how the distribution of a key epigenetic regulator called NURF is affected by histone post-translational modifications. We will determine how NURF then alters the positions of nucleosomes. By changing the position of nucleosomes NURF can affect the interaction of RNA polymerase (the enzyme that "expresses" genes) with DNA. To do this we will use a technique called chromatin immunoprecipitation (ChIP) to fish-out regions of the Drosophila genome to which NURF is targeted and identify these by determining their DNA sequence using a DNA sequencer that can sequence millions of DNA fragments in one go. We will examine whether NURF recruitment correlates with the presence of histone-modifications. Subsequently we will determine whether NURF recruitment to these regions is able to affect the activities of RNA polymerase by using the same technique of ChIP-Seq to map the distribution of RNA polymerase on genes in cells that contain or lack the NURF regulator and the histone modifications to which NURF can bind.